WO2024086739A1 - Methods and compositions of il12 muteins and il2 muteins - Google Patents

Methods and compositions of il12 muteins and il2 muteins Download PDF

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WO2024086739A1
WO2024086739A1 PCT/US2023/077331 US2023077331W WO2024086739A1 WO 2024086739 A1 WO2024086739 A1 WO 2024086739A1 US 2023077331 W US2023077331 W US 2023077331W WO 2024086739 A1 WO2024086739 A1 WO 2024086739A1
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amino acid
polypeptide
hil12m
alternatively
seq
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PCT/US2023/077331
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French (fr)
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Patrick L. LUPARDUS
Deepti ROKKAM
Zhenya KOLIESNIK
Martin Oft
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Synthekine, Inc.
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Priority claimed from PCT/US2022/078465 external-priority patent/WO2023070056A2/en
Application filed by Synthekine, Inc. filed Critical Synthekine, Inc.
Publication of WO2024086739A1 publication Critical patent/WO2024086739A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12

Definitions

  • Receptors for cytokines are typically multimers of cell surface expressed proteins that stimulate signaling via the interaction of their intracellular domains. Cytokines act as specific ligands for the extracellular domains of cytokine receptor subunits and facilitate the multimerization of such receptor subunits so as to bring the intracellular domains of such cytokine receptor subunits into proximity such that intracellular signaling may occur. Certain cytokine receptor subunits are shared among different cytokines and the nature of the cytokine determines which receptor subunits are multimerized to form the cytokine receptor complex and the intracellular signaling pattern that results. Cytokines thus act to bridge individual receptor subunits into a receptor complex that results in intracellular signaling.
  • the intracellular domains of cytokine receptor subunits possess JAK binding domains which are typically located in the boxl/box2 region of the intracellular domain of the cytokine receptor subunit near the interior surface of the cell membrane.
  • Intracellular JAK kmases associate with these JAK binding domains.
  • the JAKs phosphorylate each other.
  • the phosphorylation of the JAK induces a conformational change in the JAK providing the ability to further phosphorylate other intracellular proteins which initiates a cascade that results in activation of multiple intracellular factors which transduce the intracellular signal associated with the receptor.
  • the resulting intracellular responses, such as gene transcription, are frequently collectively referred to as downstream signaling.
  • the proteins which are phosphorylated by the JAKs are members of the signal transducer and activator of transcription (STAT) protein family. Seven members of the mammalian STAT family have been identified to date: STAT1, STAT2, STAT3, STAT4, STAT5a STAT5b, and STAT6. Delgoffe, et al., (2011) Curr Opin Immunol. 23(5): 632-8; Levy and Darnell (2002) Nat Rev Mol Cell Biol. 3(9):651-62 and Murray, (2007) J Immunol. 178(5):2623-9.
  • the selective interplay of activated JAK and STAT proteins collectively referred to as the JAK/STAT pathway, provide for a wide variety of intracellular responses observed in response to cytokine binding.
  • Interleukin- 12 (IL 12)
  • Human interleukin- 12 is a heterodimeric cytokine comprised of the human P35 (also referred to as hIL12A, Uniprot Ref. 29459) and human P40 (also referred to as hIL12B, Uniprot Ref. 29460) subunits.
  • the hIL12 heterodimer is also referred to as p70.
  • hIL12 is produced by dendritic cells, macrophages and neutrophils.
  • hIL12 is typically identified as a T cell stimulating factor which can stimulate the proliferation and activation of T cells.
  • hIL12 stimulates the production of IFNy and TNFa and modulates the cytotoxic activity of NK and CD8+ cytotoxic T cells.
  • hIL12 was first identified and referred to as cytotoxic lymphocyte maturation factor. Stem, et al (1990) Proc Natl Acad Sei USA 87:6808-6812 and Gately, et al. United States Patent No 6,683,046 issued January 27, 2004. hIL12 is also involved in immune cell differentiation in particular the differentiation of naive T cells into Thl (CD4+) cells. hIL12 is also reported to provide anti-angiogenic activity. Since its discovery more than 30 years ago, hIL12 has been proposed and evaluated for use in the treatment of a variety of neoplastic diseases, viral and bacterial infections. See, e.g. Lasek, et al (2014) Cancer Immunol Immunother (2014) 63:419- 435.
  • hIL12 binds to the hIL12 receptor, a heterodimeric complex of hIL12 receptor subunit beta-1 (IL12R[31, also referred to in the scientific literature as IL12RB1 or CD212, Uniprot Ref. P42701) and hIL12 receptor subunit beta-2 (ML12R ⁇ 2 also referred to in the scientific literature as hIL12RB2, Uniprot Ref. Q99665).
  • IL12R ⁇ 1 and ML12R ⁇ 2 are members of the class I cytokine receptor family and have homology to gpl30.
  • the expression of hIL12R ⁇ 1 and HL12R ⁇ 2 are upregulated in response to hIL12 with the majority of ML12R ⁇ 2 being found on activated T cells.
  • hIL12Rpl is a constitutively expressed type I transmembrane protein that belongs to the hemopoietin receptor superfamily. hIL12R ⁇ 1 binds with low affinity to hIL12. hIL12R ⁇ 1 is required for binding to the hIL12P40 subunit and it is associated with the Janus kinase (Jak) family member Tyk-2. The binding IL12p40 and IL12P35 subunits of IL12 toIL12R ⁇ 1 and IL12R ⁇ 2, respectively, results in the dimerization of IL12R ⁇ 1 and IL12R ⁇ 2.
  • Jak-2 and Tyk-2 are transphosphorylated, further activating Jak2 and Tyk2 kinase activity which results in phosphorylation of the IL12R ⁇ 1 and IL12R ⁇ 2 intracellular domains.
  • the phosphorylated intracellular signaling domain of IL12R ⁇ 2 provides a binding site for STAT4.
  • STAT4 binds to phosphorylated IL12R ⁇ 2 and is subsequently phosphorylated.
  • Phosphorylated STAT4 induces dimerization with another phosphorylated STAT4 molecule.
  • the phosphorylated STAT4 homodimers translocate to the nucleus resulting in, among other activities, the promotion of IFN-y gene transcription. IFN-y induces the activity and proliferation of macrophages, NK cells, and T cells, which also secrete IL12.
  • IL 12 has many properties which suggested its use in the treatment of cancers including the stimulation of IFNy production by NK cells, enhancement of the cytolytic properties of NK cells and cytotoxic T cells, and inhibition of angiogenesis.
  • IL 12 exhibited significant antitumor activity in animal models which led to its evaluation in Phase I and Phase II clinical trials in the treatment of a variety of cancers in the late 1990s. Lasek, et al., supra. While beneficial effects were observed, the significant adverse events observed resulted in termination of the clinical trials.
  • an hIL12 partial agonists of the present disclosure comprises a modified P40 subunit polypeptide that provides intracellular signaling characteristic of wt hIL12 on desired cell types, while providing significantly less intracellular signaling on undesired cell types.
  • IL12 partial agonists comprising a heterodimeric hIL12M-Fc mutein with a modified binding affinity for hIL12R ⁇ 1, or causing different Emaxfor hIL12R ⁇ 1 as compared to the binding affinity of wild-type or parental hIL12P40 polypeptide for hIL12R ⁇ 1.
  • Glassman, et al. (2021) Cell 184(4):983-999 describe the crystal structure of the IL12 and IL23 receptors and describe residues of P40 that interact with the IL12R ⁇ 1 receptor.
  • IL 12 partial agonists comprising a modified P40 subunit that preserved CD8+ T cell IFNg induction and tumor cell killing but exhibit reduced activation and cytokine production from NK cells.
  • the stimulation of NK cells is associated with significant systemic side effects such as capillary leak syndrome.
  • IL12 partial agonists that selectively activate CD8+ T cells without significant upregulation of NK cells retain the beneficial antitumor effects of IL12 while mitigating systemic toxicity associated with the activation of NK cells.
  • the IL 12 partial agonists produced potent antitumor immunity with reduced toxicity relative to IL 12 in preclinical mouse tumor models.
  • rhILl 2 recombinant human IL 12
  • IL2 recombinant human IL 12
  • Atkins, et al (1997) Clinical Cancer Research 3:409-417.
  • the toxicity associated with wt hIL12 treatment has been an impediment to the development of extended delivery forms of IL 12.
  • sustained and targeted delivery of IL 12 has been evaluated but has not yet provided a successful IL12 therapeutic agent.
  • Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus the biopharmaceutical product can require less frequent administration.
  • Engineered Fc domains have been extensively investigated in the context of therapeutic antibodies, particularly bi-specific antibodies, with numerous Fc engineered antibodies being developed and commercialized. See, e.g. Czajkowsky, et al. (2012) EMBO Mol Med 4: 1015-1028.
  • Fc binds to the neonatal Fc receptor (FcRn) on endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, keeping the molecule in circulation longer.
  • FcRn neonatal Fc receptor
  • Fc domain properties of the Fc domain are believed to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc-fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates.
  • Fc engineering modifications to the Fc domain(s), referred to as Fc engineering, have been developed that provide for particular beneficial features to the Fc domain as modulation of effector function (Wang, et al. (2016) Protein Cell 9(1 ): 63-73)
  • Zalevsky, et al. describe the amino acid substitutions M428L and N434S (EU numbering), frequently referred to as “LS” modification, to extend half-life. Zalevsky, et al. (2010) Nature Biotechnology 28: 157-159.
  • Interleukin-2 IL2
  • Interleukin-2 is a pluripotent cytokine produced by antigen activated T cells sand exerts a wide spectrum of effects on the immune system. IL2 promotes the proliferation and expansion of activated T lymphocytes, induces proliferation and activation of naive T cells, potentiates B cell growth, and promotes the proliferation and expansion of NK cells.
  • Human interleukin 2 is a 4 alpha-helix bundle cytokine of 133 amino acids.
  • IL2 is a member of the IE2 family of cytokines which includes IL2, IL-4, IL-7, IL 9, IL-15 and IL21.
  • IL2 function of IL2 is non-redundant, evidenced by genetic knockouts in mice (Schorle, et al. (1991) Nature 352(6336): 621-624).
  • the amino acid sequence of hIL2 is found in Genbank under accession locator NP_000577.2.
  • IL2 exerts its effect on mammalian immune cells through interaction with three different cell surface proteins: (1) CD25 (also referred to as the IL2 receptor alpha, IL2Ra, p55), (2) CD122 (also referred to as the interleukin-2 receptor beta, IL2R0, IL15RJ3 and p70-75), and (3) CD132 (also referred to as the interleukin 2 receptor gamma, IL2Ry; or common gamma chain as it is a component of other multimeric receptors in the IL2 receptor family).
  • CD25 also referred to as the IL2 receptor alpha, IL2Ra, p55
  • CD122 also referred to as the interleukin-2 receptor beta, IL2R0, IL15RJ3 and p70-75
  • CD132 also referred to as the interleukin 2 receptor gamma, IL2Ry; or common gamma chain as it is a component of other multimeric receptors in the IL
  • CD25 is a 55 kD polypeptide that is constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation.
  • hIL2 binds to hCD25 with a Kd of approximately 10' 8 M.
  • CD25 is also referred to in the literature as the "low affinity" IL2 receptor.
  • the human CD25 (“hCD25”) is expressed as a 272 amino acid pre-protein comprising a 21 amino acid signal sequence which is post-translationally removed to render a 251 amino acid mature protein.
  • Amino acids 22-240 amino acids 1-219 of the mature protein
  • Amino acids 241-259 correspond to transmembrane domain.
  • Amino acids 260-272 correspond to intracellular domain.
  • Human CD25 nucleic acid and protein sequences may be found as Genbank accession numbers NM_000417 and NP_0004Q8 respectively.
  • the intracellular domain of CD25 is comparatively small (13 amino acids) and has not been associated with any independent signaling activity nor has the IL2/CD25 complex has not been observed to produce a detectable intracellular signaling response.
  • CD 122 is a single pass type I transmembrane protein.
  • the human CD 122 (hCD122) is expressed as a 551 amino acid pre-protein, the first 26 amino acids comprising a signal sequence which is post-translationally cleaved in the mature 525 amino acid protein.
  • Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain
  • amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain
  • amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain.
  • CD 122 includes naturally occurring variants of the CD122 protein including the CD122 variants comprising the S57F and D365E substitutions (as numbered in accordance with the mature hCD122 protein).
  • hCD122 is referenced at UniProtKB database as entry Pl 4784.
  • Human CD 122 nucleic acid and protein sequences may be found as Genbank accession numbers NM_000878 and NP_000869 respectively.
  • CD 132 is a type 1 cytokine receptor and is shared by the receptor complexes for IL-4, IL-7, IL-9, IL- 15, and IL21, hence it being referred to in the literature as the “common” gamma chain.
  • Human CD 132 (hCD132) is expressed as a 369 amino acid pre-protein comprising a 22 amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21 amino acid transmembrane domain, and amino acids 284- 369 (ammo acids 262-347 of the mature protein) correspond to the intracellular domain.
  • hCD132 is referenced at UniProtKB database as entry P31785. Human CD132 nucleic acid and protein sequences may be found as Genbank accession numbers: NM_000206 and NP_000197 respectively.
  • 3y an “intermediate affinity” dimeric IL2 receptor comprising CD122 and CD132
  • 3y an “intermediate affinity” dimeric IL2 receptor comprising CD122 and CD132
  • 3y a “high affinity” trimeric IL2 receptor complex comprising the CD25, CD 122 and CD 132 proteins
  • hIL2 exhibits a significantly higher affinity for the trimeric receptor relative to the dimeric receptor.
  • hIL2 exhibits a Kd of approximately 10' 9 M with respect to the dimeric intermediate affinity CD122/CD132 (IL2
  • hIL2 exhibits a Kd of approximately 10 -11 M with respect to the high IL2 affinity receptor complex approximately 2-logs higher than the affinity for the intermediate affinity dimeric receptor).
  • Monomeric IL2 forms a complex with both the trimeric “high affinity” form of the IL2 receptor and the dimeric intermediate affinity receptor (Wang, etal. (2005) Science 310: 159-1163) through binding to the extracellular domains of the receptor components expressed on the cell surface.
  • the binding of IL2 to CD25 induces a conformational change in IL2 facilitating increased binding to CD 122.
  • IL2 mutants, mimicking the CD25 binding-induced conformational change demonstrate increased binding to CD122 (Levin, et al.
  • CD 132 provides formation of the dimeric intermediate-affinity or trimeric high- affinity receptor complexes which are associated with intracellular signaling.
  • JAK/STAT pathway e.g. phosphorylation of STAT5
  • soluble CD25 soluble protein
  • sCD25 soluble CD25
  • IL2 receptor The various forms of the IL2 receptor are expressed on the surface of most lymphatic cells, in particular on T cells, NK cells, and B cells, however the expression of each receptor cell type varies with a variety of factors including cell type and whether the cell has been “activated” by the binding of MHC1 to a cognate antigen.
  • Non-activated T cells and NK cells express almost exclusively the intermediate-affinity dimeric IL2 receptor, consisting of the two signaling receptors, CD122 and CD132 and demonstrate comparatively low responsiveness to IL2 since the intermediate affinity CD122/CD132 complex which comparatively low affinity for IL2 relative to the trimer CD25/CD 122/CD 132 high affinity receptor.
  • Tregs In contrast, activated T cells, and regulatory T cells (Tregs) express the trimeric high-affinity IL2 receptor consisting of CD25, CD122 and CD132. TCR-activated T cells (z.e., so called “antigen experienced” T cells) express the high-affinity trimeric IL2 receptor. While Tregs constituitively express CD25, the level of expression of CD25 on Tregs is substantially lower than the level of CD25 expression on activated T-cells where CD25 expression is substantially (>100-fold) induced upon activation .
  • TCR- activated T cells i.e., so called “antigen experienced” T cells
  • TILs tumor infiltrating T cells
  • TCR tumor recognizing cells
  • TCR T cell receptor
  • Tregs constitutively express CD25
  • TCR-activated T cells express higher levels of the trimeric receptor than Tregs.
  • hIL2 Recombinant hIL2 is approved for the treatment of human adults with metastatic melanoma (and metastatic renal cell carcinoma by the United States Food and Drug Administration (USFDA).
  • USFDA United States Food and Drug Administration
  • HD-hIL2 High Dose hIL2
  • HD-hIL2 therapy is associated with severe dose limiting toxicity, including impaired neutrophil function, fever, hypotension, diarrhea and requires expert management.
  • HD-hIL2 treatment activates most lymphatic cells, including naive T cells and NK cells, which predominantly express the intermediate affinity receptor (CD122/CD132) and CD25+ regulatory' T cells (Tregs), which express the high affinity trimeric receptor (CD25/CD122/CD132).
  • HD-hIL2 monotherapy may also induce generalized capillary leak syndrome which can lead to death. This limits the use of HD-IL2 therapy to mostly younger, very healthy patients with normal cardiac and pulmonary function.
  • HD-IL2 therapy is typically applied in the hospital setting and frequently requires admission to an intensive care unit.
  • IL2 variants have been developed and introduced into clinical development, which are designed to avoid binding to CD25 and possess enhanced binding to the intermediate affinity CD122/CD132 receptor to activate NK cells and quiescent CD8+ T cells.
  • IL2 muteins are often referred to in the literature as “non-a-IL2” or “p/y-I L2” muteins.
  • non-a-IL2 or “p/y-IL2” muteins, by virtue of their reduced binding to CD25, also avoid binding to the antigen activated T cells which have been identified as the primary mediators of anti-tumor T cell response (Peace, D. J. and Cheever, M. A. (1989) J Exp Med 169(1): 161-173).
  • NK cells are the dominant mechanism for IL2 mediated acute toxicity'. Assier E, et al. (2004) J Immunol 172:7661-7668. As NK cells express the intermediate affinity (CD122/CD132; p/y) IL2 receptor, the nature of such p/y-IL2 muteins is to enhance the proliferation of such NK cells which may lead to enhanced toxicity. Additionally, although Tregs are associated with down-regulation of CD8+ T cells, Tregs have also been shown to limit the IL2 mediated off-tumor toxicity (Li, et al. (2017) Nature Communications 8(1): 1762).
  • nitric oxide synthase inhibitors have been suggested to ameliorate the symptoms of VLS
  • the common practice when VLS is observed is the withdrawal of IL2 therapy.
  • low-dose IL2 regimens have been tested in patients. While low dose IL2 treatment regimens do partially mitigate the VLS toxicity, this lower toxicity was achieved at the expense of optimal therapeutic results in the treatment of neoplasms.
  • IL2 muteins that retain certain desirable features of the native molecule while minimizing undesirable features, depending on the therapeutic context, remain an active area of research.
  • IL12 has been evaluated in a clinical trials alone by a variety of routes of administration, dosing schedules, both alone and when combined a variety of other agents, (see e.g., Lasek, et al. (2104) Cancer Immunol Immunotherapy 63(5): 419-435).
  • the clinical experience has demonstrated that the toxicity' has been a barrier to therapeutic use in humans (Cohen, J. (1995;) Science 270:908; Leonard et al., . (1997).) Blood 90: 2541-2548 ).
  • there has yet to be an IL12 treatment modality that has demonstrated acceptable levels toxicity to permit marketing approval from a major regulatory agency.
  • IL- 12 toxicity is mediated by NK cells when co-administered with IL-2 (Carson et al., . (1999) J. Immunol. 162: 4943-4951).
  • the clinical experience with the administration of hIL12 agents in combination hIL2 agents to human subjects has demonstrated that, in addition to their potentially beneficial properties, significant dose limiting toxicities.
  • Proleukin® aldesleukin, desAlal/C125S hIL2
  • Proleukin was approved by the US FDA in 1992 for treatment of adults (>18 years old) with metastatic renal cell carcinoma and in 1998 for the treatment of metastatic melanoma. Nevertheless, the Proleukin package insert current provides warnings of potentially life- threatening side effects . There is no IL 12 agent currently approved by a maj or regulatory authority nor for its use in combination therapy including in combination with hIL2.
  • the present disclosure is direct to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of human disease.
  • the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of neoplastic disease in a mammalian subject.
  • wild-type hIL2 and wild-type hIL12 molecules are independently associated with significant toxicity when administered to a mammalian subject (e.g.
  • the applicants have surprisingly found that the administration to a mammalian subject an hIL2M of the present disclosure in combination with an hIL12M of the present disclosure results in enhanced anti -tumor effect in a mammalian subject relative to either of the hIL2 or hIL12 administered independently and that the administration of an efficacious dose of the hIL2M in combination with an hIL12M does not result in unacceptable toxicity.
  • the present disclosure is directed to the use of a human IL2 mutein in combination with a human IL 12 mutein in the treatment of neoplastic disease in a human being suffering from a neoplastic disease. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of cancer in a mammalian subject. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of a human subject that exhibits one or more clinical indicia of the presence of cancer in the subject.
  • the present disclosure is directed to the use of human IL2 muteins in combination with human IL12 muteins in the treatment of a human subject that exhibits one or more clinical indicia of the presence of a neoplasm in the subject.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
  • hIL12M hIL12M
  • hIL12M hIL12M
  • P35 hIL 12P35
  • P40M hIL12P40M
  • the HL12P35 subunit [has at least 95% sequence identity to mature wild type human ML12P35 (SEQ ID NO:2);
  • the HL12P40M subunit of the hIL12M [has at least 95% sequence identity to mature wild type human HL12P40 (SEQ ID NO:4), the ML12P40M subunit further comprising] comprises one or more amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40; and
  • step (b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutem (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132, and wherein step (a) is performed in combination with step (b).
  • hIL2M hIL2 mutem
  • SEQ ID NO: 182 wild type human IL2
  • the IL12p40M comprises one or more the amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL 12M to the extracellular domain of the hIL 12Rb 1 receptor compared to the wild type human IL12p40.
  • positions of amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild ty pe human IL12p40 is an amino acid substitution at one or more of the residues selected from the group consisting of W37, P39.
  • positions of amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is an amino acid substitution or set of amino acid substitutions selected from the group consisting of: W37A; P39A; D40A; E81A; E81N; E81D; E81C; E81Q; E81E; E81P; E81W; E81Y; F82A; F82R; F82E; F82H; F82K; F82P; F82W; F82Y; K106A; K106N; D109A; K217A; K219A; E81A/F82A; W37A/E81
  • 81A/F82A/K106A/E108A/D115A E81F/F82A; E81K/F82A, E81L/F82A; E81H/F82A; E81S/F82A; E81A/F82A/K106N; E81A/F82A/K106Q; E81A/F82A/K106T;
  • the amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is the set of amino acid substitutions E81A/F82A/K106A.
  • the h hIL12M is an hIL12M-Fc heterodimer wherein the HL12P35 and hIL12P40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein:
  • LI and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present);
  • UH1 and UH2 are each an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering);
  • Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fc2;
  • Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fcl; and wherein the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.
  • the GSA linker is a polypeptide having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19 or 20 amino acids the polypeptide comprised of amino acids selected from the group consisting of glycine, serine and alanine, optional a glycine-serme polymer of the structure (GGGGSm)n (SEQ ID NO: 192), (GGGS m )n (SEQ ID NO: 193), (GGGA m ) n (SEQ ID NO: 194) and (GGGGA m )n (SEQ ID NO: 195), and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4.
  • the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS : 27-79. In some embodiments, the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS: 36, 37 and 65.
  • Fcl and Fc2 is each a naturally occurring upper hinge region of a human immunoglobulin selected from the UH regions of human IgGl, human IgG2, human IgG3 and human IgG4 upper hinge domains.
  • the upper hinge region is selected from the group consisting of EPKSC (SEQ ID NO: 11) and EPKSS (SEQ ID NO: 12).
  • Fcl and Fc2 comprise amino acid substitutions that promote heterodimerization between Fcl and Fc2.
  • one of Fcl and Fc2 comprises the amino acid substitutions S364H/T394F and the other of Fcl and Fc2 comprises the amino acid substitutions Y349T/F405A numbered in accordance with the EU numbering system
  • one of Fcl and Fc2 comprises amino acid substitutions T350V/L351Y/F405 A/Y407V and the other of Fcl and Fc2 comprises the amino acid substitutions T350V/T366L/K392L/T394W.
  • one of Fcl and Fc2 comprises amino acid substitutions K360E/K409W and the other of Fcl and Fc2 comprises the ammo acid substitutions Q347R/D399V/F405T numbered in accordance with the EU numbering system.
  • one of Fcl and Fc2 comprises amino acid substitutions to provide a knob and the other of Fcl and Fc2 comprises amino acid substitutions provide a hole, optionally wherein the amino acid substitution to provide a knob is the T366W and the acid substitutions to provide a hole is the set of amino acid substitutions T366S/L368A/Y407V numbered in accordance with the EU numbering system.
  • Fcl and Fc2 are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds , optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) C96 of the ML12P35 and C199 of the hIL12P40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution numbered in accordance with the EU numbering system.
  • the hIL12M-Fc comprises one or more ammo acid substitutions to reduce effector function.
  • the hIL12M is a hIE12M-Fc wherein either or both of the hIE12P35 and hIL12P40M subunits of the heterodimeric hIL12M- Fc mutein comprise one or more amino acid substitutions to reduce effector function.
  • the hIL12P35 and/or hIL12P40M polypeptides comprise a set of amino acid substitutions selected from the group consisting of: (a) L234A/L235A/P329A (“LALAPA”); L234A/L235A/P329G (“LALAPG”); L234A/L235E/G237A/A330S/P331S (“AEASS”); and L234F/L235E/P331S (“FES”).
  • LALAPA L234A/L235A/P329A
  • LALAPG L234A/L235A/P329G
  • AEASS L234A/L235E/G237A/A330S/P331S
  • FES L234F/L235E/P331S
  • the amino acid substitutions to reduce effector function selected from the group consisting of: E234E;E234A/E235A; L234A/E235A/P329A; and L234A/L235A/P329G numbered in accordance with the EU numbering system.
  • Fcl and Fc2 are human IgG4 Fc domains and one or both of Fcl and Fc2 comprise one or more amino acid substitutions to eliminate N- or 0 linked glycosylation sites, optionally wherein the modification to eliminate N- or 0 linked glycosylation sites is selected from the group consisting of N297Q and N297G numbered in accordance with the EU numbering system.
  • the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises amino acid substitutions in the Fc domain at positions M428 and/or N434 (EU numbering).
  • the amino acid substitutions at positions M428 and/or N434 are M428L and/or N434S.
  • hIL12M-Fc, Fcl and Fc2 comprise a deletion of: (a) the lysine residue at position 447 or (b) a deletion of both the glycine at position 446 and the lysine residue at position 447 numbered in accordance with the EU numbering system.
  • the hIL12M is PEGylated.
  • the PEG has a molecular mass greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 5 OkDa.
  • the hIL12P40M subunit comprises an ammo acid substitution at position 282 (numbered in accordance with the hIL12P40 precursor, SEQ ID NOG) selected from the group consisting of K282G, K282A, K282N, K282QG or K282.
  • the hIL12M is recombinantly expressed in a mammalian host cell, optionally wherein the mammalian host cell is selected from HEK293 cells and CHO cells.
  • the binding affinity ofhIL12M forthe extracellular domain (ECD) of IL 12RJ31 is reduced by at least 5%, optionally by at least 10%, optionally by at least 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, optionally by at least 70%, relative to the binding affinity of wild type hIL12 for the extracellular domain (ECD) of IL12R. ⁇ 1 as determined by surface plasmon resonance.
  • the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
  • the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in in signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
  • the hIL12M decreased STAT-4 mediated signaling wherein the STAT4 signaling in NK cells is decreased by at least 10%, optionally by at least a 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, or optionally by at least 70% signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
  • the hIL12M is an IL12M-Fc comprising a polypeptide of the formula [1] selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153.
  • the hIL12M is an IL12M- Fc comprising a polypeptide of the formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
  • the hIL12M is an IL12M-Fc comprising a polypeptide of the formula [1] is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 and a polypeptide of the formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
  • the hIL12M is an IL12M-Fc comprising a first polypeptide the formula #1 is a polypeptide having an amino acid sequence:
  • the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to eliminate the sulfhydryl side chain.
  • the substitution at position C220 is C220S (EU numbering) substitution.
  • the hIL12M is a hIL12M-Fc wherein n either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises amino acid deletions in the Fc domain at positions G446 and/or K447 (EU numbering).
  • the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and HL12P40M subunits of the heterodimeric hIL12M-Fc mutem are PEGylated. In some embodiments, either or both of the hIL12P35 and hIL12P40M subunits are PEGylated via the sulfhydryl side chain of amino acid C220 of the upper hinge.
  • the hIL12M is a hIL12M-Fc wherein hIL12M-Fc mutein: (i) induces hIL-12 signaling in CD8+ T cells; and (ii) has decreased (for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decreased) hIL-12 signaling in NK cells compared to a wildtype hIL-12comprising a P40 polypeptide lacking the one or more amino acid substitutions.
  • the hIL12M is a hIL12M-Fc that activates interferon gamma (IFNy) in CD8+ T cells and has decreased IFNy signaling in CD8+ T cells, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, compared to the wildtype IL 12 comprising a P40 subunit lacking such amino acid substitutions.
  • the hIL12M is a hIL12M-Fc that has a reduced binding affinity, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduction, for hIL-12R. ⁇ 1 compared to the binding affinity of a wildtype IL12.
  • the hIL12M is a hIL12M-Fc that has decreased STAT-4 mediated signaling, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, compared to wildtype hIL12 in a when evaluated in a mammalian cell-based assay.
  • the present disclosure further provides a nucleic acid sequence encoding a polypeptide of the formula [1]:
  • hIL12P40M is an human P40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with wild-type pre- human P40 (SEQ ID NO:3); LI is a GSA linker and a is selected from 0 (absent) or 1 (present); UH1 is an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl , I
  • the present disclosure further provides a nucleic acid sequence encoding a polypeptide of the formula [2] : hIL12P35- L2 b -UH2— Fc2 [2] wherein: HL12P35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99%, or 100% sequence identity to SEQ ID NO:2; L2 is a GSA linker and b is selected from 0 (absent) or 1 (present); UH2 is an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); and Fc2 is a polypeptide comprising the lower hinge, CH2
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject by administration of an human IL2 mutein in combination with a human IL2 mutein.
  • the human IL2 mutein comprising one or more amino acid substitutions or deletions at positions 1, 2, 3, 4, 5, 6, 18, 22, 125, and 126 numbered in accordance with mature wild type human IL2.
  • the one or more supplementary therapeutic agents supplementary therapeutic agent is a biased IL2 mutein having reduced affinity for the CD132 subunit of the IL2 receptor as described in are Emmerich, et al., PCT International Application Number PCT/U S2021/013456 published July 22, 2021 as WO2021/146436A2; Emmerich, et al PCT/U S2021/013514 published July 22, 2021 as WO2021/146481A1 and Garcia, et al PCT/US2018/062122 published May 31, 2019 as W02019/104092A1, the entire teachings of which are hereby incorporated by reference.
  • human IL2 mutein comprising amino acid substitutions at positions 18, 22 and 126 numbered in accordance with mature wild type human IL2.
