Novel PD1-targeted IL-15 immunocytokines and VITOKINE fusions

Abstract

The present disclosure provides novel PD1 Ab-IL-15 immunocytokines and VitoKine compositions that aim to target attenuated or bioactivatable IL-15 directly to tumor-infiltrating lymphocytes to reduce systemic mechanism-based toxicity and lead to broader therapeutic utility of IL-15 for the treatment of cancer. The attenuated potency of IL-15 in the PD1 Ab-IL-15 immunocytokine improves target selectivity, facilitates the establishment of a stoichiometric balance between the cytokine and antibody arms, and helps to alleviate pathway overactivation and mitigate antigen sink and target-mediated deposition. The IL-15 in PD1 Ab-IL-15 VitoKine remains activated locally by proteases upregulated in diseased tissues, preventing overactivation of pathways and reducing undesirable "on-target" and "off-tissue" toxicity, as well as significantly reducing potential antigen or target sinks, and therefore resulting in a prolonged in vivo half-life and improved biodistribution, bioavailability, and therapeutic efficacy. In both PD1 Ab-IL-15 immunocytokine and VitoKine, PD1 antibodies with the ability to block PD1 and reverse T cell anergy or exhaustion may further cooperate with IL-15 anti-cancer immune responses.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 404,619, filed September 8, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (SeqListing-CUGENE PD1 AB-IL-15.xml; size: 181 kb; created on: September 5, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field While cancer has traditionally been treated with chemotherapy, radiation, targeted therapy, and surgery, the recent emergence of immunotherapy, a fifth pillar of cancer treatment, has transformed the fight against cancer. The benchmark for immunotherapeutic drugs has been established with the development of T cell checkpoint (CTLA-4 and PD1 / PD-L1) inhibitors. These treatments have been demonstrated to 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. [Background technology]

[0004] Recently, interleukin-15 (IL-15), a member of the four α-helical bundle family of cytokines, has emerged as a potential immunomodulatory agent for the treatment of cancer. IL-15 binds to its specific receptor, IL-15Rα, and transactivates a heterodimeric receptor complex composed of IL-15Rβ and the common cytokine receptor γ chain (γc) on responding cells to initiate signal transduction. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B, and natural killer (NK) cells. It also inhibits CD8 + Enhanced cytolytic activity of T cells and prolonged antigen-experienced CD8 + CD44 hiInduce memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces the maturation of dendritic cells. Therefore, it has been hypothesized that boosting IL-15 activity may enhance innate and adaptive immunity to fight tumors, making IL-15 a promising agent for anti-cancer therapy (Steel et al., Trends Pharm Sci 33:35-41, 2012). Recombinant IL-15 and IL-15 in various fusion formats are being tested in several ongoing oncology clinical trials, but none have been approved for use to date.

[0005] Despite these new advances in using IL-15 as a cancer immunotherapeutic agent to augment immune responses, limitations remain to the effective use of IL-15 as a therapeutic agent. For example, IL-15 has a short half-life (<40 minutes), resulting in 1) low bioavailability that hinders its in vivo antitumor efficacy, and 2) the requirement for high dose administration to achieve adequate therapeutic exposure, resulting in toxicity.

[0006] Several approaches have been taken to overcome the inherent challenges of IL-15 immunotherapy. One such approach to combat systemic toxicity involves localizing cytokine activity to cancer cells and their surrounding tissues through tumor-targeted IL-15 immunocytokines, constructed by fusing IL-15 to antibodies specific for tumor-associated antigens. However, this strategy lacks the ability to specifically target effector T cells within the tumor microenvironment (TME), which are relevant for anti-cancer immunity. This gap in intratumoral T cell targeting can be filled by fusing IL-15 to anti-programmed cell death protein 1 (PD1) antibodies. PD1 (also known as CD279) is highly expressed on tumor-infiltrating lymphocytes (TILs), and PD1 antibody-IL-15 immunocytokines allow IL-15 to be directly targeted to TILs. It has shown increased avidity for intratumoral CD8+ T cells, but not for Treg cells or peripheral CD4+ and CD8+ T cells. This strategy therefore further improves IL-15 anti-cancer immunity while reducing systemic toxicity.

[0007] In addition to directly targeting IL-15 to TILs to improve IL-15 anti-cancer immunity, PD1 antibodies capable of blocking PD1 and reversing T cell anergy or exhaustion can cooperate with IL-15 to further boost the anti-cancer immune response. Therefore, it is desirable to construct a PD1 Ab-IL-15 immunocytokine with a PD1 antibody that has excellent target binding and PD1 blocking capabilities. Among the various globally available PD1 blocking antibodies that have revolutionized the field of cancer immunotherapy, pembrolizumab (Keytruda®; Merck Sharp & Dohme Corp.) has received significant attention due to its high efficacy and approval for treating various cancer types. Although pembrolizumab exhibits excellent target binding and blocking capabilities, it has several sequence disadvantages, including a relatively low degree of affinity, which may raise immunogenicity concerns, and high hydrophobicity, which tends to increase its aggregation tendency. Therefore, it is preferable to optimize pembrolizumab to mitigate these sequence disadvantages while fully maintaining its biological activity. The resulting optimized sequence is expected to improve the developability of PD1 Ab-IL-15 immunocytokines.

[0008] Importantly, fusion of PD1 Ab with a fully active IL-15 moiety can abolish the intended antibody-mediated targeting, causing the fusion protein to localize to IL-15 receptor-expressing cells in the periphery instead of TILs in the tumor. Therefore, to improve target specificity and selectivity, one approach is to prepare fusions using an IL-15 moiety with attenuated IL-15Rβγ activity to establish a stoichiometric balance between the cytokine and antibody components. Additionally, reducing cytokine potency could potentially alleviate pathway overactivation as well as alleviate antigen sink and target-mediated deposition.

[0009] Another related but more sophisticated strategy for improving target specificity and selectivity is to apply the VitoKine platform disclosed by the present inventors in WO 2019246392 and WO 2021119516. In the VitoKine construct, the activity of the IL-15 moiety remains inactive or minimal until locally activated by proteases upregulated in or around the tumor. By doing so, binding of the IL-15 moiety to its receptor in the periphery or on the cell surface of non-diseased cells can be significantly limited. This can help prevent pathway overactivation and reduce undesirable "on-target" and "off-tissue" toxicity, and VitoKine's improved safety profile may allow for human dose levels within the effective range of PD1 antibodies. Additionally, the inactivity of the IL-15 moiety prior to protease activation significantly reduces potential antigens or target sinks and therefore results in a prolonged in vivo half-life and improved biodistribution and bioavailability at the intended site of treatment. Summary of the Invention

[0010] Disclosure of the Invention In one embodiment, the present invention provides a novel PD1-targeting, bioactivatable IL-15 immunocytokine (referred to herein as PD1 Ab-IL-15 VitoKine) designed to target bioactivatable IL-15 directly to tumor-infiltrating lymphocytes. The activity of the IL-15 moiety remains largely inactive or minimal until locally activated by proteases upregulated in the tumor, which limits the binding of the IL-15 moiety to receptors in the periphery or on the cell surface of non-diseased cells or normal tissues. This can help prevent pathway overactivation and reduce undesired "on-target" and "off-tissue" toxicity, as well as minimize unwanted target sinks.

[0011] In another aspect, the present invention provides a novel PD1-targeting IL-15 immunocytokine that aims to directly target the activity-modulated IL-15 domain to tumor-infiltrating lymphocytes. The attenuated IL-15 activity is expected to facilitate the establishment of a stoichiometric balance between the cytokine and antibody arms, help alleviate pathway overactivation, and mitigate antigen sink and target-mediated deposition.

[0012] The strategy specifically targets effector T cells within the tumor microenvironment (TME) that are relevant to anti-cancer immunity. By implementing this strategy, the ability of IL-15 to expand lymphocyte populations and increase their effector function is combined with the function of PD1-blocking antibodies in reversing T cell anergy or exhaustion. This approach, particularly when attenuated or bioactivatable IL-15 is used, reduces systemic mechanism-based toxicity, leading to broader therapeutic utility of IL-15 for cancer treatment, as well as improving biodistribution and bioavailability at the intended site of treatment.

[0013] In various embodiments, the PD1-targeting bioactivatable IL-15 immunocytokine is referred to herein as a PD1 Ab-IL-15 VitoKine. In various embodiments, the VitoKine platform disclosed by the inventors in WO 2019246392 and WO 2021119516 is defined by the construct depicted in Figures 1A and 1B. In various embodiments, the PD1 Ab-IL-15 VitoKine of the present invention is more particularly defined by the construct shown in Figures 1C and 1D. Referring to Figure 1C, the PD1 Ab-IL-15 VitoKine comprises a PD1-blocking antibody (targeting partial domain; D1), a dimeric IL-15 domain (active partial domain; D2) whose N-terminus is fused to the C-terminus of the homodimeric heavy chain of the PD1 antibody via an L1 linker, and whose C-terminus is fused to the N-terminus of the IL-15Rα sushi domain (hidden partial domain; D3) via an L2 linker. Referring to Figure ID, PD1 Ab-IL-15 VitoKine was similarly constructed, except that the PD1 antibody contained a knob-into-hole heterodimeric heavy chain pair and a monomeric IL-15 domain was fused to the knob heavy chain. In various embodiments, a proposed method of VitoKine activation is depicted in Figure 2.

[0014] In various embodiments, the variable domains of the PD1-blocking antibodies of the invention were optimized from the variable domain of pembrolizumab by introducing germline sequence substitutions into CDR residues, introducing germline sequence substitutions into framework somatic mutations, and / or adopting the most prevalent and better-behaved VH3 human germline family sequences as acceptor frameworks. In various embodiments, the PD1-blocking antibodies have high affinity for the human PD1 protein set forth in SEQ ID NO: 1, function to inhibit PD1 with equal or comparable potency to pembrolizumab, exhibit a higher sequence similarity score to its closest human germline sequence than pembrolizumab, thereby indicating an improved degree of humanity, and are predicted to have lower hydrophobicity than pembrolizumab, which in turn reduces the tendency to aggregate.

[0015] In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:7. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:9. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:11. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:13. In various embodiments, a PD1 blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:18.

[0016] In various embodiments, the PD1-targeted IL-15 immunocytokine is defined by the construct depicted in Figure 3. In various embodiments, the PD1-targeted IL-15 immunocytokine comprises an IL-15RαSushi+ domain having the sequence set forth in SEQ ID NO: 165 non-covalently complexed with IL-15. In various embodiments, the potency-modulating IL-15 of the PD1-targeted IL-15 immunocytokine is an IL-15 variant (or mutant) comprising a sequence derived from the sequence of a mature human IL-15 polypeptide (also referred to herein as huIL-15 or IL-15 wild-type (w / t) set forth in SEQ ID NO: 116) that contains one or more amino acid substitutions, deletions, or insertions. In various embodiments, the IL-15 variant has reduced signaling activity (EC) compared to the native IL-15 polypeptide. 50 and / or E max) The amino acid changes can include one or more amino acid substitutions, deletions, or insertions in the IL-15 polypeptide, for example, in the domains of IL-15 that interact with IL-15Rβ and / or the common cytokine receptor gamma chain (γc). In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 30, 32, 63, 68, 108, 109, or 112 of SEQ ID NO:116. In various embodiments, the amino acid change is a substitution of D for T at position 30 of the mature human IL-15 sequence, a substitution of H for D or E or N or Q at position 32, a substitution of V for F or A or K or R at position 63, a substitution of I for F or H or D or K or Q or G at position 68, a substitution of Q for A or D or E or F or H or K or L or M or N or S or T or Y at position 108, a substitution of M for A or H or R at position 109, a substitution of N for D or G or P or R at position 112, or any combination of these substitutions. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4 amino acids at the N-terminus of SEQ ID NO:116. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids at the C-terminus of SEQ ID NO:116. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions. In various embodiments, the attenuated IL-15 portion is selected from the group of sequences set forth in SEQ ID NOs: 117-163.

[0017] In various embodiments, the active portion of the PD1 Ab-IL-15 VitoKine is an IL-15 domain comprising the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO: 116. In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO: 116, comprising one or more amino acid substitutions, deletions, or insertions. In various embodiments, the IL-15 variant demonstrates increased signaling activity compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant demonstrates reduced signaling activity compared to the native IL-15 polypeptide. The amino acid changes can include one or more amino acid substitutions, deletions, or insertions in the IL-15 polypeptide, for example, in the domains of IL-15 that interact with IL-15Rβ and / or γc. In various embodiments, the amino acid changes are one or more amino acid substitutions at positions 30, 32, 63, 68, 108, 109, or 112 of SEQ ID NO: 116. In various embodiments, the amino acid change is a substitution of D for T at position 30 of the mature human IL-15 sequence, a substitution of H for D or E or N or Q at position 32, a substitution of V for F or A or K or R at position 63, a substitution of I for F or H or D or K or Q or G at position 68, a substitution of Q for A or D or E or F or H or K or L or M or N or S or T or Y at position 108, a substitution of M for A or H or R at position 109, a substitution of N for D or G or P or R at position 112, or any combination of these substitutions. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4 amino acids at the N-terminus of SEQ ID NO:116. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids at the C-terminus of SEQ ID NO:116. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions.In various embodiments, the VitoKine construct utilizes an IL-15 variant with optimally attenuated potency, thereby leading to a reduction in the intrinsic basal activity of the corresponding VitoKine construct. In various embodiments, the IL-15 variant in the VitoKine construct can tune the IL-15 VitoKine intrinsic basal activity to achieve an optimal balance between desired anti-tumor efficacy and unwanted systemic toxicity. In various embodiments, the IL-15 domain of the PD1 Ab-IL-15 VitoKine is selected from the group of sequences set forth in SEQ ID NOs: 116-163.

[0018] In various embodiments, the cryptic partial domain of the PD1 Ab-IL-15 VitoKine is the cognate receptor / binding partner or any binding partner identified for IL-15. In various embodiments, the cryptic partial domain is the IL-15Rα extracellular domain or a functional fragment thereof. In various embodiments, the IL-15Rα extracellular domain or a functional fragment thereof is the IL-15Rα Sushi+ domain having the sequence set forth in SEQ ID NO: 165.

[0019] In various embodiments, the L1 linker and L2 linker of the PD IL-15 VitoKine are both protease-cleavable peptide linkers. In various embodiments, the L1 linker is a protease-cleavable peptide linker, and L2 is a non-cleavable peptide linker. In various embodiments, the L1 linker is a non-cleavable peptide linker, and L2 is a protease-cleavable peptide linker. In various embodiments, the L1 linker and L2 linker of the PD1 Ab-IL-15 VitoKine construct are both protease-non-cleavable peptide linkers. In various embodiments, the non-cleavable linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, or 90% or more of the amino acids in the linker are G or S). Each peptide linker sequence can be independently selected. In various embodiments, the protease-cleavable linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77. In various embodiments, the protease-cleavable linker can have an 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 ends of the cleavable linker. In various embodiments, the L1 and L2 linkers of the PD1 Ab-IL-15 VitoKine construct are both protease-non-cleavable peptide linkers. In various embodiments, the protease-cleavable linker having a peptide spacer of variable length on either the N-terminus or the C-terminus or on both ends 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 various lengths.

[0020] In various embodiments, the IL-15 domain (D2) and IL-15Rα domain (D3) of the VitoKine construct are placed C-terminal to the PD1 Ab domain (D1) as depicted in Figure 1A. In various embodiments, the D2 and D3 domains of the VitoKine construct are placed N-terminal to the PD1 Ab domain (D1) as depicted in Figure 1B.

[0021] In various embodiments, the PD1 blocking Ab, IL-15 domain, and IL-15Rα domain of the PD1 Ab-IL-15 VitoKine construct can be monomeric, or dimeric (as shown in FIG. 1C), or a combination of dimeric and monomeric, e.g., the PD1 blocking Ab is dimeric, and the IL-15 domain and IL-15Rα domain are monomeric (as shown in FIG. 1D).

[0022] 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 composition of the present invention to a subject in need of cancer or cancer metastasis treatment.In one embodiment, the subject is a human subject.In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

[0023] In another aspect, the present disclosure provides a method of treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second treatment selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy including chimeric antigen receptor (CAR)-T, CAR-NK, induced pluripotent stem cell (iPS)-derived CAR-T or iPS-derived CAR-NK, and a vaccine, e.g., Bacillus Calmette-Guerin (BCG). In various embodiments, the combination therapy includes treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using agonist, antagonist, or blocking antibodies against costimulatory 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; treatments using bispecific T cell engaging antibodies (BiTE®), such as blinatumomab; treatments involving administration of biological response modifiers, such as IL-12, IL-151, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatments using therapeutic vaccines, such as sipuleucel-T; 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 immunostimulants, such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccines, such as BCG; the combination treatment provides increased effector cell killing of tumor cells, i.e., synergism exists between the pharmaceutical composition of the present invention and the immunotherapy when administered in combination.

[0024] In another aspect, the present disclosure provides a use of a pharmaceutical composition of the present invention for the preparation of a medicament for the treatment of cancer.

[0025] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the pharmaceutical composition of the present disclosure. In another aspect, the present disclosure provides a vector comprising a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method of producing a pharmaceutical composition of the present disclosure by culturing a host cell under conditions that promote expression of a protein or polypeptide is provided.

[0026] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated pharmaceutical composition of the present invention in admixture with a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 depicts representative VitoKine construct formats. (A) A VitoKine construct in which the active (D2) and masking (D3) domains are placed C-terminal to the targeting domain (D1). (B) A VitoKine construct in which the D2 and D3 domains are placed N-terminal to the D1 domain. (C) A representative PD1 Ab dimeric IL-15 VitoKine format. (D) A representative PD1 Ab monomeric IL-15 VitoKine format. [Figure 2]Figure 2 depicts the proposed activation mechanism for the PD1 Ab-IL-15 VitoKine construct. The exemplary VitoKine construct contains two protease-cleavable linkers; protease 1 activation resulting from cleavage of the L1 linker results in active form 1; protease 2 activation resulting from cleavage of the L2 linker results in active form 2; and activation by both proteases resulting from cleavage of the L1 and L2 linkers results in active form 3. After protease cleavage, the IL-15Rα Sushi domain (D3) remains noncovalently complexed with the IL-15 domain (D2). When the L1 linker is the only protease-cleavable linker, active form 1 is the only activated format. Similarly, when the L2 linker is the only protease-cleavable linker, active form 2 is the single activated format. [Figure 3] Figure 3 depicts the structure of a PD1-targeted IL-15 immunocytokine with the IL-15 domain as either a monomer (A) or dimer (B), and an IL-15 Fc fusion protein with the IL-15 domain as either a monomer (C) or dimer (D). All configurations contain IL-15Rα noncovalently complexed with IL-15. [Figure 4] Figure 4 depicts a comparison of PD1-blocking activity between a reference antibody (P-0734) and a pembrolizumab (PBL) biosimilar in a luciferase reporter assay. Figures 4A and 4B depict the dose-dependent increase in luminescence signal and fold induction, respectively. P-0734 and the PBL biosimilar share identical variable domains and have IgG1 and IgG4 isotypes, respectively. [Figure 5]Figure 5 depicts (A) ELISA binding and (B-C) PD1 blocking activity of PD1 blocking antibodies P-1148, P-1150, P-1151, and P-1153 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figure 5B and Figure 5C depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 6] Figure 6 depicts the PD1-blocking activity of PD1-blocking antibodies P-1127, P-1129, and P-1174 compared to a reference antibody (P-0734), which were tested in a luciferase reporter assay and show a dose-dependent increase in luminescence signal. [Figure 7] Figure 7 depicts the PD1-blocking activity of PD1-blocking antibodies, P-1175 and P-1181, compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 7A and 7B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 8] Figure 8 depicts the PD1-blocking activity of PD1-blocking antibodies P-1175, P-1176, P-1177, and P-1178 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 8A and 8B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 9] Figure 9 depicts the PD1-blocking activity of PD1-blocking antibodies P-1198, P-1199, and P-1201 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 9A and 9B depict the dose-dependent increase in luminescence signal and fold induction, respectively. A non-targeting germline antibody was included as a negative control. [Figure 10]Figure 10 depicts the PD1-blocking activity of PD1-blocking antibodies P-1194, P-1201, and P-1238 compared to a reference antibody (P-0734) when tested in a luciferase reporter assay. Figures 10A and 10B depict the dose-dependent increase in luminescence signal and fold induction, respectively. [Figure 11] Figure 11 depicts the binding of PD1-blocking antibodies P-1174, P-1193, P-1198, P-1199, and P-1201 to PD1+ HEK293 cells compared to a reference antibody (P-0734). Figures 11A and 11C depict the dose-dependent increase in the percentage of positive cells, and Figures 11B and 11D depict the dose-dependent increase in mean fluorescence intensity (MFI). [Figure 12] Figure 12 depicts the evaluation of the activity of P-0234 and P-0313 by analyzing their effect on inducing Ki67 expression in CD8+ T cells from fresh human PBMCs using flow cytometry. P-0234 and P-0313 are dimeric IL-15 Fc fusion proteins containing wild-type IL-15 and the S58D variant, respectively. Recombinant Fc proteins serve as negative controls. [Figure 13] Figure 13 depicts the activity assessment of various IL-15 variants by analyzing their effect on inducing Ki67 expression in CD8+ T cells of fresh human PBMCs. These IL-15 variants contain amino acid substitutions that target interaction with IL-15Rβ, including A) a single amino acid substitution at position I68, B) a single amino acid substitution at position V63, and C) a combined mutation at positions V63 and I68 along with counterparts with individual amino acid changes. P-0313, a well-characterized dimeric IL-15 S58D Fc fusion protein, serves as a dimeric wild-type IL-15 control. [Figure 14]Figure 14 depicts the activity assessment of various IL-15 deletion mutants by analyzing their effect on inducing Ki67 expression in CD8+ T cells from fresh human PBMCs. P-0866, P-0867, and P-0868 contain deletions of 1, 2, and 3 amino acids, respectively, at the N-terminus of IL-15. P-0234 is a dimeric wild-type IL-15 control. [Figure 15] Figure 15 depicts a comparison of the effects of IL-15 variants in (A) inducing Ki67 expression in CD8+ T cells from fresh human PBMCs and (B) maintaining proliferation of mouse-derived CTLL-2 cells. These IL-15 variants contain either single amino acid substitutions at positions V63 (P-0771) or I68 (P-0737) or combined mutations at positions V63 and I68 (P-0768, P-0772, and P-0773). [Figure 16] Figure 16 depicts a comparison of the effects of IL-15 variants (A) on inducing Ki67 expression in CD8+ T cells from fresh human PBMCs and (B) on maintaining proliferation of CTLL-2 cells. P-0867 and P-0868 contain deletions of two and three amino acids, respectively, at the N-terminus of IL-15. P-0234 is a dimeric wild-type IL-15 control. [Figure 17] Figure 17 depicts the activity assessment of various IL-15 variants by analyzing their effect on inducing Ki67 expression in CD8+ T cells from fresh human PBMCs. These variants contain an amino acid substitution at position Q108 that targets interaction with the common gamma receptor (γc), including A-C) a single amino acid substitution at Q108, and D) a Q108N mutation combined with another amino acid change at V63 or I68 that interferes with the IL-15Rβ interface. P-0217 is a monomeric wild-type IL-15 control. [Figure 18]Figure 18 depicts a comparison of the effects of IL-15 variants in (A) inducing Ki67 expression in CD8+ T cells from fresh human PBMCs and (B) maintaining proliferation of CTLL-2 cells. These IL-15 variants contain the Q108M ​​mutation to disrupt interaction with γc, along with other amino acid changes at V63 or I68 that interfere with the IL-15Rβ interface. P-0217 is a monomeric wild-type IL-15 control. [Figure 19] Figure 19 depicts the activity assessment of IL-15 variants by analyzing their effect on inducing Ki67 expression in CD8+ T cells from fresh human PBMCs. These variants contain an acid substitution at position N112 that targets the interaction with γc. P-0217 is a monomeric wild-type IL-15 control. [Figure 20] Figure 20 depicts a comparison of the activity of IL-15 variants in different fusion formats, specifically Fc fusions and PD-1 Ab fusions, based on their effect on inducing Ki67 expression on CD8+ T cells in fresh human PBMCs. (A) Both P-0773 and P-0870 are dimeric IL-15 V63A / I68H variant fusion proteins; P-0773 is an Fc fusion, and P-0870 is a PD1 Ab fusion. (B) P-0867 and P-0888 are a similar pair of Fc and antibody fusions, each containing a two-amino acid deletion at the N-terminus of IL-15. P-0234 and P-0313 serve interchangeably as dimeric wild-type IL-15 controls. [Figure 21] Figure 21 depicts a comparison between P-1352, a PD1 Ab-IL15 immunocytokine, and P-1271, the PD1 blocking component of P-1352, in terms of A) PD-1 binding and B) PD-L1 binding inhibition using two ELISA assays. P-1260, a non-targeting germline antibody, was used as a negative control. [Figure 22]Figure 22 depicts the impact of IL-15 valency on the activity of PD1 Ab-IL-15 immunocytokine by analyzing its effect on inducing Ki67 expression in CD8+ T cells of fresh human PBMCs. P-0869 is a PD1 Ab-IL15 immunocytokine containing the dimeric IL-15 V63AA / I68H variant, and P-1266 is its monomeric IL-15 equivalent. [Figure 23] Figure 23 depicts ex vivo activity comparison among three murine PD1 Ab-IL15 immunocytokines, P-1266, P-1295, and P-1296, by analyzing Ki67 expression in CD8+ T cells of fresh human PBMCs. They contain monomeric IL-15 variants with V63A / I68H, Q108N, and I68H / Q108N mutations, respectively, and show varying degrees of reduced activity. P-1284 serves as a format-matched control featuring monomeric wild-type IL-15. [Figure 24] Figure 24 depicts a comparison of two murine PD1 Ab-IL15 immunocytokines, P-1266 and P-1295, based on their effects on peripheral lymphocytes in C57B / L6 mice after a single intraperitoneal injection: A) Ki67 expression in CD8 T cells, B) CD8 T cell counts, C) Granzyme B expression in CD8 T cells, D) Ki67 expression in NK cells, and E) increasing NK cell counts. Blood samples were collected on days 0, 5, 7, 10, and 10 for lymphocyte phenotyping using FACS analysis. A comparison of the effects of these two immunocytokines on mouse body weight is shown in Figure 24F. Data are presented as the mean ± standard error of the mean (SEM). [Figure 25]Figure 25 depicts a comparative analysis of serum concentrations of two murine PD1 Ab-IL15 immunocytokines, P-1266 and P-1295, after a single intraperitoneal injection in C57B / L6 mice. P-1266 and P-1296 contain monomeric IL-15 variants with V63A / I68H and I68H / Q108N mutations, respectively. Blood samples were collected at multiple time points after dosing, and serum concentrations of the compounds were determined using an ELISA assay. [Figure 26] Figure 26 depicts a comparison of the pharmacodynamic effects of a 1.5 mg / kg (mpk) dose of P-1266 and 1.5 and 3.0 mpk doses of P-1296 on peripheral lymphocytes in MC38 tumor-bearing mice. P-1266 and P-1296 are murine PD1 Ab-IL-15 immunocytokines containing monomeric IL-15 variants with V63A / I68H and I68H / Q108N mutations, respectively. Blood samples for analysis were collected 5 days after a single intraperitoneal injection to assess A) increases in Ki67 expression in CD8 T cells, B) increases in Ki67 expression in NK cells, C) CD8 T cell expansion, and D) NK cell expansion. Each group consisted of four mice. [Figure 27] Figure 27 depicts a comparison of the pharmacodynamic effects of 1.5 mpk doses of P-1266 and P-0869 on peripheral lymphocytes in MC38 tumor-bearing mice. P-0869 is a murine PD1 Ab-IL15 immunocytokine containing the dimeric IL-15 V63AA / I68H variant, and P-1266 is its monomeric IL-15 equivalent. Blood samples for analysis were collected 5 days after a single intraperitoneal injection to assess A) the increase in Ki67 expression in CD8 T cells, B) the increase in Ki67 expression in NK cells, C) the expansion of CD8 T cells, and D) the expansion of NK cells. Each group consisted of four mice. [Figure 28]Figure 28 depicts the dose-dependent antitumor efficacy of P-0869, a murine PD1 Ab-IL-15 immunocytokine containing the dimeric IL-15 V63A / I68H variant, in both CT26 and MC38 mouse tumor models. Figure 28A shows the mean CT26 tumor volume ± SEM over time for various treatment groups after two doses of Q10D at varying dosage levels (0.3, 1.0, and 2.0 mg / kg). Additionally, Figure 28A also shows the absence of tumor recurrence after rechallenge implantation of CT26 cells in previously tumor-free mice. This contrasts with the successful regrowth of the same type of tumor in age-matched naive mice as controls. Figure 28B shows the mean MC38 tumor volume ± SEM over time for each treatment group after two doses of Q12D at 0.3 and 1.0 mg / kg. In both tumor models, a vehicle group was included for reference. [Figure 29] Figure 29 depicts a comparison of the antitumor efficacy of the murine PD1 Ab-IL15 V63A / I68H immunocytokine dimeric and monomeric pair, P-0869 and P-1266, in both CT26 and MC38 mouse tumor models. The mean tumor volume ± SEM over time for each group after two doses of Q12D at 1.5 mpk is shown for A) the CT26 model and B) the MC38 model. In both tumor models, a vehicle group was included for reference. [Figure 30] Figure 30 depicts a comparison of the antitumor efficacy of murine PD1 Ab-IL-15 immunocytokines, P-1266 and P-1295, which contain monomeric IL-15 variants with V63A / I68H and Q108N mutations, respectively. The mean tumor volume ± SEM over time for each group after two doses of Q12D at 1.5 mpk is shown for A) the CT26 model and B) the MC38 model. In both tumor models, a vehicle group was included for reference. [Figure 31]Figure 31 depicts the anti-tumor efficacy of P-1296, a murine PD1 Ab-IL-15 immunocytokine containing the monomeric IL-15 I68H / Q108N variant, after two doses of Q12D at different dosage levels. The mean tumor volume ± SEM over time for each group is shown for A) the CT26 model at dosages of 1.5 and 3.0 mpk and B) the MC38 model at dosages of 1.0 and 3.0 mpk. Both tumor studies include a vehicle group for comparison purposes. [Figure 32] Figure 32 depicts an evaluation of the ability of the IL-15 VitoKine platform to conceal functionality by comparing VitoKine activity with their counterpart non-VitoKine fusion proteins in a human PBMC assay using flow cytometry. Exemplary Fc VitoKine (P-0315) and PD1 Ab VitoKine (P-875) and their respective non-VitoKine counterparts, P-0313 and P-0870, were tested for stimulation of Ki67 expression on CD8+ T cells (A and C) and CD56+ NK cells (B and D). [Figure 33] Figure 33 depicts size-exclusion chromatograms of five PD1 Ab-IL-15 VitoKines (P-0874, P-1077, P-1083, P-1084, and P-1085) compared to their non-VitoKine counterparts, P-0869. The five VitoKines differ only in the length and composition of the L2 linker connecting the IL-15 and IL-15Rα Sushi+ domains. [Figure 34] Figure 34 depicts the dose-dependent induction of Ki67 expression on A) CD8+ T cells and B) NK cells after treatment with PD1 Ab-IL-15 VitoKine in fresh human PBMCs. Five PD1 Ab-IL-15 VitoKines (P-0874, P-1077, P-1083, P-1084, and P-1085) differ only in the length and composition of the L2 linker. P-0869 is their non-VitoKine counterpart. [Figure 35]Figure 35 depicts a side-by-side evaluation of the activity of PD1 Ab-IL-15 VitoKines, P-1265 and P-1263, compared to their corresponding non-VitoKine counterparts, P-1266 and P-1295, based on their effect on inducing Ki67 expression on A) CD8+ T cells and B) NK cells of fresh human PBMCs. P-1284 serves as a format-matched control featuring monomeric wild-type IL-15. [Figure 36] Figure 36 depicts a comparison between P-1340, a PD1 Ab-IL15 VitoKine, and P-1271, the PD1 blocking component of P-1340, in terms of A) PD-1 binding and B) PD-L1 binding inhibition using two ELISA assays. P-1260, a non-targeting germline antibody, was used as a negative control. [Figure 37] Figure 37 depicts flow cytometry analysis of the dose-dependent induction of Ki67 expression on A) CD8+ T cells and B) NK cells in human PBMCs, as well as C) protease cleavage and activation of PD1 Ab-IL-15 VitoKine P-0875 using reducing SDS-PAGE gel analysis. P-0875 and P-0870 are a PD1 Ab VitoKine and non-VitoKine counterpart pair containing the dimeric IL-15 V63A / I68H variant. [Figure 38] Figure 38 depicts a comparative analysis of the activity of murine PD1 Ab-IL-15 VitoKine, P-1265, at varying dosage levels (3, 6, and 12 mpk) compared to its non-VitoKine counterpart, P-1266, dosed at 1.5 mpk in C57B / L6 mice. Comparisons are based on effects on peripheral lymphocytes after a single intraperitoneal injection: A) Ki67 expression in CD8 T cells, B) CD8 T cell counts, C) Ki67 expression in NK cells, and D) increasing NK cell counts. Blood samples were collected on days 0, 3, 5, 7, 10, and 11 for lymphocyte phenotyping using FACS analysis. Data are presented as mean ± standard error of the mean (SEM). [Figure 39]Figure 39 depicts a comparative analysis of the activity of the murine PD1 Ab-IL-15 VitoKine, P-1265, compared to its dimeric VitoKine equivalent, P-1085, and its non-VitoKine counterpart, P-1266, in C57B / L6 mice. The comparison is based on the effects on peripheral lymphocytes after a single intraperitoneal injection on A) Ki67 expression in CD8 T cells, B) CD8 T cell counts, C) Ki67 expression in NK cells, and D) increasing NK cell counts. The dosage for the two VitoKines was 12 mpk, and the dosage for P-1266 was 1.5 mpk. Blood samples were collected on days 0, 3, 5, 7, 10, and 10 for lymphocyte phenotype analysis using FACS analysis. Data are presented as mean ± standard error of the mean (SEM). [Figure 40] Figure 40 depicts a comparative analysis of the activity of murine PD1 Ab-IL-15 VitoKine, P-1263, at varying dose levels (6, 12, and 24 mpk) compared to its non-VitoKine counterpart, P-1295, dosed at 1.5 mpk in C57B / L6 mice. The comparison is based on the effect on peripheral lymphocytes after a single intraperitoneal injection on A) Ki67 expression in CD8 T cells, B) CD8 T cell counts, C) Ki67 expression in NK cells, and D) increasing NK cell counts. Blood samples were collected on days 0, 3, 5, 7, 10, and 11 for lymphocyte phenotyping using FACS analysis. Data are presented as mean ± standard error of the mean (SEM). [Figure 41]Figure 41 depicts a comparative analysis of the activity of the murine PD1 Ab-IL-15 VitoKine, P-1263, compared to its non-cleavable VitoKine equivalent, P-1264, and non-VitoKine counterpart, P-1295, in C57B / L6 mice. The comparison is based on the effect on peripheral lymphocytes after a single intraperitoneal injection: A) Ki67 expression in CD8 T cells, B) CD8 T cell counts, C) Ki67 expression in NK cells, and D) increasing NK cell counts. The dosage for the two VitoKines was 12 mpk, and for P-1295, the dosage was 1.5 mpk. Blood samples were collected on days 0, 3, 5, 7, 10, and 10 for lymphocyte phenotyping using FACS analysis. Data are presented as mean ± standard error of the mean (SEM). [Figure 42] Figure 42 depicts the in vivo antitumor efficacy and pharmacodynamic effects of P-0874 (murine PD1 Ab-IL-15 VitoKine) in mice bearing established CT26 murine tumors compared to its non-cleavable VitoKine counterpart, P-0878, after two doses of 10 mg / kg Q12D. Analyses include A) mean tumor volume ± SEM over time for each treatment group, B) CD8 T cell expansion 5 days after dosing, and C) NK cell expansion 5 days after dosing. [Figure 43] Figure 43 depicts a comparative analysis of the antitumor efficacy of the murine PD1 Ab-IL-15 VitoKine, P-1265, compared to its dimeric VitoKine equivalent, P-1085, and their respective non-VitoKine counterparts, P-1266 and P-0869, in an established MC38 tumor model. The analysis includes mean tumor volume ± SEM over time for A) P-1085 dosed at 3 and 6 mpk and P-0869 dosed at 1.5 mpk, and B) P-1265 dosed at 3, 6, and 12 mpk and P-1266 dosed at 1.5 mpk. A component PD1 antibody, P-0722, dosed at 12 mpk was included for comparison, along with a vehicle group. [Figure 44]Figure 44 depicts the antitumor effect of P-1263, a murine PD1 Ab-IL-15 VitoKine, in an established MC38 mouse colon cancer model after two doses of Q12D. The component murine PD1 antibody, P-0722, administered at dosages of 6 and 18 mpk was included for comparative analysis. The mean MC38 tumor volume ± SEM over time for each treatment group is shown in Figure 44A. MC38 tumor growth curves in individual mice are presented for B) 6 mg / kg P-0722, C) 18 mg / kg P-722, D) 6 mg / kg P-1263, E) 9 mg / kg P-1263, and F) 18 mg / kg P-1263. For comparison, the mean tumor volume ± SEM over time for the vehicle group is plotted as a dotted line. The change in body weight over time for each treatment group is shown in Figure 44G. DETAILED DESCRIPTION OF THE INVENTION

[0028] In one embodiment, the present invention provides a PD1 Ab-IL-15 VitoKine construct comprising an optimized PD1 blocking antibody as the TIL targeting moiety, IL-15 or an IL-15 variant as the active moiety, and an IL-15 RaSushi domain as the masking moiety. Importantly, the IL-15 RaSushi domain has the ability to mask or attenuate the functional activity of the IL-15 domain until it is activated at the site of intended treatment.

[0029] The PD1-blocking antibody guides VitoKine to TILs in the tumor microenvironment and locally restricts VitoKine activation, improving its therapeutic index. The PD1 antibody was optimized from the variable domain of pembrolizumab by germline sequence substitution of CDR residues, germline sequence substitution of framework somatic mutations, and adoption of the most prevalent and better-behaved VH3 human germline family sequence as the acceptor framework. It is predicted to have high affinity for PD1, function to inhibit PD1 with equal or similar efficacy to pembrolizumab, have a higher sequence similarity score to its closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab, and have lower hydrophobicity and a resulting reduced tendency to aggregate compared to pembrolizumab. The PD1-blocking antibody with the optimized sequence is expected to improve the potential for development of PD1 IL-15 immunocytokines.

[0030] In another embodiment, the IL-15 domain in the PD1 Ab-IL-15 VitoKine construct is the active moiety but remains inactive until activated locally by proteases upregulated in diseased tissue; this limits binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells or tissues, preventing pathway overactivation and reducing undesirable "on-target" "off-tissue" toxicity. Additionally, the inactivity of the VitoKine active moiety prior to protease activation significantly reduces potential antigen sinks and therefore prolongs in vivo half-life, resulting in improved biodistribution, bioavailability, and efficacy at the intended site of treatment.

[0031] In various embodiments, the incorporation of a potency-attenuated IL-15 variant as an active moiety domain (such an IL-15 variant is achieved by disrupting the IL-15Rβγ interaction) can further fine-tune the intrinsic basal activity and activity of VitoKine after activation. In various embodiments, such VitoKine with a potency-attenuated IL-15 variant as an active moiety domain may additionally expand its therapeutic index.

[0032] The unique and non-signaling α-subunit of the IL-15 receptor is used as a masking moiety via a protease-cleavable linker to reversibly mask cytokine activity in the PD1 Ab-IL-15 VitoKine. The masking α-subunit may be complexed with the activated cytokine, preferably through a non-covalent association, after protease cleavage of the linker.

[0033] The three domains in the PD1 Ab-IL-15 VitoKine construct are linked using linkers of variable length and rigidity linked to protease-cleavable sequences, which are peptide substrates for specific protease subtypes with elevated or deregulated expression in the disease site, thereby allowing the functional IL-15 domain to appear or be released at the site of disease. The length and composition of the linker were optimized to drive best possible concealment of the IL-15 domain's accessibility to the receptor, reducing its systemic involvement, while maintaining VitoKine's stability in the blood circulation and allowing efficient cleavage after encountering specific proteases at the intended disease site.

[0034] In another aspect, the present disclosure provides novel PD1-targeting IL-15 immunocytokines intended to target the activity-modulated IL-15 domain directly to tumor-infiltrating lymphocytes. In various embodiments, the activity-modulated IL-15 domain (dimer) is fused to the C-terminus of a PD1 antibody heavy chain. In various embodiments, the activity-modulated IL-15 domain (monomer) is fused to the C-terminus of a heterodimeric PD1 antibody heavy chain. In various embodiments, the PD1-targeting IL-15 immunocytokine comprises an IL-15RαSushi+ domain having the sequence set forth in SEQ ID NO: 165 non-covalently complexed with IL-15.

[0035] In one embodiment, the IL-15 domain in a PD1-targeted IL-15 immunocytokine is expected to have attenuated IL-15Rβγ activity and facilitate the establishment of a stoichiometric balance between the cytokine and antibody arms, helping to mitigate pathway overactivation and mitigating antigen sink and target-mediated deposition. In various embodiments, the use of potency-attenuated IL-15 variants (such variants having impaired interaction with γc) in a PD1-targeted IL-15 immunocytokine may confer an additional benefit in mitigating antigen sink and, in turn, may result in an extended in vivo half-life, possibly due to the influence of the γc receptor in the signaling cascade leading to cell proliferation.

[0036] definition Unless otherwise defined herein, scientific and technical terms used in the context of the present invention have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms include plurals, and plural terms include the singular. Generally, the technical terms used in the context of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and these techniques, are commonly used and well known in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references referenced and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications as commonly accomplished in the art or as described herein. The technical terms used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medical 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.

[0037] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide," "polypeptide," and "protein" is a chain of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (the amino terminus) thus has a free amino group, and the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide or the α-amino group (the amino group participating in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" (abbreviated as C-terminus) refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics, e.g., those in which amino acids are joined by ether bonds rather than amide bonds.

[0038] Polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, such as, for example, (1) to reduce susceptibility to proteolysis, (2) to reduce susceptibility to oxidation, (3) to alter binding affinity for forming protein complexes, (4) to alter binding affinity, and (5) to impart or modify other physicochemical or functional properties.

[0039] As used herein, an amino acid "substitution" refers to the replacement of one amino acid in a polypeptide at a specific 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) can be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain that forms intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid with a functionally similar amino acid in a polypeptide. The following six groups each contain amino acids that are conservative substitutions for each other: 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)

[0040] "Non-conservative amino acid substitutions" refer to the replacement of a member of one of these classes with 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 is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0041] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on hydropathic index, various embodiments include substitution of amino acids whose hydropathic index is within ±2, in various embodiments within ±1, and in various embodiments within ±0.5.

[0042] It is also understood in the art that substitutions of like amino acids can be usefully made on the basis of hydrophilicity, particularly when the resulting biologically functional protein or peptide 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 adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[0043] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, various embodiments include substitutions of amino acids whose hydrophilicity values ​​are within ±2, various embodiments include substitutions within ±1, and various embodiments include substitutions within ±0.5.

[0044] Exemplary amino acid substitutions are listed in Table 1. TIFF2025531805000001.tif176170

[0045] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In various embodiments, those skilled in the art can identify suitable sections of the molecule that can be changed without destroying activity by targeting regions that are not believed to be important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In further embodiments, even sections that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0046] Additionally, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one skilled in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues in the similar polypeptide that are important for activity or structure. One skilled in the art can select substitutions of chemically similar amino acids for such predicted important amino acid residues.

[0047] Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence relative to that structure in similar polypeptides. Taking such information into account, those skilled in the art can predict the alignment of amino acid residues of a polypeptide relative to the three-dimensional structure of the polypeptide. In various embodiments, those skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the polypeptide, because such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art may generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a specific amino acid residue results in destroyed, undesirably reduced, or unsuitable activity, the variant with such a change can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0048] The terms "polypeptide fragment" and "truncated polypeptide," as used herein, refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length protein. In various embodiments, the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids in length. In various embodiments, fragments can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. A fragment can further comprise one or more additional amino acids at either or both of its termini, 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).

[0049] The terms "polypeptide variant," "hybrid polypeptide," and "polypeptide mutant," as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to another polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present disclosure include fusion proteins.

[0050] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugated to another chemical moiety, e.g., polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylated, and glycosylated.

[0051] 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 as 80% sequence identity determined by a defined algorithm, meaning that a given sequence is at least 80% identical to another sequence. In various embodiments, the identity % is selected from, for example, 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 percent identity is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0052] 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 is the same as 80% sequence homology determined by a defined algorithm, and thus, a homolog of a given sequence has a sequence homology of more than 80% over a certain length of the given sequence. In various embodiments, the homology % is selected from, for example, 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 the given sequence. In various embodiments, the percent homology is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0053] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, the BLAST series of programs published on the NCBI website on the Internet, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN.Also see Altschul et al., J. Mol. Biol. 215:403-10, 1990 (particularly referring to the published default settings, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997.Sequence searches are typically performed using the BLASTP program, where a given amino acid sequence is compared with the amino acid sequences in GenBank Protein Sequences and other public databases.The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against the amino acid sequences in GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.

[0054] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, 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 that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0055] The term "alteration" (modification), as used herein, refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of a polypeptide (eg, glycosylation).

[0056] The term "knobs-into-hole modification" as used herein refers to a modification in the interface between two immunoglobulin heavy chains in the CH3 domain.In one embodiment, the "knobs-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 antibody heavy chain.Knobs-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0057] The term "bioactivatable drug" or "VitoKine," as used herein, refers to a compound that is a drug precursor, which, after administration to a subject, releases the drug in vivo through some chemical or physiological process such that the bioactivatable drug is converted into a product that is active on the target tissue. A bioactivatable drug is any compound that undergoes bioactivation before exhibiting its pharmacological effect. A bioactivatable drug can therefore be viewed as a drug containing specialized, non-toxic protecting groups that are used in a transient manner to modify or eliminate undesirable properties in the parent molecule.

[0058] The term "immunoconjugate" or "fusion protein," as used herein, refers to a molecule comprising an antibody or its antigen-binding fragment directly or indirectly conjugated (or linked) to an effector molecule. The effector molecule can be a detectable label, immunotoxin, cytokine, chemokine, therapeutic agent, or chemotherapeutic agent. The antibody or its antigen-binding fragment may be conjugated to the effector molecule via a peptide linker. The immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment; for example, the antibody or antigen-binding fragment has approximately the same, or only slightly reduced, ability to bind to an 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 may also be referred to as "chimeric molecules."

[0059] A "linker" refers to a molecule that joins two other molecules covalently or through ionic, van der Waals, or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes at its 5'-end to one complementary sequence and at its 3'-end to another complementary sequence, thereby joining two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded, digested, or otherwise cleaved to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases. Cleavable linkers may also be cleaved by environmental cues, such as changes in temperature, pH, salt concentration, and the like.

[0060] The term "peptide linker," as used herein, refers to a peptide comprising one or more amino acids, typically about 1 to 30 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4 (SG4) n It includes a peptide linker. "n" is generally a number from 1 to 10, typically from 2 to 4.

[0061] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of an agent effective to produce the intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate-buffered saline solution, a 5% aqueous solution of dextrose, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0062] 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 may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of the disease state, and improvement in remission or prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative, palliative, and preventative treatment.

[0063] The term "effective amount" or "therapeutically effective amount" as used herein refers to the amount of a compound or composition that is sufficient to treat a specified disorder, condition or disease, for example, to ameliorate, alleviate, reduce, and / or delay one or more of its symptoms.With respect to cancer or other unwanted cell proliferation, an effective amount includes: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and preferably stop the cancer cell invasion into peripheral organs to some extent; (iv) inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumor; and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.An effective amount can be administered in one or more administrations.

[0064] The phrases "administering" or "causing to be administered" refer to the act of managing and / or allowing the administration of an agent / compound of interest to a patient by a medical professional (e.g., a physician) or someone managing the medical care of a patient. Causing to be administered can involve determining a diagnosis and / or an appropriate treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing can include, for example, issuing a prescription, annotating a medical record, and the like. Where administering is described herein, "causing to be administered" is also envisioned.

[0065] The terms "patient," "individual," and "subject" may be used interchangeably and may refer to a mammal, preferably a human or non-human primate, but may also refer to domestic mammals (e.g., canines or felines), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., equines, bovines, porcines, ovines). In various embodiments, a patient may be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other healthcare professional in a hospital, psychiatric care facility, outpatient setting, or other clinical setting. In various embodiments, a patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS; cancer and transplant patients receiving certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have high rates of mutations (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0066] The term "immunotherapy" refers to treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using costimulatory or co-inhibitory molecules (immune checkpoints), such as CTLA-4, PD1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec treatments using agonist, antagonist, or blocking antibodies against CD15, VISTA, CD276, CD272, TIM-3, and B7-H4; treatments using bispecific T cell engaging antibodies (BiTE®), such as blinatumomab; treatments involving administration of biological response modifiers, such as IL-15, IL-4, IL-7, IL-10, IL-12, IL-15, IL-151, IL-152, GM-CSF, IFN-α, IFN-β, and IFN-γ, TGF-β antagonists, or TGF-β traps; treatments using therapeutic vaccines, such as sipuleucel-T; treatments using therapeutic viruses, including, but not limited to, oncolytic viruses, such as T-vec; dendritic cell vaccines, or tumor antigen peptides or neoantibodies. Treatments using original vaccines; treatments using NK cells; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using DCs or T cells; treatments using iPS-induced NK cells; treatments using iPS-induced T cells, as well as treatments using vaccines, such as Bacillus Calmette-Guerin (BCG); treatments using tumor-infiltrating lymphocytes (TIL); treatments using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-T cells); treatments using TALL-104 cells; and treatments using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and imiquimod.

[0067] "Resistant or refractory cancer" refers to tumor cells or cancers that do not respond to prior anti-cancer treatments, including, for example, 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 start of treatment or that initially respond to treatment for a short period but then become unresponsive. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but do not respond to subsequent rounds of treatment. For purposes of the present invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with an anti-cancer therapy but recur up to five years, sometimes up to ten years, or even longer after treatment has ceased. Anti-cancer treatments can use chemotherapy alone, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it is understood that refractory tumor cells can be interchangeable with resistant tumors.

[0068] The term "neoantigen" refers to cell surface antigens to which the immune system has not previously been exposed, e.g., that are selectively expressed by cancer cells or overexpressed in cancer cells relative to most normal cells, particularly those that result from alteration of host antigens by radiation, chemotherapy, viral infection, neoplastic transformation / mutation, drug metabolism, etc.

[0069] The term "antibody," as used herein, is used in the broadest sense and encompasses a variety of antibody structures (IgG1, 2, 3, or 4, IgM, IgA, IgE), including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or bifunctional antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0070] The term "antibody fragment," as used herein, refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.

[0071] The term "Fab fragment," as used herein, refers to an immunoglobulin fragment containing the VL domain and the constant domain of the light chain (CL), and the VH domain and the first constant domain of the heavy chain (CH1).

[0072] The term "variable region" or "variable domain," as used herein, refers to the domain of an immunoglobulin or antibody heavy or light chain that is generally responsible for binding the immunoglobulin or antibody to an antigen. The variable domains of the heavy and light chains of immunoglobulins or antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR).

[0073] The term "complementarity determining region" or "CDR" refers to the antigen-contacting residues ("antigen contacts"). Generally, antibodies contain six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). The CDRs are located at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs 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).

[0074] A "single-chain antibody" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in WO 88 / 01649, U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0075] A "human immunoglobulin," as used herein, is an immunoglobulin having an amino acid sequence that corresponds to that of an immunoglobulin produced by a human or human cell, or derived from a non-human source that utilizes the human immunoglobulin repertoire or other human immunoglobulin-coding sequences. This definition of a human immunoglobulin specifically excludes humanized immunoglobulins that contain non-human antigen-binding residues.