  • the one or more supplementary therapeutic agents suppl ementary therapeutic agent is a biased human IL2 mutein comprising amino acid substitutions 18R, Q22E and Q126K.
  • the one or more supplementary therapeutic agents is a biased IL2 mutein comprising amino acid substitutions t positions 18, 22 and 126 numbered in accordance with mature wild type human IL2 that is PEGylated.
  • the present disclosure is directed to the use of a human IL2 mutein in combination with a human IL 12 mutein in the treatment of neoplastic disease in a human being suffering from a neoplastic diseas the method comprising the step of administering to the subject a therapeutically effective amount of an hIL2 mutein comprising one or more amino acid substitutions relative to the wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132.
  • the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 (numbered in accordance with mature human wild type (wt hIL2; SEQ ID NO: 182).
  • the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD 132 are selected from amino acid substitutions at positions 18, 22, and 126 are selected from the amino acid substitution at position 18 selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T; the amino acid substitution at position 22 selected from the group consisting of Q22F, Q22E, Q22G, Q22A, Q22L, Q22M.
  • the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 are selected from the groups of amino acid substitutions consisting of L18R/Q22E/Q126K (“REK”); L18R/Q22E/Q126H (“REH”); L18A/Q22E/Q126H (“AEH”); L18A/Q22E/Q126K (“AEK”); L18D/Q22E/Q126H (“DEH”); L18E/Q22E/Q126H (“EEH”); L18E/Q22E/Q126K (“EEK”); L18F/Q22E/Q126H (“FEH”); L18G/Q22E/Q126H (“GEH”); L18H/Q22E/Q126H (“HEH”); L18H/Q22E/Q126K (“HEK”); L18I/Q22E/Q126H (“IEH”); L18I/Q22E/Q126H (“
  • the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 (numbered in accordance with mature human wild type (wt hIL2; SEQ ID NO: 182), hIL2M may optionally further comprise one or more amino acid substitutions selected from the group consisting of T3C, T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, K35E; R38W, R38G; M39L, M39V; H55Y; V69A; Q74P, Q74N, Q74H, Q74S; M104A; D109C or a non-natural ammo acid with an activated side chain at position 109; AA113, T113N; AA125 is C125A or C125S; S130T, S130G and S130R.
  • the hIL2M may optionally comprise a deletion of one or more N-terminal amino acid selected from the group or deletions: des-Al; des-Al/des-P2; des-Al/des-P2/des-T3; des-Al/des-P2/des-T3/des-S4; des- Al/des-P2/des-T3/des-S4/des-S5; des-Al/des-P2/des-T3/des-S4/des-S5/des-S6; des-Al/des- P2/des-T3/des-S4/des-S5/des-S6/des-T7.
  • the hIL2M comprises a des Alai /L18R/Q22E/Q126K hIL2M having the amino acid sequence:
  • the hIL2M comprises a des Alai /L18R/Q22E/Q126K hIL2M having the amino acid sequence:
  • hIL2M useful in the practice of the present disclosure exhibits at least a 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of reduction in the binding affinity to CD 132 of wild-type hIL2 as determined by surface plasmon resonance.
  • hIL2M useful in the practice of the present disclosure exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of the binding affinity to CD25 of wild-type hIL2 as determined by surface plasmon resonance.
  • hIL2M useful in the practice of the present disclosure exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90%, optionally at least 100% of the binding affinity to CD25 of wild-type hIL2 as determined by surface plasmon resonance.
  • hIL2M is covalently linked to a earner molecule that provides for an extended duration of action in a mammalian subject.
  • the carrier molecule is selected from the group consisting of Fc polypeptides, hydrophilic polymers (e g. PEG), hydrophobic polymers (e.g. fatty acid molecules) acylated), human serum albumin.
  • the hydrophilic polymer is polyethylene glycol.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
  • hIL12M hIL12M-Fc heterodimer wherein the hIL12P35 and HL12P40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide having the amino acid sequence:
  • hIL2M comprises a polypeptide having the amino acid sequence:
  • the administration of the hIL2M and hIL12M to a mammalian subject may be achieved by administration to the subject of: (a) a pharmaceutically acceptable formulation comprising an hIL2M in combination with ) a pharmaceutically acceptable formulation comprising an hIL12M;(b) a pharmaceutically acceptable formulation comprising an hIL2M and an hIL2M (co-formulated for administration; (c) a nucleic acid molecule(s) or vector(s) comprising a nucleic acid sequence(s) encoding a heterodimeric hIL2M in combination with a nucleic acid molecule(s) or vector(s) comprising a nucleic acid sequence(s) encoding a hIL12M; (d) a recombinantly modified cell comprising a nucleic acid molecules encoding a heterodimeric hIL2M and hIL12M.
  • the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are dosed simultaneously.
  • the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are dosed contemperanously.
  • the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are modified to promote extended half-life in vivo.
  • the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M-PEG in combination with an hIL12M-Fc, wherein the HL2M-PEG is dosed about every two weeks, alternatively about every three weeks, alternatively about ever ⁇ ' 4 weeks, and the hIL12M-Fc is dosed about ever ⁇ ' two weeks, alternatively about ever ⁇ ' three weeks, or alternatively about every 4 weeks.
  • the disclosure provides a method of treating a neoplastic disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M-PEG in combination with an hIL12M-Fc.
  • the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M and at least one supplementary agent .
  • Figure 1 provides the results of an evaluation of interferon gamma induction (vertical axis) with respect to increasing concentrations of the test agent (horizontal axis) in CD8, CD4 and NK cells.
  • Panels A, B and C illustrate the effect of wild type hIL12 in comparison to hIL12M molecules comprising amino acid substitutions E81A/F82A indicated as “2xAla”, E81A/F82A/K106A indicated as “3xAla” and substitution W37A, on CD8, CD4 and NK cells, respectively.
  • Panels D, E and F provide the results of IFNy induction of (knob-into-hole (KiH) heterodimeric hIL12M-Fc muteins comprising wild type hIL12 in comparison to KiH heterodimeric hIL12M-Fc muteins comprising amino acid substitutions E81A/F82A indicated as “2xAla Fc”, E81A/F82A/K106A indicated as “3xAla Fc” and substitution W37A (W37A Fc), on CD8, CD4 and NK cells, respectively.
  • KiH knock-into-hole
  • Figure 2 provides the results of an evaluation of interferon-y and STAT4 induction (vertical axis) with respect to increasing concentrations of test agents indicated on CD8+ T cells (Panel A), CD4+ T cells (Panel B), and NK cells from two different human donors (Panels C and D).
  • Figure 3 provides spider plots of the of the tumor volume over time of mice treated with various murine IL12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below. Tumor volume is provided on the Y axis and time is on the X axis.
  • Figure 4 provides body weights (Y axis) of mice treated over time (X axis) of mice treated with various murine IL 12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below.
  • Figure 5 provides the survival data (probability of survival on Y axis) of mice treated overtime (X axis) of mice treated with various murine IL 12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below.
  • Figure 6 provides data with respect to tumor volume (y axis) with respect to days after treatment initiation (x axis) of with mIL12 proteins in Panel A and those IL 12 protein subunits in a heterodimeric Fc format in Panel B.
  • Figure 7 provides an amino acid sequence alignment of the wild type murine and human P40 (IL12Ra) proteins with the signal peptide sequence highlighted.
  • Figure 8 provides an amino acid sequence alignment of the wild type murine and human P35 (IL12Rb) proteins.
  • Figure 9 provides a graphical representation of the concentration in picograms per milliliter (pg/mL) of murine interferon gamma (y-axis) measured in serum obtained from blood samples taken over time (x-axis) in the MC38 tumor model, the design of which is provided in Table 12.
  • Panel A provides the concentration in picograms per milliliter (pg/mL) of murine interferon gamma in serum of treatment groups A-E of Table 12 at 0 hours (pretreatment) and 4 hours, 1 day and 7 days following treatment with the test agent.
  • Panel B relates to the murine interferon gamma levels in serum of treatment groups F, G, and H of Table 12 at 0 hours (pretreatment) and 4 hours, 1 day and 7 days following treatment with the test agent.
  • Figure 10 is a graphical representation of the percentage of lymphocytes (y-axis) as determined by FACS analysis with respect to each of the treatment groups of Table 12 (x-axis) in spleen and tumor tissue.
  • Figure 11 provides the results of a phenotypic FACS analysis of NK cells obtained from spleen in the treatment groups and study described in Table 12. T-bet is measured on the vertical axis and intracellular Granzyme B on the horizontal axis. The graphs are labeled with the treatment groups (A, F, G and H of Table 12).
  • Figure 12 provides a series of spider plots resulting from the CT26 tumor study as described herein with tumor volume provided on the vertical axis and time (study days) on the horizontal axis. Each panel of the figure indicates the test agent provide and the dosing schedule of the test agent in accordance with the study design provided in Table 13.
  • Figure 13 provides the results of body weight measurements of mice evaluated in the CT26 tumor study with percent change in bodyweight on the vertical axis and time (study days) on the horizontal axis.
  • Figure 14 provides results of a FACS analysis of cells obtained from the CT26 tumor study study sorted by the presence of various markers as indicated by the arrows and various doses of test agent as indicated by the figure legends.
  • Figure 15 provides the results of the efficacy of the various test agents indicated by the figure legends in response to depletion of NK cells in the study summarized in Table 14.
  • Figure 16 provides the results of evaluation of percent change in body weight (vertical axis) over the course of the study (horizontal axis) in response to the various test agent conditions provided in the MC38 tumor study to evaluate the effects of NK and CD8 cell depletion as provided in detail herein, the study design of which is summarized in Table 15.
  • Figure 17 provides a series of spider plots with respect to the antitumor efficacy (tumor volume on the vertical axis) over the course of the study (horizontal axis) in response to the various test conditions identified in Table 15.
  • Figure 18 provides the results of antitumor efficacy of IL12 test agents against MC38 tumors in various types of mice, B6 mice in the two figures in the first column, RAG2 knockout mice in the two figures in the second column and RAG2/CD132 double knockout mice in the third column. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis).
  • the figure legends identify the various test conditions provided in Table 15.
  • Figure 19 provides the results of a study evaluating the anti -tumor efficacy of a heterodimeric mIL12M-Fc polypeptide in combination with a PD1 inhibitor in the treatment of MC38 tumors in mice. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis). The figure legends correspond to the treatment groups summarized in Table 16 herein.
  • Figure 20 provides the results of a study evaluating the anti-tumor efficacy of a heterodimeric mIL12M-Fc polypeptide in combination with an mIL2 mutein polypeptide in the treatment of MC38 tumors in mice. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis). The figure legends correspond to the treatment groups summarized in Table 17 herein.
  • Figure 21 provides data relating to STAT4 signaling in NK cells of human and murine surrogate IL12-Fcs.
  • the upper Panel A provides data relating to the induction of STAT4 signaling (y-axis) of varying concentrations (x-axis) of mIL-12 Fc (Compound 1) and mIL12M-Fc (Compound 2) molecules on murine NK cells are described in Example 5 herein.
  • the lower Panel A provides data relating to the induction of STAT4 signaling (y-axis) of varying concentrations (x-axis) of hIL12-Fc and hIL12M-Fc molecules on human NK cells as more fully described in Example 5.
  • Figure 22 provides the results of body weight measurements obtained from of animals treated in the MC38 tumor study described in more detail in Example 6 herein wherein the percent of initial bodyweight is provided on the y-axis and time (Study Days) is presented on the x-axis wherein each line represents an individual animal treated.
  • Figure 23 provides a spider plot of the results of tumor volume measurements obtained from animals treated in the MC38 tumor study described in more detail in Example 6 herein. Tumor volume (y-axis) versus study day (x-axis) is provided with respect to individual animal tumor volumes is provided wherein each line represents an individual animal treated.
  • Figure 24 provides the results of body weight measurements obtained from of animals treated in the MC38 tumor study described in more detail in Example 7 herein wherein the percent of initial bodyweight is provided on the y-axis and time (Study Days) is presented on the x-axis wherein each line represents an individual animal treated.
  • Figure 25 provides a spider plot of the results of tumor volume measurements obtained from animals treated in the MC38 tumor study described in more detail in Example 7 herein. Tumor volume (y-axis) versus study day (x-axis) is provided with respect to individual animal tumor volumes is provided wherein each line represents an individual animal treated.
  • HL12P35 Residue Numbering In the present disclosure, the numbering of amino acid residues of human P35 is made in reference to the number of the precursor or “pro” form of HL12P35 as provided in SEQ ID NO: 1.
  • hP40 Residue Numbering In the present disclosure, the numbering of amino acid residues of human P40 is made in reference to the number of the precursor or “pro” form of hP40 as provided in (SEQ ID NO: 3). In reference to the hP40 muteins, substitutions are designated herein by the one letter amino acid code followed by the pro-hP40 (SEQ ID NO: 3) amino acid position followed by the one letter amino acid code which is substituted. For example, an hP40 mutein having the modification “E81 A” refers to a substitution of the glutamic acid (E) residue at position 81 of the (SEQ ID NO: 3) with an alanine (A) residue at this position. A deletion of an amino acid reside is referred to as “des” or the symbol “A” followed by the amino acid residue and its position.
  • hP40 Residue Numbering In the present disclosure, the numbering of amino acid residues of human IL2 is made in reference to the number of the mature wild type human IL2 as provided in (SEQ ID NO: 182). In reference to a hIL2 mutein (hIL2M), substitutions are designated herein by the one letter amino acid code followed by the mature hIL2 amino acid position followed by the one letter amino acid code which is substituted. For example, an hIL2M having the modification “L18R” refers to a substitution of the leucine (L) residue at position 18 of the mature wild type human IL2 (SEQ ID NO: 182) with an arginine (R) residue at this position.
  • a deletion of an amino acid reside is referred to as “des” or the symbol “A” followed by the amino acid residue and its position in the mature wild type human IL2 (SEQ ID NO: 182).
  • the deletion of the N-terminal alanine (A) residue at position 1 of the mature wild type human IL2 is referred to as “des-Alal”
  • Immunoglobulin, Upper Hinge and Fc Residue Numbering There are a variety of numbering conventions that are employed with respect to the numbering of amino acid residues of immunoglobulins including Rabat numbering, Chothia numbering, EU numbering and IMGT numbering conventions.
  • the numbering of amino acid residues of immunoglobulin molecules including domains thereof including the upper hinge and Fc domain is made in accordance with EU Numbering conventions.
  • EU Numbering conventions is readily understood by those of skill in the art. Dondelinger, et al.
  • an “M” suffix may be added to a polypeptide number (e.g. DR1535M) to identify such sequence as a “mature” molecule lacking a signal sequence so as to distinguish the polypeptide from the precursor molecule containing the signal peptide which precursor form may be identified with a “P” suffix such as DR1535P.
  • the term “about” refers to a value that is plus or minus 10% of a numerical value described herein, such as plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of numerical value described herein.
  • the term “about” also applies to all numerical ranges described herein. All values described herein are understood to be modified by the term “about” whether or not the term “about” is explicitly recited in reference to a given value.
  • Activate is used in reference to a receptor or receptor complex to reflect a biological effect, directly and/or by participation in a multicomponent signaling cascade, arising from the binding of an agonist ligand to a receptor responsive to the binding of the ligand.
  • the term activate is also used in reference to a cell that expresses a receptor wherein one more biological activities of the cell are modulated (e.g. upregulation or downregulation of STAT signaling, in response to binding of a ligand for such receptor.
  • Activity is used with respect to a molecule to describe a property of the molecule with respect to a test system (e.g., an assay) or biological or chemical property (e.g., the degree of binding of the molecule to another molecule) or of a physical property of a material or cell (e.g., modification of cell membrane potential).
  • test system e.g., an assay
  • biological or chemical property e.g., the degree of binding of the molecule to another molecule
  • a physical property of a material or cell e.g., modification of cell membrane potential
  • Examples of such biological functions include but are not limited to catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, and the ability to modulate immunological activity such as inflammatory response.
  • “Activity” is typically expressed as a level of a biological activity per unit of agent tested such as [catalytic activity]/[mg protein], [immunological activity]/[mg protein], international units (IU) of activity, [STAT3 phosphorylation]/[mg protein], [STAT4 phosphorylation]/[mg protein] [proliferation]/[mg protein], plaque forming units (pfu), etc.
  • proliferative activity refers to an activity that promotes cell proliferation and replication, including dysregulated cell division such as that observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis.
  • Administer/Administration The terms ‘‘administration” and “administer” are used interchangeably herein to refer the act of contacting a subject, including contacting a cell, tissue, organ, or biological fluid of the subject in vitro, in vivo or ex vivo with an agent
  • Administration of an agent may be achieved through any of a variety of art recognized methods including but not limited to the topical administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g respiratory inhalers including dry-powder inhalers), intraocular injection, intraabdominal injection, intralesional injection, intraovanan injection, intracerebral infusion or injection, intracerebroventricular injection
  • affinity refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor) and is measured by the equilibrium dissociation constant (KD), a ratio of the dissociation rate constant between the molecule and its target (Koff) and the association rate constant between the molecule and its target (Kon).
  • KD equilibrium dissociation constant
  • Koff ratio of the dissociation rate constant between the molecule and its target
  • Kon association rate constant
  • agonist refers a first agent that specifically binds a second agent (“target”) and interacts with the target to cause or promote an increase in the activation of the target.
  • agonists are activators of receptor proteins that modulate cell activation, enhance activation, sensitize cells to activation by a second agent, or up-regulate the expression of one or more genes, proteins, ligands, receptors, biological pathways, that may result in modulation of cell proliferation or pathways or the cell cycle.
  • an agonist is a modified form of a cognate ligand that binds to its cognate receptor and alters the state of the cognate receptor in a biological response that mimics the biological effect of the interaction of the naturally occurring cognate ligand with its cognate receptor.
  • the term “agonist” includes partial agonists, full agonists and superagonists.
  • An agonist may be described as a “full agonist” when such agonist which leads to a substantially full biological response (i.e. the response associated with the naturally occurring ligand/receptor binding interaction) induced by receptor under study, or a partial agonist.
  • a "superagonisf 1 is a type of agonist that can produce a maximal response greater than the endogenous agonist for the target receptor, and thus has an activity of more than 100% of the native ligand.
  • a super agonist is typically a synthetic molecule that exhibits greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% of the response in an evaluable quantitative or qualitative parameter of the naturally occurring form of the molecule when evaluated at similar concentrations in a comparable assay. It should be noted that the biological effects associated with the full agonist may differ in degree and/or in kind from those biological effects of partial or superagonists.
  • antagonists may specifically bind to a receptor but do not result in the signal cascade typically initiated by the receptor and may modify the actions of an agonist at that receptor.
  • Inverse agonists are agents that produce a pharmacological response that is opposite in direction to that of an agonist.
  • Antagonist refers to a molecule that opposes the action(s) of an agonist.
  • An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist.
  • Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, biological pathway including an immune checkpoint pathway, or cell.
  • an antagonist may be a mutein of the naturally occurring ligad such that binding to receptor is maintained but there is no downstream signaling.
  • Biological Sample refers to a sample obtained (or derived) from a subject.
  • a biological sample comprises a material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin enriched or cell-type specific enriched fractions derived from one or more of such tissues.
  • body fluids e.g., blood, whole blood, plasma, serum, mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin enriched or
  • Comparable is used to describe the degree of difference in two measurements of an evaluable quantitative or qualitative parameter. For example, where a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter do not deviate beyond a range that the skilled artisan would recognize as not producing a statistically significant difference in effect between the two results in the circumstances, the two measurements would be considered “comparable.” In some instances, measurements may be considered “comparable” if one measurement deviates from another by less than 35%, alternatively by less than 30%, alternatively by less than 25%, alternatively by less than 20%, alternatively by less than 15%, alternatively by less than 10%, alternatively by less than 7%, alternatively by less than 5%, alternatively by less than 4%, alternatively by less than 3%, alternatively by less than 2%, or by less than 1%. In particular embodiments, one measurement is comparable to a reference standard if it deviates by less than 15%, alternatively by less than less than
  • correspondence or “corresponding to” in the context of an amino acid or nucleic acid sequence refers to the equivalent position of a reference sequence that is aligned with one or more other sequences to maximize the percentage of sequence identity.
  • corresponding to is used in the context of generating amino acid substitutions of a mutein from a first species to generate a mutein of another species such as muteins of human sequences for generating murine surrogate muteins.
  • the term “derived from” is used in the context of a variant polyprpyifr or nucleic acid to indicate that a variant polypeptide or nucleic acid has a sequence that is based on but differs from that of a reference polypeptide or nucleic acid.
  • the term derived is not meant to be limiting as to the source or method by which the variant protein or nucleic acid is made.
  • Effective Concentration As used herein, the terms “effective concentration” or its abbreviation “EC” are used interchangeably to refer to the concentration of an agent in an amount sufficient to effect a change in a given parameter in a test system.
  • the abbreviation “E” refers to the magnitude of a given biological effect observed in a test system when that test system is exposed to a test agent. When the magnitude of the response is expressed as a factor of the concentration (“C”) of the test agent, the abbreviation “EC” is used.
  • Emax refers to the maximal magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent.
  • the subscript refers to the percentage of the Emax of the biological response observed at that concentration.
  • concentration of a test agent sufficient to result in the induction of a measurable biological parameter in a test system that is 30% of the maximal level of such measurable biological parameter in response to such test agent, this is referred to as the “EC30” of the test agent with respect to such biological parameter.
  • EC100 is used to denote the effective concentration of an agent that results in the maximal (100%) response of a measurable parameter in response to such agent.
  • EC50 refers to the concentration of the agent sufficient to result in the half-maximal (about 50%) change in the measurable parameter.
  • concentration refers to the maximum possible quantity of a test agent that can dissolve in a standard volume of a specific solvent (e.g., water) under standard conditions of temperature and pressure.
  • a saturating concentration of a drug is typically used to denote the concentration sufficient of the drug such that all available receptors are occupied by the drug, and EC50 is the drug concentration that provides the half-maximal effect.
  • enriched refers to a sample comprising a species of interest (e.g. a molecule or cell) wherein the sample is non-naturally manipulated so that a species of interest is present in: (a) a greater concentration (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, or alternatively at least 1000-fold greater) than the concentration of the species in the starting sample, such as a biological sample (e.g., a sample in which the molecule naturally occurs or in which it is present after administration); or (b) a concentration greater than the environment in which the molecule was made (e.g., a recombinantly modified bacterial or mammalian cell).
  • a greater concentration e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, or alternatively at least 1000
  • Extracellular Domain refers to the portion of a cell surface protein which is external to the plasma membrane of the cell on which it is expressed.
  • a cell surface protein comprising and ECD may be a transmembrane protein, a cell surface or membrane associated protein that comprising a domain associated with the cell membrane but which lacks an intracellular domain.
  • Identity refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same amino acid or nucleotide then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res.
  • HSPs high scoring sequence pairs
  • W the query sequence
  • T some positive-valued threshold score “T” when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul, et al., supra).
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (the reward score for a pair of matching residues; always >0) and “N” (the penalty score for mismatching residues; always ⁇ 0).
  • M the reward score for a pair of matching residues; always >0
  • N the penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: (a) the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached.
  • the BLAST algorithm parameters “W”, “T”, and “X” determine the sensitivity and speed of the alignment.
  • W word size
  • E expectation
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Hemkoff & Hemkoff, (1989) PNAS(USA) 89:10915-10919).
  • the term “in combination w ith” when used in reference to the administration of multiple agents to a single subject refers to the administration of a first agent (e.g. an hIL12M) and a second agent (e.g. an hIL2M), optionally further administering an additional (i.e. third, fourth, fifth, etc.) supplementary agent to a subject, simultaneously, contemporaneously or sequentially.
  • a first agent e.g. an hIL12M
  • a second agent e.g. an hIL2M
  • an additional i.e. third, fourth, fifth, etc.
  • a first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously , contemporaneously or sequentially.
  • a first agent is administered “simulatenously” with a second agent if first and second agents are administered within about 30 minutes of each other.
  • a first agent is administered “contemporaneously” with a second agent if first and second agents are administered within about 24 hours of each another, alternatively within about 12 hours of each other, alternatively within about 6 hours of each other, alternatively within about 2 hours of each other, or alternatively within about 60 minutes of each other.
  • a first agent and a second agent(s) are administered sequentially, e.g., where one agent is administered to the subject at least 24 hours prior to the administration of the second agent.
  • the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the biological effect of the first agent persists in the subject at the time of administration of the second agent.
  • administration of the first agent may provide a therapeutic effect over an extended time and the administration of the second agent administered while the therapeutic effect of the first agent persists in the subject such that the second agent is considered to be administered in combination with the first agent, even though the first agent may have been administered at a point in time significantly distant (e.g. days or weeks) from the time of administration of the second agent.
  • the administration of the first agent and the second agent may be separated by a significant period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer) may result in the maintenance of a therapeutically effective amount of the first agent over a period of weeks such that the administration of the second agent at time point days or weeks after the administration of the first agent such that the second agent would be considered to be administered in combination with the first agent.
  • a significant period of time e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer
  • the biological effect of the administration of a first therapeutic agent may persist for a period of time beyond the point where there is a detectable level of the first therapeutic agent in the subject.
  • one agent e.g., an hIL12M
  • a second agent e.g. an hIL2M
  • the determination of whether that therapeutic effect of the first agent persists after the agent is no longer detectable can be established through the upregulation or down-regulation of biological markers that are characteristically modulated in response to the agent.
  • One of skill in the art is capable of performing pharmacokinetic studies to determine the in vivo duration of action of the hIL12M or hIL2M molecules described herein. Studies in primates, e.g. cynomolgus monkeys, chimpanzees, rhesus monkeys, are commonly used to provide information relating to the duration of action and toxicity indicative of the response in human subjects.
  • the skilled artisan will be able to establish a therapeutically effective dose of the hIL12M in combination with an hIL2M by evaluation of pharmacokinetic data, indicators of response and/or toxicity and other factors known to the clinician.
  • the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the initial blood or serum concentration of the first agent persists in the subject at the time of administration of the second agent.
  • the initial blood or serum concentration of the first agent is measured at a time within 30 minutes, alternatively within 1 hour, alternatively within 2 hours, alternatively within 4 hours, alternatively within 6 hours, alternatively within 24 hours following the administration of the first agent.
  • the phrase "in an amount sufficient to cause a response” is used in reference to the amount of an agent sufficient to provide a detectable change in the level of an indicator measured before (e.g., a baseline level) and after the application of a test agent to a test system.
  • the test system is a cell, tissue or organism.
  • the test system is an in vitro test system such as a fluorescent assay.
  • the test system is an in vivo system which involves the measurement of a change in the level a parameter of a cell, tissue, or organism reflective of a biological function before and after the application of the test agent to the cell, tissue, or organism.
  • the indicator is reflective of biological function or state of development of a cell evaluated in an assay in response to the administration of a quantity of the test agent.
  • the test system involves the measurement of a change in the level an indicator of a cell, tissue, or organism reflective of a biological condition before and after the application of one or more test agents to the cell, tissue, or organism.
  • the term “in an amount sufficient to effect a response” may be sufficient to be a therapeutically effective amount but may also be more or less than a therapeutically effective amount.
  • prevention refers to a judgment made by a physician or other caregiver with respect to a subject that the subject requires or will potentially benefit from preventative care. This judgment is made based upon a variety of factors that are in the realm of a physician’s or caregiver’s expertise.
  • prevention refers to reducing, forestalling or delaying the onset of a particular disease, or reducing forestalling or delaying a recurrence of a particular disease, for example, after an initial course of treatment for the disease.
  • a recurrence of a disease does not necessarily have to be after initial cure, complete remission or partial remission of a disease. It is sufficient to have one or more clinical symptoms of the initial disease reappear in a subject after a period devoid of those symptoms for the disease to be considered as recurring in the subject.
  • a subject in need of treatment has been diagnosed with a disease or condition, for example, a neoplastic disease, an autoimmune disorder or an infection.
  • Inhibitor refers to a molecule that decreases, blocks, prevents, delays activation of, inactivates, desensitizes, or down-regulates, e.g., a gene, protein, ligand, receptor, or cell.
  • An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity of a cell or organism.
  • Intracellular domain refers to the portion of a cell surface protein (e.g., a cell surface receptor or receptor subnit) which is inside of the plasma membrane of a cell.
  • a cell surface protein comprising an ICD may be a transmembrane protein or a cell surface or membrane associated protein comprising a domain associated with the cell membrane but which lacks an extracellular domain.
  • the ICD may include the entire cytoplasmic portion of a transmembrane protein or membrane associated protein.
  • Isolated As used herein the term “isolated” when used in reference to a molecule that, if naturally occurring, is in an environment different from that in which it naturally occurs. “Isolated” is meant to include molecule that are within samples that are substantially enriched for the molecule of interest and/or in which the molecule of interest is partially or substantially purified. Where the molecule is not naturally occurring, “isolated” indicates that the molecule has been separated from an environment in which it was synthesized. For example a polypeptide may be isolated from a recombinant cell culture comprising cells engineered to express the polypeptide or by a solution resulting from solid phase or cell free synthesis.
  • Ligand refers to a molecule that specifically binds a receptor and where such binding causes a change in the receptor sufficient to effect a change in the activity of the receptor or results in a response in cell that expresses that receptor.
  • the term “ligand” refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor.
  • the complex of a ligand and receptor is termed a “ligand-receptor complex” (for example, the [hIL2/CD25/CD122/CD132] receptor complex).
  • the term “cognate ligand” and “cognate receptor” are used to denote a naturally occurring ligand and the receptor to which such ligand exhibits selective binding in a naturally occurring biological systems.
  • Modulate As used herein, the terms “modulate”, “modulation” and the like refer to the ability of an agent, for example, a test agent, to cause a response, either positive or negative or directly or indirectly, in a system, including a biological system, or biochemical pathway.
  • modulator includes both agonists (including partial agonists, full agonists and superagonists) and antagonists.
  • Mutein As used herein, the term “mutein” is used to refer to a variant of a wild type polypeptide comprising modifications to the primary structure (i.e. amino acid sequence) of such polypeptide.
  • a mutein may have have at at least 99% sequence identity, alternatively at least 98% sequence identity, alternatively at least about 97% sequence identity, alternatively at least 96% sequence identity, alternatively at least 95% sequence identity, alternatively at least about 94% identical, alternatively at least 93% sequence identify, alternatively at least 92% identical, alternatively at least 91% sequence identify, or alternatively at least 90% sequence identify, to the parent polypeptide from which the mutein was derived.
  • nucleic Acid The terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.
  • Operably Linked is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, which are arranged in a construct such that the functions of the component molecules is retained although the operable linkage may result in the modulation of the activity, either positively or negatively, of the individual components of the construct.
  • the operable linkage of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct where the biological activity of the protein (e.g., Emax) is diminished relative to the to the wild-type molecule, however the two are nevertheless considered operably linked.
  • PEG polyethylene glycol
  • the multiple nucleic acid sequences when combined into a single nucleic acid molecule that, for example, when introduced into a cell using recombinant technology, provides a nucleic acid which is capable of effecting the transcription and/or translation of a particular nucleic acid sequence in a cell.
  • the nucleic acid sequence encoding a signal sequence may be considered operably linked to DNA encoding a polypeptide if it results in the expression of a preprotein whereby the signal sequence facilitates the secretion of the polypeptide; a promoter or enhancer is considered operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is considered operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • the term "operably linked" means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader or associated subdomains of a molecule, contiguous and in reading phase.
  • certain genetic elements such as enhancers may function at a distance and need not be contiguous with respect to the sequence to which they provide their effect but nevertheless may be considered operably linked.
  • Parent Polypeptide As used herein, the terms "parent polypeptide” or “parent protein” are used interchangeably to designate the source of a second polypeptide (e.g., a derivative, mutant or variant) which is modified with respect to a first “parent” polypeptide. In some instances, the parent polypeptide is a wild-type or naturally occurring form of a polypeptide. In some instance, the parent polypeptide may be a modified form a naturally occurring protein that is further modified. The term parent polypeptide can also be used interchangeably with “reference polypeptide.”
  • Partial Agonist refers to a molecule (e.g., a ligand) that specifically binds to and activates a given receptor but possesses only partial activation of the receptor relative to a full agonist. The activation of the receptor may be assessed by modulation of intracellular signaling a cell expressing a receptor (e.g. modulation of intracellular levels of phospho-STAT4). Partial agonists may display both agonistic and antagonistic effects.
  • the partial agonist acts as a competitive antagonist by competing with the full agonist for the receptor binding resulting in net decrease in receptor activation relative to the contact of the receptor with the full agonist in the absence of the partial agonist.
  • Partial agonists can be used to activate receptors to give a desired submaximal response in a subject when inadequate amounts of the endogenous ligand are present, or they can reduce the overstimulation of receptors when excess amounts of the endogenous ligand are present.
  • the maximum response (Emax) produced by a partial agonist is called its intrinsic activity and may be expressed on a percentage scale where a full agonist produced a 100% response.
  • An partial agonist may have greater than 10% but less than 100%, alternatively greater than 20% but less than 100%, alternatively greater than 30% but less than 100%, alternatively greater than 40% but less than 100%, alternatively greater than 50% but less than 100%, alternatively greater than 60% but less than 100%, alternatively greater than 70% but less than 100%, alternatively greater than 80% but less than 100%, or alternatively greater than 90% but less than 100%, of the activity of the reference polypeptide when evaluated at similar concentrations in a given assay system.
  • Polypeptide As used herein the terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones.
  • polypeptide include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with amino acid sequences that facilitate purification such as chelating peptides; fusion proteins with immunologically tagged proteins; fusion proteins comprising a peptide with immunologically active polypeptide fragment (e.g. , diphtheria toxin or tetanus toxin fragments) and the like.
  • fusion proteins including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with amino acid sequences that facilitate purification such as chelating peptides; fusion proteins with immunologically tagged proteins; fusion proteins comprising a peptide with immunologically active polypeptide fragment (e
  • Prevent refers to a course of action initiated with respect to a subj ect prior to the onset of a disease, disorder, condition or symptom thereof so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof
  • a course of action to prevent a disease, disorder or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder, or condition due to genetic, experiential, or environmental factors of developing a particular disease, disorder or condition.
  • the terms “prevent”, “preventing”, “prevention” are also used to refer to the slowing of the progression of a disease, disorder, or condition from an existing state to a more deleterious state.
  • “prevent” is used in the context of prevention of the recurrence of a disease or symptom thereof wherein a prior course of therapy may have partially or completely eliminated the evidence of the disease as measured by conventional clinical testing.
  • Receptor refers to a polypeptide having a domain that specifically binds a ligand that binding of the ligand results in a change to at least one biological property of the polypeptide.
  • the receptor is a cell membrane associated protein that comprises an extracellular domain (ECD) and a membrane associated domain which serves to anchor the ECD to the cell surface.
  • ECD extracellular domain
  • the receptor is a membrane spanning polypeptide comprising an intracellular domain (ICD) and extracellular domain (ECD) linked by a membrane spanning domain referred to as a transmembrane domain (TM).
  • the binding of a ligand to the ECD of the receptor results in a conformational change in the receptor resulting in a measurable biological effect such as a change in the activity of the receptor or the binding affinity of the receptor for another protein.
  • the receptor is a membrane spanning polypeptide comprising an ECD, TM and ICD
  • the binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD.
  • a receptor is a component of a multi- component complex to facilitate intracellular signaling.
  • the ligand may bind a cell surface receptor that is not associated with any intracellular signaling alone but upon ligand binding facilitates the formation of a heteromultimeric (including heterodimeric, heterotrimeric, etc.) or homomultimeric (including homodimeric, homotrimeric, homotetrameric, etc.) complex that results in a measurable biological effect in the cell such as activation of an intracellular signaling cascade (e.g., the Jak/STAT pathway).
  • a receptor is a membrane spanning single chain polypeptide comprising ECD, TM and ICD domains wherein the ECD, TM and ICD domains are derived from the same or differing naturally occurring receptor variants or synthetic functional equivalents thereof (chimeric receptor).
  • Recombinant As used herein, the term “recombinant” is used as an adjective to refer to the method by which a polypeptide, nucleic acid, or cell was modified using recombinant DNA technology.
  • a “recombinant protein” is a protein produced using recombinant DNA technology and is frequently abbreviated with a lower case “r” preceding the protein name to denote the method by which the protein was produced (e.g., recombinantly produced human growth hormone is commonly abbreviated “rhGH”).
  • a cell is referred to as a “recombinant cell” if the cell has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like) using recombinant DNA technology.
  • exogenous nucleic acids e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like.
  • exogenous nucleic acids e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like.
  • the techniques and protocols for recombinant DNA technology are well known in
  • response for example, of a cell, tissue, organ, or organism, encompasses a quantitative or qualitative change in a evaluable biochemical or physiological parameter, (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation) where the change is correlated with the activation, stimulation, or treatment, with or contact with exogenous agents or internal mechanisms such as genetic programming.
  • a biochemical or physiological parameter e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation
  • activation refers to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms “inhibition”, “down-regulation” and the like refer to the opposite effects.
  • a “response” may be evaluated in vitro such as through the use of assay systems, surface plasmon resonance, enzymatic activity, mass spectroscopy, amino acid or protein sequencing technologies.
  • a “response” may be evaluated in vivo quantitatively by evaluation of objective physiological parameters such as body temperature, bodyweight, tumor volume, blood pressure, results of X-ray or other imaging technology or qualitatively through changes in reported subjective feelings of well-being, depression, agitation, or pain.
  • the level of activation of T cells in response to the administration of a test agent may be determined by flow cytometric methods.
  • a response can be measured by determining the level of STAT (e.g., STAT3, STAT4) phosphorylation, or IFNy production, in accordance with methods well known in the art.
  • STAT e.g., STAT3, STAT4
  • the term “exhibits significantly reduced binding” is used with respect to a variant of a first molecule (e.g., a ligand) which exhibits a significant reduction in the affinity for a second molecule (e.g., receptor) relative to the parent form of the first molecule.
  • a first molecule e.g., a ligand
  • a second molecule e.g., receptor
  • a the mutein “exhibits significantly reduced binding” if the mutein binds to a receptor with an affinity of less than 50%, alternatively less than about 40%, alternatively less than about 30%, alternatively less than about 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the parent ligand from which the murein was derived.
  • binding pairs e.g., ligand/receptor
  • a first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the first molecule of the binding pair does not bind in a significant amount to other components present in the sample.
  • a first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the affinity of the first molecule for the second molecule is at least two-fold greater, alternatively at least five times greater, alternatively at least ten times greater, alternatively at least 20-times greater, or alternatively at least 100-times greater, or alternatively at least 500-times greater or alternatively at least 1000-times greater than the affinity of the first molecule for other components present in the sample.
  • Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on- bead ligand binding assays, on-column ligand binding assays, and filter assays)) and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 with commercially available instrumentation such as the Biacore 8K, Biacore 8K+, Bia
  • Subject The terms “recipient”, “individual”, “subject”, and “patient”, are used interchangeably herein and refer to any mammal for whom, the opinion of a skilled artisan (e.g. physicician or veterinarian), treatment is desired.
  • mammal any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.
  • subject refers to a human being.
  • substantially pure indicates that a component of a composition makes up greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition.
  • a protein that is “substantially pure” comprises greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition comprising the protein.
  • the term “suffering from” refers to a determination made by a physician with respect to a subject based on the available objective or subjective information accepted in the field for the identification of a disease, disorder or condition including but not limited to X-ray, PET scans, CT-scans, conventional laboratory diagnostic tests (e.g., blood count, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or will benefit from treatment.
  • the term suffering from is typically used in conjunction with a particular disease state such as “suffering from a neoplastic disease” refers to a subject which has been diagnosed with the presence of a neoplasm.
  • T-cell As used herein the term “T-cell” or “T cell” is used in its conventional sense to refer to a lymphocytes that differentiates in the thymus, possess specific cell-surface antigen receptors, and include some that control the initiation or suppression of cell-mediated and humoral immunity and others that lyse antigen-bearing cells.
  • the T cell includes without limitation naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ T cells, helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TRI, Tregs, inducible Tregs; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs) and engineered variants of such T-cells including but not limited to CAR-T cells, recombinantly modified TILs and TCR-engineered cells.
  • the T cell is a T cell expressing the IL 12 receptor referred to interchangeably as IL12R cell, IL12R+ cell, IL12R T cell, or IL12R+ T cell.
  • Terminus/Terminal As used herein in the context of the structure of a polypeptide, “N- terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C- terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N- terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C- terminus, respectively.
  • N-terminal refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the N-terminus of the polypeptide.
  • immediately C-terminal refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the C-terminus of the polypeptide.
  • the “5 ’-terminus” (or “five-prime terminus”) and “3’- terminus” (or “carboxyl terminus”) refer to the extreme ends of the nucleic acid sequence, respectively, while the terms “5”’ and “3”’ refer to relative positions in the nucleic acid sequence of the polypeptide toward the 5’-terminus and the 3’-terminus, respectively, and can include the residues at the 5’-terminus and 3’-terminus, respectively.
  • Therapeutically Effective Amount refers to the quantity of an agent when administered to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a treatment regimen comprises multiple doses, provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition.
  • a therapeutically effective amount can be ascertained by measuring one or more relevant physiological effects, and it may be adjusted in connection with a dosing regimen and in response to diagnostic analy sis of the subject’s condition.
  • the parameters for evaluation to determine a therapeutically effective amount of an agent are determined by the physician using art accepted diagnostic criteria including but not limited to indicia such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computerized tomography, X-ray, and the like.
  • a therapeutically effective amount of an agent may be monitored to determine if a therapeutically effective amount of an agent has been administered to the subject such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of the disease, disorder or condition, biomarkers (such as inflammatory cytokines, IFN-y, granzyme, and the like), reduction in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), improvement in Immune-Related Response Criteria (irRC), increase in duration of survival, extended duration of progression free survival, extension of the time to progression, increased time to treatment failure, extended duration of event free survival, extension of time to next treatment, improvement objective response rate, improvement in the duration of response, reduction of tumor burden, complete response, partial response, stable disease, and the like that that are relied upon by clinicians in the field for the assessment of an improvement in the condition of the subject in response to administration of an agent.
  • biomarkers such as inflammatory cytokines,
  • a therapeutically effective amount is an amount of an agent when used alone or in combination with another agent provides an provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition and does not result in non-reversible serious adverse events in the course of administration of the agent to the mammalian subject.
  • Treat: The terms “treat”, “treating”, treatment” and the like refer to a course of action (such as administering to the subject a pharmaceutical composition comprising a hIL-12M in combination with a pharmaceutical composition comprising a hIL2M, optionally in combination with one or more supplementary agents) that is initiated with respect to a subject in response to a diagnosis that the subject is suffering from a disease, disorder or condition, or a symptom thereof, the course of action being initiated so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of: (a) the underlying causes of such disease, disorder, or condition afflicting a subject; and/or (b) at least one of the symptoms associated with such disease, disorder, or condition.
  • a course of action such as administering to the subject a pharmaceutical composition comprising a hIL-12M in combination with a pharmaceutical composition comprising a hIL2M, optionally in combination with one or more supplementary agents
  • treating includes a course of action taken with respect to a subject suffering from a disease where the course of action results in the inhibition (e.g., arrests the development of the disease, disorder, or condition), prevents the recurrence of disease or ameliorates one or more symptoms associated with the presence of the disease in the subject.
  • variant The terms “variant”, “protein variant” or “variant protein” or “variant polypeptide” are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide by virtue of at least one amino acid modification, substitution, or deletion.
  • the parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide or may be a modified version of a WT polypeptide.
  • WT wild-type
  • the variant polypeptide comprises from about one to about ten, alternatively about one to about eight, alternatively about one to about seven, alternatively about one to about five, alternatively about one to about four, alternatively from about one to about three alternatively from one to two amino acid modifications, substitutions, or deletions, or alternatively a single amino acid amino acid modification, substitution, or deletion compared to the parent polypeptide.
  • a variant may be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which the variant is derived.
  • Wild Type By "wild type” or “WT” or “native” when used in reference to a polypeptide or nucleic acid sequence herein is meant to refer to a polypeptide having amino acid sequence or a nucleotide sequence, respectively, that is found in nature, including allelic variations.
  • a wild- type protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been modified by the hand of man.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
  • hIL12M hIL12M
  • hIL 12P35 P35 subunit
  • hIL12P40M P40M subunit
  • the HL12P35 has at least 95% sequence identity to mature wild type human ML12P35 (SEQ ID NO:2);
  • the hIL12P40M has at least 95% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the hIL12P40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12P40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12P40; and
  • hIL2M an hIL2 mutein
  • the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (hIL2, SEQ ID NO: 182) that result in reduced binding affinity of the hIL2M to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182).
  • Wild type human IL 12 (wt hIL12) is a covalently disulfide linked heterodimeric protein comprising two wild type subunits, hP40 and hIL12P35.
  • the naturally occurring form of hIL12 comprises an interchain disulfide linkage between residue C96 of P35 (numbered in accordance with SEQ ID NO:1) and residue C199 of P40 (numbered in accordance with SEQ ID NO:3).
  • the wild type human P35 monomer (hIL12P35) is expressed as a 219 amino acid pro- protein (SEQ ID NO: 1) comprising a 22 amino acid signal sequence which is post-translationally removed to render a 197 amino acid mature protein (SEQ ID NO:2).
  • Wild type hIL12P35 (wt HL12P35) contains two intrachain disulfide linkages, the first between residues C64 and C196 and the second between residues C85 and C123 (numbered in accordance with SEQ ID NO: 1).
  • the canonical amino acid sequence of the human pro- hIL12P35 protein (UniProt Reference No. P29459) with the signal sequence (underlined) is:
  • the mature form of the wild-type human P35 (hIL12P35) less the 22 amino acid signal sequence is expressed as a 197 amino acid mature protein having the amino acid sequence:
  • Wild type human P40 (hIL12P40) is expressed as a 328 amino acid pro-protein (SEQ ID NO:3) comprising a22 amino acid signal sequence which is post-translationally removed to render a 306 amino acid mature protein (SEQ ID NO: 4).
  • hIL12P40 contains four intrachain disulfides between residues C50 and C90, C131 and C142, C170 and C193, and C300 and C327 (numbered in accordance with SEQ ID NO:3).
  • the canonical amino acid sequence of the hIL12P40 pro- protein (UniProt Reference No. P29460) with the signal sequence (underlined) is:
  • hIL12P40 The mature form of the wild-type human hIL12P40 less the 22 amino acid signal sequence (hIL12P40) is expressed as a 306 amino acid mature protein (SEQ ID NO:4)
  • the IL12 receptor comprises the IL12R(31 and IL12R02 subunits.
  • IL12 receptor activation results from the binding of IL 12 cytokine ligand to both IL 12RJ31 and IL12R02.
  • the binding of the IL 12 cytokine ligand to the IL 12 receptor complex activates the Janus tyrosine kinases, Tyk2 and Jak2, associated with IL12R ⁇ 1 and IL12R02, respectively, to phosphorylate the cytoplasmic tails of the receptors. This results in the recruitment of signal transducer and activator of transcription 4 (STAT4).
  • STAT4 signal transducer and activator of transcription 4
  • the hIL12M comprises: (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) e HL12P40M subunit of the hIL12M having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the hIL12P40M subunit further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL 12.
  • the hIL12M comprises: (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) e HL12P40M subunit of the hIL12M having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the HL12P40M subunit further comprising one or more amino acid substitutions that reduce the binding affinity of the HL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40.
  • the IL12p40M and IL12P35 are brought into functional, non- cov al ent association wherein the IL12p40M and IL12P35 are covalently attached to a first and second additional molecule (e.g., a polypeptide) wherein the first and second additional molecules provide stable non-covalent association.
  • a first and second additional molecule e.g., a polypeptide
  • Polypeptide domains which exhibit stable non-covalent formation of dimeric polypeptide pairs are well known in the art (e.g.
  • the hIL12M comprises a IL12p40M subunit (hIL12P40M) and IL12P35 subunit (IL12P35) wherein the ML12P40M is covalently linked the ML12P35 via at least one disulfide bond alone or in addition the presence of polypeptide domains that facilitate the non-covalent association of the IL12p40M and IL12P35.
  • the hIL12M comprises (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising one or more amino acid substitutions at a position corresponding to an amino acid residue selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 numbered in accordance with the human IL12p40 precursor (SEQ ID NO: 3), wherein the HL
  • the hIL12M comprises (a) an HL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising one or more amino acid substitutions at a position corresponding to an amino acid residue selected from the group consisting of P39, D40, E81, F82, KI 06, K217, and K219 of numbered in accordance with SEQ ID NO: 3.
  • the one or more amino acid substitutions at residues W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 is independently selected from the group consisting of an alanine (A) substitution, an arginine (R) substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a lysine substitution, a glutamine (Q) substitution, a glutamic acid (E) substitution, a serine (S) substitution, and a threonine (T) substitution, and combinations of any thereof.
  • A alanine
  • R arginine
  • N asparagine
  • D aspartic acid
  • L leucine
  • K lysine
  • F phenylalanine
  • T threonine
  • the hIL12M comprises (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising an amino acid substitution(s) selected from the group consisting of (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), trypto
  • the IL12M comprises an IL12p40M subunit (IL12p40M), the IL12p40M having an amino acid having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human hIL12P40 (SEQ ID NO: 4) , the IL12p40M further comprising an amino acid substitution(s) selected from the group consisting of (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), tryptophan (W), and tyrosine (Y (e) F82 is selected from the group consisting of alanine (A), arginine (R), asparagine (N),
  • the IL12p40M comprises the amino acid substitutions of E81A/F82A/K106A (numbered in accordance with SEQ ID NO:3). In one embodiment of the disclosure, the IL12p40M comprises an amino acid sequence:
  • the IL12p40M comprising one or more amino acid substitutions selected from the group consisting of the amino acid substitutions: (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), tryptophan (W), and tyrosine (Y (e) F82 is selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), histidine (H), lysine (K), phenylalanine (F) proline (P), tryptophan (W), and tyrosine (Y) (f) K106A, (g) D109A, (h) K217A,
  • P39A/D40A/E81A/F82A upon association with hIL12P35, forms a dimer (hIL12M) that (i) induces IL-12 signaling in CD8+ T cells; and (ii) has decreased IL-12 signaling in NK cells compared to an hIL12 molecule comprising a wildtype hIL-12p40 polypeptide.
  • the hIL12Ms described herein provide cell-type biased signaling of the downstream signal transduction mediated through the IL 12 receptor compared to a reference polypeptide (e.g., wild type hIL12).
  • a reference polypeptide e.g., wild type hIL12
  • the reduced binding affinity the hP40 mutein of the hIL12M to I L 12RJ31 results in a reduction in STAT4-mediated signaling compared to a reference polypeptide (wt hIL12).
  • heterodimeric hIL12M-Fc muteins of the present disclosure are partial agonists.
  • the heterodimeric hIL12M- Fc muteins described herein are partial agonists of STAT3 -mediated signaling (“STAT3 signaling”) and/or STAT4 mediated signaling (“STAT4 signaling”).
  • STAT3 signaling partial agonists of STAT3 -mediated signaling
  • STAT4 signaling STAT4 signaling
  • the hIL12M has reduced STAT3-mediated signaling compared to a reference polypeptide (wthIL12).
  • the STAT3 signaling and/or STAT4 signaling is determined by an assay selected from the group consisting of by a gene expression assay, a phospho-flow signaling assay, and an enzyme-linked immunosorbent assay (ELISA).
  • a hIL12M comprising the hP40 mutems described herein provide selective activation of certain cell types which provides beneficial properties, such as anti-inflammatory properties, and/or have reduced undesirable properties, such as pro-inflammatory side effects compared to wt hIL12.
  • the heterodimeric hIL12M-Fc muteins comprising the hP40 muteins described herein provide cell-type biased signaling of the downstream signal transduction mediated through the IL 12 receptor compared to a reference polypeptide (e.g., wild type hIL12).
  • an hIL12M of the present disclosure retain the property of wild-type hIL12 to stimulate or activate IL12 signaling in CD8+ T cells but exhibit a reduction of IFNy and/or STAT4- mediated signaling in natural killer (NK) cells.
  • the cell-type biased signaling of the hIL12M comprising the hP40 muteins described herein of the present disclosure includes the ability to provide substantial IL12 signaling (e.g., at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%) of the activity of wt hIL12 in CD8+ T cells.
  • the heterodimeric hIL12M-Fc mutems described herein exhibit increased STAT4 signaling in CD8+T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater and decrease STAT4 signaling in NK cells, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, as compared to a reference polypeptide (wt hIL12).
  • the heterodimeric hIL12M-Fc muteins described herein activate interferon gamma (IFNy) in CD8+ T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% and decreased IFNy signaling in NKT cells by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to a reference polypeptide (wt hIL12).
  • IFNy interferon gamma
  • an hIL12M comprising the hP40 muteins described herein exhibit reduced activation of NK cells while retaining the ability to stimulate CD8+ T cells.
  • the hIL12M is a hIL12M-Fc. In some embodiments the hIL12M-
  • Fc is a heterodimeric hIL12M-Fc.
  • the present disclosure provides heterodimeric hIL12M-Fc muteins comprising P40 muteins which have improved pharmacological or therapeutic properties, and methods of using such compositions.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula #1: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein: a) ML12P35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% or alternatively 100% sequence identity to SEQ ID NO:2; b) hIL12P40M is an human P40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40,
  • Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fcl, wherein the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.
  • the polypeptide of formula [1] is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 or any hIL12P40M-Fc sequence in the informal sequence listing.
  • the polypeptide of formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124 or any hIL12P35-Fc sequence in the informal sequence listing.
  • LI and L2 are independently selected from the group consisting SEQ ID NOS: 27-79. In some embodiments, LI and L2 are independently selected from the group consisting SEQ ID NOS: 36, 37 and 65.
  • UH1 and UH2 are selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a first poly peptide selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 and a second polypeptide selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 80 and a polypeptide of SEQ ID NO: 81.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 121 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 83 and a polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 141 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 144 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 129 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 147 and a polypeptide of SEQ ID NO: 82. [0195] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 150 and a polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 153 and a polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 135 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 138 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a a polypeptide of SEQ ID NO: 80 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 81.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a a polypeptide of SEQ ID NO: 121 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 83 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 141 and a polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 144 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 147 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 150 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 153 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 135 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
  • the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 138 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
  • the hIL12M muteins of the present disclosure comprise a modified human P40 polypeptide or “hP40 mutein” (also abbreviated “P40M” or “hIL12P40M”) comprising one or more amino acid substitutions, modifications and/or deletions at the interface with the extracellular domain of IL 12R
  • the binding affinity of the hP40 mutein for the extracellular domain of hIL12R ⁇ 1 is reduced by about 10%, alternatively by about 20%, alternatively by about 30%, alternatively by about 40%, alternatively by about 50%, alternatively by about 60%, alternatively by about 60%, alternatively by about 70%, alternatively by about 80%, alternatively by about 900%, alternatively to about 100% compared to binding affinity of a reference polypeptide (wt hP40) as determined by surface plasmon resonance (SPR) spectroscopy.
  • SPR surface plasmon resonance
  • the hIL12P40M is a modified wild type human hp40 polypeptide having at least 70% sequence identity to SEQ ID NO:4 (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4).
  • the hIL12P40M comprises one or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3.
  • the hIL12P40M comprises one or more amino acid substitutions at residues selected from the group consisting of E81, F82, KI 06, and K217 numbered in accordance with SEQ ID NO:3.
  • the one or more amino acid substitutions at positions W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 are selected from the group consisting of P39A, D40A, E81A, F82A, K106A, D109A, K217A, K219A.
  • the hP40 mutein comprises two or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3.
  • the hIL12P40M comprises two or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217 numbered in accordance with SEQ ID NO:3.
  • the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3.
  • the two or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: E81A/F82A, E81K/F82A, E81L/F82A, E81H/F82A and E81S/F82A.
  • the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of E81, F82, KI 06, and K217 numbered in accordance with SEQ ID NO:3.
  • the hIL12P40M comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3.
  • the hP40 mutein comprises three or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219
  • the three or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: W37A/E81A/F82A; E81A/F82A/K106A; E81A/F82A/K106A/K219A, E81A/F82A/K106N, E81A/F82A/K106Q, E81A/F82A/K106T, and E81A/F82A/K106R.
  • the hP40 mutein comprises four or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3.
  • the hP40 mutein comprises four or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219
  • the four or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: E81A/F82A/K106A/K217A, 81A/F82A/K106A/E108A/D115A and
  • the hIL12P40M comprises the set of amino acid substitutions E81A/F82A and is referred to herein as “2xAla” (SEQ ID NO:6).
  • the HL12P40M comprises the set of amino acid substitutions E81A/F82A/K106A and is referred to herein as “3xAla” (SEQ ID NO:8).
  • the hIL12P40M comprises the set of amino acid substitutions E81A/F82A/K106A/K217A and is referred to herein as “4xAla” (SEQ ID NO: 10).
  • the binding affinity of heterodimeric hIL12M-Fc muteins of the present disclosure comprising one or more, optionally two or more, optionally three or more, or optionally 4 or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 (numbered in accordance with SEQ ID NO:3) for the extracellular domain (ECD) of IL12R ⁇ 1 is reduced by at least 5%, optionally by at least 10%, optionally by at least 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, optionally by at least 70%, relative to the binding affinity of wild ty pe hP40 (SEQ ID NO:4) for the extracellular domain (ECD) of IL12R. ⁇ 1 as determined by surface plasmon resonance.
  • SEQ ID NO:4 wild ty pe h
  • Fc domain fusions incorporating a P40 mutein and/or P35 may optionally contain a GSA linker molecule between the P40 mutein and the upper hinge.
  • GSA linker refers to a polypeptide having 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19 or 20 amino acids comprised of amino acids selected from the group consisting of glycine, serine and alanine.
  • the polypeptide linker is a glycine-serine polymer of the structure (GGGGSm)n (SEQ ID NO: 192), (GGGS m )n (SEQ ID NO: 193), (GGGA m )n (SEQ ID NO: 194) and (GGGGA m ) n (SEQ ID NO: 195), and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4.
  • Exemplary glycine-serine linkers include but are not limited to the monomers: GGGGS (referred to as “G4S”) (SEQ ID NO: 65), GGGGA (referred to as “G4A”) (SEQ ID NO: 53), GGGS (referred to as “G3S”) (SEQ ID NO: 50) and GGGA (referred to as “G3A”) (SEQ ID NO: 43), or homopolymers (e.g. “GGGGSGGGGS” also referred to as (G4S)? (SEQ ID NO: 36)) or heteropolymers thereof.
  • Exemplary' GSA linkers are provided in Table 2 below:
  • the heterodimeric hIL12M-Fc muteins of the present disclosure are heterodimers comprising polypeptides of the formulae [1] and [2], which each incorporate an upper hinge region of a human immunoglobulin molecule.
  • the term “upper hinge” or “UH” refers to an amino acid sequence corresponding to amino acid residues 216-220 (EU numbering) of a human immunoglobulin molecule.
  • the upper hinge region is a naturally occurring upper hinge region of a human immunoglobulin selected from the LH regions of human IgGl, human IgG2, human IgG3 and human IgG4 upper hinge domains.
  • the upper hinge region is the upper hinge region of a human IgGl immunoglobulin. In some embodiments, the upper hinge region is the upper hinge region of a human IgGl immunoglobulin comprising the pentameric amino acid sequence: EPKSC (SEQ ID NO: 11).
  • the upper hinge region contains an unpaired cysteine residue at position 220 (EU numbering) that typically, in a complete immunoglobulin molecule, binds to a cysteine on a light chain.
  • EU numbering typically, in a complete immunoglobulin molecule, binds to a cysteine on a light chain.
  • the unpaired cysteine in the hinge domain creates the potential of the formation of improper disulfide bonds. Consequently, in some embodiments the cysteine at position 220 (C220, numbered in accordance with EU numbering) is substituted with an amino acid that does not promote disulfide bonding.
  • the Fc domain comprises a C220S mutation having the amino acid sequence EPKSS (SEQ ID NO: 12).
  • heterodimeric hIL12M-Fc muteins of the present disclosure are heterodimers comprising polypeptides of the formulae [1] and [2], which each incorporate an Fc region (Fcl and Fc2) of a human immunoglobulin molecule modified to promote heterodimerization.
  • Fc and “Fc monomer” are used interchangeably herein to designate the monomeric polypeptide subunit of an Fc dimer.
  • An Fc comprises an amino acid sequence (from amino to carboxy terminal) comprising a lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule.
  • the Fc monomer is a polypeptide comprising the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule domains of human IgGl, human IgG2, human IgG3 and human IgG4 hinge domains.
  • the CH2 domain of hlgGl corresponds to amino acid residues 231-340 (EU numbering) and is provided as SEQ ID NO: 14.
  • the CH3 domain of hlgGl corresponds to amino acid residues 341-447(EU numbering) and is provided as SEQ ID NO: 15.
  • polypeptides of the formulae [1] and [2] each incorporate a lower hinge region of a human immunoglobulin.
  • the term “lower hinge” or “LH” refers to an amino acid sequence corresponding to amino acid residues 221-229 (EU numbering) of a human immunoglobulin molecule.
  • the lower hinge region is a naturally occurring lower hinge region of a human immunoglobulin selected from the LH regions of IgGl , IgG2, IgG3 and IgG4 lower hinge domains.
  • the lower hinge region is the lower hinge region of a human IgGl immunoglobulin.
  • the lower hinge region is the lower hinge region of a human IgGl immunoglobulin comprising the decameric amino acid sequence: DKTHTCPPCP (SEQ ID NO: 13).