[0076] 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 CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions taken from the donor framework, and such substitutions are referred to herein as back mutations. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0077] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. The CH3 region herein may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "protuberance" ("knob") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; 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 described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0078] The term "effector function," as used herein, refers to a biological activity attributed to the Fc region of an immunoglobulin, which varies with immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0079] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured either through enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology.

[0080] The term "affinity" or "binding affinity" as used herein refers to the strength of the total 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 can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). A particular method for measuring affinity is SPR.

[0081] 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, including, for example, a human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T cells from a subject mount an immune response to an administered antibody. The T cells then recruit B cells to produce specific "anti-antibody" antibodies.

[0082] The term "immune cell," as used herein, refers to any cell of the hematopoietic lineage that is involved in regulating the immune response to an antigen (e.g., an autoantigen). In various embodiments, the immune cell is, for example, a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, a Langerhans cell, or a Kupffer cell.

[0083] The term "reduced binding," as used herein, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0084] The term "polymer," as used herein, generally includes, but is not limited to, homopolymers; copolymers, such as block, graft, random, and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" includes all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0085] "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 nucleic acid analogs containing non-naturally occurring bases, nucleotides containing bases that are linked to other nucleotides in linkages other than naturally occurring phosphodiester bonds, or that are attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, without limitation, for example, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). 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 short polynucleotides, generally of about 50 nucleotides or less. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0086] 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 a nascent RNA transcript is referred to as the transcription direction. A 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 the mRNA transcribed from that DNA and which are located 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; and 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."

[0087] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Therefore, the two molecules can be described as complementary, and further, the characteristics of the contacting surfaces 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.

[0088] 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), in which additional DNA segments can be introduced into the viral genome. Certain vectors have the ability to autonomously replicate in host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell 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 selected polynucleotide.

[0089] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or 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, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence.

[0090] A "host cell" is a cell that can be used to express a polynucleotide of the present disclosure. A host cell can be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., 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 nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level until a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the specific subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due, for example, to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0091] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) refers to a molecule that, by reason 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 from other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be assayed by numerous means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0092] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60%-75% of a sample represents a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein typically comprises about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more usually about 95% w / w, and is preferably greater than 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 staining the gel with stains well known in the art to visualize a single polypeptide band. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0093] The term "label" or "labeled," as used herein, refers to the incorporation of another molecule into an antibody. In one embodiment, the label is the incorporation of a detectable marker, such as a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide 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, such as 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 the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, e.g., gadolinium chelates, toxins, e.g., pertussis toxin, taxanes, Examples of suitable anti-cancer drugs include, but are not limited to, cyclohexyl 1, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. In various embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0094] The term "heterologous," as used herein, refers to a composition or condition that is not native or not found in nature, e.g., that may be achieved by replacing an existing natural composition or condition with a composition or condition derived from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and heterologous protein.

[0095] It is understood that aspects and embodiments of the disclosure described herein include aspects and embodiments that "consist of" and / or "consist essentially of."

[0096] Reference herein to "about" a value or parameter includes (and describes) variations directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0097] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that embodiments and variations of the disclosure described herein include "consisting of" and / or "consisting essentially of" embodiments and variations.

[0098] PD1 blocking antibodies In one embodiment, the PD1-blocking antibody guides the IL-15 portion of VitoKine to TILs in the tumor microenvironment (TME) and locally restricts VitoKine activation, improving the therapeutic index. In another embodiment, the PD1-blocking antibody guides the IL-15 portion of the immunocytokine to TILs in the TME. In various embodiments, the PD1-blocking antibody was optimized through modifications in the variable domain of pembrolizumab by germline sequence substitution of CDR residues, germline sequence substitution of framework residues, and adoption of the most prevalent and better-performing VH3 human germline family sequence as the acceptor framework. In various embodiments, these modifications were implemented individually or in combination to develop an optimized PD1-blocking antibody. In various embodiments, these optimized PD1 blocking antibodies are predicted to exhibit high binding affinity to PD1, function to inhibit PD1 with equal or comparable potency to pembrolizumab, have a higher sequence similarity score to its closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab, and have lower hydrophobicity, leading to a reduced aggregation tendency compared to pembrolizumab. In various embodiments, PD1 Ab-IL-15 VitoKine constructs and PD1-targeting IL-15 immunocytokines based on these optimized PD1 blocking antibodies are predicted to have enhanced developability. In various embodiments, the PD1 antibody comprises a light chain variable region having a sequence selected from the group of sequences set forth in SEQ ID NOS: 3-5 and a heavy chain variable region having a 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 NOS: 44 and a heavy chain having a sequence selected from the group of sequences set forth in SEQ ID NOS: 45-49.

[0099] IL-15 domain Interleukin-15 (IL-15) is a cytokine identified by two independent groups based on its ability to stimulate proliferation of the IL-2-dependent CTLL-2 T cell line in the presence of neutralizing anti-IL-2 antibodies (Steel et al., Trends Pharm Sci, 33:35-41, 2012). IL-15 and IL-2 share similar biological properties in vitro, consistent with their shared receptor (R) signaling component (IL-15Rβγc). However, the specificity of IL-15 compared to IL-2 is provided by a unique, private α-chain receptor that completes the IL-15Rαβγ and IL-2Rαβγ heterotrimeric high-affinity receptor complex and thereby allows differential responsiveness depending on the expressed ligand and high-affinity receptor. Interestingly, both IL-15 and IL-15Rα transcripts have a much broader tissue distribution than IL-2 / IL-2Rα. Furthermore, multiple complex post-transcriptional regulatory mechanisms tightly control IL-15 expression. Therefore, in addition to complex regulation, the essential in vivo functions of this receptor / ligand pair may differ from those of IL-2 and IL-2Rα, based on differential patterns of IL-15 and IL-15Rα expression. Research examining IL-15 biology to date has identified several key non-redundant roles, such as the importance of IL-15 during the development and function of natural killer (NK) cells, NK-T cells, and intestinal intraepithelial lymphocytes. The role of IL-15 during autoimmune processes, such as rheumatoid arthritis, and malignancies, such as adult T-cell leukemia, suggests that dysregulation of IL-15 may result in detrimental effects for the host (Fehniger et al., Blood, 97:14-32, 2001).

[0100] As used herein, the terms "native IL-15" and "native interleukin-15" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 amino acid sequence, including immature or precursor and mature forms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various species of native mammalian interleukin-15 include NP_032383 (mouse (Mus musculus), immature form), AAB60398 (rhesus monkey (macaca mulatta), immature form), NP_000576 (human, immature form), CAA62616 (human, immature form), AAI00964 (human, immature form), and AAH18149 (human). In various embodiments of the present invention, native IL-15 is the immature or precursor form of naturally occurring mammalian IL-15. In other embodiments, native IL-15 is the mature form of naturally occurring mammalian IL-15. In various embodiments, native IL-15 is the precursor form of naturally occurring human IL-15. In various embodiments, native IL-15 is the mature form of naturally occurring human IL-15. In various embodiments, the IL-15 in the VitoKine and immunocytokine constructs of the present invention is derived from the amino acid sequence of the mature human IL-15 sequence set forth in SEQ ID NO: 116: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 116)

[0101] In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-15 polypeptide set forth in SEQ ID NO: 116. In various embodiments, the IL-15 variant comprises an amino acid sequence that differs from the native (or wild-type) IL-15 protein. In various embodiments, the IL-15 variant binds to an IL-15Rα polypeptide and functions as an IL-15 agonist or antagonist. In various embodiments, the IL-15 variant can function as an IL-15 agonist or antagonist independently of its association with IL-15Rα. An IL-15 agonist is exemplified by equivalent or increased biological activity compared to wild-type IL-15. An IL-15 antagonist is exemplified by decreased biological activity compared to wild-type IL-15 or the ability to inhibit IL-15-mediated responses. In various embodiments, the IL-15 variant binds to the IL-15Rβγc receptor with increased or decreased activity. In various embodiments, the sequence of the IL-15 variant has at least one amino acid change, e.g., a substitution or deletion, compared to the native IL-15 sequence, which results in IL-15 agonist or antagonist activity. In various embodiments, the amino acid substitution / deletion is at least one amino acid change, e.g., a substitution or deletion, at the IL-15Rβ and / or γ CThe amino acid substitution / deletion is located in the domain of IL-15 that interacts with IL-15Rα. In various embodiments, the amino acid substitution / deletion does not affect binding to the IL-15Rα polypeptide or the ability to produce an IL-15 variant. Suitable amino acid substitutions / deletions for generating IL-15 variants can be identified through rational or random mutagenesis and functional assays, or other empirical methods, as provided herein, based on known IL-15 structure, comparison of IL-15 with homologous molecules, e.g., IL-15s with known structures. Additionally, suitable amino acid substitutions can be conservative or non-conservative changes and insertions of additional amino acids. In various embodiments, the amino acid change is one or more amino acid substitutions at positions 30, 32, 63, 68, 108, 109, or 112 of SEQ ID NO:116. In various embodiments, the amino acid change is a substitution of D for T at position 30 of the mature human IL-15 sequence, a substitution of H for D or E or N or Q at position 32, a substitution of V for F or A or K or R at position 63, a substitution of I for F or H or D or K or Q or G at position 68, a substitution of Q for A or D or E or F or H or K or L or M or N or S or T or Y at position 108, a substitution of M for A or H or R at position 109, a substitution of N for D or G or P or R at position 112, or any combination of these substitutions. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4 amino acids at the N-terminus of SEQ ID NO:116. In various embodiments, the amino acid change is a deletion of 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 amino acids at the C-terminus of SEQ ID NO:116. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions. In various embodiments, the IL-15 variant is selected from the group of sequences set forth in SEQ ID NOs: 117-163.

[0102] IL-15Rα domain The IL-15 receptor is a type I cytokine receptor consisting of beta (β) and gamma (γ) subunits shared with the IL-2 receptor, and an alpha (α) subunit that binds IL-15 with high affinity. Full-length human IL-15Rα is a type I transmembrane protein with a 32-AA signal peptide, a 173-AA extracellular domain, a 21-AA transmembrane domain, a 37-AA cytoplasmic tail, and multiple N- or O-linked glycosylation sites (Anderson et al., J. Biol Chem, 270:29862-29869, 1995). It has previously been demonstrated that the native soluble form of the IL-15Rα chain, corresponding to the entire extracellular domain of IL-15Rα, behaves as a high-affinity IL-15 antagonist. However, in sharp contrast to that finding, it was demonstrated that a recombinant soluble sushi domain of IL-15R alpha, which is responsible for most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta / gamma heterodimer, while not affecting IL-15 binding and function of the ternary IL-15R alpha / beta / gamma membrane receptor. These results suggested that such a soluble sushi domain, when naturally produced, may be involved in the IL-15 transpresentation mechanism (Mortier et al., J. Biol. Chem. 281:1612-1619, 2006).

[0103] As used herein, the terms "native IL-15Rα" and "native interleukin-15 receptor alpha" in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-15 receptor alpha ("IL-15Rα") amino acid sequence, including immature or precursor and mature forms and naturally occurring isoforms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various native mammalian IL-15Rα include NP_002180 (human), ABK41438 (rhesus monkey), NP_032384 (mouse), Q60819 (mouse), CA141082 (human). In various embodiments, native IL-15Rα is the full-length form of a naturally occurring mammalian IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the immature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the mature form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, native IL-15Rα is the full-length form of a naturally occurring human IL-15Rα polypeptide. In various embodiments, the native IL-15Rα protein or polypeptide is isolated or purified. In various embodiments, the IL-15Rα domain is derived from the amino acid sequence of the human IL-15Rα sequence set forth in SEQ ID NO: 164: MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 164)

[0104] In various embodiments, the functional IL-15Rα domain is an IL-15RαSushi+ domain comprising the amino acid sequence set forth in SEQ ID NO: 165: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO: 165)

[0105] In one aspect, the PD1 Ab-IL-15 VitoKine construct of the present invention contains a covalently linked IL-15Rα as a masking partial domain. In various embodiments, the masking partial domain is the IL-15Rα extracellular domain or a functional fragment thereof. In various embodiments, the masking partial domain is the IL-15Rα Sushi+ domain comprising the amino acid sequence set forth in SEQ ID NO: 165. In various preferred embodiments, the masking partial domain is a variant of the IL-15Rα Sushi+ domain. The masking partial domain is primarily used to reversibly mask the activity of the IL-15 domain in the VitoKine construct.

[0106] In various embodiments, IL-15RαSushi+ (SEQ ID NO: 165), a truncated cognate coreceptor for IL-15 that largely reproduces the binding affinity of full-length IL-15Rα (SEQ ID NO: 164), was covalently linked to the IL-15 domain in PD1 Ab-IL-15 VitoKine to mask the activity of IL-15. In various embodiments, the effectiveness of masking IL-15 activity can be further tuned by fine-tuning the length and composition of the linker connecting the IL-15 and IL-15RαSushi+ domains. As will be appreciated by those skilled in the art, the masking domain (D3) can vary from the sequence set forth in SEQ ID NO: 164, as long as it is a functional fragment that can largely reproduce the binding activity of full-length IL-15Rα (SEQ ID NO: 164). The uniqueness of the IL-15 VitoKine design relies on its full exploitation of the unique features of the IL-15 pathway, including the exceptionally high affinity (30 pM) between IL-15 and IL-15α, and the fact that complexation of IL-15α enhances the activity of IL-15 in vivo. Following cleavage of the linker connecting IL-15 and IL-15αSushi+ by proteases upregulated at disease sites, IL-15RαShushi+ or any functional fragment derived from IL-15Rα is expected to remain noncovalently associated with IL-15 and enhance IL-15 activity.

[0107] In another embodiment, the PD1-targeting IL-15 immunocytokine of the present invention comprises a non-covalently associated IL-15RαSushi+ domain. Non-covalent complexation of IL-15RαSushi+ with IL-15 was achieved in cell culture co-expression due to the exceptionally high affinity (30 pM) between IL-15 and IL-15α. As disclosed by the inventors in WO2019246379, covalent complexation of IL-15α with IL-15 significantly enhances the developability profile of the corresponding fusion protein.

[0108] L1 and L2 linkers in PD1 Ab-IL-15 VitoKine Cleavable Linker Cleavable linker, or linker sensitive to disease-related enzymes, may contain a moiety, such as a protein substrate, that has the ability to be specifically cleaved by proteases that are present at elevated levels in diseased tissues compared to non-diseased tissues.The literature contains multiple reports on the increased levels of enzymes with known substrates in various types of cancer, such as solid tumors.See, for example, La Rocca et al., Brit.J.Cancer 90:1414-1421 and Ducry et al., Bioconjug.Chem.21:5-13,2010 (each of which is incorporated herein by reference in its entirety). In various embodiments, the protease capable of cleaving the protease-cleavable linker is selected from the group consisting of metalloproteinases, e.g., matrix metalloproteinases (MMPs) 1-28, serine proteases, e.g., urokinase-type plasminogen activator (uPA) and matriptase, cysteine ​​proteases, e.g., legumain, aspartic acid proteases, and cathepsin proteases. Exemplary proteases are provided in Table 2: TIFF2025531805000002.tif215170

[0109] Exemplary protease substrate peptide sequences that can be used as protease-cleavable linkers, with or without a peptide spacer, are provided in Table 3: TIFF2025531805000003.tif147170

[0110] In various embodiments, the protease is MMP-9 or MMP-2. In further specific embodiments, the protease is matriptase. In further specific embodiments, the protease is MMP-14. In further specific embodiments, 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). In various 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).

[0111] In various embodiments, peptide spacers may be incorporated on either side of the protease-cleavable sequence, adjacent to both sides of the protease-cleavable sequence, or as a non-cleavable linker without a protease substrate site. The peptide spacer serves to position the cleavable linker, making it more accessible to the enzyme responsible for cleavage. The length and composition of the peptide spacer can be fine-tuned to balance accessibility for enzymatic cleavage with the spatial constraints required to reversibly conceal the D2 domain so that it does not exert its biological activity. The peptide spacer may contain 1 to 100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues, such as glycine and serine. In various embodiments, the peptide spacer can contain 1 to 12 amino acids including the motifs G, S, GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGS (SEQ ID NO: 111), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). In other embodiments, the peptide spacer can contain 1 to 12 amino acids including the motifs G, S, GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGSGS (SEQ ID NO: 111), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). n (where n is an integer from 1 to 10). In other embodiments, the peptide spacer may also contain amino acids other than glycine and serine. The peptide spacer is stable under physiological conditions as well as at disease sites, such as cancer sites.

[0112] Exemplary protease-cleavable linkers having a peptide spacer adjacent to a protease substrate peptide (underlined) are provided in Table 4: TIFF2025531805000004.tif110170

[0113] Non-cleavable linkers Non-cleavable linkers provide a covalent linkage between protein domains as well as additional structural and / or spatial flexibility. As 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 linkers may contain 1 to 100 amino acids. In various embodiments, the spacer can contain a GS (SEQ ID NO: 116), GGS (SEQ ID NO: 117), GGGGS (SEQ ID NO: 118), GGSG (SEQ ID NO: 119), or SGGG (SEQ ID NO: 120) motif. In other embodiments, the linker can contain a (GGGGS) (SEQ ID NO: 118) n motif, where n is an integer between 1 and 10. In other embodiments, the linker can also contain amino acids other than glycine and serine. In another embodiment, the non-cleavable linker can be a simple chemical bond, such as an amide bond (e.g., via chemical conjugation of PEG). The non-cleavable linker is stable under physiological conditions as well as at disease sites, such as cancer sites.

[0114] Exemplary non-cleavable linkers are provided in Table 5: TIFF2025531805000005.tif109170

[0115] Combinations of cleavable and non-cleavable linkers In various embodiments, the L1 and L2 linkers can both be cleavable or a combination of cleavable and non-cleavable linkers, resulting in different forms of the active portion of the IL-15 domain to meet specific therapeutic objectives, optimize the risk-to-benefit ratio, or match diverse properties of the cytokine. Exemplary active forms released by linker cleavage are depicted in Figure 2. Active Form 1, derived from cleavage of the L1 linker, and Active Form 3, derived from cleavage of the L1 and L2 linkers, are both short-acting cytokines due to their release from the targeting antibody after proteolysis. These two active forms contain either a covalently linked IL-15RαSushi+ domain or a non-covalently complexed IL-15RαSushi+ domain and exhibit distinct activities in the local environment. After acting locally, the short-acting active form can be rapidly cleared from the systemic circulation, leading to reduced toxicity. In contrast, the active form 2 derived from cleavage of the L2 linker is functionally fully restored IL-15 fused to PD1 Ab at or near the disease site. This active form has the ability to cis-activate IL-15R signaling in PD1-expressing T cells at or near the disease site, which synergistically enhances the two pathways and boosts anti-cancer immune responses while minimizing systemic toxicity.

[0116] Polynucleotides In another aspect, the present disclosure provides isolated nucleic acid molecules comprising the disclosed polynucleotides IL-15, IL-15 variants, IL-15Rα, PD1-blocking antibodies, antibody fragments, or PD1-targeting IL-15 immunocytokines, or PD1 Ab-IL-15 VitoKine constructs. 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. Synthetic DNA can be obtained by chemical synthesis of overlapping oligonucleotide fragments followed by assembly of the fragments to reconstitute part or all of the coding region and flanking sequences. RNA can also be obtained from prokaryotic expression vectors that direct high-level synthesis of mRNA, such as vectors using a T7 promoter and RNA polymerase. DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. Full-length genes may also include sequences encoding an N-terminal signal sequence. Such nucleic acids may be used, for example, in methods for producing novel VitoKine constructs.

[0117] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein, hi various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0118] In various embodiments, the recombinant nucleic acid 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. Thus, 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, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcriptional start and stop sequences, translational start and stop sequences, and an enhancer or activator sequence. Constitutive or inducible promoters, as known in the art, are contemplated by the present disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0119] In another embodiment of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding the pharmaceutical composition of the present invention 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 nucleic acid molecules are inserted into the vector using standard recombinant DNA techniques. Such vectors can contain a variety of expression control sequences that control the expression of DNA sequences when operably linked, and the expression control sequences can be used in these vectors to express DNA sequences encoding PD1-targeting IL-15 immunocytokine constructs or PD1 Ab-IL-15 VitoKine constructs. Useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate-early promoter, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter, whose expression is directed by T7 RNA polymerase, the phage lambda major operator and promoter region, the control region for the fd coat protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter for acid phosphatase (e.g., PhoS), the promoter for yeast a-mating factor, the polyhedron promoter of baculovirus systems, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It should be understood that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Additionally, the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.Exemplary expression vectors suitable for expression of the pharmaceutical compositions of the invention are pDSRa, and derivatives thereof, containing a polynucleotide encoding the pharmaceutical composition of the invention, as well as any additional suitable vectors known in the art or described below.

[0120] The recombinant nucleic acids of the present disclosure can be produced by ligating the cloned gene, or a portion thereof, into a suitable vector for expression in either or both prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian cells). Expression vehicles for the production of immunocytokines or VitoKine constructs include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: 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.

[0121] Some mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. 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 bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205), can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. Various methods used in preparing plasmids and transforming host organisms are well known in the art. For general recombinant procedures, as well as other suitable expression systems for both prokaryotic and eukaryotic cells, 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 cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., the B-gal-containing pBlueBac III).

[0122] In various embodiments, vectors are designed for production of the subject constructs in Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.) As will be apparent, the subject gene constructs can be used to cause expression of the subject constructs in cells propagated in culture to produce proteins, including fusion or variant proteins, for example, for purification.

[0123] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding the amino acid sequence for one or more of the subject constructs. The host cell may be any prokaryotic or eukaryotic cell. For example, the constructs 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.

[0124] Thus, the present disclosure further relates to methods for producing the subject constructs. For example, host cells transfected with an expression vector encoding an immunocytokine construct or a VitoKine construct can be cultured under appropriate conditions to allow expression of the VitoKine construct to occur. The construct may be secreted and isolated from a mixture of cells and a medium containing the VitoKine construct. Alternatively, the construct may be retained in the cytoplasm or in a membrane fraction, and the cells may be harvested, lysed, and the protein isolated. Cell cultures include host cells, medium, and other by-products. Suitable media for cell culture are well known in the art.

[0125] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques involve, at one level, crude fractionation of protein and non-protein fractions. Once the peptide polypeptide has been separated 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 terms "isolated polypeptide" or "purified polypeptide," as used herein, are intended to refer to a composition isolable from other components, in which the polypeptide has been purified to any degree relative to its naturally obtainable state. A purified polypeptide therefore also refers to a polypeptide that has been freed from the environment in which the polypeptide may naturally occur. Generally, "purified" refers to a polypeptide composition that has been subjected to fractionation to remove various other components, wherein the composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, e.g., comprises about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0126] Various techniques suitable for use in purification are well known to those skilled in the art.These include, for example, ammonium sulfate, PEG, and antibody (immunoprecipitation), or heat denaturation followed by centrifugation precipitation; chromatography, for example, affinity chromatography (protein A column), 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, the order of carrying out various purification steps may be changed, or certain steps may be omitted, and still be considered to result in a suitable method for preparing substantially purified polypeptide.

[0127] Pharmaceutical Composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an immunocytokine construct or a VitoKine construct mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been described extensively (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included for purposes such as modifying, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Such pharmaceutical compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants; flavoring agents and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); lolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0128] 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, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, the composition may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulating agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Additionally, therapeutic compositions may be formulated as lyophilizates using appropriate excipients, such as sucrose. The optimal pharmaceutical composition can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage.

[0129] When parenteral administration is contemplated, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired polypeptide construct in a pharmaceutically acceptable medium. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical preparations suitable for injection 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 carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and allow for the preparation of highly concentrated solutions.

[0130] In various embodiments, therapeutic pharmaceutical compositions may be formulated for targeted delivery using colloidal dispersion systems.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.Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, which is known in the art.

[0131] In various embodiments, it is envisaged that pharmaceutical compositions are orally administered.The pharmaceutical compositions administered in this manner can be formulated with or without the carriers that are commonly used in the preparation of solid dosage forms, such as tablets and capsules.In the solid dosage forms for oral administration (such as capsules, tablets, pills, dragees, powders and granules), one or more therapeutic compounds of the present disclosure can be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as acacia. The pharmaceutical compositions may be mixed with any of the following: (1) agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (2) dissolution retardants, such as paraffin; (3) absorption enhancers, such as quaternary ammonium compounds; (4) wetting agents, such as cetyl alcohol and glycerol monostearate; (5) absorbents, such as kaolin and bentonite clay; (6) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (7) coloring agents. For capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft- and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols. 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 form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0132] In various embodiments, topical administration of the pharmaceutical composition to either the skin or mucous membranes is contemplated. Topical formulations may further include one or more of a variety of agents known to be effective as skin or stratum corneum permeation 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 be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known to those skilled in the art, may also be included. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers, or propellants. The ointments, pastes, creams and gels may contain, in addition to the subject compounds of the present disclosure (e.g., VitoKine constructs), excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0133] Additional pharmaceutical compositions contemplated for use herein include formulations involving polypeptides in sustained- or controlled-delivery formulations. In various embodiments, the pharmaceutical compositions may be formulated as slow-release hydrogels, nanoparticles, or incorporated into oncolytic viruses. Methods for preparing such nanoparticles include, for example, encapsulation in nanoparticles composed of polymers with hydrophobic backbones 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 the charged combination polyester, 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 taxanes.See, for example, United States Patent No. 5,916,596; United States Patent No. 6,506,405; United States Patent No. 6,749,868; United States Patent No. 6,537,579; United States Patent No. 7,820,788; and United States Patent No. 7,923,536.Abraxane (registered trademark), an albumin-stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 and subsequently in various other countries for the treatment of metastatic breast cancer.

[0134] 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.