  • Fcl and Fc2 are derived from a polypeptide corresponding to amino acids 221-447 (EU numbering) of the human IgGl immunoglobulin having the amino acid sequence (EU numbering indicated, SEQ ID NO: 16):
  • NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID NO : 16
  • the C-terminal residue of the wild-type form of the IgGl Fc domain is a lysine, referred to as K447 in accordance with EU numbering.
  • the K447 is inconsistently removed by the producer cell during recombinant product.
  • the population of recombinant Fc monomers may be heterogenous in that some fraction of the recombinantly produced Fc monomers will contain K447 and others will not.
  • Such inconsistent proteolytic processing by producer cells may therefore result in a heterogenous population of hIL12M-Fc .
  • such heterogeneity of the active pharmaceutical ingredient is to be avoided.
  • the present disclosure provides Fc monomers that further comprising a deletion of the C-terminal K447 or a deletion of G446 and K447 and nucleic acid sequences encoding Fc monomers comprising a: (a) a deletion of the lysine residue at position 447 (K447,EU numbering, abbreviated as AK447 or des-K447), or (b) deletion of both the glycine at position 456 (G446 EU numbering, abbreviated as des-G446) and K447 (this double deletion of G446 and K447 being referred to herein as des-G446/des-K447 or AG446/AK447).
  • the modifications to the Fc domains of the heterodimeric hIL12M- Fc mutein to promote heterodimerization are complementary “knob-into-hole” mutations.
  • the modifications of the Fc domains to promote heterodimerization of the hIL12P35 and hIL12P40M-Fc domains comprises the amino acid substitution T366W (“knob”) in the first domain and the amino acid substitutions T366S/L368A/Y407V (“hole”) in the second domain.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein wherein the hIL12P35-Fc and hIL12P40M-Fc polypeptides of the heterodimeric hIL12M- Fc mutein are covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds.
  • the ML12P35 and hIL12P40M-Fc are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C96 of the hP35 domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C199 of the hP40M domain of the hIL12P40M-Fc.
  • the hIL12P35 and hIL12P40M are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C226 of the lower hinge domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C226 of the lower hinge domain of the hIL12P40M-Fc.
  • the hIL12P35-Fc and hIL12P40M are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C229 of the lower hinge domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C229 of the lower hinge domain of the hIL12P40M-Fc.
  • a first Fc domain comprises the amino acid substitution S354C
  • the second Fc domain comprises the amino acid substitution Y349C.
  • the heterodimeric hIL12M-Fc mutein comprises a first Fc domain comprising the amino acid substitution S354C and the second Fc domain comprising the amino acid substitution Y349C and wherein the hIL12P35-Fc and hIL12P40M domains are linked via a disulfide bond between the S354C of the first Fc domain and Y349C of the second Fc domain.
  • the hIL12P35-Fc andhIL12P40M-Fc of the heterodimeric hIL12M-Fc mutein are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds, optionally four or more disulfide bonds between the side chains of the following cysteine residue pairs: (a) C96 of the hP35 and Cl 99 of the hP40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution.
  • the heterodimeric IL12Fc muteins of the present disclosure comprise an amino acid substitution of the lysine (K) residue at position 260 (K260) of the mature form of the human P40 polypeptide (SEQ ID NO: 4 corresponding to position 282 of the human P40 precursor polypeptide SEQ ID NO: 3).
  • K lysine
  • SEQ ID NO: 4 corresponding to position 282 of the human P40 precursor polypeptide SEQ ID NO: 3
  • the human P40 polypeptide of the heterodimeric IL12Fc muteins of the present disclosure comprise a substitution of the lysine at position K282 (numbered in accordance with SEQ ID NO:3) polypeptide with a non-basic amino acid.
  • the non-basic amino acid is selected from the group consisting of alanine, glycine, asparagine or glutamine.
  • the P40 polypeptide of the heterodimeric IL12Fc muteins of the present disclosure comprise a mutation at position K282 (numbered in accordance with SEQ ID NO: 3) selected from the group consisting of K282G, K282A, K282N, K282Q (numbered in accordance with SEQ ID NO:3.
  • the heterodimeric IL12Fc muteins of the present disclosure comprise the human P40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A/F82A/K106A/K282G, E81A/F82A/K106A/K282A,
  • the heterodimeric IL12Fc muteins of the present disclosure comprise the human P40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A/F82A/K282G, E81A/F82A/K282A, E81A/F82A/K282N, and E81 A/F82A/K282Q (numbered in accordance with SEQ ID NO:3).
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 156 ) .
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 157 ) .
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 158 ) .
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
  • the Fcl and Fc2 monomers of the dimeric Fc contain amino acid substitutions that promote heterodimerization between Fcl and Fc2.
  • a variety of techniques are established for the promotion of heterodimerization of Fc domains. See, e.g. Kim, et al., United States Patent No. 11,087,249, issued August 3, 2021.
  • the modifications to promoter heterodimerization of the Fcl and Fc2 monomers are the HF-TA mutations and the HA-TF mutations as described in Moore, et al (2011) mAbs 3(6):546-557.
  • the HF-TA method employs the S364H/T394F substitutions on one Fc monomer and the Y349T/F405A substitutions on the complementary Fc monomer.
  • the (HA-TF) method employs the S364H/F405A substitutions on one Fc monomer and the Y349T/T394F substitutions on the complementary Fc monomer.
  • the Fcl and Fc2 monomers are modified to promote heterodimerization by the ZW1 heterodimerization method which employs the T350V/L35 IY/F405A/Y407V substitutions on one Fc monomer and the T350V/T366L/K392L/T394W substitutions on the complementary Fc monomer.
  • the Fcl and Fc2 monomers are modified to promote heterodimerization by the EW-RVT heterodimerization method which employs the K360E/K409W substitutions on one Fc monomer and the Q347R/D399V/F405T substitutions on the complementary Fc monomer. Choi , et al (2015) Molecular Immunology 65(2):377-83.
  • Fcl and Fc2 are modified to promote heterodimerization by the employment of the “knob-into-hole” (abbreviated KiH) modification as exemplified herein.
  • KiH modification comprises one or more amino acid substitutions in a first Fc monomer (e.g. Fcl) that create a bulky “knob” domain on a first Fc and one or more amino acid substitutions on a second Fc monomer (e.g. Fc2) that create a complementary pocket or “hole” to receive the “knob” of the first Fc monomer.
  • the Fc domain comprises two Fc monomers wherein the CH3 domain of a first Fc monomer wherein the threonine at (EU numbering) position 366 is modified with a bulky residue (e.g. a T366W) create a “knob” and the substitution, and a second Fc monomer comprising one or more substitutions in complementary residues of the CH3 domain of the second Fc monomer to create a pocket or “hole” to receive the bulky residue, for example by amino acid substitutions such as T366S, L368A, and/or Y407V.
  • a bulky residue e.g. a T366W
  • a second Fc monomer comprising one or more substitutions in complementary residues of the CH3 domain of the second Fc monomer to create a pocket or “hole” to receive the bulky residue, for example by amino acid substitutions such as T366S, L368A, and/or Y407V.
  • the Fcl monomer of formula 1 is a “knob” modified Fc monomer comprising the amino acid substitution T366W and the Fc2 monomer of formula 2 is a “hole” modified Fc comprising the set of amino acid substitutions T366S/L368A/Y407V.
  • the Fcl monomer of formula 1 is a “hole” modified Fc monomer comprising the set of amino acid substitutions T366S/L368A/Y407V and the Fc2 monomer of formula 2 is a “knob” modified Fc monomer comprising the amino acid substitution T366W.
  • heterodimeric hIL12M-Fc muteins of the present disclosure are provided as a complementary heterodimeric pair of polypeptides of the formulae [1] and [2] wherein the first and second polypeptide are linked by at least one disulfide bond.
  • the incorporation of a disulfide bond between the polypeptides of formulae [1] and [2] may be achieved by cysteine substitutions at particular points within the Fcl and Fc2 domains.
  • the Fcl domain of the polypeptide of formula [1] is derived from the Fc domain of hlgGl comprising an amino acid substitution S354C (EU numbering) and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hlgGl comprising an amino acid substitution Y349C (EU numbering) to provide a disulfide bond between the S354C of Fcl and Y349C of Fc2.
  • the Fcl domain of the polypeptide of formula [1] is derived from the Fc domain of hlgGl comprising an amino acid substitution Y349C (EU numbering) and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hlgGl comprising an amino acid substitution S354C (EU numbering) to provide a disulfide bond between the S354C of Fcl and Y349C of Fc2.
  • the hIE12P40M-Fc and hIL12P40M of the heterodimeric hIL12M-Fc mutein are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds , optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) C96 of the hIL12P35 and C199 of the hIL12P40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution.
  • Further examples of complementary KiH engineered heterodimeric Fc pairs that may be used in the practice of the present
  • the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to reduce effector function.
  • the Fc domain may be modified to substantially reduce binding to Fc receptors (FcyR and FcR) which reduces or abolishes antibody directed cytotoxicity (ADCC) effector function. Modification of Fc domains to reduce effector function are well known in the art. See, e.g., Wang, et al. (2018J IgGFc engineering to modulate antibody effector functions, Protein Cell 9(l):63-73.
  • the Fc domains (Fcl and Fc2) of the compositions of the present invention may comprises the amino acid substitutions L234A/L235A/P329A (EU numbering) referred to as the “LALAPA” substitutions or L234A/L235A/P329G (EU numbering) referred to as the “LALAPG” substitutions.
  • the Fc domains (Fcl and Fc2) of the compositions of the present disclosure may comprises the amino acid substitutions E233P/L234V/L235A/AG237 (referred to in the scientific literature as the PVAdelG mutation).
  • the Fc domains (Fcl and Fc2) of the compositions of the present disclosure are from hIgG4.
  • the Fc domains of the heterodimeric IL12muteins are derived from hIgG4
  • attenuation of effector function may be achieve by introduction of the S228P and/or the L235E mutations (EU numbering).
  • the IgG4 Fc incorporates an amino acid substitution at postion 297 (e.g. N297G).
  • N297G amino acid substitution at postion 297
  • the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to incorporate amino acid substitutions which extend the duration of action of the molecule and prevent clearance.
  • modifications to the Fc monomer include the amino acid substitutions M428L and N434S (EU numbering) referred to as the “LS” modification.
  • the LS modification may optionally be combined with amino acid substitutions to reduce effector function and provide for disulfide bonds between Fcl and Fc2.
  • Table 6 below provides exemplary Fcl and Fcl heterodimeric pairs possessing complementary sequence modifications to promote heterodimerization that may be employed in the design of the Fcl and Fc2 polypeptides of the formulae [1] and [2],
  • the Fc domains (Fcl and Fc2) of the compositions of the present disclosure are from hIgG4.
  • the Fc domains of the heterodimeric IL12muteins are derived from hIgG4, heterodimerization of the Fcl and Fc2 domains by the introduction of the mutations K.370E, K409W and E357N, D399V, F405T (EU numbering) in the complementary Fc sequences that comprise the heterodimeric Fc domain.
  • the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to eliminate N-linked or O-linked glycosylation sites.
  • Aglycosylated variants of Fc domains, particularly of the IgG4 subclass are known to be poor mediators of effector function.
  • Jefferies et al. 1998, Immol. Rev., vol. 163, 50- 76 It has been shown that glycosylation at position 297 (EU numbering) contributes to effector function of IgG4 Fc domains.
  • the Fc domains of the compositions of the present disclosure comprise one or modifications to eliminate N- or 0 linked glycosylation sites.
  • modifications at N297 to eliminate glycosylation sites in the IgG4 Fc domain include the amino acid substitutions N297Q and N297G.
  • the present disclosure provides heterodimeric hIL12M-Fc muteins, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]:
  • the hIL12M-Fc muteins of the present disclosure are the heterodimeric hIL12M-Fc muteins provided in Table 8 below.
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula #1:
  • polypeptide of formula [2] comprises the amino acid sequence:
  • the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]:
  • KSLSLSPG SEQ ID NO : 153
  • polypeptide of formula [2] comprises the amino acid sequence:
  • the hIL2 and/or hIL12M the present disclosure may be conjugated to one or more polyethylene glycol molecules or “PEGylated.”
  • PEGylated the method or site of PEG attachment to the binding molecule may vary, in certain embodiments the PEGylation does not alter, or only minimally alters, the activity of the binding molecule.
  • the present disclosure provides PEGylated heterodimeric hIL12M-Fc muteins.
  • both the hIL12P35-Fc and the hIL12P40M-Fc of the heterodimeric hIL12M- Fc mutein are PEGylated.
  • conjugation of the PEG moiety to the hIL2 and/or hIL12M may be accomplished via a sulfhydryl (-SH) group of a cysteine residue.
  • the PEGylation of the d heterodimeric hIL12M-Fc muteins is provided at one or both of the naturally occurring cysteine residues at position 220 (C220, EU Numbering) of the upper hinge region of the hIL12P35-Fc and/or the hIL12P40M-Fc heterodimeric hIL12M-Fc muteins.
  • PEGs suitable for conjugation to a polypeptide sequence of either or both of hIL12M and hIL2M molecules of the present disclosure are generally soluble in water at room temperature, and have the general formula
  • R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000.
  • R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure.
  • PEGylation of hIL2 and/or hIL12M may be facilitated by the incorporation of one or more non-natural amino acids having side chains to facilitate selective PEG conjugation.
  • Specific PEGylation sites can be chosen such that PEGylation of the binding molecule does not affect its binding to the target receptors.
  • PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure. [0269
  • a molecular weight of the PEG used in the present disclosure is not restricted to any particular range.
  • the PEG component of the binding molecule can have a molecular mass greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50kDa.
  • the molecular mass is from about 5kDa to about lOkDa, from about 5kDa to about 15kDa, from about 5kDa to about 20kDa, from about lOkDa to about 15kDa, from about lOkDa to about 20kDa, from about lOkDa to about 25kDa, or from about lOkDa to about 30kDa.
  • Linear or branched PEG molecules having molecular weights from about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons.
  • the PEG is a 40kD branched PEG comprising two 20 kD arms.
  • Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to resolve conjugate fractions, and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
  • PEGs suitable fbr conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000.
  • R is a protective group, it generally has from 1 to 8 carbons.
  • mPEGs Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15: 100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. US Patent No. 5,650,234), which react preferentially with the side chain of lysine residues to form a carbamate linkage but are also known to react with histidine and tyrosine residues.
  • SC-PEG succinimdyl carbonate PEG
  • BTC-PEG see, e.g., Dolence, et al. US Patent No. 5,650,234
  • PEG-aldehyde linker targets a single site on the N-terminus of a polypeptide through reductive amination.
  • Pegylation frequently occurs at the a-amino group at the N-terminus of the polypeptide, the epsilon amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant polypeptides possess a single alpha and a number of epsilon amino and imidazole groups, numerous positional isomers can be generated depending on the linker chemistry. General PEGylation strategies known in the art can be applied herein.
  • the PEG can be bound to a binding molecule of the present disclosure via a terminal reactive group (a “spacer”) which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol.
  • a terminal reactive group a “spacer” which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol.
  • the PEG having the spacer which can be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating succinic acid ester of polyethylene glycol with hi- lly droxysuccinylimide.
  • the PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure.
  • PEGs useful in the practice of the present disclosure include a lOkDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), lOkDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), a 20kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), a 20kDa linear PEG- NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-
  • a linker can used to join the PEG molecule to the heterodimeric hIL12M-Fc mutein .
  • Suitable linkers include “flexible linkers” which are generally of sufficient length to permit some movement between the modified polypeptide sequences and the linked components and molecules.
  • the linker molecules are generally about 6-50 atoms long.
  • the linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
  • Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids. Examples of flexible linkers are described in Section IV. Further, a multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or SO- SO) of these linker sequences may be linked together to provide flexible linkers that may be used to conjugate two molecules.
  • the linker is a GSA linker as described above.
  • the linker can be a chemical linker, e.g., a PEG-aldehyde linker.
  • the binding molecule is acetylated at the N-terminus by enzymatic reaction with N-terminal acetyltransferase and, for example, acetyl CoA.
  • the binding molecule can be acetylated at one or more lysine residues, e.g., by enzymatic reaction with a lysine acetyltransferase. See, for example Choudhary et al. (2009) Science 325 (5942):834 840.
  • the heterodimeric hIL12M-Fc muteins of the present disclosure provide cell-type biased signaling of the downstream signal transduction mediated through the IL12 receptor compared to a reference polypeptide (e.g., wild type hIL12).
  • a reference polypeptide e.g., wild type hIL12
  • the heterodimenc hIL12M-Fc muteins of the present disclosure retain significant hIL-12 signaling in CD8+ T cells and have decreased hIL-12 signaling in NK cells compared to a wildtype hIL-12.
  • the selective activation of CD8+ T cells versus NK cells can be evaluated by the activation of interferon gamma (IFNy) while providing a reduction in STAT4 signaling in NK cells.
  • IFNy interferon gamma
  • the heterodimeric hIL12M-Fc muteins of the present disclosure activate interferon gamma (IFNy) in CD8+ T cells and have decreased IFNy signaling in CD8+ T cells compared to the wildtype IL 12.
  • IFNy interferon gamma
  • the heterodimeric hIL12M-Fc muteins of the present disclosure activate IFNy in CD8+ T cells and have decreased IFNy signaling in CD8+ T cells compared to the wildtype IL12.
  • Reduced STAT4 Signaling in NK cells
  • the heterodimeric hIL12M-Fc muteins describe herein exhibit substantially full or increased STAT4 signaling in CD8+T cells and decreased STAT4 signaling in NK cells compared to a reference polypeptide (wt hIL12).
  • wt hIL12 a reference polypeptide
  • a series of experiments were performed to evaluate the effect of various IL12 muteins comprising hP40 muteins, both as proteins and as Fc fusions, of STAT4 signaling in CD8+ T cells, CD4+ T cells relative to STAT4 signaling in NK cells.
  • the evaluation of STAT4 was performed in substantial accordance with the teaching of Example 2.
  • test articles evaluated were wt hIL12 and hIL12 proteins comprising a P40 subunit with the substitutions E81A/F82A indicated as “2xAla”, E81A/F82A/K106A indicated as “3xAla” and KiH heterodimeric hIL12M-Fc constructs comprising wild type hIL12P35 and hP40 (wt Fc), E81A/F82A indicated as “2xAla Fc”, E81 A/F82A/K106A indicated as “3xAla Fc” on CD8, CD4 andNK cells, respectively.
  • the results of these experiments are provided in Figure 2 of the attached drawings.
  • the heterodimeric hIL12M-Fc muteins of the present disclosure provide differential STAT4 signaling on CD8+ T cells relative to NK cells. Additionally, the data provided in Figure 2, particularly Panel A and Panel B demonstrate that the heterodimeric hIL12M-Fc muteins of the present disclosure act as IL 12 partial agonists on T cells.
  • the heterodimeric hIL12M-Fc muteins described herein are useful in the treatment of neoplastic disease.
  • surrogate murine IL12Fc muteins containing analogous mutations to the human molecules were generated to evaluate the effects in anMC38 mouse tumor model.
  • a sequence alignment of the naturally occurring human and mouse P40 and P35 polypeptides are provided in Figures 7 and 8, respectively, of the attached drawings.
  • a description of the heterodimeric mIL12Fc test agents used in the MC38 tumor study are summarized in the Tables 9 and 10 below:
  • mice approximately IxlO 6 MC38 cells in Matrigel were implanted subcutaneously into 6-8 week old C57BL/6 mice and the tumors permitted to attain an average tumor volume at the initiation of treatment of approximately 100 mm 3 - 120mm 3 , The mice were separated into individual treatment groups. The mice were treated by intraperitoneal administration of the various test agents at the doses and dosing schedule indicated in the Table 11 below. In this study bodyweight (BW), an indication of toxicity, and tumor volume (TV) as an indicator of anti-tumor efficacy were measured twice per week.
  • BW bodyweight
  • TV tumor volume
  • Figure 3 provides a spider plot summary of the effect on tumor volume with respect to each animal in each study group. As demonstrated by the data presented, the murine surrogate of the heterodimeric IL12Fc mutein as described herein was effective in the control of tumor growth in this study.
  • FIG. 4 provides the average body weight of the animals during the course of the above study.
  • the wild type IL 12 Fc test agents evaluated in this study demonstrated an inhibition of tumor growth
  • the data presented in Figure 4 indicates that such wild type IL12 Fc fusions are associated with significant toxicity as indicated by a significant loss of bodyweight (see, e g. Figure 4, Panels B, C, and E).
  • the heterodimeric IL12Fc mutein evaluated in group H comprising the 2xAla mutations in the mP40 domain did not suggest significant toxicity. This is particularly noteworthy as the IL12Fc mutein comprising the 2xAla P40 mutations was administered at a dose at more than 50 fold higher than the other wild type IL12Fc conjugates evaluated.
  • the Fc conjugated heterodimeric mIL12-Fc molecules comprising the amino acid substitutions (i.e., "2x Ala” and “3xAla”) demonstrated a significant delay in the induction of interferon gamma in relation to the other treatment groups, in particular in relation to the IL 12 Fc comprising the wild type P40 sequence. This delay in the induction of IFNg results in a decrease in the acutue toxicity associated with IL 12 treatment.
  • T-bet is required for NK cell effector function and NK cell cytolyitic activity.
  • Figure 11 of the attached drawings with T-bet on the vertical axis and granzyme B on the horizontal axis.
  • FIG. 14 provides FACS analysis which indicates that wild type IL 12 Fc results in NK cell degranulation but that 3xAla IL 12 Fc does not substantially induce NK cell degranulation.
  • NK cells were depleted using an the NK 1.1 antibody 6-8 week old C57BL/6 treated with an NK cell depleting antibody (aNKl. l/IL12) followed by IL-12 administration.
  • PBS or aNKl.l was administered on days -3, 0, 3 and 7.
  • IL12 was administered on days 0, 4, and 8 (see Table 14).
  • NK depletion alleviates toxicity as evaluated by body weight loss and prevents mortality after 1 ,6ug IL-12WT Fc treatment. NK cell depletion it also substantially reduces mortality and morbidity in at the dose of 4.8ug. Additionally, NK cell depletion is transient and NK cells rebound at d6 after the last NK depletion dose.
  • a heterodimeric hIL12M-Fc compising a hIL12P40M-Fc polypeptide is efficacious in the treatment of cancers and possesses a substantially reduced toxicity relative to that observed with IL 12 agents comprising a wild-type P40 polypeptide.
  • B6 mice were used as a control group relative to the RAG2 knockoout (KO) mice which lack T and B cells and the RAG2/CD132 double knockout mice which lack T, B and NK cells. Briefly, the approximately IxlO 6 MC38 cells were implanted s.c.
  • mice were randomized into treatment groups as described in Table 16 below.
  • the test articles and contols were administered in accordance with the schedule describe in Table 16 below. Micd were evaluated for weight loss and tumor volume twice weekly.
  • the murine IL2 mutem was developed for in vivo studies in mice to correlate activity between the rodent (mouse) and primate (human) environments for human IL2 muteins comprising amino acid substitutions at positions 18, 22 and 126 numbered in accordance with mature wild type hIL2, in particular an hIL2 mutein comprising the amino acid substitutions L18R/Q22E/Q126K.
  • the amino acid sequence of the murine IL2 (mIL2) polypeptide used in this study is:
  • APTSSSTSSSTAEAQQQQQHLEQLRMDLEELLSRMENYRNLKLPRMLTF KFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRV TVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCHSIISTSPQ (SEQ ID NO: 181) and is N-terminally PEGylated with a 40kD branched chain PEG with a linker and is referred to as PEG-mREH.
  • the results of this study are presented in Figures 19 (PD1) and 20 (mIL2 mutein).
  • the combination of the heterodimeric Fc P40M mutein provides an enhanced antitumor effect in this model.
  • the combination of the heterodimeric Fc P40M mutein and the anti-PDl antibody desmonstrates a significantly enhanced effect leading to complete response.
  • the study design for MC38 Tumor Study #3 is provided in Table 21 and Example 7.
  • the data generated of MC38 Tumor Study #3 studies are presented in Figures 23 and 24 of the attached drawings.
  • the administration of Compound 3, a murine surrogate of an hIL2M mutein of the present disclosure and Compound 2, in combination with a murine surrogate of hIL12M mutein of the present disclosure generates superior anti-tumor to the administration of either agent alone without unacceptable toxicity.
  • heterodimeric wild type IL12 Fc and the anti-PDl antibody was observed to alleviate the toxicity previously observed with the heterodimeric wild type IL 12 Fc in this model.
  • the foregoing data demonstrates that the heterodimeric hIL2 IL12 muteins of the present disclosure are useful in the treatment of neoplastic disease in combination with supplementary therapeutic agents, particularly IL2, IL2 muteins and checkpoint inhibitors such as anti-PDl antibodies.
  • the hIL2M and hIL12M molecules of the present disclosure comprise polypeptides.
  • the hIL2M and hIL12M of the present disclosure comprise anon- peptidyl components such as a PEG molecule. The process for PEGylation of proteins is discussed elsewhere herein.
  • hIL2M and hIL12M of the present disclosure such including the hIL12P40M (or hIL12P40M-Fc) and hIL12P35 (or hIL12P35-Fc) polypeptide subunits as well as the recombinant production of the heterodimeric hIL12M-Fc muteins muteins of the present disclosure.
  • hIL2M and hIL12M polypeptide and/or ML12P40M (hIL12P40M-Fc) and hIL12P35 (hIL12P35-Fc) subunit domains muteins may be produced by conventional methodology for the construction of polypeptides including recombinant or solid phase syntheses as described in more detail below.
  • polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be prepared by chemical synthesis.
  • the chemical synthesis may proceed via liquid-phase or solid-phase.
  • Solid-phase peptide synthesis allows the incorporation of unnatural amino acids and/or peptide/protein backbone modification.
  • SPPS Solid-phase peptide synthesis
  • Various forms of SPPS are available for synthesizing polypeptide domains of hIL2M and hIL12M molecules of the present disclosure are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J.A. et al., (2005) Protein Pept Lett. 12:723-8).
  • the alpha functions and any reactive side chains may be protected with acid-labile or base-labile groups that are stable under the conditions for linking amide bonds but can readily be cleaved without impairing the peptide chain that has formed.
  • either the N-terminal or C-terminal amino acid may be coupled to a suitable support material.
  • suitable support materials are those which are inert towards the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and which do not dissolve in the reaction media being used.
  • Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers; and the like.
  • the successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.
  • the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups.
  • the peptide obtained can be purified by various chromatographic methods including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high pressure liquid chromatography (HPLC) and reversed-phase HPLC.
  • polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be produced by recombinant DNA technology'.
  • the techniques and materials for recombinant production of polypeptide in procaryotic and eucaryotic cells are well established and known to those of skill in the art.
  • a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will be accomplish, the nucleic acid sequence being operably linked to one or more expression control sequences encoding by the vector and functional in the target host cell.
  • the recombinant protein may be recovered through disruption of the host cell or from the cell medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide.
  • the recombinant protein may be purified and concentrated for further use including incorporation.
  • polypeptide domains of hIL2M and hIL12M molecules of the present disclosure are produced by recombinant methods using a nucleic acid sequence encoding the polypeptide domains of hIL2M and hIL12M (or fusion proteins thereof).
  • the nucleic acid sequence encoding the desired polypeptide domains of hIL2M and hIL12M molecules can be synthesized by chemical means using an oligonucleotide synthesizer by techniques well known in the art.
  • nucleic acid molecules encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may contain naturally occurring sequences or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide.
  • These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids.
  • the nucleic acid molecules can be double-stranded or single-stranded (i.e., either a sense or an antisense strand).
  • the nucleic acid sequence encoding the wild type human P40 signal peptide and the hIL12P40M polypeptide is selected from the group consisting of SEQ ID NOS: 94, 97, 99, 100, 102, 105, 118, 120, 126, 128, 131, 134, 137, 140, 143, 146, 149, and 152.
  • the nucleic acid sequence encoding the wild type human P35 signal peptide and the hIL12P40M polypeptide is selected from the group consisting of SEQ ID NOS:95, 96, 98, 103, 104, 106, and 123. [O3 IO
  • the nucleic acid sequence encoding polypeptide domains of hIL12M molecules of the present disclosure is modified to encode a signal peptide to facilitate secretion of the hIL12M.
  • the signal peptide may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector.
  • a heterologous signal peptide is employed, it is preferably a signal peptide that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal peptide is selected from the group consisting of human serum albumin signal peptide, prolactin albumin signal peptide, the human IL2 signal peptide, human trypsinogen-2, human CD-5, the human immunoglobulin kappa light chain, human azurocidin, Gaussia luciferase and functional derivatives thereof. Particular amino acid substitutions to increase secretion efficiency using signal peptides are described in Stem, et al.
  • the signal peptide may be a synthetic sequence prepared in accordance established principles. See e.g., Nielsen, et al. (1997) Protein Engineering 10(1 ): 1 -6 (Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites)' ,' Bendtsen, et al (2004) J. Mol.
  • the signal peptide the hIL12P40M and hIL12P35 polypeptide subunits of the heterodimeric hIL12M-Fc mutein is the naturally occurring hP40, hP19, and hIL12P35 signal peptide, respectively (i.e. the human hP40 and hIL12P35 signal sequences).
  • the signal peptide of the hIL12P35 sequence is the naturally occurring wild type human P35 sequence having the amino acid sequence MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 179).
  • the signal peptide of the HL12P40M sequence is the naturally occurring wild type human P40 sequence having the amino acid sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 180).
  • a signal peptide depends on whether it is desired to secrete the heterodimeric hIL12M-Fc mutein from the recombinant cells in which it is made. If the chosen host cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the alpha mating factor secretion signal peptide may be employed to achieve extracellular secretion as described in Singh, United States Patent No. 7,198,919 Bl.
  • transition metal chelating polypeptides useful in the practice of the present disclosure binding molecule are polypeptides comprising 3-6 contiguous histidine residues (SEQ ID NO: 197) such as a six-histidine (His)e peptide (SEQ ID NO: 198) and are frequently referred to in the art as “His-tags.”
  • a hemagglutinin tag may be incorporated into the chimeric protein to facilitate purification of protein expressed in eukaryotic cells.
  • first and second it should not be understood as limiting to the orientation of the elements of the fusion protein and a heterologous polypeptide can be linked at either the N-terminus and/or C-terminus of the polypeptide domains of heterodimeric hIL12M-Fc mutein .
  • the N-terminus may be linked to a targeting domain and the C-terminus linked to a hexa-histidine tag purification handle (SEQ ID NO: 198).
  • the nucleic acid sequence encoding polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be “codon optimized” to facilitate expression in a particular host cell type.
  • Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast and bacterial host cells, are well known in the and there are online tools to provide for a codon optimized sequences for expression in a variety of host cell types. See e.g., Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David hacker (Human Press New York). Additionally, there are a variety of web based on-line software packages that are freely available to assist in the preparation of codon optimized nucleic acid sequences.
  • nucleic acid sequences encoding polypeptide domains of hIL2M and hIL12M molecules of the present disclosure prepared may be operably linked to suitable genetic control elements that are capable of effecting expression of the polypeptide in the host cell to be transformed with the expression vector.