[0135] The effective amount of a pharmaceutical composition used therapeutically depends, for example, on the therapeutic context and purpose. Those skilled in the art will appreciate that appropriate dosage levels for treatment will therefore vary, in part, depending on the molecule being delivered, the condition for which the polypeptide is being used, the route of administration, and the patient's size (body weight, body surface, or organ size) and condition (age and general health). Thus, clinicians may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.0001 mg / kg to about 100 mg / kg or more, depending on the factors discussed above. Polypeptide compositions may be injected or administered preferably intravenously. Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly, biweekly, triweekly, monthly, or for even longer durations, depending on the half-life and clearance rate of the particular formulation. The frequency of dosing depends on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the 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 (of the same or different concentrations / dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely performed. The appropriate dosage may be ascertained through the use of appropriate dose-response data.

[0136] The pharmaceutical composition may be administered according to known methods, for example, orally, via injection, intravenously, intraperitoneally, intratumorally, intracerebrally (intraparenchymally), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, intralesionally, intramedullary, intrathecally, intraventricularly, intravesically, transdermally, subcutaneously, or intraperitoneally; as well as intranasally, intestinal, topically, sublingually, urethrally, vaginally, or rectally, by sustained-release system, or by implantation device. If desired, the composition 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 suitable material into which the desired molecule has been absorbed or encapsulated. When 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.

[0137] therapeutic use The present disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as monotherapy or in a combination treatment regimen) of a pharmaceutical composition of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the immunocytokines or VitoKine constructs of the present disclosure are 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, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, and parathyroid glands, and their distant metastases, lymphoma, sarcoma, 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 cancer, as well as bronchial adenoma and pulmonary pleuroblastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, neuroblastoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumors. 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 uterine sarcoma. Tumors of the gastrointestinal tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, liver, breast, pancreatic, rectal, small intestine, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular-cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal carcinoma and lip cancer. Lymphoma includes, but is not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.Sarcomas include, but are not limited to, sarcoma of 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.

[0138] In various embodiments, the pharmaceutical compositions of the present disclosure may be used as a single agent for the treatment of all types of cancer, 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.

[0139] A "therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated.

[0140] The therapeutically effective dose is IC 50 The dose can be estimated initially from cell culture assays by determining the IC (half maximal inhibitory concentration). The dose can then be calculated using the IC as determined in cell culture. 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range including the range of 0.1 to 1.5 mg / kg of circulating plasma. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be chosen by the individual physician in consideration of the subject's condition.

[0141] 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, or several divided doses (multiple or repeated 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 particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are primarily dictated by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0142] Therefore, those skilled in the art will understand based on the disclosure provided herein that dosage and dosage regimen will be adjusted according to the method well known in the therapeutic field.That is, the maximum acceptable dosage can be easily established, and the effective amount that provides detectable therapeutic benefit to the subject can also be determined, as well as the time requirement for administering each agent to provide detectable therapeutic benefit to the subject.Therefore, although certain dosage and dosage regimen are exemplified herein, these examples do not limit the dosage and dosage regimen that can be provided to the subject in the implementation of the present disclosure.

[0143] It should 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. For any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or implementation of the claimed compositions. Furthermore, dosage regimens using the compositions of the present disclosure may be based on various factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, and route of administration. Thus, dosage regimens can vary widely but can be routinely determined 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. Therefore, the present disclosure encompasses intra-subject dose escalation as determined by those skilled in the art. It will be appreciated that determination of appropriate dosages and regimens is well known in the relevant art and could be accomplished by one of ordinary skill in the art once provided with the teachings disclosed herein.

[0144] Exemplary, non-limiting daily dosage ranges for therapeutically or prophylactically effective amounts of the compositions of the present disclosure are 0.0001-100 mg / kg, 0.0001-90 mg / kg, 0.0001-80 mg / kg, 0.0001-70 mg / kg, 0.0001-60 mg / kg, 0.0001-50 mg / kg, 0.0001-40 mg / kg, 0.0001-30 mg / kg, 0.0001-20 mg / kg, 0.0001-10 mg / kg, 0.0001~5mg / kg, 0.0001~4mg / kg, 0.0001~3mg / kg, 0.0001~2mg / kg, 0.0001~1mg / kg, 0.001~50mg / kg, 0.001 ~40mg / kg, 0.001~30mg / kg, 0.001~20mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~4mg / kg, 0.001~3mg / kg, 0.001 ~2mg / kg, 0.001~1mg / kg, 0.010~50mg / kg, 0.010~40mg / kg, 0.010~30mg / kg, 0.010~20mg / kg, 0.010~10mg / kg, 0.01 0~5mg / kg, 0.010~4mg / kg, 0.010~3mg / kg, 0.010~2mg / kg, 0.010~1mg / kg, 0.1~50mg / kg, 0.1~40mg / kg, 0.1~30mg / k The dosage may be 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 1-50 mg / kg, 1-40 mg / kg, 1-30 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, or 1-1 mg / kg body weight. It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens will 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 compositions.

[0145] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure are, for example, LD 50 (a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutically effective doses is the therapeutic index, which is the ratio LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0146] The dosage frequency of the pharmaceutical composition of the present disclosure depends on the nature of treatment and the specific disease being treated.Subject can be treated at regular intervals, for example, weekly or monthly, until desired therapeutic results are achieved.Exemplary dosage frequencies include but are not limited to: once a week without interruption; once a week, every other week; once every two weeks; once every three weeks; once a week for two weeks without interruption, then monthly; once a week for three weeks without interruption, then monthly; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or yearly.

[0147] Combination treatment As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to a pharmaceutical composition of the present disclosure and one or more other therapeutic agents, are intended to mean, and refer to and include, the simultaneous administration of a polypeptide construct of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated together in a single dosage form that releases said components at substantially the same time in said subject; the substantially simultaneous administration of a composition of the present disclosure and a combination of therapeutic agents to a subject in need of treatment, where such components are formulated separate from one another in separate dosage forms that are taken by said subject at substantially the same time and that release said components at substantially the same time in said subject; Sequential administration of a combination of a polypeptide construct of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated apart from one another to be in separate dosage forms taken by the subject at successive times with a significant time interval between each administration, and where such components are released to the subject at substantially different times; and sequential administration of a combination of a polypeptide construct of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated together to be in a single dosage form that releases the components in a controlled manner, where they are released to the subject at the same and / or different times in a concurrent, sequential, and / or overlapping manner, where each portion may be administered by the same or different routes.

[0148] 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 composition of the present invention in combination with a second treatment, including but not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation.For example, such a method can be used in preventive cancer prevention, preventing cancer recurrence and metastasis after surgery, and as an adjuvant for other conventional cancer treatments.The present disclosure recognizes that the effectiveness of conventional cancer treatments (such as chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combined methods described herein.

[0149] A wide range of conventional compounds have been shown to have anti-neoplastic activity.These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignant tumors.Although chemotherapy has been effective in treating various types of malignant tumors, many anti-neoplastic compounds induce undesirable side effects.It has been shown that when two or more different treatments are combined, the treatments can function synergistically and allow the dosage of each treatment to be reduced, thereby reducing the harmful side effects caused by each compound at higher dosages.In other cases, malignant tumors that are refractory to treatment can respond to the combined treatment of two or more different treatments.

[0150] In various embodiments, a second anti-cancer agent, e.g., a chemotherapeutic agent, is administered to the patient. A list of exemplary chemotherapeutic agents includes 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, These include, but are not limited to, combinations of agents such as, but not limited to, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyurea taxanes (e.g., paclitaxel and docetaxel) and / or anthracycline antibiotics, as well as DA-EPOCH, CHOP, CVP, or FOLFOX. In various embodiments, the dosage of such chemotherapeutic agents is about 10 mg / m 2 , 20 mg / m 2 , 30 mg / m 2 , 40 mg / m 2 , 50 mg / m 2 , 60 mg / m 2 , 75 mg / m 2 , 80 mg / m 2 , 90 mg / m 2 , 100 mg / m 2 , 120 mg / m 2 , 150 mg / m 2 , 175 mg / m 2 , 200 mg / m 2 , 210 mg / m 2 , 220 mg / m 2 , 230 mg / m 2 , 240 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 , and 300 mg / m 2 This includes, but is not limited to, any of the following:

[0151] In various embodiments, the combination treatment methods of the present disclosure include treatments using depleting antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using antibodies against specific tumor antigens, such as CTLA-4, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec treatments using agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), including, but not limited to, antibodies against VISTA, CD276, CD272, TIM-3, and B7-H4; treatments using bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; treatments involving the administration of biological response modifiers, such as IL-2, IL-7, IL-10, IL-12, GM-CSF, IFN-α, IFN-β, IFN-γ, TGF-β antagonists, or TGF-β traps; treatments using therapeutic vaccines, including, but not limited to, oncolytic viruses, such as T-vec, or therapeutic vaccines, such as sipuleucel-T; treatments using dendritic cell vaccines, or tumor antigen peptide or neoantigen vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatment using iPS-induced CAR-T or iPS-induced CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TIL); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-T cells); treatment using TALL-104 cells; and treatment using immunostimulants, such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9, and vaccines, such as Bacillus Calmette-Guerin (BCG), and imiquimod; the combination treatment provides increased effector cell killing of tumor cells, i.e., synergism exists between the VitoKine construct and the immunotherapy when administered in combination.

[0152] In various embodiments, the combination therapy involves simultaneously administering the polypeptide composition of the present disclosure and the second agent composition, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the polypeptide composition and the second agent composition are administered sequentially, i.e., the immunocytokine or VitoKine construct composition is administered either before or after the administration of the second agent composition. In various embodiments, the administration of the immunocytokine or VitoKine construct composition and the second agent composition is concurrent, i.e., the administration periods of the immunocytokine or VitoKine construct composition and the second agent composition overlap with each other. In various embodiments, the administration of the immunocytokine or VitoKine construct composition and the second agent composition is non-concurrent. For example, in various embodiments, the administration of the immunocytokine or VitoKine construct composition is terminated before the administration of the second agent composition. In various embodiments, the administration of the second agent composition is terminated before the administration of the immunocytokine or VitoKine construct composition.

[0153] The following examples are given to more fully illustrate the present disclosure, but are not to be construed as limiting the scope of the invention. [Example]

[0154] Example 1 Optimization of PD1 blocking antibody sequence The present invention aims to optimize the variable domain sequence of pembrolizumab to enhance its similarity score to human germline sequences, an indicator of "humanity." This enhancement could potentially reduce the risk of immunogenicity. Additionally, the inventors used human VH3 family germline sequences, which are less homologous but more prevalent and behave better as alternative acceptor frameworks. This was done with the goal of improving the biophysical properties of the resulting humanized antibody, ensuring full activity, and enhancing its sequence humanity.

[0155] Pembrolizumab was humanized by CDR grafting technology using the most homologous human antibody sequence available in the RCSB Protein Data Bank as the acceptor human framework. The frameworks found in GenBank 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., U.S. Patent No. 8,354,509 B2). However, pembrolizumab only shares 79.6% sequence identity with the closest human germline IGHV1-2, according to a comparison of the variable region exons using the International Immunogenetics Information System (IMGT) DomainGapAlign tool (www.imgt.org). Similarity scores to human germline sequences were proposed as a definitional criterion for therapeutic antibodies by the WHO's International Nonproprietary Names (INN) group in 2014, likely based on the notion that higher similarity may indicate reduced immunogenicity. The low similarity, or "degree of humanness," score of the heavy chain of pembrolizumab (Abhinandan KR et al., J Mol Biol (2007) 369:852-62) may be due to the low level of conservation between the mouse CDRs and their human sequence counterparts, the need to retain a few structurally important mouse framework residues to recapitulate antigen binding, and the conservation of unique somatic mutations in the human framework sequence AB063829.

[0156] To enhance the degree of humanization of pembrolizumab, certain CDR residues were targeted for replacement with equivalent residues from the closest human germline sequence. This method is referred to herein as CDR germlining. While avoiding CDR perturbations has traditionally been a central principle in humanized Ab design, only a limited number of CDR residues are involved in direct antigen interactions. Therefore, certain CDR residues can be replaced without impairing antibody activity. 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).

[0157] Regarding the CDR3 sequence, a portion of the light chain CDR3 (CDR-L3) and the entire heavy chain CDR3 (CDR-H3) were not part of the variable region exon V region of the germline sequence. Consequently, no human germline residues were available to replace the mouse CDR counterparts. Additionally, CDR3, especially CDR-H3, is highly variable and is crucial for antigen binding and functional activity, making it essential to preserve its conformation. Therefore, both the CDR-L3 (QHSRDLPLT; SEQ ID NO: 25) and CDR-H3 (RDYRFDMGFDY; SEQ ID NO: 33) of pembrolizumab were excluded from the CDR germlining process.

[0158] The CDR-L1 and CDR-L2 sequences of pembrolizumab were aligned to their counterparts from the closest human germline, IGKV3D-11 (GenBank accession # X17264; SEQ ID NO: 39). The alignment is shown in Table 6A. Similarly, the alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with the closest human germline sequence, IGHV1-2 (GenBank accession # X62106; SEQ ID NO: 37), is shown in Table 6B. TIFF2025531805000006.tif92170

[0159] Several pembrolizumab CDR residues are directly involved in polar interactions with PD1, such as hydrogen bonds and salt bridges (Horita, S. et al. Sci. Rep (2016). 6:35297). Contact residues in or near CDR1 and CDR2 of both VL and VH are located in CDR-L1. L Ser28, L Tyr30, just before CDR-L2 L Tyr49 in CDR-L2 L Tyr53 in CDR-H1 H Tyr33, H Tyr35 in CDR-H2 H Asn52, H Ser53, H Asn54, H Thr57, H Asn58 (where superscript "L" denotes light chain and "H" denotes heavy chain). CDR residues not shown L With the exception of Tyr49, all antigen-interacting CDR residues listed above are in bold and italic in Tables 6A and 6B. Among the CDR residues that differ from the germline sequence are the CDR-L1 residues L Lys27, L His34, CDR-L2 residue L Leu54, L Glu55, CDR-H2 residue H Phe59, H Asn60, H Glu61, H Lys64, and H Asn65 (underlined in Tables 6A and 6B) was selected for CDR germlining, with the following amino acid substitutions, either individually or in combination: L K27Q, L H34A, L L54R, L E55A, H F59Y, H N60A, H E61Q, H K64Q, and HThey were replaced with their respective human germline equivalents with N65G. Other CDR residues were preserved to avoid any disruption of antigen-interacting residues.

[0160] For CDR-H1 (NYYMY; SEQ ID NO: 26), a single residue: H Only Asn31 is eligible for CDR germlining and is replaced with its equivalent residue in the human germline sequence, glycine. The other residues are involved in direct antigen interaction with PD1 ( H Tyr33 and H Tyr35), or conserved between mouse and human germline sequences ( H Tyr32 and H However, considering the short length of CDR-H1, which is only 5 amino acids, and the fact that two residues have direct interactions with the antigen, any amino acid changes could potentially disrupt the CDR conformation and affect the activity of the antibody. H Asn31 is unaltered throughout CDR germlining, and CDR-H1 is completely conserved.

[0161] In addition to the low level of conservation between the mouse CDRs and their corresponding human germline sequences, the pembrolizumab heavy chain framework (FR) also contains multiple non-germline residues, which arise due to the retention of unique somatic mutations in the acceptor framework sequence AB063829. H Val9, FR-H3 H Thr76, H Lys82a, H Gln83, H in Phe84 and FR-H4 H These somatic mutations, including Thr108, ​​are not believed to be structurally significant. H V9A, H T76S, H K82aS, H Q83R, H F84S,H The substitution with T108L leads to a significant improvement in the similarity score to the human germline sequence without disrupting the CDR conformation or altering the activity of the antibody.

[0162] Furthermore, we used IGHV3-23 (SEQ ID NO: 38) 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 resulting humanized antibody without compromising functional activity. IGHV3-23 belongs to the human antibody heavy chain germline VH3 family, the most common VH family in the human repertoire. It is also the most prevalent among commercially available human monoclonal antibodies and is widely recognized for its excellent drug-like properties. Considering that CDR conformation is highly sensitive to the chemical environment of the surrounding framework, a small number of structurally significant framework residues in pembrolizumab that differ from their VH3 germline family equivalents were selected for backmutation to their corresponding pembrolizumab equivalents. Furthermore, to improve the similarity score to the human germline sequence, several CDR-H2 residues were targeted for CDR germlining using IGHV3-23 CDR-H2 as a template.

[0163] The alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23 is shown in Table 6C. The six CDR-H2 residues: H Phe59, H Asn60, H Glu61, H Lys62, H Phe63, and H Asn65 (underlined in Table 6C; superscript "L" represents the light chain and "H" refers to the heavy chain) with the following amino acid substitution: H F59Y, H N60A, H E61D, H K62S, H F63V, andH It was selected for CDR germlining at N65G. CDR-H1 was excluded from the CDR germlining process for the reasons mentioned above. TIFF2025531805000007.tif48170

[0164] two framework residues, H Thr30 and H Arg94 is considered structurally significant and its corresponding germline equivalent, H Ser30 and H Lys94 was preserved without modification. Five additional IGHV3 framework residues that belong to the Vernier zone (U.S. Pat. Nos. 5,821,337 and 5,859,205) and may have structural significance: H Val48, H Ser49, H Ile69, H Arg71, and H Asn73 to their corresponding pembrolizumab residues, either individually or in combination, H Met48, H Gly49, H Leu69, H Thr71, and H The amino acid sequence was reverted to Ser73. The importance of specific framework amino acid residues was evaluated experimentally. The number of backmutations was minimized to ensure the highest similarity score to the germline sequence without negatively affecting antibody activity.

[0165] All optimized antibody sequences were expressed as full-length antibodies with a kappa light chain constant region containing the sequence set forth in SEQ ID NO: 34 and a modified IgG1 heavy chain constant region containing the sequence set forth in SEQ ID NO: 35. Table 7 lists the VL, VH, CDR-L1, CDR-L2, and CDR-H2 SEQ ID NOs for exemplary optimized PD1-blocking antibodies, along with a reference antibody (P-0734) containing the VL and VH sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 6, respectively. All antibodies of the invention contain the same CDR-L3 (SEQ ID NO: 25), CDR-H1 (SEQ ID NO: 26), and CDR-H3 (SEQ ID NO: 33). TIFF2025531805000008.tif146170

[0166] Example 2 Construction, production, and purification of optimized PD1-blocking antibodies All genes were codon-optimized for expression in mammalian cells and were synthesized and subsequently subcloned into recipient mammalian expression vectors through the services of GenScript. Protein expression was driven by a CMV promoter, and a synthetic SV40 polyA signal sequence was placed at the 3' end of the coding sequence. A leader sequence was engineered at the N-terminus of the construct to ensure proper signal transduction and processing for secretion.

[0167] Antibodies were produced by co-transfecting vectors harboring the light and heavy chains in a 1:1 ratio in ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. On the day of transfection, ExpiCHO cells were grown at 6 x 10 in ExpiCHO™ Expression Medium (ThermoFisher). 6The cells were diluted to 1000 cells / mL. A total of 0.8 μg of DNA expression vector per mL of culture volume was mixed with cold OptiPRO™ medium (40 μL per mL of cell culture). After adding ExpiFectamine™ CHO reagent at 3.2 μL per mL of cell culture, the solution was gently mixed and subsequently incubated at room temperature for 5 minutes. The ExpiFectamine™ CHO / plasmid DNA complex was then gently transferred to the cells and incubated at 130 rpm in a 37°C shaker incubator with an 8% CO2 atmosphere. ExpiFectamine™ CHO Enhancer (6 μL per mL of cell culture) and ExpiCHO™ Feed (240 μL per mL of cell culture) were added to the flask with gentle swirling 18-22 hours after transfection. After 8 days of culture, the supernatant was collected for purification by centrifugation at 2200 rpm for 20 min, followed by sterile filtration using a 0.22 μm filter (Corning).

[0168] Secreted antibodies were purified from cell culture supernatants using Protein A affinity chromatography. The cell culture supernatant was loaded onto a MabSelect SuRe 5 mL column (Cytiva) equilibrated with 5 column volumes (CV) of phosphate-buffered saline (pH 7.2) (ThermoFisher). Unbound proteins were removed by washing with 5 CV of PBS (pH 7.2), and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride buffer (pH 3.2). The antibody solution was neutralized by adding 3% 1 M Tris buffer (pH 10.2), followed by concentration and buffer exchange into PBS (pH 7.2) using an Amicon® Ultra-15 Ultracel (Merck Millipore) with a 10 KDa MWCO.

[0169] The purity and molecular weight of the purified antibodies were analyzed by SDS-PAGE with and without reducing agents 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 instructions. The aggregate content of the antibodies was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected onto an AdvanceBio size-exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) using 150 mM sodium phosphate buffer (pH 7.0) as the mobile phase at 25 °C.

[0170] The antibody concentration of purified protein samples was determined by measuring absorbance at 280 nm using a Nanodrop spectrophotometer (ThermoFisher) and dividing it by the molar extinction coefficient calculated based on the amino acid sequence. Endotoxin levels of purified protein samples were measured using Endosafe nexgen-PTS (Charles River) according to the manufacturer's instructions.

[0171] Example 3 Assays to evaluate the biological activity of optimized PD1-blocking antibodies The antibodies of the present 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 μg of antigen per well (100 μL / well). After triple washing with PBS / 0.05% Tween 20, the plates were incubated with SuperBlock (ThermoFisher) for 2 hours at room temperature to block nonspecific binding. PD1 antibodies serially diluted 3-fold in blocking buffer (PBS containing 1% bovine serum albumin) were added to the washed plates (100 μL / well) and incubated for 1 hour at room temperature. After another wash, the antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (ThermoFisher) diluted 1:5000 in blocking buffer (100 μL / well) for 1 hour at room temperature. After the final wash, 100 μL / well of TMB substrate (ThermoFisher) was added. The plate was sealed and incubated in the dark for 5-20 minutes. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50 μL / well), and the absorbance was measured at 450 nm using a plate reader. Curves were plotted and the half-maximal effective concentration (EC) was calculated using GraphPad Prism software. 50 ) values ​​were calculated.

[0172] Additionally, HEK 293T cells (Crown Bioscience) stably expressing the human PD1 gene were used to determine the cell-based binding strength of the optimized PD1 antibodies by flow cytometry. After harvesting, HEK293-hPD1 cells were cultured at 1 × 10 in 96-well U-bottom plates. 5Cells were seeded at 100 μL per well and 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 3-fold serial dilutions of each antibody in FACS buffer at concentrations ranging from 0.01 to 100 nM for 30 minutes at 4°C. Subsequently, cells were washed twice with FACS buffer to remove unbound molecules, and 40 μL of 1:100 diluted PE-labeled goat anti-human Fc secondary antibody (eBiosciences) was added to the cells. After a 30-minute incubation at 4°C and another double wash with FACS buffer, cell-bound antibodies were detected by flow cytometry (BD ACCURI-C6) using the PE-labeled secondary antibody, and EC was analyzed using GraphPad Prism software. 50 The value was calculated.

[0173] Furthermore, we measured the efficacy of PD1 antibodies in blocking PD1 interactions using a thaw-and-use format of the Promega luciferase reporter assay, a biologically relevant mechanism-based assay. Cell thawing and plating procedures were followed exactly as described in the manufacturer's protocol.

[0174] Briefly, one vial (0.5 mL) of PD-L1 aAPC / CHO-K1 cells was thawed and mixed with 14.5 mL of cell recovery medium (90% Ham's F12 / 10% FBS). Next, 100 μL of this cell suspension was added to the inner 60 wells of two 96-well flat-bottom assay plates, with the surrounding wells receiving 100 μL of cell recovery medium. After overnight incubation at 37°C and 5% CO2, the medium was discarded. The inner wells received 40 μL of 3-fold serially diluted compounds, and the surrounding wells received 80 μL of assay buffer (99% RPMI 1640 / 1% FBS). Subsequently, one vial (0.5 mL) of PD1 effector cells was thawed and mixed with 5.9 mL of assay buffer, and 40 μL of this mixture was added to the inner wells. After 6 hours of incubation at 37°C, 5% CO2, and 7 minutes of equilibration at room temperature, 80 μL of Bio-Glo™ reagent was added to all wells. The plate was then incubated for 10 minutes at room temperature with shaking. The resulting luminescence was measured using a luminescence plate reader (BioTek synergy h1).

[0175] Background was calculated by averaging the relative light units (RLU) of surrounding wells. Fold induction was determined by subtracting the background from the RLU of the antibody sample and dividing by the RLU of the control sample (no antibody) minus the background, or Fold induction = RLU (antibody - background) / (RLU (no antibody control - background). Finally, EC50 values ​​were determined using the fitted curves using GraphPad Prism software.

[0176] Example 4 Evaluation of PD1 antibodies containing germline modifications based on the closest human germline sequence First, we compared the efficacy of P-0734 with that of the pembrolizumab (PBL) biosimilar in blocking PD1 / PD-L1 interaction. P-0734 and the PBL biosimilar share identical variable domains, but they differ in their heavy chain constant regions. PBL contains an IgG4 constant chain (SEQ ID NO: 36) containing the S228P mutation, while P-0734 has an IgG1 constant chain (SEQ ID NO: 35) with L234A / L235A / G237A mutations to prevent Fc effector function. As shown in Figure 4, P-0734 and the PBL biosimilar were equally effective 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 sequence, not by immunoglobulin class. P-0734 faithfully reproduced the efficacy of the PBL biosimilar in blocking the PD1 / PD-L1 interaction and is referred to herein as the reference antibody.

[0177] in CDR-L1 using antibodies with slightly different mutational contexts L In H34A and CDR-H2 H The impact of the F60Y CDR germline substitution was evaluated. These antibodies, P-1148, P-1150, P-1151, and P-1153, all had a CDR-L1 germline substitution of F60Y. L K27Q, CDR-L2 L L54R, L E55A and CDR-H2 H N60A, H E61Q, H K64Q, H P-1150 contains the germline substitution N65G. L P-1151 contains an H34A substitution and an additional H F59Y substitution, and P-1153 L H34A and H Table 8 provides a list of CDR germline substitutions for these exemplary PD1 blocking antibodies. TIFF2025531805000009.tif45170

[0178] As shown in Figures 5B and 5C and summarized in Table 9, CDR-L1 germline substitutions L The H34A is H Regardless of the presence (P-1151 vs. P-1153) or absence (P-1148 vs. P-1150) of the F59Y substitution, E max PD1 blockade efficacy (EC 50 ) consistently led to an approximately 3.5-fold reduction in H The impact of the F59Y germline substitution was similarly assessed, and the data are shown in FIGS. 5B and 5C and summarized in Table 9. L regardless of the presence (P-1150 vs. P-1153) or absence (P-1148 vs. P-1151) of the H34A substitution. H The F59Y CDR germline substitution increases potency (EC 50 ; approximately 1.8-fold reduction) and signal (E max Compared to P-0734, the cumulative CDR germline substitutions in P-1153 resulted in a small but consistent reduction in both PD1 blockade potency (EC 50 ) and an almost 20-fold decrease in E max This ultimately led to a 50% reduction in L H34A and H Both substitutions, F59Y and F59Y, are considered deleterious in this particular framework, and the original CDR residues, L His34 and H Phe59 will be conserved.

[0179] However, despite significant differences in potency of blocking the PD1 / PD-L1 interaction, all four optimized PD1 antibodies and the reference antibody, P-0734, had EC values ​​close to 100 pM. 50 The binding strengths were almost identical with the values ​​(Fig. 5A and Table 9). TIFF2025531805000010.tif60170

[0180] This body of data suggested that mechanism-based functional assays have the ability to discern subtle changes in activity that are not detectable by ELISA binding assays. Therefore, the luciferase PD1 / PD-L1 reporter assay is used herein as the primary tool to characterize and rank PD1-blocking antibodies derived from pembrolizumab via germline substitutions. Derivative antibodies that maintain full functional activity are expected to exhibit the same in vivo efficacy as pembrolizumab.

[0181] CDR-L2 germline substitutions by comparing P-1127 and P-1129 L The potential negative effects of E55A were further evaluated. Both of these molecules contain nucleotides in the light chain CDRs. L K27Q and L Contains the K54E germline substitution, with the only sequence difference being an additional CDR-L2 substitution at P-1129, L As demonstrated in Figure 6A, P-1129 showed a slight but noticeable reduction in potency (EC 3.0 for P-1127 and P-1129, respectively). 50 = 0.39 nM and 0.58 nM) and E max showed a slight 10% decrease in L The E55A amino acid substitution was considered deleterious, and the original residue, L Glu55 will be conserved.

[0182] A total of six CDR germline substitutions, L K27Q, L L54R, H N60A, H E61Q, H K64Q, and H P-1174, harboring N65G, exhibited identical PD1 blocking activity to P-1127 and P-0734, with EC values ​​of 0.64 nM, 0.54 nM, and 0.67 nM for P-0734, P-1127, and P-1174, respectively.50 (Figure 6B). Additionally, P-1174 contains one CDR germline substitution, L P-1174 was derived from P-114 by eliminating E55A. When compared to P-0734, P-1174 exhibited higher potency than P-1148 (see Figures 5B and 6B). This portion of the data demonstrates that the original CDR residues, L They further concurred in the conclusion that Glu55 should not be altered.

[0183] 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. H Val9, FR-3 H Thr76, H Lys82a, H Gln83, H in Phe84 and FR-4 H These non-germline residues, including Thr108, ​​originating from conserved unique somatic mutations in the acceptor framework sequence, are not considered structurally significant. To further enhance the sequence similarity score or degree of similarity to the germline, these non-germline residues in the P-1174 framework were compared with their respective germline counterparts. H V9A, H T76S, H K82aS, H Q83R, H F84S, H As expected, P-1271 exhibited the same PD1 blocking activity as the reference antibody, P-0734 (Figure 6C), with EC values ​​of 0.66 nM for P-1271 and 0.70 for P-0734, respectively. 50 was the value.

[0184] In conclusion, CDR germline substitutions at P-1174 and P-1271, L K27Q, L L54R, H N60A, H E61Q,H K64Q, and H N65G enhanced the degree of humanization of the antibody sequence without compromising potency in blocking the PD1 / PD-L1 interaction. Six additional framework germline substitutions in P-1271 further increased the similarity score to the closest human germline sequence. Table 10 lists the germline substitutions and similarity scores to the closest human germline sequence of P-1174 and P-1271 compared to the reference antibody, P-0734. TIFF2025531805000011.tif64170

[0185] Example 5 Evaluation of PD1 antibodies containing germline modifications based on the more prevalent human germline family (VH3) The incorporation of framework germlining substitutions based on the human antibody heavy chain germline IGHV3-23 (SEQ ID NO: 38) was investigated to test whether antibody frameworks with substantially lower sequence homology but superior biophysical properties could enhance the drug-like properties of the resulting antibodies while fully retaining functional activity. Of the 33 framework germlining substitutions (Table 11A), five Vernier zone residues: H V48, H S49, H I69, H R71, and H The importance of N73, their respective pembrolizumab equivalents, individually or in combination, H V48M, H S49G, H I69L, H R71T, and H This was experimentally evaluated by backmutation to N73S. Additionally, six CDR-H2 residues, H F59Y, H N60A, H E61D, H K62S, H F63V, and HN65G was selected for CDR germline substitution with their corresponding residue in IGHV3-23. Table 11B provides a summary of VH3 germline substitutions in exemplary antibodies. TIFF2025531805000012.tif149170

[0186] Figure 7 depicts the PD1 blocking activity of P-1175 and P-1181, which differ only in the CDR-H2 germline substitution (as shown in Table 11B). Compared to P-0734, both P-1175 and P-1181 exhibited substantially reduced potency in blocking PD1 interactions. Notably, P-1174 exhibited significantly reduced potency (EC 50 ) and a 10-fold reduction in E max This corresponds to a 15-fold decrease in potency and a 25% decrease in E max and a 40-50% reduction in fold induction (shown in Figures 7A and 7B and summarized in Table 12). P-1181 showed a more drastic reduction in activity, and its two separate CDR germline substitutions, H K62S, H F63V is considered deleterious and therefore the original CDR residue, H Lys62 and H Phe63 will be conserved. The significance of individual CDR residues needs to be evaluated experimentally; these findings suggest that even CDR residues that are close to the interface or not immediately adjacent to antigen contact residues can negatively affect activity.

[0187] Two to five framework residue backmutations were introduced into P-1175 to yield P-1176, P-1177, and P-1178, as detailed in Table 11. As indicated by the data in Figure 8, the backmutations in P-1176, H I69L, H R71T, and HThe combination of N73S effectively restored PD1 blocking activity, nearly matching the level of P-0734. Similarly, the activity was not as effective as in P-1176, but the reversion mutation, H V48M and H The combination of S49G and S49G significantly restored the phenotype in P-1177. Nevertheless, the reversion of these two mutations to P-1176 ( H V48M and H Incorporation of the S49G) did not lead to a further increase in activity for the resulting antibody, P-1178 (P-1178 vs. P-1176 in Figure 8 and Table 12). TIFF2025531805000013.tif62170

[0188] P-1198( H N73S), P-1199( H R71T, H N73S), and P-1201( H I69L, H R71T, H By comparing the PD1 blocking activity of the three FR backmutations (N73S), H I69L, H R71T, and H The significance of each of the N73S mutations was further evaluated. As demonstrated in Figure 9, each added back mutation led to a small but significant cumulative increase in PD1 blocking activity. Only the combination of all three back mutations in P-1201 led to a near-complete restoration of functional activity (EC values ​​of 1.28 nM and 0.78 nM for P-1201 and P-0734, respectively). 50 value). Therefore, all three back mutations, H I69L, H R71T, H N73S is considered essential and will be incorporated.

[0189] Furthermore, the PD1 inhibitory activity of P-1194, P-1201, and P-1238 was compared and shown in Figures 10A and 10B. One additional CDR germline substitution, HP-1194 and P-1201, which differ only by F59Y, showed identical PD1 blocking potency when the IGHV1-2 germline sequence was employed. H This suggests that, contrary to previous observations that the F59Y germline substitution was deleterious, this particular substitution did not negatively affect activity. Therefore, it is conceivable that the impact of individual CDR germline substitutions depends on the context of the surrounding framework sequences. P-1238 was equipotent to the reference antibody, P-0734, with EC values ​​of 0.73 nM and 0.70 nM, respectively. 50 Compared with P-1194, two additional framework reversion mutations in P-1238, H V48M, and H S49G contributed to a small but discernible improvement in activity.

[0190] In the final evaluation, P-1174, P-1193, P-1198, P-1199, and P-1201 were classified as PD1. + The PD1-expressing antibodies P-1174, which fully preserved its PD1-blocking potency (Fig. 6C and 6D), exhibited binding affinity to PD1-expressing cells comparable to that of the reference antibody, P-0734 (Fig. 11A and 11B). P-1198, P-1199, and P-1201, which contain one to three framework backmutations, exhibited subtle but significant differences in potency in blocking PD1 interactions (Fig. 9), but no such variation in activity was detected in cell-based binding assays. All three compounds inhibited PD1. + P-1193 demonstrated equal potency as P-0734 in binding to cells (Figures 11C and 11D and Table 13). However, as shown in Figures 11C and 11D and Table 13, the cell-based binding assay was able to distinguish P-1193, which does not contain framework backmutations, from the other compounds. However, the degree of reduction was less pronounced than that observed in the blocking assay. The data further confirm our previous observation that mechanism-based PD1 / PD-L1 blocking assays are more sensitive than binding assays in identifying subtle activity differences. TIFF2025531805000014.tif56170

[0191] In summary, the optimized PD1-blocking antibodies, P-1194, P-1201, and P-1238, built on a VH framework (IGHV3-23) with substantially lower sequence homology but superior biophysical properties, can fully or nearly fully retain the functional activity of the antibodies and demonstrate improved similarity scores to the closest human germline sequence (IGHV3-23). ​​The mutation details and similarity scores for each antibody are summarized in Table 14. TIFF2025531805000015.tif87170

[0192] Example 6 Germlined substitutions led to reduced hydrophobicity of optimized PD1-blocking antibodies Among 23 FDA- and EMA-approved therapeutic mAbs, pembrolizumab was the most hydrophobic 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 coefficient (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 have a significant risk of hydrophobic interactions. The probabilities of hydrophobic interactions for its VH and VL are 0.97 and 0.61, respectively. An antibody is predicted to have a high risk of hydrophobic interactions if the probability is 0.5 or greater (1 being the maximum possible value).

[0193] While the focus of germline substitutions was to enhance the degree of "humanity" in the antibody sequence, the process also resulted in a significant reduction in the probability of hydrophobic interactions for several optimized antibody sequences. Table 15 provides a summary of the predicted probability of hydrophobic interactions for the variable domains of exemplary optimized PD1-blocking antibodies, as estimated by SSH2.0. TIFF2025531805000016.tif84170

[0194] Two light chain CDR germline substitutions, as shown in Table 15: L K27Q and L L54R significantly reduced the hydrophobicity probability of the VL from 0.607 for P-0734 to 0.131. These two amino acid changes were applied to the VL in all optimized PD1-blocking antibodies listed in Table 15. CDR germline substitutions in the heavy chain resulted in only a slight reduction in hydrophobicity, with the hydrophobicity probability changing from 0.971 for (P-0734) to 0.848 for (P-1174) and to about 0.8 for antibodies whose VHs are based on the VH-3 family framework. However, germline substitutions ( H V9A, H T76S, H K82aS, H Q83R, H F84S, H T108L) was implemented into 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.

[0195] Hydrophobic patches on the surface of antibodies are often implicated as one of the main contributors to their tendency to aggregate. Furthermore, these hydrophobic patches can cause high viscosity. Consequently, exemplary PD1-blocking antibodies with significantly reduced hydrophobic potential are expected to exhibit improved biophysical properties. PD1-targeting IL-15 immunocytokines and VitoKine fusions constructed using these optimized PD1-blocking antibodies are also expected to have enhanced developability profiles.

[0196] Example 7 Design and methods for assessing the in vitro activity of IL-15 variants Identifying IL-15 variants with optimally attenuated potency is crucial for constructing PD1-targeting immunocytokines to achieve a balance between cytokine and antibody components. In its native form, IL-15 exhibits significant disparities in potency and molecular weight compared to antibodies. Additionally, when designing PD1 Ab-IL15 VitoKines, incorporating IL-15 variants of specific potency facilitates precise tuning of both the intrinsic basal activity of the resulting VitoKine and its post-proteolytic function. Additionally, immunocytokines and VitoKine design require IL-15 variants with different potency levels. All IL-15 variants were initially produced as Fc fusions, and their activity was assessed using functional assays.

[0197] IL-15 variant Fc fusions were constructed by fusing a specific IL-15 variant to the C-terminus of the Fc chain (SEQ ID NO: 166), resulting in a dimeric IL-15 moiety. Alternatively, IL-15 variants were linked to the C-terminus of the knob chain from a knob-into-hole heterodimeric Fc chain pair (SEQ ID NOs: 167 and 168) to generate a monomeric IL-15 component. In both configurations, a flexible GS linker "GGGGSGGGGSGGGGS" (SEQ ID NO: 115) was used, and the IL-15Rα Sushi domain (SEQ ID NO: 165) was noncovalently complexed with each IL-15 domain (as depicted in Figures 3C and 3D). Noncovalent complexation of the IL-15Rα Sushi domain was demonstrated in our application PCT / US2019 / 038210 to significantly enhance the developability of IL-15 fusion proteins.

[0198] The functional activity of IL-15 variants was assessed using an established ex vivo human peripheral blood mononuclear cell (PBMC) assay. This was performed to evaluate their ability to stimulate cell proliferation by measuring Ki67 expression in CD8 T cells and NK cells. Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from buffy coats purchased from the Blood Oklahoma Institute. Purified PBMCs were then treated with increasing doses of IL-15 variants and incubated at 37°C for 5 days. On day 5, cells were washed with FACS buffer (1% fetal bovine serum in phosphate-buffered saline, pH 7.2) and first stained with Fc blocker (BioLegend) and surface marker antibodies, such as anti-human CD8-APC (BioLegend) and anti-human CD56-FITC, at a 1:50 dilution. After 30 minutes of incubation and washing, cells were treated with standard fixation and permeabilization solution (ThermoFisher) for an additional 30 minutes at room temperature in the dark. After centrifugation, cells were treated with permeabilization buffer (ThermoFisher) containing anti-human Ki67-PE antibody (BD Life Sciences). After a final 30-minute incubation, cells were collected, washed, and resuspended in FACS buffer before analysis using an Attune NxT flow cytometer (ThermoFisher). Data are expressed as the percentage of Ki67-positive cells in the gated population.

[0199] To determine cross-reactive IL-15 variants in mice using an in vitro method, a CTLL-2 cell proliferation assay was used. CTLL-2, a cytotoxic murine T cell line derived from C57BL / 6 mice, is commonly used to assess the biological activity of IL-2 and IL-15 by measuring the degree of cell proliferation. Briefly, CTLL-2 cells in logarithmic growth phase were washed three times with PBS buffer and incubated at 5 × 10 5The cells were resuspended in basal medium (RPMI medium containing 10% fetal bovine serum, 2 mM L-glutamate, and 1 mM sodium pyruvate) at a density of 5 x 10 cells / mL. After 4 hours of incubation at 37°C, 100 µL of these cells was added to an equal volume of serially diluted IL-15 fusion protein in basal medium in each well of a 96-well plate. 4 A final density of cells / well was achieved. After 48 hours of incubation at 37°C, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. EC 50 The results were analyzed using GraphPad Prism software to derive values.

[0200] P-0234 and P-0313 are used interchangeably as dimeric wild-type IL-15 controls. P-0313 contains the IL-15 S58D mutation (SEQ ID NO: 117), but its activity is consistently equivalent or slightly enhanced when compared to P-0234, which has wild-type IL-15 (SEQ ID NO: 116) (Figure 12). P-0217 is the monomeric equivalent of P-0234 and serves as a control for monomeric wild-type IL-15. Table 16 lists exemplary IL-15 variants in Fc fusion constructs. TIFF2025531805000017.tif241170

[0201] Example 8 Attenuation of IL-15 activity by substituting IL-15 residues that interact with IL-15Rβ Selection of IL-15 mutations that disrupt the IL-5Rβ interface was guided by examination of the IL-15 / IL-15R co-crystal structure (PDB ID: 4GS7 and Ring et al., 2012, Nat. Immunol. 13:1187-1195). Although residue I68 only forms a van der Waals interaction with IL-15Rβ, the impact of its substitution is surprisingly diverse. In particular, amino acid substitutions at position I68 resulted in variants with a wide range of potency in stimulating CD8 T cell proliferation. EC 50 Values ​​varied from 0.4 nM to approximately 300 nM, recording a significant difference of 700-fold. Compared to the control molecule, P-0313, the level of activity reduction ranged from 10-fold to roughly 7000-fold. The results are shown in Figure 13A and summarized in Table 17A. TIFF2025531805000018.tif69170

[0202] In contrast to residue I68, V63 does not directly interact with IL-15Rβ residues but is in close proximity to critical contact residues, D61 and N65, which form the receptor binding interface (see Ring et al., 2012, Nat. Immunol. 13:1187-1195). As expected, substitutions at position V63 resulted in only a moderate attenuation (approximately 4- to 10-fold reduction) of IL-15's ability to stimulate CD8 T cell proliferation (shown in Figure 13B). The EC of these variants in stimulating CD8 T cell proliferation was significantly lower than that of the IL-15Rβ variants. 50 The values ​​can be found in Table 17B along with their fold reduction compared to P-0313. TIFF2025531805000019.tif50170

[0203] Although the change in the amino acid at position V63 only resulted in a small reduction in IL-15 activity, it provides an approach for fine-tuning the level of attenuation by pairing it with other IL-15 amino acid changes that interfere with IL-15 receptor interaction. Exemplary combination mutants incorporating V63A and one of the mutations at I68, including V68H, I68Q, and I68G, were constructed and their activity was evaluated in human PBMCs. These combined mutations that disrupt IL-15Rβ interaction incrementally reduced the potency in stimulating CD8 T cell proliferation (detailed in Table 17C). Compared to P-0313, the V63A substitution resulted in an approximately 4.2-fold reduction in potency. Consistently, within the context of the I68H, I68Q, and I68G mutations, the V63A substitution resulted in a 2.1-fold, 4.3-fold, and 3.8-fold reduction in CD8 T cell proliferation potency, respectively. The data are also shown in Figure 13C. TIFF2025531805000020.tif81170

[0204] In addition to modifying IL-15 residues at the interface with the IL-15Rβ receptor subunit for targeted attenuation of activity, truncating N-terminal residues offers an alternative strategy. The N-terminus of IL-15 is an alpha-helix containing key residues that engage with IL-15Rβ, such as Ser7, Asp8, and Lys10 (Ring et al., 2012, Nat. Immunol. 13:1187-1195). Three dimeric IL-15 Fc fusion proteins, P-0866, P-0867, and P-0868, each with one, two, and three amino acid deletions, respectively, at the IL-15 N-terminus, were assayed using a human PBMC assay. The results are depicted in Figure 14. For P-0866, the single amino acid deletion did not affect its ability to stimulate CD8 T cell proliferation compared to the wild-type control, P-0234. Rather, a small 2-fold increase in potency was observed (EC of 289 pM for P-0234). 50 EC of 130 pM compared to 50Introduction of the second amino acid deletion in P-0867 resulted in a significant decrease in potency, nearly a 100-fold reduction (EC of 24,800 pM compared to 289 pM for P-0234). 50 The addition of one further deletion in P-0868 did not further reduce its functional activity.

[0205] A CTLL-2 cell proliferation assay (described in Example 7) was subsequently used to evaluate cross-reactive IL-15 variants in mice. In particular, five compounds, P-0771, P-0773, P-0772, P-0737, and P-0768, each with a distinct IL-15Rβ interfering mutation, were compared for their biological activity in terms of promoting human CD8 T cell proliferation and supporting mouse-derived CTLL-2 cell growth. The results are shown in Figures 15A and 15B and summarized in Table 18. From P-0771 to P-0773 to P-0772 / P-0737, there was an approximately 10-fold incremental decrease in the induction of Ki67 expression in human CD8 T cells. The potency gap between P-0771 and P-0768 was a significant 350-fold (EC of 0.132 nM and 48 nM, respectively). 50 In the CTLL-2 cell proliferation assay, P-0771 and P-0773 retained the biological activity observed in the human PBMC assay and exhibited EC 50 The results showed a 10-fold difference in the expression levels of P-0772 and P-0737. However, P-0772 and P-0737 exhibited a significant reduction in their ability to sustain CTLL-2 proliferation. This reduction was disproportionate to their potency in stimulating Ki67 expression in human CD8 T cells (approximately a 5-log reduction compared to a 100-fold reduction). Furthermore, P-0768 completely lost its ability to sustain CTLL-2 proliferation. TIFF2025531805000021.tif60170

[0206] The disproportionate decrease in activity observed in mouse cells compared to human cells was also seen in two N-terminal deletion mutants, P-0867 and P-0868. As shown in Figure 16A, there was an almost 100-fold reduction in stimulation of CD8 T cell proliferation compared to the wild-type control, P-0234. However, these mutants did not show a robust EC of 32.7 nM with P-0234. 50 In contrast, there was an almost complete loss of efficacy in supporting CTLL-2 cell proliferation (Fig. 16B).

[0207] To ensure that the absence of CTLL-2 activity correlated with in vivo activity in mice, P-0768 was administered to naive Balb / C mice to assess its impact on peripheral CD8 and NK cell proliferation. Consistent with the results of the CTLL-2 assay, there were no observable pharmacodynamic effects on peripheral lymphocytes, including CD8 T and NK cells. This contrasted sharply with the significant expansion of peripheral blood CD8 T and NK cells upon administration of either P-0313 or P-0773. Both of these compounds exhibit CTLL2 activity consistent with their activity in human cells, as detailed in Table 18.

[0208] The reduced or complete loss of cross-reactivity to mouse receptors makes it difficult to evaluate compounds with desirable in vitro potency in human cells, such as P-0772 and P-0768, for in vivo pharmacodynamic effects and antitumor efficacy in well-established syngeneic mouse tumor models, including CT26 and MC38. Alternative mutational strategies are needed.

[0209] Example 9 Modulation of IL-15 activity by substituting IL-15 residues at the interface with γc The Q108 residue of IL-15 is one of the hotspots at the interface with several key γc residues (Ring et al., 2012, Nat. Immunol. 13:1187-1195). Several IL-15 variants containing amino acid substitutions at Q108 were constructed and evaluated for their impact on cell proliferation, particularly by measuring Ki67 expression in CD8 T cells from fresh human PBMCs. Exemplary fusion proteins of IL-15 variants containing mutations at Q108 can be found in Table 16.

[0210] As shown in Figure 17A, various agonistic activities on CD8 T cell proliferation result from amino acid substitutions at position Q108 of IL-15, including Q108M ​​in P-0836, Q108N in P-1202, Q108T in P-1203, and Q108D in P-1204, Q108F in P-1205, Q108L in P-1206, and Q108Y in P-1207. 50 The values ​​range widely, from 2.7 nM for P-1202 (IL-15 Q108N) to 164 nM for P-1207 (IL-15 Q108Y). Furthermore, P-1204 (IL-15 Q108D) shows a complete loss of activity. These results are consistent with an EC of 0.35 nM for the wild-type counterpart, P-0217. 50 be compared to the value.

[0211] Figures 17B and 17C further depict the ex vivo activity of additional IL-15 Q108 variants. P-0793 (IL-15 Q108A) and P-0764 (IL-15 Q108S) showed a significant decrease in potency, with EC values ​​ranging from 20 to 50 nM. 50 Their signaling strength was also significantly reduced, and the amplitude of signaling was significantly lower than that of wild-type E maxThe agonistic activity of P-0796 (IL-15 Q108K) was reduced to 30-40% of the maximum possible effect (Figure 17B), demonstrating the characteristics of a partial agonist. Similar to P-1204 (IL-15 Q108D) and P-0684 (IL-15 Q108E; data not shown), P-0796 (IL-15 Q108K) also completely lost its agonistic potential. Shown in Figure 17C is a comparison of P-1059 and P-1061, both Fc fusions containing dimeric IL-15 domains. The IL-15 Q108H mutation in P-1061 exhibited slightly better potency than the Q108N mutation in P-1059, with EC values ​​of 0.23 nM and 0.39 nM, respectively. 50 Exemplary IL-15 variants (consisting of monomeric IL-15 unless otherwise noted) with a change at the Q108 residue and their agonist potency (EC < 0.05 in inducing CD8 T cell proliferation) were compared. 50 A summary of the values ​​can be found in Table 19. TIFF2025531805000022.tif114170

[0212] In summary, IL-15 mutations at the Q108 residue can be divided into distinct categories based on the extent to which the substitution affects CD8 T cell proliferation potency. Mutations in category 1, exemplified by Q108H and Q108N, cause only a slight to small reduction in IL-15 agonist activity. Mutations in category 2, represented by Q108A, Q108L, Q108M, Q108S, and Q108T, involve amino acids with either non-aromatic hydrophobic side chains or polar, uncharged side chains, leading to substantial decreases in activity. In category 3, mutants exemplified by Q108F and Q108Y involve aromatic amino acid substitutions and show a more drastic reduction in activity compared to category 2 mutations. Mutants in category 4, exemplified by Q108D, D108E, and Q108K, involve substitutions with charged amino acids and show complete inhibition of activity. Within each category, mutations with similar characteristics are expected to demonstrate comparable activity levels, for example, IL-15 variants with Q108I or Q108V mutations (residues with non-aromatic hydrophobic side chains) are expected to exhibit similar activity to other variants in category 2.

[0213] The potency of IL-15 can be further tuned by pairing it with other mutations that interfere with receptor interactions. For example, combination mutants were constructed that incorporated modifications affecting IL-15Rβ interactions, such as V63A, V63K, V68H, and V68F, along with changes affecting γc interactions, such as Q108N and Q108M. When tested for CD8 T cell proliferation in human PBMCs, these combined substitutions demonstrated incremental reductions in IL-15 agonist activity. This concept was exemplified by comparing the activity of P-1242 and P-1243 with that of P-1202 in stimulating CD8 T cell proliferation (Figure 17D). All these IL-15 variants contain the Q108N mutation, but P-1242 adds the V63K mutation, and P-1243 incorporates I68H. EC values ​​for P-1242 and P-1243 were 0.01. 50The values ​​were 41 nM and 27 nM, respectively, compared to 5.0 nM for P-1202, indicating a 5-8 fold reduction due to the additional amino acid changes, consistent with the impact observed for these two mutations (see Table 17).