  • suitable genetic control elements capable of effecting expression of the polypeptide in the host cell to be transformed with the expression vector.
  • the specific type of control elements necessary to effect expression will depend upon the cell type to be transformed.
  • the cell to be transformed is a mammalian T-cell.
  • control elements refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation which affect the replication, transcription and translation of the polypeptide coding sequence in a recipient cell.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell. [0316
  • the nucleic acid sequences encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure is/are operably linked to a promoter sequence.
  • promoter is used in its conventional sense to refer to a nucleotide sequence at which the initiation and rate of transcription of a coding sequence is controlled.
  • the promoter contains the site at which RNA polymerase binds and also contains sites for the binding of regulatory factors (such as repressors or transcription factors). Promoters can be naturally occurring or synthetic.
  • the promoter can be constitutively active, activated in response to external stimuli (inducible), active in particular cell type or cell state (tissue specific or tumor specific) promoters, and/or regulatable promoters.
  • inducible promoter refers to promoters that facilitate transcription of the Bioactive polypeptide preferably (or solely) under certain conditions and/or in response to external chemical or other stimuli.
  • inducible promoters are known in the scientific literature (see, e.g., Yoshida et al., Biochem. Biophys. Res. Comm., 230:426-430 (1997); lida et al., J. Virol., 70(9): 6054-6059 (1996); Hwang et al., J. Virol., 71(9): 7128-7131 (1997); Lee et al., Mol. Cell. Biol., 17(9): 5097-5105 (1997); and Dreher et al., J. Biol. Chem., 272(46): 29364-29371 (1997).
  • radiation inducible promoters include the EGR-1 promoter. Boothman et al., volume 138, supplement pages S68-S71 (1994).
  • each polypeptide subunit may be operably linked to an expression control sequence (monocistronic) or multiple polypeptides may be encoded by a polycistronic construct where multiple polypeptides are expressed under the control of a single expression control sequence.
  • an expression control sequence monocistronic
  • multiple polypeptides may be encoded by a polycistronic construct where multiple polypeptides are expressed under the control of a single expression control sequence.
  • IRES internal ribosome entry site
  • FMVD2A foot and mouth disease vims protein 2A
  • IRES elements include the picomavirus IRES of poliovirus, rhinovirus, encepahlomyocardits virus, the aphthovirus IRES of foot and mouth disease virus, the IRES cricket paralysis virus (CrPV) the hepatitis A IRES of hepatitis A virus, the hepatitis C IRES of hepatitis C virus, the pestivirus IRES of swine fever or bovine diarrhea viruses, the cripavirus IRES, and mammalian IRES elements such as the fibroblast growth factor-1 IRES, the fibroblast growth factor-2 IRES, PDGF IRES, VEGF IRES, IGF-2 IRES.
  • IRES elements include the picomavirus IRES of poliovirus, rhinovirus, encepahlomyocardits virus, the aphthovirus IRES of foot and mouth disease virus, the IRES cricket paralysis virus (CrPV) the hepatitis A IRES of hepati
  • the use of IRES elements typically results in significantly lower expression of the second protein of the polycistronic message.
  • the use of the FMDV2A system results in more efficient production of the downstream proteins as the multiple proteins are first expressed as a fusion protein which contains the autoproteolytic FMDV2A domain which cleaves the polyprotein into functional subunits.
  • Ryan and Drew (1994) EMBO J. 13(4): 928-933 the use of the FMDV2A system frequently may in the addition of a small number amino acids to the carboxy terminus of the upstream protein.
  • nucleic acid sequences encoding the hIL12P35-Fc and hIL12P40M-Fc subunits of the heterodimeric hIL12M-Fc muteins of the present disclosure may be provided in a bicistronic expression cassette to provide for co-expression of the subunits in a mammalian host cell.
  • the present disclosure provides bicistronic nucleic acids arranged as illustrated below:
  • nucleic acid sequence encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure will be inserted into an vector.
  • An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are examples of non-viral vectors.
  • the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that are functional in the chosen expression host.
  • Expression vectors typically contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
  • Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques.
  • vectors can contain origins of replication, and other genes that encode a selectable marker.
  • neomycin-resistance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells.
  • marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B- phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).
  • CAT chloramphenicol acetyltransferase
  • ADA adenosine deaminase
  • DHFR dihydrofolate reductase
  • HPH hygromycin-B- phosphotransferase
  • HPH hygromycin-B- phosphotransferase
  • TK thymidine kinase
  • lacZ encoding beta-galactosidase
  • the expression cassete comprising the CMV promoter and nucleic acid sequence encoding the ML12P35 (or hIL12P35-Fc) and ML12P40M (or hIL12P40M-Fc) polypeptides is inserted into a pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA).
  • the expression cassete comprising the CMV promoter and nucleic acid sequence encoding the ML12P35 and hIL12P40M polypeptides is inserted into the multiple cloning site of the pExSyn2.0 expression vector as prepared in accordance with the teaching of Example 1.
  • Host cells are typically selected in accordance with their compatibility with the chosen expression vector, the toxicity of the product coded for by the DNA sequences, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells.
  • the hIL12P40M, and hIL12P35 polypeptide subunits of the heterodimeric hIL12M-Fc muteins muteins and biologically active variants and fragments thereof can also be made in eukaryotes, such as yeast or human cells.
  • eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.
  • yeast cells examples include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al.
  • pCDM8 Seed (1987) Nature 329:840
  • pMT2PC Kaufman et al. (1987) EMBO J. 6: 187:195)
  • Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL-2648), Expi293 cells, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA.
  • ATCC CCL 2 canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
  • [032 ⁇ S] iVector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory' Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non- viral vector.
  • conditions which facilitate uptake of foreign nucleic acid by mammalian cells include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).
  • the nucleic acid sequences encoding the hIL12P35 (or ML12P35- Fc) and hIL12P40M (or IL12P40M-Fc) are each provided a separate expression vectors which are then co-transfected into the host cell.
  • a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in a mammalian cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M operably linked to a promoter functional in a mammalian cell are co-transfected into a mammalian host cell.
  • the promoter functional in a mammalian cell of the first and second recombinant expression vectors is the CMV promoter.
  • first and second recombinant expression vectors are pCDNA3.4 mammalian expression vectors (Life Technologies, Carlsbad, CA). In some embodiments the first and second recombinant expression vectors are pExSyn2.0 expression vectors as prepared in accordance with the teaching of Example 1.
  • the present disclosure provides a recombinant mammalian host cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in the recombinant mammalian host cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a promoter functional in the recombinant mammalian host cell.
  • the nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) and hIL12P40M (or IL12P40M-Fc) further encodes a signal peptide, in some embodiments.
  • the signal peptide for the hIL12P35 (or HL12P35- Fc) polypeptide is the wild type human P35 signal peptide (SEQ ID NO: 179) .
  • the signal peptide for the hIL12P40M (or IL12P40M-Fc) polypeptide is the wild type human P40 signal peptide (SEQ ID NO: 180).
  • the nucleic acid sequence encoding the hIL12P35 (or hIL12P35-Fc) and hIL12P40M (or IL12P40M-Fc) signal peptide In some embodiments, recombinant mammalian host cell is selected from the group consisting of CHO and 293 cells.
  • the present disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding HL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in a CHO cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a promoter functional in a CHO cell.
  • the present disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a CMV promoter and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a CMV.
  • the first and second recombinant expression vectors are pCDNA3.4 mammalian expression vectors (Life Technologies, Carlsbad, CA).
  • the first and second recombinant expression vectors are pExSyn2.0 expression vectors as prepared in accordance with the teaching of Example 1.
  • the present disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding a hIL12P40M-Fc selected from the group consisting of SEQ ID NOS: 80, 83, 121, 141, 144, 129, 135, 138, 147, 150, and 153 and a second expression vector comprising a nucleic acid sequence encoding a hIL12P35-Fc selected from the group consisting of SEQ ID NOS: 81, 82, and 124.
  • the cell for production of the hIL12M-Fc is a CHO cell.
  • Host cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Mammalian host cells may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary' supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.
  • Recombinantly-produced polypeptides can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is employed. Alternatively, the recombinant polypeptides can also be recovered from host cell lysates.
  • a protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) may be employed during the recovery phase from cell lysates to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • PMSF phenyl methyl sulfonyl fluoride
  • Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural specific binding of one molecular species to separate and purify a second species from a mixture.
  • Antibodies are commonly used in affinity chromatography.
  • Size selection steps may also be used, e.g., gel filtration chromatography (also known as size-exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size.
  • a protein solution is passed through a column that is packed with semipermeable porous resin.
  • the semipermeable resin has a range of pore sizes that determines the size of proteins that can be separated with the column.
  • at least one step in the puficiation of the hIL12M- Fc is a column chromatographic purification step employing a Protein A column. Protein A purification of Fc conjugated polypeptides is well known in the art using commercially available equipment and reagents.
  • the substantially purified forms of the heterodimeric hIL12M-Fc mutein can be used, e.g., as therapeutic agents, as described herein.
  • the biological activity of the heterodimeric hIL12M-Fc mutein produced in accordance with the foregoing can be confirmed by assay using procedures well known in the art including but not limited to competition ELISA, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fiting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009) with instrumentation commercially available from GE Healthcare Bio-Sciences such as the Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100
  • the hIL2M and hIL12M molecules are be incorporated into compositions, including pharmaceutical compositions.
  • pharmaceutical compositions include pharmaceutical compositions.
  • pharmaceutical formulation pharmaceutical formulation
  • pharmaceutical composition comprises the hIL2M and/or hIL12M and at least one pharmaceutically acceptable carrier, buffer, dispersant, preservatives or tonicity agent.
  • pharmaceutical composition comprises the hIL2M and at least one pharmaceutically acceptable carrier.
  • pharmaceutical composition comprises the hIL12M and at least one pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the hIL2M and/or hIL12M is formulated to be compatible with its intended route of administration and is compatible with the therapeutic use for which the heterodimenc hIL2M and/or hIL12M is to be administered to the subject in need of treatment or prophyaxis.
  • Carriers include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • buffers includes buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and/or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the pharmaceutical formulations for parenteral administration to a subject should be sterile and should be fluid to facilitate easy syringability. It should be stable under the conditions of manufacture and storage and are preserved against the contamination. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • a pharmaceutical formulation comprising a the hIL2M and/or hIL12M molecules to a subject in need of treatment or prophyaxis by a variety of routes of administration, including parenteral administration, oral, topical, or inhalation routes.
  • the methods of the present disclosure involve the parenteral administration of a pharmaceutical formulation comprising a the hIL2M a and/or nd hIL12M molecules to a subject in need of treatment.
  • the methods of the present disclosure involve the parenteral administration of a pharmaceutical formulation comprising a heterodimeric the hIL2M and a pharmaceutical formulation hIL12M to a subject in need of treatment.
  • parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration.
  • Parenteral formulations comprise solutions or suspensions used for parenteral application can include vehicles the carriers and buffers.
  • compositions for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the formulation is provided in a prefilled syringe presentation.
  • a pharmaceutical formulation comprising a heterodimeric the hIL2M and a pharmaceutical formulation hIL12M is administered to a subject in need of treatment in a formulation to provide extended release.
  • extended release formulations of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the hIL2M and/or hIL12M molecules are formulated with earners that will protect the hIL2M and/or hIL12M against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques known to those of skill in the art of pharmaceutical formulation.
  • delivery of the hIL2M and/or hIL12M to a subject in need of treatment is achieved by the administration of a nucleic acid encoding the hIL2M and/or hIL12M.
  • Methods for the administration of a nucleic acids encoding the hIL2M and/or hIL12M to a subject is achieved by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010), or Putnam (Am. J. Health Syst. Pharm.
  • hIL2M and/or hIL12M is administered to a subject by the administration of a pharmaceutically acceptable formulation of recombinant expression vector comprising a nucleic acid sequence encoding the hIL2M and/or hIL12M operably linked to one or more expression control sequences operable in a mammalian subject.
  • the expression control sequence may be selected that is operable in a limited range of cell types (or single cell type) to facilitate the selective expression of the hIL2M and/or hIL12M in a particular target cell type.
  • the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments the recombinant viral vector is a recombinant adenoassociated virus (rAAV) or recombinant adenovirus (rAd), in particular a replication deficient adenovirus derived from human adenovirus serotypes 3 and/or 5.
  • rAAV recombinant adenoassociated virus
  • rAd recombinant adenovirus
  • delivery of the hIL2M and/or hIL12M to a subject in need of treatment is achieved by the administration of recombinant host cells modified to express the hIL2M and/or hIL12M, which may be administered in the therapeutic and prophylactic applications described herein.
  • the recombinant host cells are mammalian cells, e.g., human cells.
  • the present disclosure provides methods of use of hIL2M in combination with hIL12M of in the treatment of subjects suffering from a neoplastic disease disorder or condition by the administration of a therapeutically effective amount hIL2M and hIL12M, nucleic acids encoding hIL2M and hIL12M, recombinant vector(s) encoding hIL2M and hIL12M or a recombinant cell expressing hIL2M and hIL12M as described herein.
  • the recombinatnt vector is a recombinant viral vector encoding hIL2M and hIL12M.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of
  • hIL12M hIL12 mutein
  • hIL12p35 p35 subunit
  • hIL12p40M p40M subunit
  • the hIL12p35 has at least 95% sequence identity to mature wild type human hIL12p35 (SEQ ID NO:2);
  • the hIL12p40M has at least 95% sequence identity to mature wild type human hIL12p40 (SEQ ID NO:4), the hIL12p40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12p40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40; and
  • step (b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutem (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182), wherein step (a) is performed in combination with step (b).
  • hIL2M hIL2 mutem
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject, the method comprising the steps of:
  • hIL2M hIL2 mutein
  • compositions and methods of the present disclosure are useful in the treatment of subject suffering from a neoplastic disease characterized by the presence neoplasms, including benign and malignant neoplasms, and neoplastic disease.
  • Examples of benign neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to adenomas, fibromas, hemangiomas, and lipomas.
  • Examples of pre-malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to hyperplasia, atypia, metaplasia, and dysplasia.
  • malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to carcinomas (cancers arising from epithelial tissues such as the skin or tissues that line internal organs), leukemias, lymphomas, and sarcomas typically derived from bone fat, muscle, blood vessels or connective tissues). Also included in the term neoplasms are viral induced neoplasms such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular disease including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion and the like.
  • carcinomas cancers arising from epithelial tissues such as the skin or tissues that line internal organs
  • leukemias arising from lymphomas
  • sarcomas typically derived from bone fat, muscle, blood vessels or connective tissues.
  • viral induced neoplasms such as warts and EBV induced
  • neoplastic disease includes cancers characterized by solid tumors and non- solid tumors including but not limited to breast cancers; sarcomas (including but not limited to osteosarcomas and angiosarcomas and fibrosarcomas), leukemias, lymphomas, genitourinary cancers (including but not limited to ovarian, urethral, bladder, and prostate cancers); gastrointestinal cancers (including but not limited to colon esophageal and stomach cancers); lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical carcinoma-in-situ; intestinal polyposes; oral leukoplakias; histiocytoses, hyperprofroliferative scars including ke
  • neoplastic disease includes carcinomas.
  • carcinoma refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • neoplastic disease includes adenocarcinomas.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • hematopoietic neoplastic disorders refers to neoplastic diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative/myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ambiguous lineage.
  • Exemplary myeloid disorders amenable to treatment in accordance with the present disclosure include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML).
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin’s Lymphoma, and immunodeficiency-associated lymphoproliferative disorders.
  • Exemplary lymphic disorders amenable to treatment in accordance with the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • the hematopoietic neoplastic disorder arises from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia).
  • the term "hematopoietic neoplastic disorders” refers malignant lymphomas including, but are not limited to, non-Hodgkins lymphoma and variants thereof, peripheral T cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
  • the determination of whether a subject is “suffering from a neoplastic disease” refers to a determination made by a physician with respect to a subject based on the available information accepted in the field for the identification of a disease, disorder or condition including but not limited to X-ray, CT-scans, conventional laboratory diagnostic tests (e.g. blood count, etc ), genomic data, protein expression data, immunohistochemistry, that the subject requires or will benefit from treatment.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of: (a) administering to the mammalian subject a therapeutically effective amount of a first pharmaceutical composition comprising a hIL12 mutein (hIL12M), the hIL12M comprising a p35 subunit (hIL 12p35) and p40M subunit (hIL12p40M) wherein: (a) the hIL12p35 has at least 95% sequence identity to mature wild type human hIL12p35 (SEQ ID NO:2); and the hIL12p40M has at least 95% sequence identify to mature wild type human hIL12p40 (SEQ ID NON), the hIL12p40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12p40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 wherein the
  • step (b) administering to the mammalian subject a therapeutically effective amount of a second pharmaceutical composition comprising hIL2 mutein (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182), and ⁇ wherein step (a) is performed in combination with step (b).
  • hIL2M hIL2 mutein
  • SEQ ID NO: 182 wild type human IL2
  • step is performed (a) simultaneously with step (b). In one embodiment, step is performed contemporaneously with step (b). In one embodiment, step (a) is performed sequentially with step (b) and step (b) is performed prior to step (a). In one embodiment, step (a) is performed sequentially with step (b) and step (a) is performed prior to step (b). In one embodiment, step (a) is performed sequentially with step (b) and the hIL12M is provided in advance of the administration of the hIL2M. In one embodiment, step (b) is performed sequentially with step (a) and step (b), the administration of the hIL2M is performed in advance of step (a), the administration of the hIL12M.
  • step (a) is performed sequentially with step (b) and step (b), the administration of the hIL2M, is performed at least one 1 day, alternatively at least 2 days, alternatively at least 3 days, alternatively at least 4 days, alternatively at least 5 days, alternatively at least 6 days, alternatively at least one week, alternatively at least 10 days, alternatively at least 2 weeks, alternatively at least 3 weeks, alternatively at least 4 w'eeks, in advance of step (a) the administration of the hIL12M.
  • the present disclosure provides a method of treating a neoplastic disease in a mammalian subject, the method comprising the administration of a therapeutically effective amount of a hIL12M combination with a therapeutically effective amount of a hIL2M, wherein the therapeutically effective of amount of the hIL2M is a dose of approximately 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, as
  • the present disclosure provides a method of treating a neoplastic disease in a human subject, the method comprising the steps of:
  • hIL12M hIL12 mutein
  • hIL2M hIL2 mutein having the amino acid sequence:
  • step (b), the administration of the hIL2M is performed in advance of step (a), the administration of the hIL12M, and the pharmaceutical composition comprising a therapeutically effective amount of the hIL2 mutein (hIL2M) contains a dose of approximately of 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively every two weeks, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (b) is administered by subcutaneous injection,
  • Both the hIL12M (STK-026) and hIL2M (STK-012) molecules are modified to provide an extended half- life.
  • Pre-clinical pharmacokinetic studies in primates and clinical experience with STK-012 indicate that the compound persists in vivo in human beings over a period of from 2 to 4 weeks depending on the dose.
  • Pre-clinical pharmacokinetic studies in primates indicate that the STK- 026 molecule persists in vivo for a period of two weeks to more than 4 weeks depending on dose administered.
  • the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately 5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every 4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 containing a dose of approximately 5 pg/kg and the hIL12M STK-026 is dosed on schedule of of every 4 weeks, wherein the dosing of the hIL12M STK-026 is initiated two weeks after dosing of the hIL2M STK-012.
  • the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately 5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every 4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 contains a dose of approximately 250 pg/kg a to approximately 1000 pg/kg and is dosed on schedule of of every 4 weeks, wherein the dosing of the hIL12M STK- 026 is initiated three weeks after dosing of the hIL2M STK-012.
  • the method of the present disclosure may optionally include, in addition to hIL12M and hIL2M, the administration of one or more active anti -neoplastic agents (“supplementary agents”).
  • supplementary agents active anti -neoplastic agents
  • Such further anti-neoplastic agents are referred to interchangeably as “supplementary anti-neoplastic combinations” or “supplementary anti-neoplastic combination therapy” and those therapeutic agents that are used in combination with the hIL2M and hIL12M the present disclosure are referred to as “supplementary anti-neoplastic agents.”
  • the term “supplementary anti-neoplastic agents” includes anti-neoplastic agents that can be administered or introduced separately, for example, formulated separately for separate administration (e.g., as may be provided in a kit) and/or therapies that can be administered or introduced in combination with the hIL2M and hIL12M molecules.
  • the supplementary anti-neoplastic agent is a chemotherapeutic agent.
  • the supplementary agent is a “cocktail” of multiple chemotherapeutic agents.
  • the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e g. radiation therapy).
  • chemotherapeutic agents includes but is not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chiorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as
  • chemotherapeutic agents also includes anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide,
  • a supplementary anti-neoplastic agent is one or more chemical or biological agents identified in the art as useful in the treatment of neoplastic disease, including, but not limited to, a cytokines or cytokine antagonists such as INF a, or anti-epidermal growth factor receptor, irinotecan; tetrahydrofolate antimetabolites such as pemetrexed; antibodies against tumor antigens, a complex of a monoclonal antibody and toxin, a T-cell adjuvant, bone marrow transplant, or antigen presenting cells (e.g., dendritic cell therapy), anti- tumor vaccines, replication competent viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or an immunomodulator to achieve additive or synergistic suppression of tumor growth, non-steroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e
  • NSAIDs non-
  • the heterodimeric hIL12M-Fc mutein is administered in combination with BRAF/MEK inhibitors, kinase inhibitors such as sunitinib, PARP inhibitors such as olaparib, EGFR inhibitors such as osimertinib (Ahn, etal. (2016) J Thorac Oncol 11 :S115), IDO inhibitors such as epacadostat, and oncolytic viruses such as talimogene laherparepvec (T- VEC).
  • BRAF/MEK inhibitors kinase inhibitors such as sunitinib
  • PARP inhibitors such as olaparib
  • EGFR inhibitors such as osimertinib (Ahn, etal. (2016) J Thorac Oncol 11 :S115)
  • IDO inhibitors such as epacadostat
  • oncolytic viruses such as talimogene laherparepvec (T- VEC).
  • a “supplementary anti -neoplastic agent” is a therapeutic antibody (including bi-specific and tri-specific antibodies which bind to one or more tumor associated antigens including but not limited to bispecific T cell engagers (BITEs), dual affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs).
  • the therapeutic antibody is an antibody that binds to at least one tumor antigen selected from the group consisting of HER2 (e.g. trastuzumab, pertuzumab, ado-trastuzumab emtansine), nectin-4 (e.g. enfortumab), CD79 (e.g.
  • CTLA4 e.g. ipilumumab
  • CD22 e.g. moxetumomab pasudotox
  • CCR4 e.g. magamuizumab
  • IL23pl9 e.g. tildrakizumab
  • PDL1 e.g. durvalumab, avelumab, atezolizumab
  • IL17a e.g. lxekizumab
  • CD38 e.g. daratumumab
  • SLAMF7 e.g. elotuzumab
  • CD20 e.g.
  • rituximab tositumomab, ibritumomab and ofatumumab
  • CD30 e.g. brentuximab vedotin
  • CD33 e.g. gemtuzumab ozogamicin
  • CD52 e.g. alemtuzumab
  • EpCam CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate binding protein
  • GD2 e.g. dinuntuximab
  • GD3 IL6
  • silutxumab GM2
  • Le y VEGF
  • VEGFR VEGFR2
  • PDGFRa e.g. olartumumab
  • EGFR e.g. cetuximab, panitumumab and necitumumab
  • ERBB2 e.g. trastuzumab
  • ERBB3, MET IGF1R, EPHA3, TRAIL Rl, TRAIL R2, RANKL RAP, tenascin, integrin aV
  • a therapeutic antibody is an immune checkpoint modulator for the treatment and/or prevention neoplastic disease in a subject as well as diseases, disorders or conditions associated with neoplastic disease.
  • the term “immune checkpoint pathway” refers to biological response that is triggered by the binding of a first molecule (e.g. a protein such as PD1) that is expressed on an antigen presenting cell (APC) to a second molecule (e.g. a protein such as PDL1) that is expressed on an immune cell (e.g. a T-cell) which modulates the immune response, either through stimulation (e.g. upregulation of T-cell activity) or inhibition (e.g. downregulation of T-cell activity) of the immune response.
  • a first molecule e.g. a protein such as PD1
  • APC antigen presenting cell
  • PDL1 protein such as PDL1
  • T-cell an immune cell
  • stimulation e.g. upregulation of T-cell activity
  • inhibition e.g. down
  • the immune checkpoint pathway modulator is an antagonist of a negative immune checkpoint pathway that inhibits the binding of PD1 to PDL1 and/or PDL2 (“PD1 pathway inhibitor”).
  • PD1 pathway inhibitors includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and/or PDL2.
  • nivolumab Opdivo®, BMS-936558, MDX1106, commercially available from BristolMyers Squibb, Princeton NJ
  • pembrolizumab Keytruda®MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ
  • atezolizumab Tecentriq®, Genentech/Roche, South San
  • Additional PD1 pathway inhibitors antibodies are in clinical development including but not limited to durvalumab (MEDI4736, Medimmune/ AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), and avelumab (MSB0010718C, Merck Serono/Pfizer) and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in United States Patent No. 8,217,149 (Genentech, Inc) issued July 10, 2012; United States Patent No. 8,168,757 (Merck Sharp and Dohme Corp.) issued May 1, 2012, United States Patent No. 8,008,449 (Medarex) issued August 30, 2011 , United States Patent No. 7,943,743 (Medarex, Inc) issued May 17, 2011.
  • Examples of antibody therapeutics which are FDA approved and may be used as supplementary agents for use in the treatment of neoplastic disease include atezohzumab, olaratumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrohzumab, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumaxomab, panitumumab, bevacizumab, ramucirumab, siltuximab, enfortumab vedotin, polatuzumab vedotin, [fam
  • a supplementary anti-neoplastic agent is one or more non- pharmacological modalities (e.g., localized radiation therapy or total body radiation therapy or surgery).
  • the present disclosure contemplates treatment regimens wherein a radiation phase is preceded or followed by treatment with a treatment regimen comprising a hIL 12 mutein and one or more supplementary anti-neoplastic agents.
  • the present disclosure further contemplates the use of heterodimeric hIL12M-Fc mutein in combination with surgery (e.g. tumor resection).
  • the present disclosure further contemplates the use of a heterodimeric hIL12M-Fc mutein in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapy.
  • kits comprising the hIL2M and hIL12M agents of the present disclosure.
  • the kit comprises a first sterile vial comprising a pharmaceutical formulation of hIL2M and a second sterile vial comprising a pharmaceutical formulation of hIL12M agents and instructions for use.
  • the kit comprises a first sterile vial comprising a liquid pharmaceutical formulation of hIL2M-PEG and a second sterile vial comprising a liquid pharmaceutical formulation of hIL12M-Fc and instructions for use.
  • the kit comprises a first sterile vial comprising a liquid pharmaceutical formulation of hIL2M-PEG suitable for use in an autoinjector device and a second sterile vial comprising a liquid pharmaceutical formulation of hIL12M-Fc suitable for use in an autoinjector device and instructions for use.
  • the kit comprises a first sterile prefilled syringe comprising a liquid pharmaceutical formulation of hIL2M-PEG and a second sterile prefilled syringe comprising a liquid pharmaceutical formulation of hIL12M-Fc and instructions for use.
  • the kit comprises a sterile vial comprising a lyophilized pharmaceutical formulation of hIL2M-PEG and sterile vial comprising a solution for reconstiution of of the lyophilized hIL2M-PEG formulation, sterile vial comprising a pharmaceutical formulation of hIL12M-Fc and sterile vial comprising a solution for reconstiution of of the lyophilized hIL2M- PEG formulation and instructions for use.
  • the kit comprises a first sterile vial comprising a pharmaceutical formulation of STK-026 and a second sterile vial comprising a pharmaceutical formulation of STK-
  • the kit may optionally comprise a one or more codes (such as QR codes, passwords, passkeys or similar electronic access codes) which may provide access (or a license) to a remote monitoring system or a telehealth application on a mobile device.
  • codes such as QR codes, passwords, passkeys or similar electronic access codes
  • the heterodimeric IL12 muteins of the present disclosure are produced in substantial accordance with the following procedure.
  • a pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA) was modified to include additional restriction sites in the Multiple Cloning Cloning Site (MCS) and renamed pExSyn2.0.
  • MCS Multiple Cloning Cloning Site
  • Nucleic acid sequences encoding the hP40Fc and hIL12P40M polypeptides are cloned into pExSyn2.0 at the EcoRI and BamHI restriction sites, using standard molecular biology cloning techniques.
  • a nucleic acid sequence encoding the wt HL12P35, a Gly-Ser linker and an Hisx8 chelating peptide (SEQ ID NO: 199) were cloned into the pExSyn2.0 vector at the EcoRI and BamHI using standard molecular biology cloning techniques.
  • the vectors were DNA sequenced (MC Lab, South San Francisco, CA) to confirm identity.
  • the vectors encoding the hP40Fc and hIL12P35 are co-transfected into Expi293 Cells in substantial accordance with the manufacturers protocol (Life Technologies, Carlsbad, CA).
  • His-tagged hIL12 muteins (His-tag on P35 C-termmus) are captured using 0.1 ml Ni Sepharose excel resin (Cytiva, part # GE17371201), equilibrated in Phosphate Buffered Saline (PBS) containing 10 mM Imidazole.
  • PBS Phosphate Buffered Saline
  • the muteins are eluted from the Ni resin with 0.5 ml of PBS containing 250 mM Imidazole and dialyzed into PBS. Concentrations are determined with UV absorbance at 280 nm using extinction coefficients determined from the protein sequence.
  • IL12-Fc heterodimer is produced by transfection of two constructs consisting of P35-Fc and P40-Fc monomers. DNA is produced, scaled up, and expi293 or expiCHO cells transfected as described above. The Fc-tagged IL 12 complexes are captured using Protein-A resin equilibrated in PBS, and eluted from the column with 100 mM Sodium Acetate pH2.8. Elutions are neutralized and dialyzed into PBS. Further purification using standard techniques such as Size Exclusion Chromatography and/or Anion Exchange Chromatography are used to produce pure IL12-Fc heterodimer. Concentrations are determined with UV absorbance at 280 nm using extinction coefficients determined from the protein sequence.
  • IFN gamma activity in isolated human PBMCs is performed in substantial accordance with the following procedure. Isolated human whole PBMCs are removed from storage in liquid nitrogen, thawed, and counted. Cells are divided into two groups from which were isolated either Pan-T Cells or Natural Killer Cells using StemCell negative isolation kits (StemCell Technologies, Cat. #17951, Cat. #19055), per manufacturer’s protocol. Cells are then counted, resuspended in Complete Yssel’s media (IMDM, Gibco, Cat. #122440-053) containing .25% w/v Human Albumin (Sigma, Cat.