[0214] Furthermore, IL-15 variants with the Q108 mutation retained mouse cross-reactivity despite substantially reduced agonistic activity in human lymphocytes. This contrasted sharply with I68 variants such as I68Q and I68G, which lost this cross-reactivity (Figures 14A and 14B). In Figure 18, four IL-15 variants, each with one additional substitution interfering with IL-15Rβ interaction in addition to the Q108M ​​mutation, including I68H at P-0832, I68F at P-0833, V63A at P-0834, and V63K at P-0835, showed significantly reduced potency and signaling strength in stimulating CD8 T cell proliferation compared with the wild-type control, P-0217. The variability in activity among them was attributed to distinct IL-15Rβ-interfering substitutions (Figure 18A). This activity pattern was consistent across CTLL-2 assays (Figure 18B), suggesting that the Q108 mutant IL-15 variants retain the ability to cross-react with the mouse receptor. Preservation of mouse cross-reactivity streamlines translational research, enabling convenient mouse studies to investigate in vivo pharmacodynamics and antitumor efficacy, especially for targeted IL-15 variants with low potency.

[0215] In addition to Q108, other IL-15 residues at or near the γc-receptor interface, including but not limited to D30, H32, M109, and N112, can also be modified to achieve varying levels of potency attenuation. Exemplary substitutions include D30T, H32E, H32D, H32N, H32Q, M109A, M109H, M109R, N112D, N112R, N112G, and N112P. These amino acid changes resulted in variable but generally small decreases in activity. For example, in Figure 19, P-1324 (N112G) significantly reduced the EC for CD8 T cell proliferation. 50 On the other hand, the P-1293 (N112D) mutation did not cause any change in EC 50 A four-fold reduction in the EC value (EC of 0.49 nM compared to 0.12 nM for P-0217) 50 β-interfering mutations to achieve specific potency levels. As will be appreciated by those skilled in the art, any additional combination mutations are within the spirit and scope of the present invention.

[0216] In summary, the incorporation of amino acid deletions, IL-15Rβ-interfering substitutions, or γc-disrupting modifications into IL-15, either individually or in combination, led to variants that exhibited a wide range of potency levels in stimulating human cytotoxic lymphocytes. Notably, N-terminal deletions or certain substitutions that affect IL-15 interaction with IL-15Rβ can lead to loss of mouse receptor cross-reactivity if potency falls below a certain threshold. However, changes that interfere with γc preserve the ability of IL-15 to cross-react with mouse receptors, with similar potency levels.

[0217] Example 10 Construction of PD1 Ab-IL-15 immunocytokine with optimized PD1 antibody and IL-15 variants spanning different potency levels Linking an IL-15 variant to a PD1 antibody aims to deliver the IL-15 variant preferentially in cis to PD1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, to promote selective signaling. This strategy can also reduce systemic exposure to IL-15 and provide synergy by removing negative regulation and revitalizing T cells in both function and number. The use of an IL-15 variant with reduced potency helps balance the disparity in potency and molecular weight between the cytokine and the antibody arm in its native form. This balance allows for optimal dosing and preserves the function of each arm. Reduced cytokine activity is expected to minimize peripheral activation, mitigate in vivo antigen sink and target-mediated deposition, and facilitate tumor targeting via the antibody arm.

[0218] The PD1 antibodies used to construct the PD1 Ab-IL-15 immunocytokine were selected from optimized human PD1 blocking antibodies containing the light chain sequence set forth in SEQ ID NO: 44 and the heavy chain sequences set forth in SEQ ID NOs: 45-49. These optimized PD1 blocking antibodies have high affinity for the human PD1 protein and demonstrate equal or comparable potency to pembrolizumab in blocking PD1. They also have higher sequence similarity scores to their closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab. Furthermore, they are predicted to have lower hydrophobicity, which in turn is likely to reduce aggregation tendency compared to pembrolizumab. PD1-targeting IL-15 immunocytokines constructed using these optimized PD1 blocking antibodies are also predicted to have an enhanced developability profile.

[0219] Using the flexible linker, "GGGGSGGGGSGGGGS" (SEQ ID NO: 115), the IL-15 domain was fused to the C-terminus of a PD1-blocking antibody heavy chain to form a dimeric IL-15 structure (shown in FIG. 3A) or to the C-terminus of the knob chain of a knob-into-hole heterodimeric heavy chain pair, resulting in a monomeric IL-15 structure (shown in FIG. 3A). In both configurations, the IL-15Rα Sushi domain (SEQ ID NO: 165) was non-covalently complexed with the IL-15 domain through co-expression during cell culture.

[0220] As will be appreciated by those skilled in the art, any IL-15 variant exhibiting varying levels of potency as disclosed herein, including but not limited to, sequences set forth in SEQ ID NOS: 117-163, can serve as a building block in constructing PD1 Ab-IL-15 immunocytokine constructs to potentiate and / or augment PD1 antibody-based therapies for various cancers. Additionally, the use of monomeric IL-15 in these constructs is expected to circumvent avidity effects and provide an additional approach for modulating IL-15 efficacy. Table 20A lists exemplary PD1 Ab-IL-15 immunocytokine constructs. TIFF2025531805000023.tif60170

[0221] Considering that the human PD1-blocking antibodies of the present invention do not bind to mouse PD1, surrogate mouse PD1 Ab-IL-15 immunocytokines were similarly prepared in either dimeric or monomeric forms. The linker connecting the PD1 antibody heavy chain and the IL-15 domain is set forth in SEQ ID NO: 115. In both configurations, the IL-15Rα Sushi+ domain (SEQ ID NO: 165) is noncovalently complexed with each IL-15 domain (as depicted in Figures 3A and 3B). These surrogate immunocytokines were used for in vivo studies to evaluate their pharmacodynamic effects on lymphocyte stimulation in mice and antitumor efficacy in syngeneic mouse tumor models. Table 20B lists detailed information about the surrogate constructs. TIFF2025531805000024.tif55170

[0222] The construction of expression vectors, transient expression as well as subsequent purification and characterization of these immunocytokine fusions were carried out according to the procedures outlined in Example 2.

[0223] Example 11 Ex vivo characterization of PD1 Ab-IL-15 immunocytokines It is important to demonstrate that the observed potency levels of IL-15 variants in Fc fusion format remain consistent when fused to antibodies. This consistency ensures the reliability of results across different fusion formats. As depicted in Figure 20, the biological activity of the IL-15 domain remains consistent whether fused to an Fc or PD1-blocking antibody. Notably, P0773 is an Fc fusion, and P-0870 is a PD1 antibody fusion, both containing the dimeric IL-15 variant V63A / I68H, but they exhibited identical potency in stimulating a dose-dependent increase in Ki67 expression in CD8 T cells (Figure 20A). In Figure 20B, P-0867 and P-0886, a similar pair featuring two amino acid deletions at the N-terminus of IL-15, exhibited the same activity. This consistency across formats emphasizes the reliability and validity of the observed potency levels of IL-15 variants.

[0224] It is also essential that the PD1 antibody retains its binding and functional activity when incorporated into the PD1 Ab-IL-15 immunocytokine. PD1 antibodies with excellent target binding and PD1 blocking function can enhance the specificity and selectivity of TIL targeting and further cooperate with the IL-15 anti-cancer immune response by effectively reversing T cell anergy and exhaustion.

[0225] Two ELISA assays were performed to evaluate the PD1 binding and inhibitory ability of the exemplary immunocytokine P-1352 relative to its component PD1 antibody, P-1271. A non-targeting germline antibody, P-1260 (SEQ ID NOs: 171, 172, and 173), was included as a negative control. The binding ELISA procedure is outlined in Example 3. The competition ELISA followed a similar protocol. After coating, blocking, and washing steps, each well received a mixture of 0.5 μg / mL biotinylated human PD-L1-Fc (Acro Biosystems) with equal volumes of three-fold serial dilutions of either P-1271 or P-1352, starting at a concentration of 100 μM. After 1 hour of incubation at 37°C, HRP-conjugated streptavidin (ThermoFisher) diluted to 0.1 μg / mL was added to the plate and incubated at 37°C for 1 hour. Plates were subsequently developed using TMB (ThermoFisher) substrate. Absorbance was read at 450 nm with 630 nm as the reference wavelength. Half-maximal inhibitory concentration (IC50) values ​​were derived using GraphPad Prism software.

[0226] Both P-1271 and P-1352 had EC of 26.8 pM and 30.4 pM, respectively. 50 Figure 21A shows that the P-1271 and P-1352 antibodies exhibit indistinguishable PD1 binding capabilities, as demonstrated by IC values ​​of 1.42 nM for P-1271 and 1.88 nM for P-1352. Similarly, Figure 21B shows that the P-1271 and P-1352 antibodies exhibit indistinguishable PD1 binding capabilities, as demonstrated by IC values ​​of 1.42 nM for P-127 50 These findings highlight that the PD1 antibody fully retained its binding and blocking activity when incorporated into an immunocytokine construct.

[0227] Additionally, switching IL-15 from a dimeric form in P-0869 to a monomeric form in P-1266 resulted in an EC of 0.67 nM and 2.0 nM for P-0869 and P-1266, respectively. 50Figure 22 highlights that the PD1 Ab-IL-15 immunocytokines, along with the IL-15 V63A / I68H variant, resulted in a 3-fold decrease in ex vivo activity. Both P-0869 and P-1266 are PD1 Ab-IL-15 immunocytokines incorporating the IL-15 V63A / I68H variant. These findings highlight that the incorporation of monomeric IL-15 into fusion constructs may offer an alternative approach to modulating IL-15 potency, circumventing the avidity effects commonly observed with the dimeric form.

[0228] Finally, before proceeding to in vivo studies, the murine PD1 Ab-IL-15 immunocytokines, P-1266, P-1295, and P-1296, were evaluated for their activity in stimulating Ki67 expression in human CD8+ T cells. The monomeric IL-15 variants in these immunocytokines have mutations including V63A / I68H (targeting IL-15Rβ binding), Q108N (targeting γc binding), and I68H / Q108N (both interfering with IL-15Rβγ binding). As shown in Figure 23, EC 50 The potencies indicated by the values ​​are 1.94 nM for P-1266, 4.93 nM for P-1295, and 44.3 nM for P-1296. The EC of 0.13 nM for the wild-type IL-15 control, P-1284. 50 When compared with the values, the reduction in potency is by factors of 15, 38, and 340, respectively.

[0229] Example 12 Pharmacokinetic and Pharmacodynamic Effects of PD1 Ab-IL-15 Immunocytokine in Mice The pharmacodynamic effects of PD1Ab-IL-15 immunocytokine on immune cells in the peripheral blood of C57BL / 6 mice were investigated using P-1266 and P-1295. The monomeric IL-15 domain in P-1266, containing the V63A / I68H mutation, demonstrated 2-3 times greater potency than P-1295 (with the Q108N mutation) in stimulating Ki67 expression in human CD8 T cells (Figure 23).

[0230] Seven-week-old female C57BL / 6 mice were received from Charles River Laboratory and acclimated in-house prior to the study. On day 0, mice were intraperitoneally administered either vehicle (sterile PBS buffer) or a single dose of each test compound, P-1266 and P-1295, at a dosage of 1.5 mg / kg (mpk). Each group consisted of five mice. Blood samples were collected on days 0, 5, 7, 10, and 12 post-injection and subsequently processed to prepare single cell suspensions.

[0231] Briefly, red blood cells were lysed using BD Pharmingen lysis buffer, and the total number of viable mononuclear blood cells was counted after excluding dead cells with trypan blue. These lysed immune cells were then fixed and permeabilized using fixation / permeabilization buffer (eBioscience) by incubation in the dark at room temperature for 30 minutes. After washing, the fixed and permeabilized cells were stained with antibodies to identify distinct immune cell subsets using a flow cytometer (Beckton Dickinson). Additionally, cell proliferation and activation within the identified subsets were assessed using the Ki67 proliferation marker and the granzyme B cytotoxicity marker. Different immune cell subsets were identified, and the absolute number of circulating cells was quantified on the flow cytometer using the following commercially available antibodies: CD3-APC.Cy7, CD8-Percp-cy5.5, CD335-APC, Ki67-PE, and granzyme B-BV421. Data from flow cytometry were analyzed using FlowJo software and results were plotted in GraphPad Prism.

[0232] Both P-1266 and P-1295 stimulated a significant increase in the percentage of cells expressing Ki67, a marker indicative of cell proliferation, in CD8+ T (Figure 24A) and NK cells (Figure 24D). The slight differences in maximum signals are consistent with differences in their in vitro activity, suggesting that both compounds exhibit similar cross-reactivity to the mouse receptor. For NK cells, known to be more responsive to IL-15, both showed peak activity at day 5; for CD8 cells, P-1266 peaked at day 5 and P-1295 peaked at day 7, consistent with their respective potencies.

[0233] In contrast to cell proliferation, there were significant differences in cell expansion of CD8 T cells (Figure 24B) and NK cells (Figure 24E) between P-1266 and P-1295. In response to P-1266, CD8 T cells increased from 550 cells per μL of blood to a peak of 6955 cells per μL on day 7, a 12.5-fold increase. Similarly, NK cells underwent a 15-fold surge, reaching a peak of 1275 cells per μL on day 5 from an initial count of 86 cells per μL of blood. On the other hand, P-1295 led to a negligible 1.6-fold increase in CD8 T cells and a small 3-fold expansion in NK cells. This pattern was reflected in the percentage of CD8 T cells expressing the activation marker, granzyme B, which was significantly lower for P-1295 at 21% compared to 90% for P-1266 (Figure 24C). Given that discrepancies in mouse receptor cross-reactivity are unlikely to be the cause, it is plausible that the variation in cell expansion results from the Q108N mutation in P-1295 disrupting interaction with γc, while the V63A / I68H mutation in P-1266 affects binding to IL-15Rβ, which in turn affects how each receptor influences the signaling cascade leading to cell expansion.

[0234] Furthermore, P-1266 was associated with a detectable decrease in body weight on day 5, consistent with a dramatic expansion of cytotoxic lymphocytes. In contrast, P-1295 exhibited no weight loss (FIG. 24F).

[0235] The pharmacokinetic (PK) effects of P-1266 and P-1295 were compared in parallel in vivo studies. Each compound was administered intravenously at a dose of 1 mg / kg, and blood samples were collected by buccal bleeding at 4, 24, 48, 72, 120, 168, and 240 hours after injection. Each group consisted of three mice, and blood was collected either weekly or every three days, with a maximum frequency of two times per mouse.

[0236] Serum concentrations of compounds were determined using an ELISA assay. Briefly, maxisorp plates were coated with mouse PD1 protein (R&D systems) overnight at 4°C. After this, the plates were blocked with Superblock (ThermoFisher). Blood samples at various dilutions were added to the plates and incubated for 1 hour at room temperature. A biotinylated monoclonal anti-IL15 antibody (BD Bioscience) paired with HRP-conjugated streptavidin (ThermoFisher) was applied. The resulting signals were developed using Ultra TMB substrate solution, and values ​​were extrapolated from a nonlinear regression curve fit in GraphPad Prism.

[0237] As shown in Figure 25, P-1295 exhibits an improved PK profile compared to P-1266. Serum concentrations of P-1295 remained constant up to 128 hours after a 1 mg / kg dose, while P-1266 concentrations began to decline by 72 hours. For both compounds, serum concentrations approached the lower limit of quantitation (LLOQ), represented by the dotted line. The improved PK profile of P-1295 can be attributed to reduced lymphocyte expansion due to its γc-interfering mutation. This potentially leads to reduced target-mediated drug deposition and target sink.

[0238] Furthermore, in a parallel pharmacodynamic experiment, we compared the effects of P-1266 and P-1296 on peripheral lymphocytes in MC38 tumor-bearing mice (details of the tumor model are detailed in Example 13). Treatment consisted of vehicle control, P-1266 at a dose of 1.5 mg / kg, and P-1296 at doses of 1.5 and 3 mg / kg, with each group containing four mice. Five days after injection, blood samples were collected and subsequently processed to prepare single-cell suspensions. Given the observed activity pattern of P-1295 and the fact that P-1296 with significantly reduced efficacy contains the same γc-interfering mutation Q108N as P-1295, along with the I68H mutation targeting IL-15Rβ interaction, we hypothesized that P-1296 stimulates lower Ki67 expression on lymphocytes. Furthermore, a more significant decrease in cell expansion activity was expected.

[0239] As shown in Figure 26, when P-1296 was administered at dosages of 1.5 and 3 mg / kg, it stimulated significant Ki67 expression on CD8 T cells (shown in Figure 26A) and NK cells (Figure 26B) compared to vehicle controls. However, expression was still less intense than that seen with P-1266. This data confirms that P-1296 still reacts with the mouse receptor, albeit with reduced potency. A tendency to replicate the results of P-1295 (Figure 24) was seen with P-1296; in this case, expansion of both lymphocytes was significantly lower than that observed with P-1266, as shown in Figures 26C and 26D.

[0240] It is generally believed that incorporating a monomeric IL-15 domain may circumvent the avidity effect of the dimeric form, leading to lower potency. This was evidenced by the three-fold decrease in activity when IL-15 was switched from the dimeric form in P-0869 to the monomeric form in P-1266 (Figure 22). However, when these compounds were tested in MC38 tumor-bearing mice at a dose of 1.5 mg / kg, immunophenotyping outcomes assessed after 5 days contradicted this general assumption and the in vivo results. Remarkably, dimeric P-0869 led to Ki67 expression comparable to its monomeric counterpart, P-1266, but unexpectedly induced substantially reduced cell proliferation, as shown in Figure 27. In particular, P-1266 treatment led to a 26-fold surge in CD8 T cells and a 17-fold boost in NK cells, whereas P-0869 resulted in only a 6- and 8-fold increase in CD8 T and NK cells, respectively.

[0241] In summary, when compared with IL-15 variants affecting IL-15Rβ binding, IL-15 variants with γc-interfering mutations trigger Ki67 expression in mice consistent with their ex vivo potency, but with a disproportionately lower increase in cell count and an improved PK profile. Furthermore, dimeric IL-15 triggers much lower lymphocyte expansion than its monomeric counterpart, a finding that contradicts both widespread acceptance and in vitro observations.

[0242] Example 13 Antitumor efficacy of PD1 Ab-IL-15 immunocytokine in a syngeneic mouse tumor model The antitumor efficacy of PD1 Ab-IL-15 immunocytokine was examined in both MC38 and CT26 mouse colon cancer models. Both tumor models have been extensively used to evaluate the efficacy of anticancer immunotherapy and have proven to be very valuable. Notably, CT26 is considered a "cold" tumor and is less responsive to PD1 treatment than the MC38 model.

[0243] For MC38 tumors, 5 × 10 subcutaneous injections were administered into the right flank of 7- to 9-week-old female C57BL / 6 mice. 5 Approximately 75 mm of MC38 colon cancer cells were transplanted. 3 Mice bearing established MC38 tumors with an average volume of 5 × 10 were randomized into groups, with that day designated as day 0. 5 The CT26 tumor model was established by subcutaneously implanting CT26 cells into the right flank of female Balb / C mice. After 9–11 days, the average tumor volume reached approximately 75 mm. 3 Once the mice reached 100 mg / kg, they were grouped and that day was designated as day 0. Test compounds were administered via intraperitoneal injection on day 1, the day after randomization. Tumor size and body weight were checked twice a week. A caliper was used, and volume = 0.5 x (width) 2 Tumor volume (TV) was monitored, calculated as 1 / (length). Tumor growth inhibition (TGI, %) was calculated using the following formula: TGI (%) = [1 - (TV of treated group) / (TV of control group)] x 100 (%). The termination criterion for sacrificing animals was a tumor size of 1500 mm 3 was reached or exceeded and / or the tumor became necrotic.

[0244] Various dosage levels (0.3, 1.0, and 2.0 mg / kg) of P-0869 were administered to CT26 tumor-bearing mice every 10 days (Q10D) for a total of two injections on days 1 and 11. Each dose is marked with a dotted line and arrow. Vehicle (sterile PBS) was included as a negative control. The mean tumor volume for each group as a function of time is shown in Figure 28A. Mice treated with vehicle rapidly developed large subcutaneous tumors. P-0869 demonstrated potent efficacy in inhibiting tumor growth in a dose-dependent manner. Remarkably, tumors were completely eradicated in three mice: one from the 1 mg / kg dose group and two from the 2 mg / kg dose group. The observed anticancer results are remarkable given the generally reduced efficacy seen in the CT26 tumor model when subjected to PD1 treatment.

[0245] The three mice that remained tumor-free were then reintroduced with CT26 cell transplantation 67 days after the initial treatment, or 75 days after the primary transplant. As shown in Figure 28A, none of the re-challenged mice had tumor recurrence, compared to the successful engraftment observed in age-matched naive mice used as controls. It is clear that the PD1 Ab-IL-15 immunocytokine induced long-term immunity.

[0246] P-0869 also effectively suppressed MC38 tumor growth in a dose-dependent manner, as shown in Figure 28B. Two doses of P-0869 were administered at dosage levels of 0.3 and 1 mg / kg on days 1 and 13. Vehicle (sterile PBS) was included as a negative control, and each group consisted of eight mice. On day 19, tumor growth inhibition (TGI) was 64% for the group treated with 0.3 mg / kg P-0869 and reached a full 100% for the 1 mg / kg group, compared with the vehicle-treated group. Remarkably, in the group receiving the 1 mg / kg dosage, six of the eight mice experienced complete tumor eradication.

[0247] As described in Example 12, dimeric IL-15 in P-0869 triggered much lower CD8 T and NK cell expansion than its monomeric counterpart, P-1266, despite comparable potency in stimulating Ki67 expression on the same lymphocytes. Nevertheless, when considering antitumor efficacy, both P-1266 and P-0869 produced similar results in the CT26 tumor (Figure 29A) and MC38 tumor models (Figure 29B). In the CT26 tumor model, each compound was administered at two doses of 1 mg / kg Q12D. After treatment with P-0869, two of the seven mice remained tumor-free, while one of the seven mice in the P-1266 group achieved complete tumor eradication. Similarly, in the MC38 tumor model, each mouse was given two doses of 1.5 mg / kg, one and one 3. P-0869 treatment led to 3 of 7 mice being tumor-free, whereas 4 of 7 mice from the P-1266 group had complete tumor clearance. In both cases, P-1266 and P-0869 resulted in comparable TGI.

[0248] In a comparable manner, P-1295 and P-1296 were similarly evaluated in CT26 and MC38 tumor models. Both P-1295 and P-1296 contain a Q108N mutation that disrupts their interaction with the γc receptor subunit. Additionally, P-1296 has an I68H mutation that affects the IL-15Rβ interface. A comparison of their in vitro activities is shown in Figure 23. Compared to the wild-type IL-15 counterpart, P-1284, the EC 50 The values ​​varied from 0.13 nM to 1.94 nM for P-1266, 4.93 nM for P-1295, and 44.3 nM for P-1296, representing a 15-, 38-, and 340-fold reduction in potency. Immune cell expansion triggered by P-1295 and P-1296 was significantly lower than that by P-1266 (shown in Figures 24 and 25). This difference is likely attributable to different receptor subunits being affected, which subsequently alter the signaling cascade that drives cell expansion.

[0249] Despite the observed reduction in immune cell expansion, both P-1295 and P-1296 exhibited antitumor efficacy in both the MC38 and CT26 models. Notably, as depicted in Figures 30A and 30B, at two doses of 1.5 mg / kg, the efficacy of P-1295 closely matched that of P-1266. Remarkably, in MC38 tumors, both compounds achieved complete tumor elimination in all eight mice (Figure 30B). Furthermore, at doses of 1.5 and 3 mg / kg, P-1296 significantly inhibited tumor growth in the CT26 (72% and 64% TGI on day 9, as shown in Figure 31A) and MC38 models (97% and 100% TGI on day 17, with 3 and 5 of 7 mice tumor-free, as shown in Figure 31B).

[0250] In summary, PD1 Ab-IL-15 immunocytokines with a range of agonistic potencies of IL-15 variants affecting different receptor subunits all demonstrated antitumor potential. These results were consistently observed when the compounds were evaluated in CT26 and MC38 tumor models. This highlights the potential versatility and efficacy of PD1 Ab-IL-15 immunocytokines in addressing multiple tumor types.

[0251] Example 14 Tuning the endogenous basal activity of IL-15 VitoKine using IL-15 variants with varying potency levels Compared to PD1 Ab-IL15 immunocytokines with attenuated IL-15Rβγ activity, the VitoKine platform may offer a more sophisticated strategy for balancing antibody and cytokine components, preventing pathway overactivation, and minimizing target-mediated deposition in addition to antigen sinks. This enhances safety profiles and bioavailability, potentially enabling human dosing within the effective range of PD1 antibodies. The VitoKine technology was disclosed by the inventors in International Publication Nos. 2019246392 and 2021119516. In such constructs, the activity of the cytokine domain is hidden until locally activated by tumor-associated antigens. Despite over 1000-fold more efficient activity hiding using the optimal L2 linker and cryptic domain, VitoKine, which contains a highly potent IL-15 domain, may still exhibit significant intrinsic basal activity.

[0252] For example, P-0315, an Fc-dimeric IL-15 VitoKine harboring the fully active IL-15 S58D variant, had an EC of 30 nM in human PBMCs. 50 CD8 T cells at 1000 ng / mL and an EC of 11 nM 50 P-0315 has intrinsic basal activity capable of stimulating NK cells at elevated concentrations. This is present despite a greater than three-order of magnitude reduction in activity compared to its non-VitoKine counterpart, P-0313 (Figures 32A and 32B). When administered at in vivo doses higher than 1 mg / kg, the inherent basal activity of P-0315 may persistently stimulate peripheral receptors and lead to prolonged in vivo pharmacodynamic effects, potentially imposing a risk of systemic toxicity (data not shown).

[0253] Incorporating a lower potency IL-15 variant helps tune the intrinsic basal activity of the IL-15 VitoKine, which correlates proportionally with the activity of the IL-15 moiety. For example, P-0875, a PD1 Ab-IL-15 VitoKine containing the IL-15 V63A / I68H variant as the D2 domain, exhibits significantly reduced intrinsic basal activity due to the weakening of the IL-15 domain. Consistent with the 1000-2000-fold increased secretion efficiency characteristic of this IL-15 VitoKine platform, P-0875 is approximately 1000-2000-fold less potent than its non-VitoKine counterpart, P-0870. The estimated EC for P-0875 is 50 The values ​​are approximately 2 μM and 225 nM in stimulating the proliferation of human CD8 T cells and NK cells, respectively, as shown in Figures 32C and 32D. Given their higher sensitivity to IL-15 compared to CD8 T cells, NK cells were included in the evaluation to assess the ex vivo activity of IL-15 VitoKine constructs, especially those with lower intrinsic basal activity.

[0254] In a preliminary pharmacodynamic study using cynomolgus monkeys, P-0875 was administered at a dose exceeding the typical effective range of PD1 antibodies in humans. Despite this elevated dose, there was only a minimal increase in peripheral blood cytotoxic lymphocytes, and no adverse events were observed. This significantly enhanced safety profile may be due to the adoption of an attenuated active domain, which resulted in a reduction in VitoKine's intrinsic basal activity.

[0255] Importantly, the tunable basal intrinsic activity of IL-15 VitoKine allows for a precise balance between the inertness of VitoKine prior to cleavage and its potency upon activation. This facilitates achieving the desired antitumor efficacy while minimizing the risk of unintended systemic toxicity. Any IL-15 variants, each with different potency levels as disclosed herein, including those defined by SEQ ID NOS: 116-163, can be used as active partial domains to construct PD1 Ab-IL-15 VitoKine.

[0256] Furthermore, the valency of the IL-15 domain in PD1 Ab-IL-15 VitoKine can be adjusted to further tune its potency in proteolytic activation, in addition to its intrinsic basal activity. Both dimeric and monomeric PD1 Ab-IL-15 VitoKine were constructed, the structures of which are shown in Figure 1C and Figure 1D, respectively.

[0257] Example 15 Optimizing the developability and activity of IL-15 VitoKines through the employment of different L2 linkers IL-15 VitoKine constructs containing the 10-amino acid MMP-2 / 9 cleavable L2 linker "GGPLGMLSQS" (SEQ ID NO: 85) tend to have inferior protein developability profiles compared to IL-15 fusion proteins with a non-covalently associated IL-15RαSushi+ domain. Among them, P-0874 and P-0869 represent such a pair. The structure of P-0874, a PD1 Ab-IL-15 VitoKine, is shown in Figure 1C, and its molecular details can be found in Table 23B. P-0869, whose structure is shown in Figure 3A, is the non-VitoKine counterpart of P-0874, with the IL-15RαSushi+ domain (SEQ ID NO: 165) non-covalently introduced during co-expression.

[0258] Size exclusion chromatography (SEC) analysis of P-0869 and P-0874 clearly demonstrates poorer purity for P-0874, as shown in Figures 33A and 33B. After a single Protein A purification step, P-0869 contained 37.6% impurities, primarily attributable to aggregate and small fragment content, in sharp contrast to the 100% purity for P-0874. Furthermore, P-0869 was produced at significantly reduced levels compared to P-0874, in addition to its increased tendency to aggregate.

[0259] The suboptimal expression profile of IL-15 VitoKine may be attributed to the spatial constraints introduced by the L2 linker, which may lead to distorted interactions between the IL-15 domain and the IL-15Rα Sushi domain. It is conceivable that adjusting the length of the L2 linker and / or altering its sequence / flexibility may enhance the developability and biophysical attributes of the resulting IL-15 VitoKine. Given that the length and composition of the L2 linker play a crucial role in the efficiency of D3 occlusion, a balance must be struck to ensure that the inertness of VitoKine activity remains largely unchanged. Following the above considerations, the L2 linker in P-0874 was replaced with linkers of varying lengths and compositions. The resulting PD1 Ab-IL-15 VitoKine constructs are listed in Table 21. For example, the L2 linker in P-0874 has 10 amino acids, while in P-1077, P-1083, P-1084 it has 15 amino acids, and in P-1085 it has 20 amino acids. TIFF2025531805000025.tif53170

[0260] The expression levels (mg / L) and purity of Protein A-purified material, as determined by SEC chromatography, for exemplary molecules are summarized in Table 22. Additionally, their SEC chromatograms are depicted in Figure 33. Remarkably, incorporation of a 20-amino acid dual protease-cleavable L2 linker (SEQ ID NO: 93) into P-1085 significantly enhanced its developability profile. Aggregation tendency was significantly reduced, and purity improved from 62% for P-0874 to over 80% for P-1085, as indicated by SEC (Figure 33 and Table 22). Additionally, productivity increased significantly, varying from approximately 20 mg / L to 91 mg / L. Such improvement in developability was only seen when the linker was extended from 10 to 20 amino acids, but not when the linker length was increased to 15 amino acids.

[0261] Even more strikingly, P-1084, which contains a 15-amino acid L2 linker (SEQ ID NO: 92) with the same core sequence (GPLGMLSQPMAKK; SEQ ID NO: 76) as in P-1085, not only exhibited lower expression compared to RO1085 (listed in Table 22), but also exhibited minor (<10%) premature cleavage during the cell culture process (data not shown). This observation suggests that peptide spacers adjacent to cleavable linkers may introduce additional, unwanted cleavage sites in a context-dependent manner. Finally, in addition to length, the composition of the L2 linker significantly affected the developability profile of IL-15 VitoKines. This is evidenced by the data for P-1347. Although its only difference from P-1085 is the L2 linker composition, which is a 20 amino acid non-cleavable flexible linker, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 102), P-1347 exhibited poor purity of 50% and a reduced expression level of 19.2 mg / L. TIFF2025531805000026.tif59170

[0262] The biological function of the VitoKine constructs was assessed by measuring the increase in Ki67 expression in CD8 T cells and NK cells within human PBMCs. The data can be seen in Figure 34 and show that EC 50 The values ​​are summarized in Table 22. EC for the non-VitoKine fusion counterpart, P-0869 50 The EC values ​​were 1.14 nM for CD8 T cells and 0.089 nM for NK cells. Premature cleavage of the L2 linker in P-1084 was neither expected nor desirable, resulting in an unwanted increase in its basal activity. Compared to P-1083, which features an L2 linker of identical length, P-1084 exhibited 25-fold higher basal activity, with EC values ​​of 3.95 nM for P-1084 and 93.1 nM for P-1083 in stimulating NK cell proliferation. 50 was the value.

[0263] As expected, extension of the L2 linker from 10 in P-0874 to 15 in P-1083 and further to 20 amino acids in P-1085 led to incremental enhancements in activity for both CD8 T cells and NK cells (Figure 34). In direct comparison with P-0869, P-1085 demonstrated an overall cloning efficiency of approximately 350-fold. This efficiency represents a 325-fold reduction in potency for CD8 T cells and a 371-fold reduction in potency for NK cells, and is a small reduction from the 1000- to 2000-fold cloning efficiency when a 10-amino acid cleavable linker (SEQ ID NO: 85) was used as the L2 linker.

[0264] The 20-amino acid double protease-cleavable linker (SEQ ID NO: 93) in P-1085 was subsequently incorporated into two additional PD1 Ab-IL-15 VitoKines: P-1265 and P-1263. P-1265 is the monomeric counterpart of P-1085, containing the IL-15 V63A / I68H variant. P-1263 and P-1265 differ only in the IL-15 domain, with P-1263 containing the Q108N mutation. Both exhibited significantly better developability profiles compared to the IL-15 VitoKine with a 10-amino acid L2 linker, as exemplified by P-0874. Remarkably, P-1263 exhibited 90% purity, and P-1265 achieved 85% purity, and both had some expression levels exceeding 50 mg / L.

[0265] The activity of P-1265 and P-1263 was evaluated in human PBMCs for their ability to stimulate Ki67 expression in CD8 T and NK cells. As shown in Figure 35, both compounds exhibited approximately a 300-fold decrease in activity when compared to their respective non-VitoKine counterparts, i.e., P-1266 for P-1265 and P-1295 for P-1263. This decreased activity is characteristic of the cloning efficiency associated with the 20-amino acid L2 linker. Additionally, the basal activity of VitoKine correlates linearly with the activity of its L-15 moiety.

[0266] In summary, the incorporation of the 20 amino acid MMP and matriptase double cleavable linker "GGSGPLGMLSQPMAKKGGGS" (SEQ ID NO: 93) significantly improved the developability profile of IL-15 VitoKine. However, the incorporation of a longer linker slightly reduced the cloning efficiency, leading to VitoKine with a relatively higher intrinsic basal activity. If lower basal activity is preferred, tuning the level of inactivity can be achieved by incorporating a less potent IL-15 variant. Furthermore, altering the valency of the cytokine domain provides another approach to modulate the inactivity of VitoKine.

[0267] Example 16 Construction of PD1 Ab-IL-15 VitoKine with optimized components PD1 Ab-IL-15 VitoKine, which comprises a PD1-blocking antibody as the targeting domain (D1), IL-15 or an IL-15 variant as the active partial domain (D2), and the IL-15RαSushi+ domain (SEQ ID NO: 165) as the masking partial domain (D3), is shown in Figure 1C (dimeric IL-15) and Figure 1D (monomeric IL-15).

[0268] It is desirable to construct a PD1 Ab-IL-15 VitoKine with a PD1 antibody that has superior PD1 binding and blocking activity to reverse T cell anergy or exhaustion and cooperate with the IL-15 anti-cancer immune response. The PD1 antibodies used to construct the PD1 Ab-IL-15 VitoKine were selected from optimized human PD1 blocking antibodies containing the light chain sequence set forth in SEQ ID NO: 44 and the heavy chain sequences set forth in SEQ ID NOs: 45-49. These optimized PD1 blocking antibodies have high affinity for the human PD1 protein and demonstrate equal or comparable efficacy to pembrolizumab in blocking PD1. They also have higher sequence similarity scores to their closest human germline sequence, resulting in an improved degree of humanity compared to pembrolizumab. Furthermore, they are predicted to have lower hydrophobicity, which in turn is likely to reduce aggregation tendency compared to pembrolizumab. PD1-targeting IL-15 VitoKines constructed using these optimized PD1 blocking antibodies are also predicted to have an enhanced developability profile.

[0269] Both the L1 linker connecting D1 and D2 and the L2 linker connecting D2 and D3 can be cleavable or non-cleavable, but the L2 linker is preferably cleavable. Cleavage of the L2 linker leads to active form 2 (depicted in Figure 2), which is a fully functional IL-15 domain non-covalently complexed with IL-15RαSushi fused to PD1 Ab. This form can activate IL-2R signaling in PD1-expressing T cells near the disease site, enhancing both pathways and coordinating anti-cancer immune responses while reducing systemic toxicity. On the other hand, if the L1 linker is cleavable, its cleavage results in active form 1, which has a shorter half-life, exhibits reduced potency, and lacks TIL targeting ability.

[0270] By adjusting the length and composition of the L2 linker, the intrinsic basal activity of VitoKine can be fine-tuned. For example, a 10-aa MMP-2 / 9 cleavable L2 linker (SEQ ID NO: 85) typically results in a 1000- to 2000-fold increase in cloning efficiency. In contrast, a 20-aa MMP and matriptase dual-cleavable L2 linker (SEQ ID NO: 93) leads to a ∼350-fold increase in cloning efficiency. In addition to adjusting the intrinsic basal activity, the dual-cleavable L2 linker significantly enhanced the developability profile of IL-15 VitoKine. The sequence of the cleavable linker can be further refined to better suit various tumors. Furthermore, the intrinsic basal activity of IL-15 VitoKine can be modulated by incorporating IL-15 variants with varying potency. Table 23A lists exemplary PD1 Ab-IL-15 immunocytokine constructs. TIFF2025531805000027.tif54170

[0271] All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into recipient mammalian expression vectors through the services of GenScript. VitoKine constructs were produced by co-transfecting the mammalian expression vectors into Expi293 cells (ThermoFisher) according to the manufacturer's instructions. Protein purification and characterization were performed according to the procedures outlined in Example 2.

[0272] Since the human PD1-blocking antibodies of the present invention do not bind to mouse PD1, surrogate mouse PD1 Ab-IL-15 immunocytokines were constructed in a similar manner using mouse PD1 antibodies. These were generated for in vivo studies to evaluate their pharmacodynamic effects in stimulating lymphocyte proliferation and expansion in mice, as well as their efficacy in inhibiting tumor growth in syngeneic mouse tumor models. Table 23B lists exemplary surrogate mouse PD1 Ab-IL-15 VitoKine constructs. The L1 linker in all VitoKines in Table 23B is the non-cleavable "GGGGSGGGGSGGGGS" linker (SEQ ID NO: 115). In the case of P-0878, P-1347, and P-1264, they are the non-cleavable equivalents of P-0874, P-1265, and P-1264, respectively. They contained length-matched non-cleavable L2 linkers corresponding to their respective VitoKines. TIFF2025531805000028.tif95170

[0273] Example 17 In vitro activity and proteolytic activation of PD1 Ab-IL-15 VitoKine It is essential that PD1 antibodies retain their binding and functional activity when incorporated into PD1 Ab-IL-15 VitoKine. PD1 antibodies with excellent target binding and PD1 blocking function can enhance the specificity and selectivity of TIL targeting and further cooperate with IL-15 anti-cancer immune responses by effectively reversing T cell anergy and exhaustion.

[0274] Two ELISA assays were performed to evaluate the PD1 binding and inhibitory ability of the exemplary IL-15 VitoKine P-1340 relative to its component PD1 antibody, P-1271. Additionally, a non-targeting germline antibody, P-1260 (SEQ ID NOs: 171, 172, and 173), was included as a negative control. Methods for the binding ELISA are outlined in Example 3, and the competition ELISA is described in Example 11. Both P-1271 and P-1340 had EC2 binding and inhibition values ​​of 26.8 pM and 32.8 pM, respectively. 50 Figure 36A shows that the P-1271 and P-1340 antibodies exhibit indistinguishable PD1 binding capabilities, as demonstrated by IC values ​​of 1.42 nM for P-1271 and 1.51 nM for P-1340. Similarly, Figure 36B shows that the P-1271 and P-1340 antibodies exhibit indistinguishable PD1 binding capabilities, as demonstrated by IC values ​​of 1.42 nM for P-127 50 These findings highlight that the PD1 antibody fully retained its binding and blocking activity when incorporated into the VitoKine construct.

[0275] In addition to confirming that the activity of the IL-15 moiety can be efficiently masked by the IL-15RαSushi domain and remain inactive regardless of the PD1 antibody sequence composition, it is also essential to verify that the PD1 Ab-IL-15 VitoKine can be efficiently cleaved and activated to fully restore the activity of the IL-15 moiety. The presence of bulky antibodies may spatially hinder protease accessibility and prevent efficient cleavage.

[0276] An exemplary PD1 Ab-IL-15 VitoKine, P-0875, was evaluated for protease cleavage and subsequent activation of the IL-15 domain. P-0875 contains a single MMP-2 / 9 cleavable linker, "GGPLGMLSQS" (SEQ ID NO: 85), connecting the IL-15 V63A / I68H variant and the IL-15Rα Sushi domain. Briefly, 3.3 μg of latent MMP-2 (BioLegend) was first activated with APMA (Millipore Sigma) according to the manufacturer's instructions, which was then buffer-exchanged and added to 120 μg of P-0875 in 0.4 ml of the manufacturer's recommended assay buffer (100 mM Tris, 20 mM CaCl, 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5). After 2 hours of incubation at 37°C, the digested samples were then purified on Protein A resin using the bind-elute mode, and the eluted samples were analyzed in reducing SDS-PAGE gels and their biological functions were assessed in ex vivo functional assays.

[0277] As depicted in Figure 37C, the appearance of the IL-15Ra Sushi domain as a distinct band at approximately 9 kDa on the gel (boxed) confirmed efficient cleavage at the MMP-2 / 9 substrate peptide linker. The presence of the IL-15Ra Sushi domain after Protein A elution also suggested that the IL-15Ra Sushi domain, released from the covalent linkage, remained noncovalently associated with IL-15. Such association was strong enough to withstand the low pH conditions during Protein A elution. Figures 37A and 37B further demonstrate the inactivity of VitoKine and its approximately 2000-fold recovery of potency in both NK cells and CD8+ T cells after in vitro proteolytic activation, which restored activity to levels matching that of the non-VitoKine PD1 Ab-IL-15 fusion counterpart, P-0870.

[0278] Similar evaluations were performed on VitoKine P-1340 and P-1349. They also demonstrated approximately 350-fold greater cloning efficiency compared to their respective non-VitoKine counterparts, P-1380 and P-1369, with reduced activity as intact molecules and full recovery of activity upon in vitro protease cleavage. They were also confirmed to be cleavable by both MMP-2 / 9 (BioLegend) and matriptase (R&D Systems).

[0279] Example 18 PD1-Ab-IL-15 VitoKine minimized systemic pharmacodynamic effects in non-tumor-bearing mice The VitoKine platform is designed to reduce systemic toxicity and widen the therapeutic window. By maintaining active cytokines in an inactive state, interactions with receptors on healthy cells are avoided, reducing unintended cytokine pathway activation and minimizing adverse effects. To test this, healthy C57BL / 6 mice were administered P-1265 and the pharmacodynamic effects on peripheral immune cells were compared to those induced by its non-VitoKine counterpart, P-1266. This experiment was carried out according to a procedure similar to that detailed in Example 12.

[0280] As shown in Figures 38A and 38C, at a dose of 1.5 mg / kg, P-1266, the non-VitoKine counterpart of P-1265, induced maximal Ki67 expression (100%) in both CD8 T and NK cells. Expression remained consistently high from days 3 to 7 before rapidly declining to baseline by day 10. On the other hand, P-1265, a PD1 Ab-IL-15 VitoKine harboring the monomeric IL-15 V63A / I68H variant, showed a dose-dependent increase in Ki67 expression for both CD8 T and NK cells. Notably, for CD8 T cells, expression peaked at 47%, 66%, and 84% on day 7 for the substantially higher doses of 3, 6, and 12 mg / kg, respectively (Figure 38A). For NK cells, peak levels of 64%, 84%, and 94% were observed on day 5 for the 3, 6, and 12 mg / kg doses, respectively (Figure 38C).

[0281] However, P-1265 demonstrated a dramatically different profile of cell expansion than P-1266. As shown in Figures 38B and 38D and summarized in Table 24, P-1266 dosed at 1.5 mg / kg promoted a 33-fold expansion of CD8 T cells from baseline that peaked on day 7 and a 27-fold expansion of NK cells on day 5. The increase in CD8 T and NK cell numbers was significantly lower even at the 12 mg / kg dose, which was 8-fold higher than the P-1266 dose. For the lower doses of 3 and 6 mg / kg, expanded cells remained relatively constant from day 5 through the end of the study, day 12. TIFF2025531805000029.tif54170

[0282] In parallel in vivo studies using naive C57B / L6 mice, the pharmacodynamic effects of PD1 Ab-IL-15 VitoKine, P-1265, were evaluated along with its dimeric VitoKine equivalent, P-1085, and its non-VitoKine counterpart, P-1266. A single dosage of 12 mg / kg was administered for P-1265 and P-1085, while P-1266 was dosed at 1.5 mg / kg. Blood samples were collected on days 0, 5, 7, 10, and 11 for lymphocyte analysis using flow cytometry. Figure 39 reveals that both the monomeric and dimeric VitoKine formats induced a more pronounced increase in Ki67 expression on peripheral lymphocytes (Figures 39A and 39C) compared with expansion of these cells (Figures 39B and 39D). However, this increase was still substantially lower than that of their active non-VitoKine counterparts. Reflecting on the observations from Example 12 and Figure 27, dimeric IL-15, previously in immunocytokine format and now in VitoKine form, had a less pronounced effect than its monomeric counterpart, contrary to in vitro findings. Notably, at 12 mg / kg, peak Ki67 expression was 74% for P-1265 and 38% for P-1085 (Figure 39A). Meanwhile, CD8 cell counts increased from a baseline level of 551 cells per μL of blood to a peak of 1315 cells / μL for P-1265 and 962 cells / μL for P-1085 (Figure 39B). A similar pattern was observed for NK cells (Figures 39C and 39D).

[0283] Similarly, the effects of the PD1 Ab-IL-15 VitoKine, P-1263, at varying dose levels (6, 12, and 24 mpk) were compared in C57B / L6 mice with its non-VitoKine counterpart, P-1295, dosed at 1.5 mpk. Both compounds contain a monomeric IL-15 variant with a Q108N mutation that interferes with γc interactions. After a single intraperitoneal injection, blood samples were collected on days 0, 5, 7, 10, and 11 for lymphocyte analysis using flow cytometry. As shown in Figure 40, even when dosed at 24 mg / kg, 16-fold higher than the dose of P-1295, the only apparent pharmacodynamic change from P-1263 was a small increase in Ki67 expression on NK cells (Figure 40C). Ki67 expression on CD8 T cells showed only a modest increase (Figure 40A). Given that IL-15 variants with γc-interfering mutations typically led to much lower cell expansion than IL-12 variants with IL-12Rβ mutations (as seen in Example 12 and Figure 24), it is expected that VitoKine, which has γc-interfering mutations in the IL-15 domain, exhibited even lower cell expansion. These expected findings are demonstrated in Figures 40C and 40D. Remarkably, no weight loss or other signs of stress were observed with P-1263 treatment, even when dosed as high as 24 mg / kg.

[0284] Finally, in a parallel experiment, the pharmacodynamic effects of P-1263 were evaluated in C57B / L6 mice relative to its non-cleavable VitoKine® equivalent, P-1264. The only difference between P-1264 and P-1263 is the L2 linker (see Table 23B for details). P-1264 exhibited identical in vitro activity to P-1263, but the IL-15 domain in P-1264 remains hidden and inactive because the D3 domain cannot be cleaved, leading to activation. Both P-1263 and P-1264 were administered at 12 mg / kg, while the active non-VitoKine® counterpart, P-1295, was dosed at 1.5 mg / kg. Blood samples were analyzed at days 0, 5, 7, 10, and 11 for lymphocyte phenotyping. As shown in Figure 41, no discernible difference in cell growth or expansion was observed between VitoKine and its non-cleavable counterpart, suggesting that VitoKine remains intact in the peripheral blood circulation.

[0285] In summary, compared with their active IL-15 non-VitoKine counterparts, IL-15 VitoKines exhibited a significant reduction in systemic proliferation and, in particular, expansion of specific lymphocyte populations, including CD8+ T and NK cells. This highlights the effectiveness of the VitoKine format in concealing IL-15 activity, thereby preventing unwanted activation of the IL-15 pathway and mitigating the risk of unwanted "on-target" effects in "off-tissue" settings. Furthermore, the reduction in systemic pharmacodynamics was even more pronounced in VitoKines incorporating IL-15 variants with mutations that disrupt γc interactions and when the IL-15 domain is in a dimeric format. These findings provide additional methods for fine-tuning the intrinsic basal activity of VitoKines and balancing it with their post-activation potency.

[0286] Example 19 Antitumor efficacy of PD1 Ab-IL-15 VitoKine in a syngeneic mouse tumor model Using a CT26 mouse colon cancer tumor model, we investigated the essential role of VitoKine activation in antitumor efficacy by comparing P-874 and its non-cleavable VitoKine equivalent, P-0878. The only difference between P-874 and P-0878 is in the L2 linker connecting the IL-15 (D2) and IL-15Rα Sushi+ (D3) domains. P-0878 contains a length-matched non-cleavable L2 linker (see Table 23B for details). P-0878 showed identical in vitro activity to P-08874, but the IL-15 domain in P-0878 remains hidden and inactive because the D3 domain cannot be cleaved, leading to activation.

[0287] The CT26 model was established in the same manner as detailed in Example 13. P-0874 and P-0878 were administered twice at a dose of 10 mg / kg according to a Q12D dosing schedule, and a vehicle (sterile PBS) group was included for comparison. As depicted in Figure 42A, because CT26 is considered a "cold" tumor and is less responsive to PD1 treatment than the MC38 model, tumors eventually developed in all mice. Treatment with P-0878, which has an inactivatable, inactive IL-15 domain, did not result in any observable antitumor effect. In sharp contrast, administration of P-0784 at the same dose demonstrated significantly improved efficacy, showing a 67% TGI at day 25. This finding suggests that the enhanced antitumor efficacy of VitoKine molecules depends on enzymatic cleavage of the linker to release the hidden moiety, thereby activating the IL-15 domain around the tumor.

[0288] Notably, there was minimal peripheral immune cell expansion and little difference between P-0874 and P-0878 in these tumor-bearing mice 5 days after the first injection (Figures 42B and 42C). It is also worth noting that both VitoKines were well tolerated in mice when given at 10 mg / kg, with no evidence of weight loss.

[0289] The antitumor efficacy of PD1 Ab-IL-15 VitoKine, P-1265, compared with its dimeric VitoKine equivalent, P-1085, and their respective non-VitoKine counterparts, P-1266 and P-0869, was evaluated in an established MC38 tumor model. All of these compounds contain the IL-15 V63A / I68H variant, which disrupts interaction with IL-15Rβ. P-1085 was administered twice every 12 days (Q12D) at dosages of 3 and 6 mg / kg, and P-0869 was given at two doses of 1.5 mg / kg. The mean tumor volume, along with the standard error of the mean (SEM) for each group as a function of time, is shown in Figure 43A. Vehicle-treated mice rapidly developed large subcutaneous tumors, and all other treatment groups exhibited high efficacy in inhibiting tumor growth, with tumor growth inhibition (TGI) approaching 100% 43 days after the start of treatment.

[0290] P-1265, a PD1 Ab-IL15 VitoKine containing the monomeric IL15 V63A / I68H variant, was compared with its active non-VitoKine counterpart, P-1266. On day 1, the following treatments were administered: 12 mg / kg of murine PD1 antibody P-0722, varying dosage levels of P-1265 (3 mg / kg, 6 mg / kg, and 12 mg / kg), and 1.5 mg / kg of P-1265. These treatments were administered intraperitoneally Q12D for a total of two doses. Vehicle (PBS) was used as a control. As shown in Figure 43B, P-0722 slightly delayed tumor growth, while all other treatment groups exhibited high efficacy in tumor growth inhibition. Especially for the low dose VitoKine group (3 mg / kg), the initial weaker tumor growth inhibition was later reversed and ultimately resulted in 6 out of 7 mice becoming tumor-free.