  • Pan- T Cells The plates used to stimulate Pan- T Cells are coated with 5 ug/mL anti-CD3 antibody (Biolegend, Cat. #300458) in Phosphate Buffered Saline (PBS) (Coming, Cat. #12-031-CV), stored overnight at 4C, and are washed prior to cell isolations. All cells are supplemented with human IL-2 and recombinant human IL-18 (R&D Systems, Cat. #9124-IL-050/CF), final concentrations 100 pM and 50 ng/mL, respectively. Pan-T Cells are additionally supplemented with 10 ug/mL anti-CD28 antibody (Biolegend, Cat. #302934), final concentration 10 ug/mL.
  • PBS Phosphate Buffered Saline
  • Pan-T Cells are additionally supplemented with 10 ug/mL anti-CD28 antibody (Biolegend, Cat. #302934), final concentration 10 ug/mL.
  • IL-12 mutant proteins were titrated in Complete Yssel’s Media at concentrations ranging from 200nM to 2fM, 1: 10 dilutions, and are added to wells in equivalent volume to previously plated cells, the final concentrations typically ranging from lOOnM to HM. Cells are then incubated at 37C, 5% CO2 for 48 hours.
  • Compound 1 Wild Type murine IL12 Fc: Compound 1 used in the following efficacy and toxicity studies is a mIL12Fc molecule comprised of a wild-type murine IL 12P35 (mIL12R35, UniProt Ref.
  • P43431 fused to a first murine IgG2 (m!gG2) Fc domain (mIL I 2/G5-Fcl ) using a 15 amino acid GS linker (G4Sx3 (SEQ ID NO: 37)) and a wild-type murine IL12p40 (mIL12p40) fused to a second murine IgG2 (mIgG2) Fc domain (mlLl 2/G5-Fc2) wherein the Fcl and Fc were modified to promote heterodimerization.
  • the mIL12P35-Fcl subunit of the mIL12Fc is a polypeptide having the amino acid sequence (mIL12P35 underlined; GS linker italicized):
  • the mIL12p40-Fc2 subunit of the mIL12Fc is a polypeptide having the amino acid sequence (mIL12p40 underlined)
  • Compound 2 mIL12M-Fc.
  • Compound 2 used in the following efficacy and toxicity studies is a murine mIL12M Fc molecule (mIL12M-Fc) molecule comprised of: (1) a wild-type murine IL12P35 (mIL12P35, UniProt Ref.
  • mIL12-Fc Compound 1
  • mIL12M-Fc Compound 2
  • NK cells were isolated by negative selection.
  • Murine NK cells were treated with mIL2 and hILl 8 for 48 hours and then stimulated with a titration of Compound 1 or Compound 2 for 20 minutes.
  • Cells were fixed, permeabilized, and stained for STAT4 phosphorylation prior to measurement via flow cytometry. The results of this study are presented in Figure 21, Panel A.
  • the mIL12M-Fc molecule (Compound 2) exhibited significantly reduced STAT4 signaling in murine NK cells compared to the wild type mIL12-Fc molecule (Compound 1).
  • human hIL12M-Fc comprising a p40 subunit having the amino acid substitutions E81 A/F82A/K106A and wild type hIL12-Fc molecules were evaluate for their ability to stimulate STAT4 siginahng in human NK cells. Briefly, human NK cells were isolated from LRS chamber derived PBMCs and then treated with human IL 12 wild type Fc or human IL12 mutant Fc for 20 minutes.
  • amurine surrogate hIL2M compound comprising: (1) amurine IL2 polypeptide correspond to the amino acid substitutions L18R/Q22E/Q126K of the hIL2M; (2) a 2 amino acid GS linker having the sequence Gly-Ser; and (3) a chelating peptide comprising eight histidine residues (SEQ ID NO: 199), the polypeptide of the mIL2M having the amino acid sequence:
  • MC-38 tumor cells (Kerafast, Catalog #ENH204-FP) were thawed and cultured in MC-38 media (DMEM [Gibco, Catalog #11995-065] supplemented with 10% FBS [Coming, 35-016-CV] and Penicillin/Streptomycin [Gibco, Catalog #15140-122] at 37°C and 5% CO2.
  • DMEM Gibfast, Catalog #ENH204-FP
  • FBS Coming, 35-016-CV
  • Penicillin/Streptomycin Gibco, Catalog #15140-122
  • mice were randomized into treatment groups (eight mice per group) on Studay Day -1. Starting the day following randomization, Study Day 0, treatment of the animals commenced in accordance with the following schedule:
  • Tumor volumes and body weights were measured throughout the study. In those animals where tumor volume exceeded 2000 mm 3 were euthanized.
  • mice treated with DPBS control (Group 1), Compound 2 (mIL12M- Fc, Group 5), or Compound 3 (mIL2M, Group 2) indicated a retention of greater than 97% of initial body weights.
  • mice treated with Compound 1 wild type mIL12Fc, Group 3
  • mice treated with Compound 1 showed on average BW reduction of approximately 12% (88% of initial body weight).
  • Compound 3 (mIL2M) to the treatment regimen with Compounds 1 and 2 further distinguished toxicity differences between Compounds 1 (wild type mIL12Fc) and 2 (mIL12M- Fc).
  • Compound 3 when administered with Compound 1 (wild type mIL12Fc), treatment Group 4, led to decreases in body weight to an average of approximately 28% (72.1% of initial body weight) on Study Day 7.
  • treatment Group 4 all but one animal was removed from the study and euthanized due to animal welfare endpoint criteria.
  • the combination of Compound 2 with Compound 3 demonstrated average Day 7 body weight reductions of approximately 7% (92.6% of initial body weights) with no animals requiring euthanasia.
  • each of Compounds 1, 2, and 3 demonstrated some anti-tumor activity with varying degrees of complete responders (CRs): Group 2 (Panel B) 0 of 8 CRs, Group 3 (Panel B) 1 of 8 CRs and Group 5 (Panel B) 2 of 8 CRs.
  • CRs complete responders
  • B Body Weights and Tolerability: Loss of body weight and survival were evaluated as measures of toxicity. All mice were weighed, and relative body weight (BW) change normalized to the cumulative weight of each animal at the at the initiation of the study (Study Day -1) as summarized in the spider plots in Figure 24. As illustrated in Figure 24, at Study Day 7 all groups maintained, on average, greater than 99% of their initial body weights. Over the course of the study, no animals were removed from the study due to body weight loss or other clinical symptoms.

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Abstract

The present disclosure provides methods of use of hIL2 muteins in combination with hIL12 muteins in the treatment of neoplastic disease.

Description

Methods and Compositions of IL12 Muteins and IL2 Muteins
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to PCT Applicatioon No. PCT/US2022/078465, filed October 20, 2022, and U.S. Provisional Application No. 63/496,342, filed April 14, 2023, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Receptors for cytokines are typically multimers of cell surface expressed proteins that stimulate signaling via the interaction of their intracellular domains. Cytokines act as specific ligands for the extracellular domains of cytokine receptor subunits and facilitate the multimerization of such receptor subunits so as to bring the intracellular domains of such cytokine receptor subunits into proximity such that intracellular signaling may occur. Certain cytokine receptor subunits are shared among different cytokines and the nature of the cytokine determines which receptor subunits are multimerized to form the cytokine receptor complex and the intracellular signaling pattern that results. Cytokines thus act to bridge individual receptor subunits into a receptor complex that results in intracellular signaling.
[0003] The intracellular domains of cytokine receptor subunits possess JAK binding domains which are typically located in the boxl/box2 region of the intracellular domain of the cytokine receptor subunit near the interior surface of the cell membrane. Intracellular JAK kmases associate with these JAK binding domains. When the intracellular domains of receptor subunits are brought into proximity, typically by the binding of the cognate ligand for the receptor to the extracellular domains of the receptor subunits, the JAKs phosphorylate each other. Four Janus kinases have been identified in mammalian cells: JAK1, JAK2, JAK3 and TYK2. Ihle, et al. (1995) Nature 377(6550):591-4, 1995; O’Shea and Plenge (2012) Immunity 36(4):542-50. The phosphorylation of the JAK induces a conformational change in the JAK providing the ability to further phosphorylate other intracellular proteins which initiates a cascade that results in activation of multiple intracellular factors which transduce the intracellular signal associated with the receptor. The resulting intracellular responses, such as gene transcription, are frequently collectively referred to as downstream signaling.
[0004] In many instances, the proteins which are phosphorylated by the JAKs are members of the signal transducer and activator of transcription (STAT) protein family. Seven members of the mammalian STAT family have been identified to date: STAT1, STAT2, STAT3, STAT4, STAT5a STAT5b, and STAT6. Delgoffe, et al., (2011) Curr Opin Immunol. 23(5): 632-8; Levy and Darnell (2002) Nat Rev Mol Cell Biol. 3(9):651-62 and Murray, (2007) J Immunol. 178(5):2623-9. The selective interplay of activated JAK and STAT proteins, collectively referred to as the JAK/STAT pathway, provide for a wide variety of intracellular responses observed in response to cytokine binding.
Interleukin- 12 (IL 12)
[0005] Human interleukin- 12 (hIL12) is a heterodimeric cytokine comprised of the human P35 (also referred to as hIL12A, Uniprot Ref. 29459) and human P40 (also referred to as hIL12B, Uniprot Ref. 29460) subunits. The hIL12 heterodimer is also referred to as p70. hIL12 is produced by dendritic cells, macrophages and neutrophils. hIL12 is typically identified as a T cell stimulating factor which can stimulate the proliferation and activation of T cells. hIL12 stimulates the production of IFNy and TNFa and modulates the cytotoxic activity of NK and CD8+ cytotoxic T cells. hIL12 was first identified and referred to as cytotoxic lymphocyte maturation factor. Stem, et al (1990) Proc Natl Acad Sei USA 87:6808-6812 and Gately, et al. United States Patent No 6,683,046 issued January 27, 2004. hIL12 is also involved in immune cell differentiation in particular the differentiation of naive T cells into Thl (CD4+) cells. hIL12 is also reported to provide anti-angiogenic activity. Since its discovery more than 30 years ago, hIL12 has been proposed and evaluated for use in the treatment of a variety of neoplastic diseases, viral and bacterial infections. See, e.g. Lasek, et al (2014) Cancer Immunol Immunother (2014) 63:419- 435.
[0006] hIL12 binds to the hIL12 receptor, a heterodimeric complex of hIL12 receptor subunit beta-1 (IL12R[31, also referred to in the scientific literature as IL12RB1 or CD212, Uniprot Ref. P42701) and hIL12 receptor subunit beta-2 (ML12Rβ2 also referred to in the scientific literature as hIL12RB2, Uniprot Ref. Q99665). hIL12Rβ1 and ML12Rβ2 are members of the class I cytokine receptor family and have homology to gpl30. The expression of hIL12Rβ1 and HL12Rβ2 are upregulated in response to hIL12 with the majority of ML12Rβ2 being found on activated T cells.
[0007] hIL12Rpl is a constitutively expressed type I transmembrane protein that belongs to the hemopoietin receptor superfamily. hIL12Rβ1 binds with low affinity to hIL12. hIL12Rβ1 is required for binding to the hIL12P40 subunit and it is associated with the Janus kinase (Jak) family member Tyk-2. The binding IL12p40 and IL12P35 subunits of IL12 toIL12Rβ1 and IL12Rβ2, respectively, results in the dimerization of IL12Rβ1 and IL12Rβ2. In response to the dimerization of IL12Rβ1 and IL12Rβ2, Jak-2 and Tyk-2 are transphosphorylated, further activating Jak2 and Tyk2 kinase activity which results in phosphorylation of the IL12Rβ1 and IL12Rβ 2 intracellular domains. The phosphorylated intracellular signaling domain of IL12Rβ2 provides a binding site for STAT4. STAT4 binds to phosphorylated IL12Rβ2 and is subsequently phosphorylated. Phosphorylated STAT4 induces dimerization with another phosphorylated STAT4 molecule. The phosphorylated STAT4 homodimers translocate to the nucleus resulting in, among other activities, the promotion of IFN-y gene transcription. IFN-y induces the activity and proliferation of macrophages, NK cells, and T cells, which also secrete IL12.
[0008] IL 12 has many properties which suggested its use in the treatment of cancers including the stimulation of IFNy production by NK cells, enhancement of the cytolytic properties of NK cells and cytotoxic T cells, and inhibition of angiogenesis. IL 12 exhibited significant antitumor activity in animal models which led to its evaluation in Phase I and Phase II clinical trials in the treatment of a variety of cancers in the late 1990s. Lasek, et al., supra. While beneficial effects were observed, the significant adverse events observed resulted in termination of the clinical trials. In the ensuing time, a variety of approaches have been evaluated for the use of hIL12 molecules and gene therapy vectors encoding hIL12 but the toxicity associated with these agents has, so far, limited their development to Phase I and Phase II clinical trials and there are no commercially available therapeutic agents comprising IL 12.
[0009] Because different cell types respond to the binding of ligands to their cognate receptors with different sensitivities, modulation of the affinity of the heterodimeric hIL12 ligand (or its individual components) for the hIL12 receptor (or its individual components) relative to wild-type hIL12 (i.e., comprising wild-type P35 and P40) can stimulate desired activities on target cells while reducing undesired activities on non-target cells. In some embodiments, an hIL12 partial agonists of the present disclosure comprises a modified P40 subunit polypeptide that provides intracellular signaling characteristic of wt hIL12 on desired cell types, while providing significantly less intracellular signaling on undesired cell types. This is achieved, for example, by contacting the cell with IL12 partial agonists comprising a heterodimeric hIL12M-Fc mutein with a modified binding affinity for hIL12Rβ1, or causing different Emaxfor hIL12Rβ1 as compared to the binding affinity of wild-type or parental hIL12P40 polypeptide for hIL12Rβ1.
[0010] Glassman, et al. (2021) Cell 184(4):983-999 describe the crystal structure of the IL12 and IL23 receptors and describe residues of P40 that interact with the IL12Rβ1 receptor. In particular, Glassman, et al. describe IL 12 partial agonists comprising a modified P40 subunit that preserved CD8+ T cell IFNg induction and tumor cell killing but exhibit reduced activation and cytokine production from NK cells. The stimulation of NK cells is associated with significant systemic side effects such as capillary leak syndrome. IL12 partial agonists that selectively activate CD8+ T cells without significant upregulation of NK cells retain the beneficial antitumor effects of IL12 while mitigating systemic toxicity associated with the activation of NK cells. The IL 12 partial agonists produced potent antitumor immunity with reduced toxicity relative to IL 12 in preclinical mouse tumor models.
[0011] To maximize the antitumor effect of IL 12 in a subject, it is desirable to provide a sustained systemic level of the cytokine. The in vivo half-life of recombinant human IL 12 (rhILl 2), while longer than other cytokines such as IL2, remains comparatively short. The half- life of wild-type rhIL12 following intravenous bolus injection of a single 500 ng/kg dose of rhIL12, the maximum tolerated dose in the study, was observed to be between 5.3 hours and 10.3 hours. Atkins, et al (1997) Clinical Cancer Research 3:409-417. However, the toxicity associated with wt hIL12 treatment has been an impediment to the development of extended delivery forms of IL 12. As previously noted, sustained and targeted delivery of IL 12 has been evaluated but has not yet provided a successful IL12 therapeutic agent.
[0012] Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, and thus the biopharmaceutical product can require less frequent administration. Engineered Fc domains have been extensively investigated in the context of therapeutic antibodies, particularly bi-specific antibodies, with numerous Fc engineered antibodies being developed and commercialized. See, e.g. Czajkowsky, et al. (2012) EMBO Mol Med 4: 1015-1028. Fc binds to the neonatal Fc receptor (FcRn) on endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re-released into the circulation, keeping the molecule in circulation longer. These properties of the Fc domain are believed to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc-fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates. A variety of modifications to the Fc domain(s), referred to as Fc engineering, have been developed that provide for particular beneficial features to the Fc domain as modulation of effector function (Wang, et al. (2018) Protein Cell 9(1 ): 63-73) For example, Zalevsky, et al. describe the amino acid substitutions M428L and N434S (EU numbering), frequently referred to as “LS” modification, to extend half-life. Zalevsky, et al. (2010) Nature Biotechnology 28: 157-159.
[0013] Fc conjugates of IL 12 have been described in the literature. For example, Gillies, et al. described a IL12 Fc conjugate with each of the wild-type P35 and wild-type P40 subunits expressed as afusion protein with an Fc subunit. Gillies, etal. (1998) J. Immunol. 160:6195-6203 and Gillies, et al. United States Patent No. 6,838,260 issued January 4, 2005. Similarly, Kim et al, (PCT/KR2017/008676 published February 15, 2018 as WO/2018/030806; United States Patent No. 11,078,249 issued August 3, 2021; United States Patent No. 10,696,722 issued June 30, 2020) describe IL12 Fc conjugate where the P35 and P40 subunits were expressed as a fusion proteins with Fc domains wherein the Fc domains are modified to promote heterodimerization. Cheung, et al. (PCT International Patent Application no PCT/US2019/057721 published April 30, 2020 as WO/2020/086758) and Bigelow, et al (PCT International Patent Application PCT/US2021/028701 published October 28, 2021 qw WO/2021/216916) describe and IL12 Fc conjugates. Bemett, et al. (PCT International Patent Application PCT/US 19/54570 published April 9, 2020 as WO/2020/072821, United States Patent Publication US 2020/0216509 published July 9, 2020; United States Patent No. 11,358,999 issued June 14, 2022) describe IU2 Fc conjugates comprising wild-type and modified P35 and P40 subunits. See also, Epstein, et al. Chinese Patent Application Serial No CN201410597561.4A published May 4, 2016.
Interleukin-2 (IL2)
[0014] Interleukin-2 (IL2)is a pluripotent cytokine produced by antigen activated T cells sand exerts a wide spectrum of effects on the immune system. IL2 promotes the proliferation and expansion of activated T lymphocytes, induces proliferation and activation of naive T cells, potentiates B cell growth, and promotes the proliferation and expansion of NK cells. Human interleukin 2 (IL2) is a 4 alpha-helix bundle cytokine of 133 amino acids. IL2 is a member of the IE2 family of cytokines which includes IL2, IL-4, IL-7, IL 9, IL-15 and IL21. However, the function of IL2 is non-redundant, evidenced by genetic knockouts in mice (Schorle, et al. (1991) Nature 352(6336): 621-624). The amino acid sequence of hIL2 is found in Genbank under accession locator NP_000577.2.
[0015] IL2 exerts its effect on mammalian immune cells through interaction with three different cell surface proteins: (1) CD25 (also referred to as the IL2 receptor alpha, IL2Ra, p55), (2) CD122 (also referred to as the interleukin-2 receptor beta, IL2R0, IL15RJ3 and p70-75), and (3) CD132 (also referred to as the interleukin 2 receptor gamma, IL2Ry; or common gamma chain as it is a component of other multimeric receptors in the IL2 receptor family).
[0016] CD25 is a 55 kD polypeptide that is constitutively expressed in Treg cells and inducibly expressed on other T cells in response to activation. hIL2 binds to hCD25 with a Kd of approximately 10'8M. CD25 is also referred to in the literature as the "low affinity" IL2 receptor. The human CD25 (“hCD25”) is expressed as a 272 amino acid pre-protein comprising a 21 amino acid signal sequence which is post-translationally removed to render a 251 amino acid mature protein. Amino acids 22-240 (amino acids 1-219 of the mature protein) correspond to the extracellular domain. Amino acids 241-259 (amino acids 220-238 of the mature protein) correspond to transmembrane domain. Amino acids 260-272 (amino acids 239-251 of the mature protein) correspond to intracellular domain. Human CD25 nucleic acid and protein sequences may be found as Genbank accession numbers NM_000417 and NP_0004Q8 respectively. The intracellular domain of CD25 is comparatively small (13 amino acids) and has not been associated with any independent signaling activity nor has the IL2/CD25 complex has not been observed to produce a detectable intracellular signaling response.
[0017] CD 122 is a single pass type I transmembrane protein. The human CD 122 (hCD122) is expressed as a 551 amino acid pre-protein, the first 26 amino acids comprising a signal sequence which is post-translationally cleaved in the mature 525 amino acid protein. Amino acids 27-240 (amino acids 1-214 of the mature protein) correspond to the extracellular domain, amino acids 241-265 (amino acids 225-239 of the mature protein) correspond to the transmembrane domain and amino acids 266-551 (amino acids 240-525 of the mature protein) correspond to the intracellular domain. As used herein, the term CD 122 includes naturally occurring variants of the CD122 protein including the CD122 variants comprising the S57F and D365E substitutions (as numbered in accordance with the mature hCD122 protein). hCD122 is referenced at UniProtKB database as entry Pl 4784. Human CD 122 nucleic acid and protein sequences may be found as Genbank accession numbers NM_000878 and NP_000869 respectively.
[0018] CD 132 is a type 1 cytokine receptor and is shared by the receptor complexes for IL-4, IL-7, IL-9, IL- 15, and IL21, hence it being referred to in the literature as the “common” gamma chain. Human CD 132 (hCD132) is expressed as a 369 amino acid pre-protein comprising a 22 amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21 amino acid transmembrane domain, and amino acids 284- 369 (ammo acids 262-347 of the mature protein) correspond to the intracellular domain. hCD132 is referenced at UniProtKB database as entry P31785. Human CD132 nucleic acid and protein sequences may be found as Genbank accession numbers: NM_000206 and NP_000197 respectively.
[0019] In addition to the “low affinity” IL2 receptor (i.e., CD25), two additional IL2 receptor complexes have been characterized: (1) an “intermediate affinity” dimeric IL2 receptor comprising CD122 and CD132 (also referred to as “IL2R|3y”), and (2) a “high affinity” trimeric IL2 receptor complex comprising the CD25, CD 122 and CD 132 proteins (also referred to as “IL2Ra|3y”). . hIL2 exhibits a significantly higher affinity for the trimeric receptor relative to the dimeric receptor. hIL2 exhibits a Kd of approximately 10'9M with respect to the dimeric intermediate affinity CD122/CD132 (IL2|3y) receptor complex. hIL2 exhibits a Kd of approximately 10-11M with respect to the high IL2 affinity receptor complex approximately 2-logs higher than the affinity for the intermediate affinity dimeric receptor). [0020] Monomeric IL2 forms a complex with both the trimeric “high affinity” form of the IL2 receptor and the dimeric intermediate affinity receptor (Wang, etal. (2005) Science 310: 159-1163) through binding to the extracellular domains of the receptor components expressed on the cell surface. The binding of IL2 to CD25 induces a conformational change in IL2 facilitating increased binding to CD 122. IL2 mutants, mimicking the CD25 binding-induced conformational change demonstrate increased binding to CD122 (Levin, et al. (2012) Nature 484(7395): 529-533). The association of CD 132 provides formation of the dimeric intermediate-affinity or trimeric high- affinity receptor complexes which are associated with intracellular signaling. In addition to providing intracellular signaling via the JAK/STAT pathway (e.g. phosphorylation of STAT5) and other cellular systems, the interaction of hIL2 with the hIL2 high affinity trimeric receptor on a cell initiates a process by which CD122 is internalized, the membrane bound form of CD25 is released from the activated cell as a soluble protein (referred to as “soluble CD25” or “sCD25”) as well as triggering the release of IL2 endogenously produced by the activated cell which is capable of acting in an autocrine and/or paracrine fashion.
[0021] The various forms of the IL2 receptor are expressed on the surface of most lymphatic cells, in particular on T cells, NK cells, and B cells, however the expression of each receptor cell type varies with a variety of factors including cell type and whether the cell has been “activated” by the binding of MHC1 to a cognate antigen. Non-activated T cells and NK cells express almost exclusively the intermediate-affinity dimeric IL2 receptor, consisting of the two signaling receptors, CD122 and CD132 and demonstrate comparatively low responsiveness to IL2 since the intermediate affinity CD122/CD132 complex which comparatively low affinity for IL2 relative to the trimer CD25/CD 122/CD 132 high affinity receptor. In contrast, activated T cells, and regulatory T cells (Tregs) express the trimeric high-affinity IL2 receptor consisting of CD25, CD122 and CD132. TCR-activated T cells (z.e., so called “antigen experienced” T cells) express the high-affinity trimeric IL2 receptor. While Tregs constituitively express CD25, the level of expression of CD25 on Tregs is substantially lower than the level of CD25 expression on activated T-cells where CD25 expression is substantially (>100-fold) induced upon activation . TCR- activated T cells (i.e., so called “antigen experienced” T cells), including tumor infiltrating T cells (“TILs”) and tumor recognizing cells, upregulate CD25 and CD122 upon receiving a T cell receptor (TCR) signal (Kalia, et al. (2010) Immunity 32(1): 91-103. The significant upregulation of CD25 and CD 122 in response to receiving a T cell receptor (TCR) signal renders the antigen activated T cell highly sensitive to the IL2 cytokine. Although, Tregs constitutively express CD25, TCR-activated T cells express higher levels of the trimeric receptor than Tregs. As a consequence, the expansion of antigen activated T cells in antigen-challenged hosts significantly outpaces the expansion of Tregs. (Humblet-Baron, et al. (2016) J Allergy Clm Immunol 138(1): 200-209 e208). [0022] Recombinant hIL2 is approved for the treatment of human adults with metastatic melanoma (and metastatic renal cell carcinoma by the United States Food and Drug Administration (USFDA). Therapeutic application of High Dose hIL2 (HD-hIL2) induces tumor rejection in highly immune infiltrated melanomas and renal cell carcinomas (Atkins, et al. (1999) J Clin Oncol 17(7):2105-2116). However, HD-hIL2 therapy is associated with severe dose limiting toxicity, including impaired neutrophil function, fever, hypotension, diarrhea and requires expert management. Dutcher, et al. (2014) J Immunother Cancer 2(1): 26. HD-hIL2 treatment activates most lymphatic cells, including naive T cells and NK cells, which predominantly express the intermediate affinity receptor (CD122/CD132) and CD25+ regulatory' T cells (Tregs), which express the high affinity trimeric receptor (CD25/CD122/CD132). HD-hIL2 monotherapy may also induce generalized capillary leak syndrome which can lead to death. This limits the use of HD-IL2 therapy to mostly younger, very healthy patients with normal cardiac and pulmonary function. HD-IL2 therapy is typically applied in the hospital setting and frequently requires admission to an intensive care unit.
[0023] Clinical experience demonstrates that HD-IL2 treatment activates naive T cells and NK cells, which predominantly express the intermediate affinity receptor as well as CD25+ regulatory T cells (Tregs) which mediate the activity of CD8+ T cells. Due to their constitutive expression of CD25, Tregs are particularly sensitive to IL2. To avoid preferential activation of Tregs, IL2 variants have been developed and introduced into clinical development, which are designed to avoid binding to CD25 and possess enhanced binding to the intermediate affinity CD122/CD132 receptor to activate NK cells and quiescent CD8+ T cells. Such IL2 muteins are often referred to in the literature as “non-a-IL2” or “p/y-I L2” muteins. However, such “non-a-IL2” or “p/y-IL2” muteins, by virtue of their reduced binding to CD25, also avoid binding to the antigen activated T cells which have been identified as the primary mediators of anti-tumor T cell response (Peace, D. J. and Cheever, M. A. (1989) J Exp Med 169(1): 161-173).
[0024] Additionally, preclinical experiments have implicated NK cells as the dominant mechanism for IL2 mediated acute toxicity'. Assier E, et al. (2004) J Immunol 172:7661-7668. As NK cells express the intermediate affinity (CD122/CD132; p/y) IL2 receptor, the nature of such p/y-IL2 muteins is to enhance the proliferation of such NK cells which may lead to enhanced toxicity. Additionally, although Tregs are associated with down-regulation of CD8+ T cells, Tregs have also been shown to limit the IL2 mediated off-tumor toxicity (Li, et al. (2017) Nature Communications 8(1): 1762). Although nitric oxide synthase inhibitors have been suggested to ameliorate the symptoms of VLS, the common practice when VLS is observed is the withdrawal of IL2 therapy. To mitigate the VLS associated with HD IL2 treatment, low-dose IL2 regimens have been tested in patients. While low dose IL2 treatment regimens do partially mitigate the VLS toxicity, this lower toxicity was achieved at the expense of optimal therapeutic results in the treatment of neoplasms.
[0025] In light of the pluripotent effects of hIL2 and its demonstrated ability' to modulate the activities of a wide variety of cell types associated with human disease, IL2 muteins that retain certain desirable features of the native molecule while minimizing undesirable features, depending on the therapeutic context, remain an active area of research.
Clinical Experience with the Combination of hIL2 and hIL12
[0026] In view of the established anti -tumor potential of hIL12 and hIL2, a variety of preclinical and clinical studies have been conducted to evaluate the potential combinatorial effects of these agents. In preclinical models, IL-12 administration has been shown to promote tumor clearance (Ardolino et al., . (2014) J. Clin. Invest. 124: 4781—4794; Brunda et al., . (1993, ) J. Exp. Med. 178:1223-1230; Brunda et al. (1995) J. Immunother. Emphasis Tumor Immunol. 17: 71-77; Momin et al., . (2019) Sci. Transl. Med. 11, eaaw2614). Although IL12 has been evaluated in a clinical trials alone by a variety of routes of administration, dosing schedules, both alone and when combined a variety of other agents, (see e.g., Lasek, et al. (2104) Cancer Immunol Immunotherapy 63(5): 419-435). The clinical experience has demonstrated that the toxicity' has been a barrier to therapeutic use in humans (Cohen, J. (1995;) Science 270:908; Leonard et al., . (1997).) Blood 90: 2541-2548 ). In the more than 30 years of efforts since the initial clinical trials, there has yet to be an IL12 treatment modality that has demonstrated acceptable levels toxicity to permit marketing approval from a major regulatory agency. In mice, IL- 12 toxicity is mediated by NK cells when co-administered with IL-2 (Carson et al., . (1999) J. Immunol. 162: 4943-4951). The clinical experience with the administration of hIL12 agents in combination hIL2 agents to human subjects has demonstrated that, in addition to their potentially beneficial properties, significant dose limiting toxicities. As of the date of this writing, the only form of recombinant human IL2 currently approved for use in human subjects is Proleukin® (aldesleukin, desAlal/C125S hIL2). Proleukin was approved by the US FDA in 1992 for treatment of adults (>18 years old) with metastatic renal cell carcinoma and in 1998 for the treatment of metastatic melanoma. Nevertheless, the Proleukin package insert current provides warnings of potentially life- threatening side effects . There is no IL 12 agent currently approved by a maj or regulatory authority nor for its use in combination therapy including in combination with hIL2.
[0027] Consequently there remains a unmet medical need for treatment modalities that enable the use of IL2 and IL12 molecules retaining the beneficial antitumor properties of IL2 and hIL12 in the treatment in the treatment of neoplastic disease while exhibiting clinically acceptable and/or clinically manageable toxicity.
SUMMARY OF THE DISCLOSURE
[0028] The present disclosure is direct to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of human disease. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of neoplastic disease in a mammalian subject. Although wild-type hIL2 and wild-type hIL12 molecules are independently associated with significant toxicity when administered to a mammalian subject (e.g. humans), the applicants have surprisingly found that the administration to a mammalian subject an hIL2M of the present disclosure in combination with an hIL12M of the present disclosure results in enhanced anti -tumor effect in a mammalian subject relative to either of the hIL2 or hIL12 administered independently and that the administration of an efficacious dose of the hIL2M in combination with an hIL12M does not result in unacceptable toxicity.
[0029] In one embodiment, the present disclosure is directed to the use of a human IL2 mutein in combination with a human IL 12 mutein in the treatment of neoplastic disease in a human being suffering from a neoplastic disease. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of cancer in a mammalian subject. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL 12 muteins in the treatment of a human subject that exhibits one or more clinical indicia of the presence of cancer in the subject. In one embodiment, the present disclosure is directed to the use of human IL2 muteins in combination with human IL12 muteins in the treatment of a human subject that exhibits one or more clinical indicia of the presence of a neoplasm in the subject.
[0030] In one embodiments, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
(a) administering to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M), the hIL12M comprising a P35 (hIL 12P35) subunit and P40M (hIL12P40M) subunit wherein:
• the HL12P35 subunit [has at least 95% sequence identity to mature wild type human ML12P35 (SEQ ID NO:2);
• the HL12P40M subunit of the hIL12M [has at least 95% sequence identity to mature wild type human HL12P40 (SEQ ID NO:4), the ML12P40M subunit further comprising] comprises one or more amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40; and
(b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutem (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132, and wherein step (a) is performed in combination with step (b).
[0031] In one embodiment of the method of the disclosure, the IL12p40M comprises one or more the amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL 12M to the extracellular domain of the hIL 12Rb 1 receptor compared to the wild type human IL12p40. Examples of positions of amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild ty pe human IL12p40 is an amino acid substitution at one or more of the residues selected from the group consisting of W37, P39. D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, K219 and and K282 numbered in accordance with SEQ ID NO:3 (hIL12P40 precursor). Examples of positions of amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is an amino acid substitution or set of amino acid substitutions selected from the group consisting of: W37A; P39A; D40A; E81A; E81N; E81D; E81C; E81Q; E81E; E81P; E81W; E81Y; F82A; F82R; F82E; F82H; F82K; F82P; F82W; F82Y; K106A; K106N; D109A; K217A; K219A; E81A/F82A; W37A/E81A/F82A; E81A/F82A/K106A; E81A/F82A/K106A/K219A; E81A/F82A/K106A/K217A;
81A/F82A/K106A/E108A/D115A; E81F/F82A; E81K/F82A, E81L/F82A; E81H/F82A; E81S/F82A; E81A/F82A/K106N; E81A/F82A/K106Q; E81A/F82A/K106T;
E81A/F82A/K106R; and P39A/D40A/E81A/F82A. In one embodiment, the amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is selected from the group consisting of 2xAla = E81A/F82A; 3xAla = E81A/F82A/K106A; and 4xAla: E81A/F82A/K106A/K217A. In one embodiment, the amino acid substitutions that reduce the binding affinity of the hIL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is the set of amino acid substitutions E81A/F82A/K106A. [0032] In some embodiments, the h hIL12M is an hIL12M-Fc heterodimer wherein the HL12P35 and hIL12P40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein:
• LI and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present);
• UH1 and UH2 are each an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering);
• Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fc2;
• Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fcl; and wherein the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.
[0033] In some embodiments, the GSA linker is a polypeptide having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19 or 20 amino acids the polypeptide comprised of amino acids selected from the group consisting of glycine, serine and alanine, optional a glycine-serme polymer of the structure (GGGGSm)n (SEQ ID NO: 192), (GGGSm)n (SEQ ID NO: 193), (GGGAm)n (SEQ ID NO: 194) and (GGGGAm)n (SEQ ID NO: 195), and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4. In some embodiments, the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS : 27-79. In some embodiments, the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS: 36, 37 and 65.
[0034] In some embodiments ofthehIL12M-Fc, Fcl and Fc2 is each a naturally occurring upper hinge region of a human immunoglobulin selected from the UH regions of human IgGl, human IgG2, human IgG3 and human IgG4 upper hinge domains. In some embodiments, the upper hinge region is selected from the group consisting of EPKSC (SEQ ID NO: 11) and EPKSS (SEQ ID NO: 12). [0035] In some embodiments of the hIL12M-Fc, Fcl and Fc2 comprise amino acid substitutions that promote heterodimerization between Fcl and Fc2. In some embodiments of the hIL12M-Fc, one of Fcl and Fc2 comprises the amino acid substitutions S364H/T394F and the other of Fcl and Fc2 comprises the amino acid substitutions Y349T/F405A numbered in accordance with the EU numbering system In some embodiments of the hIL12M-Fc, one of Fcl and Fc2 comprises amino acid substitutions T350V/L351Y/F405 A/Y407V and the other of Fcl and Fc2 comprises the amino acid substitutions T350V/T366L/K392L/T394W. In some embodiments of the hIL12M-Fc, one of Fcl and Fc2 comprises amino acid substitutions K360E/K409W and the other of Fcl and Fc2 comprises the ammo acid substitutions Q347R/D399V/F405T numbered in accordance with the EU numbering system. In some embodiments of the hIL12M-Fc, one of Fcl and Fc2 comprises amino acid substitutions to provide a knob and the other of Fcl and Fc2 comprises amino acid substitutions provide a hole, optionally wherein the amino acid substitution to provide a knob is the T366W and the acid substitutions to provide a hole is the set of amino acid substitutions T366S/L368A/Y407V numbered in accordance with the EU numbering system.
[0036] In some embodiments of the hIL12M-Fc, Fcl and Fc2 are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds , optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) C96 of the ML12P35 and C199 of the hIL12P40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution numbered in accordance with the EU numbering system.
[0037] In some embodiments of the hIL12M-Fc, Fcl and Fc2 comprise one or more ammo acid substitutions to reduce effector function. In some embodiments, the hIL12M is a hIE12M-Fc wherein either or both of the hIE12P35 and hIL12P40M subunits of the heterodimeric hIL12M- Fc mutein comprise one or more amino acid substitutions to reduce effector function. In some embodiments, the hIL12P35 and/or hIL12P40M polypeptides comprise a set of amino acid substitutions selected from the group consisting of: (a) L234A/L235A/P329A (“LALAPA”); L234A/L235A/P329G (“LALAPG”); L234A/L235E/G237A/A330S/P331S (“AEASS”); and L234F/L235E/P331S (“FES”). In some embodiments of the hIL12M-Fc comprising one or more amino acid substitutions to reduce effector function, the amino acid substitutions to reduce effector function selected from the group consisting of: E234E;E234A/E235A; L234A/E235A/P329A; and L234A/L235A/P329G numbered in accordance with the EU numbering system. [0038] In some embodiments of the hIL12M-Fc, Fcl and Fc2 are human IgG4 Fc domains and one or both of Fcl and Fc2 comprise one or more amino acid substitutions to eliminate N- or 0 linked glycosylation sites, optionally wherein the modification to eliminate N- or 0 linked glycosylation sites is selected from the group consisting of N297Q and N297G numbered in accordance with the EU numbering system.
[0039] In some embodiments, the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises amino acid substitutions in the Fc domain at positions M428 and/or N434 (EU numbering). In some embodiments the amino acid substitutions at positions M428 and/or N434 are M428L and/or N434S.
[0040] In some embodiments of the hIL12M-Fc, Fcl and Fc2 comprise a deletion of: (a) the lysine residue at position 447 or (b) a deletion of both the glycine at position 446 and the lysine residue at position 447 numbered in accordance with the EU numbering system.
[0041] In some embodiments of the hIL12M is PEGylated. In some embodiments wherein the hIL12M where PEGylated, the PEG has a molecular mass greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 5 OkDa. In some embodiments wherein the hIL 12M where PEGylated, the hIL12 is a hIL12M-Fc heterodimer comprises of polypeptide of formula [1] and polypeptide of formula [2] and wherein and PEG covalently linked to one or both of the C220 residues ( EU Numbering) of the upper hinge regions of the polypeptide of formula [1] and polypeptide of formula [2],
[0042] In some embodiments of the hIL12M, the hIL12P40M subunit comprises an ammo acid substitution at position 282 (numbered in accordance with the hIL12P40 precursor, SEQ ID NOG) selected from the group consisting of K282G, K282A, K282N, K282QG or K282.
[0043] In some embodiments of the hIL12M, the hIL12M is recombinantly expressed in a mammalian host cell, optionally wherein the mammalian host cell is selected from HEK293 cells and CHO cells.
[0044] In some embodiments ofthehIL12M, the binding affinity ofhIL12M forthe extracellular domain (ECD) of IL 12RJ31 is reduced by at least 5%, optionally by at least 10%, optionally by at least 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, optionally by at least 70%, relative to the binding affinity of wild type hIL12 for the extracellular domain (ECD) of IL12R.β1 as determined by surface plasmon resonance. [0045] In some embodiments of the hIL12M, the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
[0046] In some embodiments of the hIL12M, the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in in signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
[0047] In some embodiments of the hIL12M, the hIL12M decreased STAT-4 mediated signaling wherein the STAT4 signaling in NK cells is decreased by at least 10%, optionally by at least a 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, or optionally by at least 70% signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
[0048] In some embodiments, the hIL12M is an IL12M-Fc comprising a polypeptide of the formula [1] selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153. In some embodiments, the hIL12M is an IL12M- Fc comprising a polypeptide of the formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124. In some embodiments, the hIL12M is an IL12M-Fc comprising a polypeptide of the formula [1] is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 and a polypeptide of the formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
[0049] In some embodiments, the hIL12M is an IL12M-Fc comprising a first polypeptide the formula #1 is a polypeptide having an amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKA AGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTI STDLTFSVKSSRGS SDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAC PAAEESLPIEVMVDAVHKLKYENYTSS FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEY PDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNASISVRAQDRYYSS SWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVLHE
ALHSHYTQKSLSLS PG ( SEQ ID NO : 129 ) , and the polypeptide of the formula [2] is a polypeptide having an amino acid sequence:
RNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLS S I YEDLKMYQVE FKTMNAKLLMDPKRQI FL DQNMLAVI DELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVT IDRVMSYLNA SGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS H E D P EVK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) , and wherein wherein the polypeptide of formula [1] is linked to the polypeptide of formula [2] by at least one disulfide bond, optionally wherein the hIL12M is recombinantly procuced in
CHO cells.
[0050] In some embodiments, the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to eliminate the sulfhydryl side chain. In some embodiments, the substitution at position C220 is C220S (EU numbering) substitution.
[0051] In some embodiments, the hIL12M is a hIL12M-Fc wherein n either or both of the hIL12P35 and hIL12P40M subunits of the heterodimeric hIL12M-Fc mutein comprises amino acid deletions in the Fc domain at positions G446 and/or K447 (EU numbering).
[0052] In some embodiments, the hIL12M is a hIL12M-Fc wherein either or both of the hIL12P35 and HL12P40M subunits of the heterodimeric hIL12M-Fc mutem are PEGylated. In some embodiments, either or both of the hIL12P35 and hIL12P40M subunits are PEGylated via the sulfhydryl side chain of amino acid C220 of the upper hinge.
[0053] In some embodiments, the hIL12M is a hIL12M-Fc wherein hIL12M-Fc mutein: (i) induces hIL-12 signaling in CD8+ T cells; and (ii) has decreased (for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decreased) hIL-12 signaling in NK cells compared to a wildtype hIL-12comprising a P40 polypeptide lacking the one or more amino acid substitutions.
[0054] In some embodiments, the hIL12M is a hIL12M-Fc that activates interferon gamma (IFNy) in CD8+ T cells and has decreased IFNy signaling in CD8+ T cells, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, compared to the wildtype IL 12 comprising a P40 subunit lacking such amino acid substitutions. [0055] In some embodiments, the hIL12M is a hIL12M-Fc that has a reduced binding affinity, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduction, for hIL-12R.β1 compared to the binding affinity of a wildtype IL12.
[0056] In some embodiments, the hIL12M is a hIL12M-Fc that has decreased STAT-4 mediated signaling, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, compared to wildtype hIL12 in a when evaluated in a mammalian cell-based assay.
[0057] The present disclosure further provides a nucleic acid sequence encoding a polypeptide of the formula [1]:
ML12P40M- Lla-UH1— Fcl [1] wherein: hIL12P40M is an human P40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with wild-type pre- human P40 (SEQ ID NO:3); LI is a GSA linker and a is selected from 0 (absent) or 1 (present); UH1 is an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl , IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization.
[0058] The present disclosure further provides a nucleic acid sequence encoding a polypeptide of the formula [2] : hIL12P35- L2b-UH2— Fc2 [2] wherein: HL12P35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99%, or 100% sequence identity to SEQ ID NO:2; L2 is a GSA linker and b is selected from 0 (absent) or 1 (present); UH2 is an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); and Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization. IL2 Muteins
[0059] In some embodiments, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject by administration of an human IL2 mutein in combination with a human IL2 mutein.
[0060] In some embodiments of the method of the present disclosure the human IL2 mutein comprising one or more amino acid substitutions or deletions at positions 1, 2, 3, 4, 5, 6, 18, 22, 125, and 126 numbered in accordance with mature wild type human IL2. In some embodiments of the methods of the present disclosure, the one or more supplementary therapeutic agents supplementary therapeutic agent is a biased IL2 mutein having reduced affinity for the CD132 subunit of the IL2 receptor as described in are Emmerich, et al., PCT International Application Number PCT/U S2021/013456 published July 22, 2021 as WO2021/146436A2; Emmerich, et al PCT/U S2021/013514 published July 22, 2021 as WO2021/146481A1 and Garcia, et al PCT/US2018/062122 published May 31, 2019 as W02019/104092A1, the entire teachings of which are hereby incorporated by reference.
[0061] In some embodiments of the methods of the present disclosure, human IL2 mutein comprising amino acid substitutions at positions 18, 22 and 126 numbered in accordance with mature wild type human IL2. In some embodiments of the methods of the present disclosure, the one or more supplementary therapeutic agents suppl ementary therapeutic agent is a biased human IL2 mutein comprising amino acid substitutions 18R, Q22E and Q126K. In some embodiments of the methods of the present disclosure, the one or more supplementary therapeutic agents is a biased IL2 mutein comprising amino acid substitutions t positions 18, 22 and 126 numbered in accordance with mature wild type human IL2 that is PEGylated.
[0062] In one embodiment, the present disclosure is directed to the use of a human IL2 mutein in combination with a human IL 12 mutein in the treatment of neoplastic disease in a human being suffering from a neoplastic diseas the method comprising the step of administering to the subject a therapeutically effective amount of an hIL2 mutein comprising one or more amino acid substitutions relative to the wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132. In some embodiments, the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 (numbered in accordance with mature human wild type (wt hIL2; SEQ ID NO: 182). In some embodiments, the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD 132 are selected from amino acid substitutions at positions 18, 22, and 126 are selected from the amino acid substitution at position 18 selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, L18I, L18Y, L18H, L18D, L18N and L18T; the amino acid substitution at position 22 selected from the group consisting of Q22F, Q22E, Q22G, Q22A, Q22L, Q22M. Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, and F; and the amino acid substitution at position 126 selected from the group consisting of Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T. In some embodiments, the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 are selected from the groups of amino acid substitutions consisting of L18R/Q22E/Q126K (“REK”); L18R/Q22E/Q126H (“REH”); L18A/Q22E/Q126H (“AEH”); L18A/Q22E/Q126K (“AEK”); L18D/Q22E/Q126H (“DEH”); L18E/Q22E/Q126H (“EEH”); L18E/Q22E/Q126K (“EEK”); L18F/Q22E/Q126H (“FEH”); L18G/Q22E/Q126H (“GEH”); L18H/Q22E/Q126H (“HEH”); L18H/Q22E/Q126K (“HEK”); L18I/Q22E/Q126H (“IEH”); L18I/Q22E/Q126K (“IEK”); L18K/Q22E/Q126H (“KEH”); L18M/Q22E/Q126H (“MEH”); L18N/Q22E/Q126H (“NEH”); L18Q/Q22E/Q126H (“QEH”); L18R/Q22A/Q126H (“RAH”); L18R/Q22D/Q126H (“RDH”); L18R/Q22E/Q126E (“REE”); L18R/Q22E/Q126M (“REM”); L18R/Q22E/Q126T (“RET”); L18R/Q22E/Q126V (“REV”); L18R/Q22E/Q126L (“REL”); L18R/Q22E/Q126F (“REF”); L18R/Q22E/Q126N (“REN”); L18R/Q22E/Q126R (“RER”); L18R/Q22E/Q126Y (“REY”); L18R/Q22F/Q126H (“RFH”); L18R/Q22G/Q126H (“RGH”); L18R/Q22H/Q126H (“RHH”); L18R/Q22I/Q126H (“RIH”); L18R/Q22K/Q126H (“RKH”); L18R/Q22L/Q126H (“RLH”); L18R/Q22M/Q126H (“RMH”); L18R/Q22N/Q126H (“RNH”); L18R/Q22R/Q126H (“RRH”); L18R/Q22S/Q126H (“RSH”); L18R/Q22T/Q126H (“RTH”); L18R/Q22T/Q126K (“RTK”); L18R/Q22V/Q126H (“RVH”); L18R/Q22W/Q126H (“RWH”); L18R/Q22Y/Q126H (“RYH”); L18S/Q22E/Q126H (“SEH”); L18T/Q22E/Q126H (“TEH”); L18V/Q22E/Q126H (“VEH”); L18V/Q22E/Q126K (“VEK”); L18W/Q22E/Q126H (“WEH”); and L18Y/Q22E/Q126H (“YEH”).
[0063] In some embodiments, the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD132 are selected from amino acid substitutions at positions 18, 22, and 126 (numbered in accordance with mature human wild type (wt hIL2; SEQ ID NO: 182), hIL2M may optionally further comprise one or more amino acid substitutions selected from the group consisting of T3C, T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, K35E; R38W, R38G; M39L, M39V; H55Y; V69A; Q74P, Q74N, Q74H, Q74S; M104A; D109C or a non-natural ammo acid with an activated side chain at position 109; AA113, T113N; AA125 is C125A or C125S; S130T, S130G and S130R. [0064] In some embodiments. hIL2M useful in the practice of the present disclosure, the hIL2M may optionally comprise a deletion of one or more N-terminal amino acid selected from the group or deletions: des-Al; des-Al/des-P2; des-Al/des-P2/des-T3; des-Al/des-P2/des-T3/des-S4; des- Al/des-P2/des-T3/des-S4/des-S5; des-Al/des-P2/des-T3/des-S4/des-S5/des-S6; des-Al/des- P2/des-T3/des-S4/des-S5/des-S6/des-T7.
[0065] In one embodiment of the present disclosure, the hIL2M comprises a des Alai /L18R/Q22E/Q126K hIL2M having the amino acid sequence:
PTS SSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFCKS I ISTLT ( SEQ ID NO : 188 ) .
[0066] In one embodiment of the present disclosure, the hIL2M comprises a des Alai /L18R/Q22E/Q126K hIL2M having the amino acid sequence:
PTS SSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN RWITFCKS I ISTLT ( SEQ ID NO : 188 ) and further comprises a 40kD branched chain PEG comprising two 20Kd arms covalently linked to the N-terminal proline residue of the polypeptide.
[0067] In some embodiments, hIL2M useful in the practice of the present disclosure exhibits at least a 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of reduction in the binding affinity to CD 132 of wild-type hIL2 as determined by surface plasmon resonance.
[0068] In some embodiments, hIL2M useful in the practice of the present disclosure exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of the binding affinity to CD25 of wild-type hIL2 as determined by surface plasmon resonance.
[0069] In some embodiments, hIL2M useful in the practice of the present disclosure exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90%, optionally at least 100% of the binding affinity to CD25 of wild-type hIL2 as determined by surface plasmon resonance.
[0070] In some embodiments, hIL2M is covalently linked to a earner molecule that provides for an extended duration of action in a mammalian subject. In some embodiments, the carrier molecule is selected from the group consisting of Fc polypeptides, hydrophilic polymers (e g. PEG), hydrophobic polymers (e.g. fatty acid molecules) acylated), human serum albumin. In some embodiments, the hydrophilic polymer is polyethylene glycol.
[0071] In one embodiment, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
(a) administering to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M) wherein: the hIL12M is an hIL12M-Fc heterodimer wherein the hIL12P35 and HL12P40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDT PEEDGITWTLDQS SEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKT FLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSS DPQGVT C G AAT L S AE RVRG DN KE Y E Y S VE C QE D S AC P AAE E S L P I E VMVD AVH KL KYENYTS S FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNAS I SVRAQDRYYS SSWSEWASVPCSGGGGSGGGGSEPKS SDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLS PG ( SEQ ID NO : 129 ) , and the polypeptide of the formula [2] is a polypeptide having an ammo acid sequence:
RNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTS EEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSC LASRKTS FMMALCLSS I YEDLKMYQVEFKTMNAKLLMDPKRQI F LDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCIL LHAFRIRAVTI DRVMSYLNASGGGGSGGGGSEPKSSDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALAAPIEKTI SKAKGQPREPQVCTLPPSRDELTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGS F FLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) , and wherein wherein the polypeptide of formula [1] is linked to the polypeptide of formula [2] by at least one disulfide bond ; and
(b) administering to the mammalian subj ect a therapeutically effective amount of an hIL2 mutein (hIL2M), comprises a polypeptide having the amino acid sequence:
PTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFY
MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCKS I ISTLT
( SEQ ID NO : 188 ) wherein the N-terminal proline residue is covalently linked to a 40kD branched chain PEG comprising two 20Kd arms.
[0072] In some embodiments of the method of the present disclosure, the administration of the hIL2M and hIL12M to a mammalian subject may be achieved by administration to the subject of: (a) a pharmaceutically acceptable formulation comprising an hIL2M in combination with ) a pharmaceutically acceptable formulation comprising an hIL12M;(b) a pharmaceutically acceptable formulation comprising an hIL2M and an hIL2M (co-formulated for administration; (c) a nucleic acid molecule(s) or vector(s) comprising a nucleic acid sequence(s) encoding a heterodimeric hIL2M in combination with a nucleic acid molecule(s) or vector(s) comprising a nucleic acid sequence(s) encoding a hIL12M; (d) a recombinantly modified cell comprising a nucleic acid molecules encoding a heterodimeric hIL2M and hIL12M.
[0073] In one aspect, the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are dosed simultaneously. In another aspect, the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are dosed contemperanously. In another aspect, the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M wherein the hIL2M and hIL12 are modified to promote extended half-life in vivo. In another aspect, the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M-PEG in combination with an hIL12M-Fc, wherein the HL2M-PEG is dosed about every two weeks, alternatively about every three weeks, alternatively about ever}' 4 weeks, and the hIL12M-Fc is dosed about ever}' two weeks, alternatively about ever}' three weeks, or alternatively about every 4 weeks. In another aspect, the disclosure provides a method of treating a neoplastic disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M-PEG in combination with an hIL12M-Fc.
[0074] In another aspect, the disclosure provides a method of treating a neoplastic, infectious or autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an hIL2M in combination with an hIL12M and at least one supplementary agent .
BRIEF DESCRIPTION OF THE FIGURES
[0075] Figure 1 provides the results of an evaluation of interferon gamma induction (vertical axis) with respect to increasing concentrations of the test agent (horizontal axis) in CD8, CD4 and NK cells. Panels A, B and C illustrate the effect of wild type hIL12 in comparison to hIL12M molecules comprising amino acid substitutions E81A/F82A indicated as “2xAla”, E81A/F82A/K106A indicated as “3xAla” and substitution W37A, on CD8, CD4 and NK cells, respectively. Panels D, E and F provide the results of IFNy induction of (knob-into-hole (KiH) heterodimeric hIL12M-Fc muteins comprising wild type hIL12 in comparison to KiH heterodimeric hIL12M-Fc muteins comprising amino acid substitutions E81A/F82A indicated as “2xAla Fc”, E81A/F82A/K106A indicated as “3xAla Fc” and substitution W37A (W37A Fc), on CD8, CD4 and NK cells, respectively.
[0076] Figure 2 provides the results of an evaluation of interferon-y and STAT4 induction (vertical axis) with respect to increasing concentrations of test agents indicated on CD8+ T cells (Panel A), CD4+ T cells (Panel B), and NK cells from two different human donors (Panels C and D).
[0077] Figure 3 provides spider plots of the of the tumor volume over time of mice treated with various murine IL12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below. Tumor volume is provided on the Y axis and time is on the X axis.
[0078] Figure 4 provides body weights (Y axis) of mice treated over time (X axis) of mice treated with various murine IL 12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below.
[0079] Figure 5 provides the survival data (probability of survival on Y axis) of mice treated overtime (X axis) of mice treated with various murine IL 12 agents and murine IL12Fc muteins in an MC38 tumor model study as described more fully below.
[0080] Figure 6 provides data with respect to tumor volume (y axis) with respect to days after treatment initiation (x axis) of with mIL12 proteins in Panel A and those IL 12 protein subunits in a heterodimeric Fc format in Panel B.
[0081] Figure 7 provides an amino acid sequence alignment of the wild type murine and human P40 (IL12Ra) proteins with the signal peptide sequence highlighted. [0082] Figure 8 provides an amino acid sequence alignment of the wild type murine and human P35 (IL12Rb) proteins.
[0083] Figure 9 provides a graphical representation of the concentration in picograms per milliliter (pg/mL) of murine interferon gamma (y-axis) measured in serum obtained from blood samples taken over time (x-axis) in the MC38 tumor model, the design of which is provided in Table 12. Panel A provides the concentration in picograms per milliliter (pg/mL) of murine interferon gamma in serum of treatment groups A-E of Table 12 at 0 hours (pretreatment) and 4 hours, 1 day and 7 days following treatment with the test agent. Panel B relates to the murine interferon gamma levels in serum of treatment groups F, G, and H of Table 12 at 0 hours (pretreatment) and 4 hours, 1 day and 7 days following treatment with the test agent.
[0084] Figure 10 is a graphical representation of the percentage of lymphocytes (y-axis) as determined by FACS analysis with respect to each of the treatment groups of Table 12 (x-axis) in spleen and tumor tissue.
[0085] Figure 11 provides the results of a phenotypic FACS analysis of NK cells obtained from spleen in the treatment groups and study described in Table 12. T-bet is measured on the vertical axis and intracellular Granzyme B on the horizontal axis. The graphs are labeled with the treatment groups (A, F, G and H of Table 12).
[0086] Figure 12 provides a series of spider plots resulting from the CT26 tumor study as described herein with tumor volume provided on the vertical axis and time (study days) on the horizontal axis. Each panel of the figure indicates the test agent provide and the dosing schedule of the test agent in accordance with the study design provided in Table 13.
[0087] Figure 13 provides the results of body weight measurements of mice evaluated in the CT26 tumor study with percent change in bodyweight on the vertical axis and time (study days) on the horizontal axis.
[0088] Figure 14 provides results of a FACS analysis of cells obtained from the CT26 tumor study study sorted by the presence of various markers as indicated by the arrows and various doses of test agent as indicated by the figure legends.
[0089] Figure 15 provides the results of the efficacy of the various test agents indicated by the figure legends in response to depletion of NK cells in the study summarized in Table 14.
[0090] Figure 16 provides the results of evaluation of percent change in body weight (vertical axis) over the course of the study (horizontal axis) in response to the various test agent conditions provided in the MC38 tumor study to evaluate the effects of NK and CD8 cell depletion as provided in detail herein, the study design of which is summarized in Table 15.
[0091] Figure 17 provides a series of spider plots with respect to the antitumor efficacy (tumor volume on the vertical axis) over the course of the study (horizontal axis) in response to the various test conditions identified in Table 15.
[0092] Figure 18 provides the results of antitumor efficacy of IL12 test agents against MC38 tumors in various types of mice, B6 mice in the two figures in the first column, RAG2 knockout mice in the two figures in the second column and RAG2/CD132 double knockout mice in the third column. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis). The figure legends identify the various test conditions provided in Table 15.
[0093] Figure 19 provides the results of a study evaluating the anti -tumor efficacy of a heterodimeric mIL12M-Fc polypeptide in combination with a PD1 inhibitor in the treatment of MC38 tumors in mice. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis). The figure legends correspond to the treatment groups summarized in Table 16 herein.
[0094] Figure 20 provides the results of a study evaluating the anti-tumor efficacy of a heterodimeric mIL12M-Fc polypeptide in combination with an mIL2 mutein polypeptide in the treatment of MC38 tumors in mice. Tumor volume is provided on the vertical axis over the course of the study (horizontal axis). The figure legends correspond to the treatment groups summarized in Table 17 herein.
[0095] Figure 21 provides data relating to STAT4 signaling in NK cells of human and murine surrogate IL12-Fcs. The upper Panel A provides data relating to the induction of STAT4 signaling (y-axis) of varying concentrations (x-axis) of mIL-12 Fc (Compound 1) and mIL12M-Fc (Compound 2) molecules on murine NK cells are described in Example 5 herein. The lower Panel A provides data relating to the induction of STAT4 signaling (y-axis) of varying concentrations (x-axis) of hIL12-Fc and hIL12M-Fc molecules on human NK cells as more fully described in Example 5.
[0096] Figure 22 provides the results of body weight measurements obtained from of animals treated in the MC38 tumor study described in more detail in Example 6 herein wherein the percent of initial bodyweight is provided on the y-axis and time (Study Days) is presented on the x-axis wherein each line represents an individual animal treated. [0097] Figure 23 provides a spider plot of the results of tumor volume measurements obtained from animals treated in the MC38 tumor study described in more detail in Example 6 herein. Tumor volume (y-axis) versus study day (x-axis) is provided with respect to individual animal tumor volumes is provided wherein each line represents an individual animal treated.
[0098] Figure 24 provides the results of body weight measurements obtained from of animals treated in the MC38 tumor study described in more detail in Example 7 herein wherein the percent of initial bodyweight is provided on the y-axis and time (Study Days) is presented on the x-axis wherein each line represents an individual animal treated.
[0099] Figure 25 provides a spider plot of the results of tumor volume measurements obtained from animals treated in the MC38 tumor study described in more detail in Example 7 herein. Tumor volume (y-axis) versus study day (x-axis) is provided with respect to individual animal tumor volumes is provided wherein each line represents an individual animal treated.
DETAILED DESCRIPTION OF THE INVENTION
[0100] To facilitate the understanding of present disclosure, certain terms and phrases are defined below as well as throughout the specification. The definitions provided herein are non- limiting and should be read in view of the knowledge of one of skill in the art.
[0101] Before the present methods and compositions are described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting.
[0102] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications, patents, published patent applications, GenBank accession numbers and UniProt reference numbers mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0104] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
[0105] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be constmed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.
[0106] Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp = base pair(s); kb = kilobase(s); pl = picoliter(s); s or sec = second(s); min = minute(s); h or hr = hour(s); AA or aa = amino acid(s); kb = kilobase(s); nt = nucleotide(s); pg = picogram; ng = nanogram; pg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; pl or pL = microliter; ml or mL = milliliter; 1 or L = liter; pM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. = intramuscular(ly); i.p. = mtrapentoneal(ly); SC or SQ = subcutaneous(ly); QD = daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbeco’s Modification of Eagle’s Medium; EDTA = ethylenediaminetetraacetic acid.