[0291] The antitumor activity of P-1263, a murine PD1 Ab-IL-15 VitoKine containing the monomeric IL-15 Q108N variant as the active domain, was evaluated in the established MC38 model. Variable dose levels (6 mg / kg, 9 mg / kg, and 18 mg / kg) of P-1263 were administered twice according to the Q12D schedule. The component PD1 antibody, P-0722, administered at doses of 6 and 18 mg / kg was included for comparative analysis.

[0292] The mean tumor volume, along with the SEM for each group, as a function of time is shown in Figure 44A. Vehicle-treated mice rapidly developed large subcutaneous tumors. PD1 antibody treatment showed a small anti-tumor growth effect, and such effect increased with increasing dosage. In contrast, the P-1263-treated group exhibited high efficacy in inhibiting tumor growth in a dose-dependent manner.

[0293] Figures 44B-44F further show tumor growth curves for individual mice for the five different treatment groups. Each line in the graph represents one mouse, with the mean tumor growth of the vehicle group represented by a dotted line. Treatment with 18 mg / kg P-1263 demonstrated the most significant and sustained effect, with five of seven mice from this group completely eradicating tumor growth by day 38 (Figure 44F). Similarly, in the group treated with 12 mg / kg P-1263, four of seven mice remained tumor-free at the end of the study (Figure 44E). On the other hand, P-1263 administered at 6 mg / kg had less efficacy, with two of seven mice remaining tumor-free (Figure 44D). Other mice in each group showed tumor growth after an initial period of delayed tumor growth. For comparison, P-0722 dosed at 18 mg / kg showed some effect in slowing tumor growth, but the treatment did not result in complete tumor eradication (FIG. 44C).

[0294] Figure 44G shows that P-1263 was well tolerated with little or no weight loss, even at dosages as high as 18 mg / kg. Remarkably, this antitumor efficacy was achieved with significantly lower peripheral lymphocyte proliferation and expansion (as seen in Figure 40). This efficacy is partially attributable to the high-dose tolerability afforded by the VitoKine format. Consequently, the PD1 Ab-IL-15 VitoKine platform offers a broader therapeutic window, allowing the antibody component to fully achieve its potential in reversing T-cell anergy and exhaustion.

[0295] Taken together, PD1 Ab-IL-15 VitoKine effectively inhibited tumor growth while minimizing peripheral lymphocyte proliferation and expansion. Consequently, challenges commonly associated with fully active cytokines, such as overstimulation of immune pathways, undesirable on-target and off-tissue toxicities, and unwanted target sinks, can be mitigated by using the VitoKine format without compromising antitumor efficacy. Importantly, the compatibility of PD1 Ab-IL-15 VitoKine with higher dosing levels ensures that the antibody arm can optimally target and reverse T cell anergy and exhaustion, enhancing existing immune responses. This results in further enhanced immune system activity against tumors.

[0296] As will be appreciated by one of skill in the art, any PD1 Ab-IL-15 VitoKine construct comprising the optimized PD1 antibodies described herein, an IL-15 variant (dimeric or monomeric) with suitable potency to balance inactivity in pre-cleavage activity and potency after activation, and appropriate L1 and L2 linker sequences is within the spirit and scope of the present invention.

[0297] All of the articles and methods disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled 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 this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes 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 may suitably be practiced in the absence of any element not specifically disclosed herein. Therefore, while the present invention has been particularly disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be undertaken by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims. Sequence Listing

[0298] The amino acid sequences listed in the accompanying sequence listing are shown using the standard one-letter code for amino acids, as defined in 37 CFR 1.822. SEQ ID NO: 1 is the amino acid sequence of the mature human PD1 polypeptide. SEQ ID NOs: 2 to 5 are the amino acid sequences of the light chain variable domains of human PD1-blocking antibodies. SEQ ID NOs: 6 to 18 are the amino acid sequences of the heavy chain variable domains of human PD1-blocking antibodies. SEQ ID NOs: 19 to 21 are the amino acid sequences of the light chain CDR1 of a human PD1 blocking antibody. SEQ ID NOs: 22 to 24 are the amino acid sequences of the light chain CDR2 of a human PD1 blocking antibody. SEQ ID NO: 25 is the amino acid sequence of the human PD1 blocking antibody light chain CDR3. SEQ ID NO: 26 is the amino acid sequence of human PD1 blocking antibody heavy chain CDR1. SEQ ID NOs: 27 to 32 are the amino acid sequences of the heavy chain CDR2 of a human PD1 blocking antibody. SEQ ID NO: 33 is the amino acid sequence of the human PD1 blocking antibody heavy chain CDR3. SEQ ID NO: 34 is the amino acid sequence of the human kappa light chain constant domain. SEQ ID NO: 35 is the amino acid sequence of a human IgG1 heavy chain constant domain containing the L234A / L235A / G237A mutations. SEQ ID NO: 36 is the amino acid sequence of the human IgG4 heavy chain constant domain containing the S228P mutation. SEQ ID NO: 37 is the amino acid sequence of human immunoglobulin germline exon HGHV1-2 (GenBank accession number: 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 number: X17264). SEQ ID NO: 40 is the amino acid sequence of the human antibody heavy chain variable domain having GenBank Accession No.: AB063829. SEQ ID NO: 41 is the amino acid sequence of the human antibody light chain variable domain having GenBank accession number: M29469. SEQ ID NO: 42 is the amino acid sequence of the light chain of the reference human PD1 blocking antibody P-0734. SEQ ID NO: 43 is the amino acid sequence of the heavy chain of the reference human PD1 blocking antibody P-0734. SEQ ID NO: 44 is the amino acid sequence of the light chain of a human PD1 blocking antibody. SEQ ID NO: 45 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1174. SEQ ID NO: 46 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1194. SEQ ID NO: 47 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1201. SEQ ID NO: 48 is the amino acid sequence of the heavy chain of the human PD1 blocking antibody P-1238. SEQ ID NO: 49 is the amino acid sequence of the heavy chain of the PD1 human blocking antibody P-1271. SEQ ID NO: 50 is the amino acid sequence of the light chain of the benchmark human PD1 blocking antibody P-0795. SEQ ID NO: 51 is the amino acid sequence of the heavy chain of the benchmark human PD1 blocking antibody P-0795. SEQ ID NO: 52 is the amino acid sequence of the light chain of the surrogate murine PD1 blocking antibody P-0722. SEQ ID NO: 53 is the amino acid sequence of the heavy chain of the surrogate murine PD1 blocking antibody P-0722. SEQ ID NOs: 54 to 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 a protease substrate peptide. SEQ ID NOs: 95-115 are the amino acid sequences of various non-cleavable linker sequences. SEQ ID NO: 116 is the human IL-15 mature form amino acid sequence. SEQ ID NOs: 117 to 163 are the amino acid sequences of human IL-15 variant polypeptides. SEQ ID NO: 164 is the human IL-15Rα amino acid sequence. SEQ ID NO: 165 is the human IL-15Rα Sushi domain plus amino acid sequence. SEQ ID NO: 166 is the amino acid sequence of human IgG1-Fc containing the L234A / L235A / G237A mutations. SEQ ID NO: 167 is the amino acid sequence of human IgG1 knob-Fc containing the L234A / L235A / G237A mutations. SEQ ID NO: 168 is the amino acid sequence of human IgG1 whole-Fc containing the L234A / L235A / G237A mutations. SEQ ID NOs: 169 and 170 are the amino acid sequences of the heterodimeric heavy chain pair of the surrogate murine PD1 blocking antibody P-0722. SEQ ID NOs: 171 and 172 are the amino acid sequences of the heterodimeric heavy chains of germline antibody P-1260. SEQ ID NO: 173 is the amino acid sequence of the light chain of germline antibody P-1260. SEQ ID NO: 174 is the amino acid sequence of the heavy chain of PD1 human blocking antibody P-1271 containing a hole mutation. SEQ ID NOs: 175-180 are the amino acid sequences of the heavy chains of various human PD1 Ab-IL-15 immunocytokines. SEQ ID NOs: 181-185 are the amino acid sequences of the heavy chains of various PD1 Ab-IL-15 VitoKines. Sequence Listing Human PD1 protein mature sequence FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 2) Human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 3) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 4) Sequence of the human PD1-blocking antibody light chain variable domain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLAWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 5) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 6) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 7) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 8) Sequence of the human PD1-blocking antibody heavy chain variable domain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 9) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 10) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 11) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 12) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 13) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 14) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 15) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 16) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 17) Sequence of the human PD1-blocking antibody heavy chain variable domain EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 18) Human PD1-blocking antibody CDR-L1 sequence RASKGVSTSGYSYLH (SEQ ID NO: 19) Human PD1-blocking antibody CDR-L1 sequence RASQGVSTSGYSYLH (SEQ ID NO: 20) Human PD1-blocking antibody CDR-L1 sequence RASQGVSTSGYSYLA (SEQ ID NO: 21) Sequence of human PD1-blocking antibody CDR-L2 YLASYLES (SEQ ID NO: 22) Sequence of human PD1-blocking antibody CDR-L2 YLASYRES (SEQ ID NO: 23) Sequence of human PD1-blocking antibody CDR-L2 YLASYRAS (SEQ ID NO: 24) Sequence of human PD1-blocking antibody CDR-L3 QHSRDLPLT (SEQ ID NO: 25) Human PD1-blocking antibody CDR-H1 sequence NYYMY (SEQ ID NO: 26) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFNEKFKN (SEQ ID NO: 27) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFAQKFQG (SEQ ID NO: 28) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNYAQKFQG (SEQ ID NO: 29) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNYADKFKG (SEQ ID NO: 30) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFADKFKG (SEQ ID NO: 31) Human PD1 blocking antibody CDR-H2 sequence GINPSNGGTNFNDSVKG (SEQ ID NO: 32) Human PD1 blocking antibody CDR-H3 sequence RDYRFDMGFDY (SEQ ID NO: 33) Human kappa light chain constant domain sequence TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34) Sequence of the human IgG1 constant domain with the L234A / L235A / G237A mutations ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 35) Sequence of the human IgG4 constant domain with the S228P mutation ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 36) Human antibody germline IGHV1-2 sequences QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO: 37) Human antibody germline IGHV3-23 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCAK (SEQ ID NO: 38) Human antibody germline IGKV3D-11 sequence EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO: 39) Human antibody GenBank number: AB063829 sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTSNAISWVRQAPGQGLEWMGWISTYKGKANYAQKFQDRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARWRAVVGRGGGLDVWGQGTTVTVSS (SEQ ID NO: 40) Human antibody GenBank number: M29469 sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNKATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSKWPLTFGGGTKVEIKG (SEQ ID NO: 41) Reference antibody P-0734 light chain sequence EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42) Reference antibody P-0734 heavy chain sequence QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 43) Sequence of the human PD1-blocking Ab light chain EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44) Sequence of the heavy chain of human PD1-blocking Ab P-1174 QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 45) Sequence of the heavy chain of the human PD1-blocking antibody P-1194 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 46) Sequence of the heavy chain of the human PD1-blocking antibody P-1201 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 47) Sequence of the heavy chain of the human PD1-blocking antibody P-1238 EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 48) Sequence of the heavy chain of human PD1-blocking Ab P-1271 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 49) Light chain sequence of the benchmark human PD1 blocking antibody P-0795 DIVMTQSPLSLPVTPGEPASITCKASQDVETVVAWYLQKPGQSPRLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYSRYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50) Sequence of the heavy chain of the benchmark human PD1 blocking antibody P-0795 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSSGVAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 51) Sequence of the light chain of the surrogate mouse PD1 blocking antibody P-0722 DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 52) Sequence of the heavy chain of the surrogate mouse PD1 blocking antibody P-0722 EVQLQESGPGLVKPSQSLSLTCSVTGYSITSSYRWNWIRKFPGNRLEWMGYINSAGISNYNPSLKRRISITRDTSKNQFFLQVNSVTTEDAATYYCARSDNMGTTPFTYWGQG TLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKP CICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG (SEQ ID NO: 53) Protease substrate peptide sequence SPLGLAGS (SEQ ID NO: 54) Protease substrate peptide sequence EPLELRAG (SEQ ID NO: 55) Protease substrate peptide sequence LSGRSDNH (SEQ ID NO: 56) Protease substrate peptide sequence GPLGIAGQ (SEQ ID NO: 57) Protease substrate peptide sequence GTAHLMGG (SEQ ID NO: 58) Protease substrate peptide sequence RIGSLRTA (SEQ ID NO: 59) Protease substrate peptide sequence SGRSENIRTA (SEQ ID NO: 60) Protease substrate peptide sequence GPLGMLSQ (SEQ ID NO: 61) Protease substrate peptide sequence GPAGMKGL (SEQ ID NO: 62) Protease substrate peptide sequence RPSASRSA (SEQ ID NO: 63) Protease substrate peptide sequence PLGLAG (SEQ ID NO: 64) Protease substrate peptide sequence LGGSGRSANAILE (SEQ ID NO: 65) Protease substrate peptide sequence GGSGRSANAI (SEQ ID NO: 66) Protease substrate peptide sequence SGRSA (SEQ ID NO: 67) Protease substrate peptide sequence AANL (SEQ ID NO: 68) Protease substrate peptide sequence GPTNKVR (SEQ ID NO: 69) Protease substrate peptide sequence GFFY (SEQ ID NO: 70) Protease substrate peptide sequence GPICFRLG (SEQ ID NO: 71) Protease substrate peptide sequence RQAGFSL (SEQ ID NO: 72) Protease substrate peptide sequence RQARAVGG (SEQ ID NO: 73) Protease substrate peptide sequence PMAKK (SEQ ID NO: 74) Protease substrate peptide sequence HSSKLQ (SEQ ID NO: 75) Protease substrate peptide sequence GPLGMLSQPMAKK (SEQ ID NO: 76) Protease substrate peptide sequence PMAKKGPLGMLSQ (SEQ ID NO: 77) Protease-cleavable linker sequence GGGSGGGGSGGGGSLSGRSDNHGGSGGGGS (SEQ ID NO: 78) Protease-cleavable linker sequence GSSSGRSENIRTAGT (SEQ ID NO: 79) Protease-cleavable linker sequence GGGGSGGGGSGGGSLGGSGRSANAILEGGSGGGGS (SEQ ID NO: 80) Protease-cleavable linker sequence GGGGSGGGGSLGGSGRSANAILEGGGGS (SEQ ID NO: 81) Protease-cleavable linker sequence GGGGSLGGSGRSANAILEGGS (SEQ ID NO: 82) Protease-cleavable linker sequence GGGSGPTNKVRGGS (SEQ ID NO: 83) Protease-cleavable linker sequence GGSGPLGMLSQGGGS (SEQ ID NO: 84) Protease-cleavable linker sequence GGPLGMLSQS (SEQ ID NO: 85) Protease-cleavable linker sequence GGGPLGMLSQGGS (SEQ ID NO: 86) Protease-cleavable linker sequence GGPTNKVRGS (SEQ ID NO: 87) Protease-cleavable linker sequence GRQARAVGGS (SEQ ID NO: 88) Protease-cleavable linker sequence GGGSGRSENIRTAGG (SEQ ID NO: 89) Protease-cleavable linker sequence SGGPGPAGMKGLPGS (SEQ ID NO: 90) Protease-cleavable linker sequence GGGGSPMAKKGGGGS (SEQ ID NO: 91) Protease-cleavable linker sequence GGPLGMLSQPMAKKS (SEQ ID NO: 92) Protease-cleavable linker sequence GGSGPLGMLSQPMAKKGGGS (SEQ ID NO: 93) Protease-cleavable linker sequence GGGPMAKKGPLGMLSQGGGS (SEQ ID NO: 94) Non-cleavable linker sequence EPKSSDKTHTSPPS (SEQ ID NO: 95) Non-cleavable linker sequence GGGSGGGSGGGS (SEQ ID NO: 96) Non-cleavable linker sequence GGGS (SEQ ID NO: 97) Non-cleavable linker sequence GSSGGSGGS (SEQ ID NO: 98) Non-cleavable linker sequence GSSGT (SEQ ID NO: 99) Non-cleavable linker sequence GGGGSGGGGSGGGS (SEQ ID NO: 100) Non-cleavable linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 101) Non-cleavable linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 102) Non-cleavable linker sequence GGGSGGGS (SEQ ID NO: 103) Non-cleavable linker sequence GS (SEQ ID NO: 104) Non-cleavable linker sequence GGS (SEQ ID NO: 105) Non-cleavable linker sequence GGGGS (SEQ ID NO: 106) Non-cleavable linker sequence GGSGG (SEQ ID NO: 107) Non-cleavable linker sequence SGGG (SEQ ID NO: 108) Non-cleavable linker sequence GSGS (SEQ ID NO: 109) Non-cleavable linker sequence GSGSGS (SEQ ID NO: 110) Non-cleavable linker sequence GSGSGSGS (SEQ ID NO: 111) Non-cleavable linker sequence GSGSGSGSGS (SEQ ID NO: 112) Non-cleavable linker sequence GSGSGSGSGSGS (SEQ ID NO: 113) Non-cleavable linker sequence GGGGSGGGGS (SEQ ID NO: 114) Non-cleavable linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 115) Sequence of the mature form of human IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 116) Sequence of human IL-15 S58D variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDADIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 117) Human IL-15 variant with a single amino acid deletion at the N-terminus WVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 118) Human IL-15 variant with two amino acid deletions at the N-terminus VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 119) Human IL-15 variant with a three-amino acid deletion at the N-terminus NVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 120) Sequence of human IL-15 V63A variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 121) Sequence of human IL-15 V63F variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTFENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 122) Sequence of human IL-15 V63K variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTKENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 123) Sequence of human IL-15 V63R variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTRENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 124) Sequence of human IL-15 I68D variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIDLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 125) Sequence of human IL-15 I68F variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIFLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 126) Sequence of human IL-15 I68G variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 127) Sequence of human IL-15 I68H variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 128) Sequence of human IL-15 I68K variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIKLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 129) Sequence of human IL-15 I68Q variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 130) Sequence of human IL-15 V63A / I68G variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIGLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 131) Sequence of human IL-15 V63A / I68H variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 132) Sequence of human IL-15 V63A / I68Q variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIQLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 133) Sequence of human IL-15 Q108A variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVAMFINTS (SEQ ID NO: 134) Sequence of human IL-15 Q108D variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVDMFINTS (SEQ ID NO: 135) Sequence of human IL-15 Q108E variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVEMFINTS (SEQ ID NO: 136) Sequence of human IL-15 Q108F variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVFMFINTS (SEQ ID NO: 137) Sequence of human IL-15 Q108H variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVHMFINTS (SEQ ID NO: 138) Sequence of human IL-15 Q108K variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVKMFINTS (SEQ ID NO: 139) Sequence of human IL-15 Q108L variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVLMFINTS (SEQ ID NO: 140) Sequence of human IL-15 Q108M ​​variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 141) Sequence of human IL-15 Q108N variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVNMFINTS (SEQ ID NO: 142) Sequence of human IL-15 Q108S variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVSMFINTS (SEQ ID NO: 143) Sequence of human IL-15 Q108T variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVTMFINTS (SEQ ID NO: 144) Sequence of human IL-15 Q108Y variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVYMFINTS (SEQ ID NO: 145) Sequence of human IL-15 V63A / Q108M ​​variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTAENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 146) Sequence of human IL-15 V63K / Q108M ​​variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTKENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 147) Sequence of human IL-15 I68F / Q108M ​​variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIFLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 148) Sequence of human IL-15 I68H / Q108M ​​variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVMMFINTS (SEQ ID NO: 149) Sequence of human IL-15 V63K / Q108N variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTKENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVNMFINTS (SEQ ID NO: 150) Sequence of human IL-15 I68H / Q108N variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIHLANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVNMFINTS (SEQ ID NO: 151) Sequence of human IL-15 D30T variant NWVNVISDLKKIEDLIQSMHIDATLYTESTVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 152) Sequence of human IL-15 H32E variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVEPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 153) Sequence of human IL-15 H32D variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVDPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 154) Sequence of human IL-15 H32N variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVNPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 155) Sequence of human IL-15 H32Q variant NWVNVISDLKKIEDLIQSMHIDATLYTESDVQPSCKVTAM...

Claims

1. An isolated interleukin-15 (IL-15) fusion protein complex comprising: (1) an IL-15 polypeptide (or variant thereof) linked to an optimized PD1-blocking antibody; and (2) an IL-15 receptor alpha ("IL-15Rα") domain non-covalently linked to the IL-15 polypeptide, thereby forming an IL-15 / IL-15Rα-PD1-blocking antibody fusion protein, wherein the optimized PD1-blocking antibody 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, and wherein the optimized PD1 blocking antibody targets the IL-15 / IL-15Rα-PD1 antibody fusion protein to tumor infiltrating lymphocytes (TILs).

2. The IL-15 / IL-15Rα-PD1 blocking antibody fusion protein of claim 1, wherein the IL-15 polypeptide is linked to the C-terminus of the PD1 blocking antibody.

3. 7. The IL-15 / IL-15Rα-PD1 blocking antibody fusion protein of any one of claims 1 to 6, wherein the IL-15 variant polypeptide is selected from the group of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 117 to 163.

4. 8. The IL-15 / IL-15Rα-PD1 blocking antibody fusion protein of any one of claims 1 to 7, wherein the IL-15Rα domain comprises the amino acid sequence set forth in SEQ ID NO: 165 or any functional fragment thereof.

5. 9. The IL-15 / IL-15Rα-PD1-blocking antibody fusion protein of any one of claims 1 to 8, wherein the IL-15 polypeptide is covalently attached to the PD1-blocking antibody by a peptide linker.

6. 10. The IL-15 / IL-15Rα-PD1 blocking antibody fusion protein of claim 9, wherein the peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54 to 115.

7. 7. The IL-15 / IL-15Rα-PD1 blocking antibody fusion protein construct of any one of claims 1 to 6, which is in a monomeric form or in a dimeric form.

8. 1. A bioactivatable polypeptide drug construct comprising, in N-terminal 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 concealment moiety D3 domain ("D3"); D1 functions to target said bioactivatable moiety to a site of intended treatment; D3 has the ability to conceal the functional activity of D2 until it is activated at said site of intended treatment; and D1 is an optimized PD1 blocking antibody, D2 is an IL-15 variant polypeptide, and D3 is an IL-15Rα domain.

9. 1. A bioactivatable polypeptide drug construct comprising, in an N-terminal to C-terminal direction (D3-D2-D1), 1) a cryptic 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 said bioactivatable moiety to a site of intended treatment; D3 is capable of hiding the functional activity of D2 until it is activated at said site of intended treatment; and D1 is an optimized PD1 blocking antibody, D2 is an IL-15 variant polypeptide, and D3 is an IL-15Rα domain.

10. 10. The bioactivatable polypeptide drug construct of any one of claims 8 to 9, wherein the optimized PD1-blocking antibody is selected from an antibody comprising: (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.

11. 11. A bioactivatable polypeptide drug construct according to any one of claims 8 to 10, wherein domain D2 is an IL-15 variant polypeptide selected from the group of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 117 to 163.

12. A bioactivatable polypeptide drug construct according to any one of claims 8 to 11, wherein domain D3 is an IL-15Rα sushi variant polypeptide having the amino acid sequence set forth in SEQ ID NO:

165.

13. 13. The construct of any one of claims 8 to 12, 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 dimers and monomers.

14. 14. The construct of any one of claims 8 to 13, 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.

15. The construct of claim 14, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.

16. The construct of claim 14, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95 to 115.

17. 17. The construct of any one of claims 8 to 16, 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.

18. 18. The construct of claim 17, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.

19. The construct of claim 17, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95 to 115.

20. The construct of any one of claims 14 to 17, wherein L1 and L2 are both protease-cleavable peptide linkers.

21. The construct of any one of claims 14 to 17, wherein L1 and L2 are both non-cleavable peptide linkers.

22. The construct of any one of claims 14 to 21, wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker.

23. The construct of any one of claims 14 to 21, wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

24. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 7 in admixture with a pharmaceutically acceptable carrier.

25. A pharmaceutical composition comprising a bioactivatable polypeptide drug construct according to any one of claims 8 to 24 in admixture with a pharmaceutically acceptable carrier.

26. A method for treating cancer or cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 24 to 25.

27. 27. The method of claim 26, wherein the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

28. 28. The method of any one of claims 26-27, further comprising a second therapeutic agent or treatment capable of treating cancer or cancer metastasis in the subject.

29. 29. The method of claim 28, wherein the second treatment is selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapy including CAR-T, CAR-NK, iPS-derived CAR-T or iPS-derived CAR-NK, and a vaccine, such as Bacillus Calmette-Guerin (BCG).

30. 30. The method of claim 29, wherein the immunotherapy is selected from the group consisting of: treatment using a depleting antibody against a specific tumor antigen; treatment using an antibody-drug conjugate; treatment using an agonist, antagonist, or blocking antibody against a costimulatory or co-inhibitory molecule (immune checkpoint), 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 a bispecific T-cell engaging antibody (BiTE®), such as blinatumomab; treatment involving administration of a biological response modifier, such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ.

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

32. 32. An expression vector comprising the nucleic acid molecule of claim 31.

33. A host cell comprising the expression vector of claim 32.

34. 34. A method for producing a fusion protein according to any one of claims 1 to 7, comprising culturing a host cell according to claim 33 under conditions promoting expression of said fusion protein and recovering said fusion protein.

35. 35. An isolated fusion protein produced by the method of claim 34.

36. 33. A method for producing a bioactivatable polypeptide drug construct according to any one of claims 8 to 23, comprising culturing a host cell according to claim 33 under conditions promoting expression of said bioactivatable polypeptide drug construct and recovering said bioactivatable polypeptide drug construct protein.

37. 37. An isolated bioactivatable polypeptide drug construct protein produced by the method of claim 36.

Images