[0107] It will be appreciated that throughout this disclosure reference is made to amino acids according to the single letter or three letter codes. For the reader’s convenience, the single and three letter amino acid codes are provided in Table 1.
Figure imgf000030_0001
[0108] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).
Nomenclature of Amino Acid Substitutions and Deletions
[0109] The present disclosure provides variant polypeptides comprising amino acid substitutions relative to the wild-type or parent polypeptide. The following nomenclature is used herein to refer to substitutions, deletions or insertions. Residues may be designated herein by the one-letter or three-letter amino acid code of the naturally occurring amino acid found in the wild- type molecule. [0110] HL12P35 Residue Numbering: In the present disclosure, the numbering of amino acid residues of human P35 is made in reference to the number of the precursor or “pro” form of HL12P35 as provided in SEQ ID NO: 1.
[0111] hP40 Residue Numbering: In the present disclosure, the numbering of amino acid residues of human P40 is made in reference to the number of the precursor or “pro” form of hP40 as provided in (SEQ ID NO: 3). In reference to the hP40 muteins, substitutions are designated herein by the one letter amino acid code followed by the pro-hP40 (SEQ ID NO: 3) amino acid position followed by the one letter amino acid code which is substituted. For example, an hP40 mutein having the modification “E81 A” refers to a substitution of the glutamic acid (E) residue at position 81 of the (SEQ ID NO: 3) with an alanine (A) residue at this position. A deletion of an amino acid reside is referred to as “des” or the symbol “A” followed by the amino acid residue and its position.
[0112] hP40 Residue Numbering: In the present disclosure, the numbering of amino acid residues of human IL2 is made in reference to the number of the mature wild type human IL2 as provided in (SEQ ID NO: 182). In reference to a hIL2 mutein (hIL2M), substitutions are designated herein by the one letter amino acid code followed by the mature hIL2 amino acid position followed by the one letter amino acid code which is substituted. For example, an hIL2M having the modification “L18R” refers to a substitution of the leucine (L) residue at position 18 of the mature wild type human IL2 (SEQ ID NO: 182) with an arginine (R) residue at this position. A deletion of an amino acid reside is referred to as “des” or the symbol “A” followed by the amino acid residue and its position in the mature wild type human IL2 (SEQ ID NO: 182). For example, the deletion of the N-terminal alanine (A) residue at position 1 of the mature wild type human IL2 is referred to as “des-Alal”
[0113] Immunoglobulin, Upper Hinge and Fc Residue Numbering: There are a variety of numbering conventions that are employed with respect to the numbering of amino acid residues of immunoglobulins including Rabat numbering, Chothia numbering, EU numbering and IMGT numbering conventions. In the context of the present disclosure, the numbering of amino acid residues of immunoglobulin molecules including domains thereof including the upper hinge and Fc domain (comprising the lower hinge, CH2 and CH3 domains) is made in accordance with EU Numbering conventions. Translation of EU numbering conventions used herein to Rabat numbering, Chothia numbering, or IMGT numbering conventions is readily understood by those of skill in the art. Dondelinger, et al. (2018) Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition Frontiers in Immunology Volume 9 Article #:2278. [0114] Additionally, in certain instances herein, an “M” suffix may be added to a polypeptide number (e.g. DR1535M) to identify such sequence as a “mature” molecule lacking a signal sequence so as to distinguish the polypeptide from the precursor molecule containing the signal peptide which precursor form may be identified with a “P” suffix such as DR1535P.
Definitions
[0115] Unless otherwise indicated, the following terms are intended to have the meaning set forth below. Other terms are defined elsewhere throughout the specification.
[0116] The term “about” refers to a value that is plus or minus 10% of a numerical value described herein, such as plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of numerical value described herein. The term “about” also applies to all numerical ranges described herein. All values described herein are understood to be modified by the term “about” whether or not the term “about” is explicitly recited in reference to a given value.
[0117] Activate: As used herein the term “activate” is used in reference to a receptor or receptor complex to reflect a biological effect, directly and/or by participation in a multicomponent signaling cascade, arising from the binding of an agonist ligand to a receptor responsive to the binding of the ligand. The term activate is also used in reference to a cell that expresses a receptor wherein one more biological activities of the cell are modulated (e.g. upregulation or downregulation of STAT signaling, in response to binding of a ligand for such receptor.
[0118] Activity: As used herein, the term “activity” is used with respect to a molecule to describe a property of the molecule with respect to a test system (e.g., an assay) or biological or chemical property (e.g., the degree of binding of the molecule to another molecule) or of a physical property of a material or cell (e.g., modification of cell membrane potential). Examples of such biological functions include but are not limited to catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, and the ability to modulate immunological activity such as inflammatory response. “Activity” is typically expressed as a level of a biological activity per unit of agent tested such as [catalytic activity]/[mg protein], [immunological activity]/[mg protein], international units (IU) of activity, [STAT3 phosphorylation]/[mg protein], [STAT4 phosphorylation]/[mg protein] [proliferation]/[mg protein], plaque forming units (pfu), etc. As used herein, the term proliferative activity refers to an activity that promotes cell proliferation and replication, including dysregulated cell division such as that observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis. [0119] Administer/Administration: The terms ‘‘administration” and “administer” are used interchangeably herein to refer the act of contacting a subject, including contacting a cell, tissue, organ, or biological fluid of the subject in vitro, in vivo or ex vivo with an agent Administration of an agent may be achieved through any of a variety of art recognized methods including but not limited to the topical administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection, intragastric infusion, intraprostatic injection, intravesical infusion (e.g., bladder), inhalation (e.g respiratory inhalers including dry-powder inhalers), intraocular injection, intraabdominal injection, intralesional injection, intraovanan injection, intracerebral infusion or injection, intracerebroventricular injection (ICVI), and the like. The term “administration” includes contact of an agent to the cell, tissue or organ as well as the contact of an agent to a fluid, where the fluid is in contact with the cell, tissue or organ.
[0120] Affinity: As used herein the term “affinity” refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor) and is measured by the equilibrium dissociation constant (KD), a ratio of the dissociation rate constant between the molecule and its target (Koff) and the association rate constant between the molecule and its target (Kon).
[0121] Agonist: As used herein, the term “agonist” refers a first agent that specifically binds a second agent (“target”) and interacts with the target to cause or promote an increase in the activation of the target. In some instances, agonists are activators of receptor proteins that modulate cell activation, enhance activation, sensitize cells to activation by a second agent, or up-regulate the expression of one or more genes, proteins, ligands, receptors, biological pathways, that may result in modulation of cell proliferation or pathways or the cell cycle. In some embodiments, an agonist is a modified form of a cognate ligand that binds to its cognate receptor and alters the state of the cognate receptor in a biological response that mimics the biological effect of the interaction of the naturally occurring cognate ligand with its cognate receptor. The term “agonist” includes partial agonists, full agonists and superagonists. An agonist may be described as a “full agonist” when such agonist which leads to a substantially full biological response (i.e. the response associated with the naturally occurring ligand/receptor binding interaction) induced by receptor under study, or a partial agonist. A "superagonisf 1 is a type of agonist that can produce a maximal response greater than the endogenous agonist for the target receptor, and thus has an activity of more than 100% of the native ligand. A super agonist is typically a synthetic molecule that exhibits greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% of the response in an evaluable quantitative or qualitative parameter of the naturally occurring form of the molecule when evaluated at similar concentrations in a comparable assay. It should be noted that the biological effects associated with the full agonist may differ in degree and/or in kind from those biological effects of partial or superagonists. In contrast to agonists, antagonists may specifically bind to a receptor but do not result in the signal cascade typically initiated by the receptor and may modify the actions of an agonist at that receptor. Inverse agonists are agents that produce a pharmacological response that is opposite in direction to that of an agonist.
[0122] Antagonist: As used herein, the term “antagonist’' or “inhibitor” refers to a molecule that opposes the action(s) of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist. Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, biological pathway including an immune checkpoint pathway, or cell. In some instances, an antagonist may be a mutein of the naturally occurring ligad such that binding to receptor is maintained but there is no downstream signaling.
[0123] Biological Sample: As used herein, the term “biological sample” or “sample” refers to a sample obtained (or derived) from a subject. By way of example, a biological sample comprises a material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin enriched or cell-type specific enriched fractions derived from one or more of such tissues.
[0124] Comparable: As used herein, the term “comparable” is used to describe the degree of difference in two measurements of an evaluable quantitative or qualitative parameter. For example, where a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter do not deviate beyond a range that the skilled artisan would recognize as not producing a statistically significant difference in effect between the two results in the circumstances, the two measurements would be considered “comparable.” In some instances, measurements may be considered “comparable” if one measurement deviates from another by less than 35%, alternatively by less than 30%, alternatively by less than 25%, alternatively by less than 20%, alternatively by less than 15%, alternatively by less than 10%, alternatively by less than 7%, alternatively by less than 5%, alternatively by less than 4%, alternatively by less than 3%, alternatively by less than 2%, or by less than 1%. In particular embodiments, one measurement is comparable to a reference standard if it deviates by less than 15%, alternatively by less than 10%, or alternatively by less than 5% from the reference standard.
[0125] Corresponding To: As used herein, the terms “correspondence” or “corresponding to” in the context of an amino acid or nucleic acid sequence refers to the equivalent position of a reference sequence that is aligned with one or more other sequences to maximize the percentage of sequence identity. As used herein, the term “corresponding to” is used in the context of generating amino acid substitutions of a mutein from a first species to generate a mutein of another species such as muteins of human sequences for generating murine surrogate muteins.
[0126] Derived From: As used herein, the term “derived from” is used in the context of a variant polyprpyifr or nucleic acid to indicate that a variant polypeptide or nucleic acid has a sequence that is based on but differs from that of a reference polypeptide or nucleic acid. The term derived and is not meant to be limiting as to the source or method by which the variant protein or nucleic acid is made.
[0127] Effective Concentration (EC): As used herein, the terms “effective concentration” or its abbreviation “EC” are used interchangeably to refer to the concentration of an agent in an amount sufficient to effect a change in a given parameter in a test system. The abbreviation “E” refers to the magnitude of a given biological effect observed in a test system when that test system is exposed to a test agent. When the magnitude of the response is expressed as a factor of the concentration (“C”) of the test agent, the abbreviation “EC” is used. In the context of biological systems, the term Emax refers to the maximal magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is provided with a subscript (e.g., EC40, EC50, etc.) the subscript refers to the percentage of the Emax of the biological response observed at that concentration. For example, the concentration of a test agent sufficient to result in the induction of a measurable biological parameter in a test system that is 30% of the maximal level of such measurable biological parameter in response to such test agent, this is referred to as the “EC30” of the test agent with respect to such biological parameter. Similarly, the term “EC100” is used to denote the effective concentration of an agent that results in the maximal (100%) response of a measurable parameter in response to such agent. Similarly, the term EC50 (which is commonly used in the field of pharmacodynamics) refers to the concentration of the agent sufficient to result in the half-maximal (about 50%) change in the measurable parameter. The term “saturating concentration” refers to the maximum possible quantity of a test agent that can dissolve in a standard volume of a specific solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, a saturating concentration of a drug is typically used to denote the concentration sufficient of the drug such that all available receptors are occupied by the drug, and EC50 is the drug concentration that provides the half-maximal effect.
[0128] Enriched: As used herein in the term “enriched” refers to a sample comprising a species of interest (e.g. a molecule or cell) wherein the sample is non-naturally manipulated so that a species of interest is present in: (a) a greater concentration (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, or alternatively at least 1000-fold greater) than the concentration of the species in the starting sample, such as a biological sample (e.g., a sample in which the molecule naturally occurs or in which it is present after administration); or (b) a concentration greater than the environment in which the molecule was made (e.g., a recombinantly modified bacterial or mammalian cell).
[0129] Extracellular Domain: As used herein the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein which is external to the plasma membrane of the cell on which it is expressed. A cell surface protein comprising and ECD may be a transmembrane protein, a cell surface or membrane associated protein that comprising a domain associated with the cell membrane but which lacks an intracellular domain..
[0130] Identity: The term "identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same amino acid or nucleotide then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W of the query sequence, which either match or satisfy some positive-valued threshold score “T” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (the reward score for a pair of matching residues; always >0) and “N” (the penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: (a) the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached. The BLAST algorithm parameters “W”, “T”, and “X” determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but uses as defaults a word size (“W”) of 28, an expectation (“E”) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Hemkoff & Hemkoff, (1989) PNAS(USA) 89:10915-10919).
[0131] In Combination With: As used herein, the term “in combination w ith" when used in reference to the administration of multiple agents to a single subject refers to the administration of a first agent (e.g. an hIL12M) and a second agent (e.g. an hIL2M), optionally further administering an additional (i.e. third, fourth, fifth, etc.) supplementary agent to a subject, simultaneously, contemporaneously or sequentially. The term “in combination with” shall also understood to apply to the situation where a first agent and a second agent are co-formulated in single pharmaceutically acceptable formulation and the co-formulation is administered to a subject. A first agent is considered to be administered in combination with a second agent if the first and second agents are administered simultaneously , contemporaneously or sequentially. A first agent is administered “simulatenously” with a second agent if first and second agents are administered within about 30 minutes of each other. A first agent is administered “contemporaneously” with a second agent if first and second agents are administered within about 24 hours of each another, alternatively within about 12 hours of each other, alternatively within about 6 hours of each other, alternatively within about 2 hours of each other, or alternatively within about 60 minutes of each other. In certain instances, a first agent and a second agent(s) are administered sequentially, e.g., where one agent is administered to the subject at least 24 hours prior to the administration of the second agent. When a first agent and second agent are administered to a subject sequentially, the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the biological effect of the first agent persists in the subject at the time of administration of the second agent. When administered sequentially, administration of the first agent may provide a therapeutic effect over an extended time and the administration of the second agent administered while the therapeutic effect of the first agent persists in the subject such that the second agent is considered to be administered in combination with the first agent, even though the first agent may have been administered at a point in time significantly distant (e.g. days or weeks) from the time of administration of the second agent. For example, in the case of molecules that have been designed to provide an extended duration of action in a subject (for example, by conjugation of the active agent to a carrier molecule such as an Fc polypeptide or polyethylene glycol (PEG) polymer), the administration of the first agent and the second agent may be separated by a significant period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer) may result in the maintenance of a therapeutically effective amount of the first agent over a period of weeks such that the administration of the second agent at time point days or weeks after the administration of the first agent such that the second agent would be considered to be administered in combination with the first agent. In some embodiments, the biological effect of the administration of a first therapeutic agent may persist for a period of time beyond the point where there is a detectable level of the first therapeutic agent in the subject. For purposes of the present disclosure, one agent (e.g., an hIL12M) is considered to be administered in combination with a with a second agent (e.g. an hIL2M) if the biological effect resulting from the administration of the first agent persists in the subject at the time of administration of the second agent such that the therapeutic effects of the first agent and second agent overlap, whether or not there is a detectable level of the first agent remaining in the subject at the time of administration of the second agent. The determination of whether that therapeutic effect of the first agent persists after the agent is no longer detectable can be established through the upregulation or down-regulation of biological markers that are characteristically modulated in response to the agent. One of skill in the art is capable of performing pharmacokinetic studies to determine the in vivo duration of action of the hIL12M or hIL2M molecules described herein. Studies in primates, e.g. cynomolgus monkeys, chimpanzees, rhesus monkeys, are commonly used to provide information relating to the duration of action and toxicity indicative of the response in human subjects. In one embodiment, the skilled artisan will be able to establish a therapeutically effective dose of the hIL12M in combination with an hIL2M by evaluation of pharmacokinetic data, indicators of response and/or toxicity and other factors known to the clinician. When a first agent and second agent are administered to a subject sequentially, the first agent is considered to be administered “in combination with” with the second agent if at least 10%, alternatively at least 20%, alternatively at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%, alternatively at least 95% of the initial blood or serum concentration of the first agent persists in the subject at the time of administration of the second agent. The initial blood or serum concentration of the first agent is measured at a time within 30 minutes, alternatively within 1 hour, alternatively within 2 hours, alternatively within 4 hours, alternatively within 6 hours, alternatively within 24 hours following the administration of the first agent.
[0132] In An Amount Sufficient Amount to Effect a Response: As used herein the phrase "in an amount sufficient to cause a response” is used in reference to the amount of an agent sufficient to provide a detectable change in the level of an indicator measured before (e.g., a baseline level) and after the application of a test agent to a test system. In some embodiments, the test system is a cell, tissue or organism. In some embodiments, the test system is an in vitro test system such as a fluorescent assay. In some embodiments, the test system is an in vivo system which involves the measurement of a change in the level a parameter of a cell, tissue, or organism reflective of a biological function before and after the application of the test agent to the cell, tissue, or organism. In some embodiments, the indicator is reflective of biological function or state of development of a cell evaluated in an assay in response to the administration of a quantity of the test agent. In some embodiments, the test system involves the measurement of a change in the level an indicator of a cell, tissue, or organism reflective of a biological condition before and after the application of one or more test agents to the cell, tissue, or organism. The term “in an amount sufficient to effect a response” may be sufficient to be a therapeutically effective amount but may also be more or less than a therapeutically effective amount.
[0133] In Need of Prevention: As used herein the term “in need of prevention” refers to a judgment made by a physician or other caregiver with respect to a subject that the subject requires or will potentially benefit from preventative care. This judgment is made based upon a variety of factors that are in the realm of a physician’s or caregiver’s expertise. In some embodiments, prevention refers to reducing, forestalling or delaying the onset of a particular disease, or reducing forestalling or delaying a recurrence of a particular disease, for example, after an initial course of treatment for the disease. A recurrence of a disease does not necessarily have to be after initial cure, complete remission or partial remission of a disease. It is sufficient to have one or more clinical symptoms of the initial disease reappear in a subject after a period devoid of those symptoms for the disease to be considered as recurring in the subject.
[0134] In Need of Treatment: The term “in need of treatment” as used herein refers to ajudgment made by a physician or other caregiver with respect to a subject that the subject requires or may potentially benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician’s or caregiver's expertise. In some embodiments, a subject in need of treatment has been diagnosed with a disease or condition, for example, a neoplastic disease, an autoimmune disorder or an infection. [0135] Inhibitor: As used herein the term “inhibitor” refers to a molecule that decreases, blocks, prevents, delays activation of, inactivates, desensitizes, or down-regulates, e.g., a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity of a cell or organism.
[0136] Intracellular Domain: As used herein the term "intracellular domain" or its abbreviation "ICD" refers to the portion of a cell surface protein (e.g., a cell surface receptor or receptor subnit) which is inside of the plasma membrane of a cell. A cell surface protein comprising an ICD may be a transmembrane protein or a cell surface or membrane associated protein comprising a domain associated with the cell membrane but which lacks an extracellular domain. The ICD may include the entire cytoplasmic portion of a transmembrane protein or membrane associated protein.
[0137] Isolated: As used herein the term “isolated” when used in reference to a molecule that, if naturally occurring, is in an environment different from that in which it naturally occurs. “Isolated” is meant to include molecule that are within samples that are substantially enriched for the molecule of interest and/or in which the molecule of interest is partially or substantially purified. Where the molecule is not naturally occurring, “isolated” indicates that the molecule has been separated from an environment in which it was synthesized. For example a polypeptide may be isolated from a recombinant cell culture comprising cells engineered to express the polypeptide or by a solution resulting from solid phase or cell free synthesis.
[0138] Ligand: As used herein, the term “ligand” refers to a molecule that specifically binds a receptor and where such binding causes a change in the receptor sufficient to effect a change in the activity of the receptor or results in a response in cell that expresses that receptor. In one embodiment, the term “ligand” refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. The complex of a ligand and receptor is termed a “ligand-receptor complex” (for example, the [hIL2/CD25/CD122/CD132] receptor complex). In some examples, the term “cognate ligand” and “cognate receptor” are used to denote a naturally occurring ligand and the receptor to which such ligand exhibits selective binding in a naturally occurring biological systems.
[0139] Modulate: As used herein, the terms “modulate”, “modulation” and the like refer to the ability of an agent, for example, a test agent, to cause a response, either positive or negative or directly or indirectly, in a system, including a biological system, or biochemical pathway. The term modulator includes both agonists (including partial agonists, full agonists and superagonists) and antagonists. [0140] Mutein: As used herein, the term “mutein” is used to refer to a variant of a wild type polypeptide comprising modifications to the primary structure (i.e. amino acid sequence) of such polypeptide. A mutein may have have at at least 99% sequence identity, alternatively at least 98% sequence identity, alternatively at least about 97% sequence identity, alternatively at least 96% sequence identity, alternatively at least 95% sequence identity, alternatively at least about 94% identical, alternatively at least 93% sequence identify, alternatively at least 92% identical, alternatively at least 91% sequence identify, or alternatively at least 90% sequence identify, to the parent polypeptide from which the mutein was derived.
[0141] Nucleic Acid: The terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like.
[0142] Operably Linked: The term “operably linked” is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, which are arranged in a construct such that the functions of the component molecules is retained although the operable linkage may result in the modulation of the activity, either positively or negatively, of the individual components of the construct. For example, the operable linkage of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct where the biological activity of the protein (e.g., Emax) is diminished relative to the to the wild-type molecule, however the two are nevertheless considered operably linked. When the term “operably linked” is applied to the relationship of multiple nucleic acid sequences encoding differing polypeptides, the multiple nucleic acid sequences when combined into a single nucleic acid molecule that, for example, when introduced into a cell using recombinant technology, provides a nucleic acid which is capable of effecting the transcription and/or translation of a particular nucleic acid sequence in a cell. For example, the nucleic acid sequence encoding a signal sequence may be considered operably linked to DNA encoding a polypeptide if it results in the expression of a preprotein whereby the signal sequence facilitates the secretion of the polypeptide; a promoter or enhancer is considered operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is considered operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, in the context of nucleic acid molecules, the term "operably linked" means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader or associated subdomains of a molecule, contiguous and in reading phase. However, certain genetic elements such as enhancers may function at a distance and need not be contiguous with respect to the sequence to which they provide their effect but nevertheless may be considered operably linked.
[0143] Parent Polypeptide: As used herein, the terms "parent polypeptide" or "parent protein" are used interchangeably to designate the source of a second polypeptide (e.g., a derivative, mutant or variant) which is modified with respect to a first “parent” polypeptide. In some instances, the parent polypeptide is a wild-type or naturally occurring form of a polypeptide. In some instance, the parent polypeptide may be a modified form a naturally occurring protein that is further modified. The term parent polypeptide can also be used interchangeably with “reference polypeptide.”
[0144] Partial Agonist: As used herein, the term “partial agonist” refers to a molecule (e.g., a ligand) that specifically binds to and activates a given receptor but possesses only partial activation of the receptor relative to a full agonist. The activation of the receptor may be assessed by modulation of intracellular signaling a cell expressing a receptor (e.g. modulation of intracellular levels of phospho-STAT4). Partial agonists may display both agonistic and antagonistic effects. For example, when both a full agonist and partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for the receptor binding resulting in net decrease in receptor activation relative to the contact of the receptor with the full agonist in the absence of the partial agonist. Partial agonists can be used to activate receptors to give a desired submaximal response in a subject when inadequate amounts of the endogenous ligand are present, or they can reduce the overstimulation of receptors when excess amounts of the endogenous ligand are present. The maximum response (Emax) produced by a partial agonist is called its intrinsic activity and may be expressed on a percentage scale where a full agonist produced a 100% response. An partial agonist may have greater than 10% but less than 100%, alternatively greater than 20% but less than 100%, alternatively greater than 30% but less than 100%, alternatively greater than 40% but less than 100%, alternatively greater than 50% but less than 100%, alternatively greater than 60% but less than 100%, alternatively greater than 70% but less than 100%, alternatively greater than 80% but less than 100%, or alternatively greater than 90% but less than 100%, of the activity of the reference polypeptide when evaluated at similar concentrations in a given assay system.
[0145] Polypeptide: As used herein the terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term polypeptide include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with amino acid sequences that facilitate purification such as chelating peptides; fusion proteins with immunologically tagged proteins; fusion proteins comprising a peptide with immunologically active polypeptide fragment (e.g. , diphtheria toxin or tetanus toxin fragments) and the like.
[0146] Prevent: As used herein the terms “prevent”, “preventing”, “prevention” and the like refer to a course of action initiated with respect to a subj ect prior to the onset of a disease, disorder, condition or symptom thereof so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject’s risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof A course of action to prevent a disease, disorder or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder, or condition due to genetic, experiential, or environmental factors of developing a particular disease, disorder or condition. In certain instances, the terms “prevent”, “preventing”, “prevention” are also used to refer to the slowing of the progression of a disease, disorder, or condition from an existing state to a more deleterious state. In some instances, “prevent” is used in the context of prevention of the recurrence of a disease or symptom thereof wherein a prior course of therapy may have partially or completely eliminated the evidence of the disease as measured by conventional clinical testing.
[0147] Receptor: As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds a ligand that binding of the ligand results in a change to at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane associated protein that comprises an extracellular domain (ECD) and a membrane associated domain which serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a membrane spanning polypeptide comprising an intracellular domain (ICD) and extracellular domain (ECD) linked by a membrane spanning domain referred to as a transmembrane domain (TM). In some instances, the binding of a ligand to the ECD of the receptor results in a conformational change in the receptor resulting in a measurable biological effect such as a change in the activity of the receptor or the binding affinity of the receptor for another protein. In some instances, where the receptor is a membrane spanning polypeptide comprising an ECD, TM and ICD, the binding of a ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, a receptor is a component of a multi- component complex to facilitate intracellular signaling. For example, the ligand may bind a cell surface receptor that is not associated with any intracellular signaling alone but upon ligand binding facilitates the formation of a heteromultimeric (including heterodimeric, heterotrimeric, etc.) or homomultimeric (including homodimeric, homotrimeric, homotetrameric, etc.) complex that results in a measurable biological effect in the cell such as activation of an intracellular signaling cascade (e.g., the Jak/STAT pathway). In some embodiments, a receptor is a membrane spanning single chain polypeptide comprising ECD, TM and ICD domains wherein the ECD, TM and ICD domains are derived from the same or differing naturally occurring receptor variants or synthetic functional equivalents thereof (chimeric receptor).
[0148] Recombinant: As used herein, the term “recombinant” is used as an adjective to refer to the method by which a polypeptide, nucleic acid, or cell was modified using recombinant DNA technology. A “recombinant protein” is a protein produced using recombinant DNA technology and is frequently abbreviated with a lower case “r” preceding the protein name to denote the method by which the protein was produced (e.g., recombinantly produced human growth hormone is commonly abbreviated “rhGH”). Similarly, a cell is referred to as a “recombinant cell” if the cell has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like) using recombinant DNA technology. The techniques and protocols for recombinant DNA technology are well known in the art such as those can be found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.
[0149] Response: The term “response,” for example, of a cell, tissue, organ, or organism, encompasses a quantitative or qualitative change in a evaluable biochemical or physiological parameter, (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation) where the change is correlated with the activation, stimulation, or treatment, with or contact with exogenous agents or internal mechanisms such as genetic programming. In certain contexts, the terms “activation”, “stimulation”, and the like refer to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms “inhibition”, “down-regulation” and the like refer to the opposite effects. A “response” may be evaluated in vitro such as through the use of assay systems, surface plasmon resonance, enzymatic activity, mass spectroscopy, amino acid or protein sequencing technologies. A “response” may be evaluated in vivo quantitatively by evaluation of objective physiological parameters such as body temperature, bodyweight, tumor volume, blood pressure, results of X-ray or other imaging technology or qualitatively through changes in reported subjective feelings of well-being, depression, agitation, or pain. In some embodiments, the level of activation of T cells in response to the administration of a test agent may be determined by flow cytometric methods. In some methods, a response can be measured by determining the level of STAT (e.g., STAT3, STAT4) phosphorylation, or IFNy production, in accordance with methods well known in the art.
[0150] Significantly Reduced Binding: As used herein, the term “exhibits significantly reduced binding” is used with respect to a variant of a first molecule (e.g., a ligand) which exhibits a significant reduction in the affinity for a second molecule (e.g., receptor) relative to the parent form of the first molecule. With respect to muteins of naturally occurring receptor ligands, for example, a hIL12P40 mutein, hIL12M mutein or hIL2 mutein described herein, a the mutein “exhibits significantly reduced binding” if the mutein binds to a receptor with an affinity of less than 50%, alternatively less than about 40%, alternatively less than about 30%, alternatively less than about 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the parent ligand from which the murein was derived.
[0151] Specifically Binds: As used herein the term “specifically binds” refers to the degree of affinity for which a first molecule exhibits with respect to a second molecule. In the context of binding pairs (e.g., ligand/receptor) a first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the first molecule of the binding pair does not bind in a significant amount to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the affinity of the first molecule for the second molecule is at least two-fold greater, alternatively at least five times greater, alternatively at least ten times greater, alternatively at least 20-times greater, or alternatively at least 100-times greater, or alternatively at least 500-times greater or alternatively at least 1000-times greater than the affinity of the first molecule for other components present in the sample. Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on- bead ligand binding assays, on-column ligand binding assays, and filter assays)) and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 with commercially available instrumentation such as the Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752).
[0152] Subject: The terms “recipient”, “individual”, “subject”, and “patient”, are used interchangeably herein and refer to any mammal for whom, the opinion of a skilled artisan (e.g. physicician or veterinarian), treatment is desired. As used herein, the term mammal any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some embodiments the term “subject” refers to a human being.
[0153] Substantially Pure: As used herein, the term “substantially pure” indicates that a component of a composition makes up greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition. A protein that is “substantially pure” comprises greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition comprising the protein.
[0154] Suffering From: As used herein, the term “suffering from” refers to a determination made by a physician with respect to a subject based on the available objective or subjective information accepted in the field for the identification of a disease, disorder or condition including but not limited to X-ray, PET scans, CT-scans, conventional laboratory diagnostic tests (e.g., blood count, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or will benefit from treatment. The term suffering from is typically used in conjunction with a particular disease state such as “suffering from a neoplastic disease” refers to a subject which has been diagnosed with the presence of a neoplasm.
[0155] T-cell: As used herein the term “T-cell” or “T cell” is used in its conventional sense to refer to a lymphocytes that differentiates in the thymus, possess specific cell-surface antigen receptors, and include some that control the initiation or suppression of cell-mediated and humoral immunity and others that lyse antigen-bearing cells. In some embodiments the T cell includes without limitation naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ T cells, helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TRI, Tregs, inducible Tregs; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs) and engineered variants of such T-cells including but not limited to CAR-T cells, recombinantly modified TILs and TCR-engineered cells. In some embodiments the T cell is a T cell expressing the IL 12 receptor referred to interchangeably as IL12R cell, IL12R+ cell, IL12R T cell, or IL12R+ T cell.
[0156] Terminus/Terminal: As used herein in the context of the structure of a polypeptide, “N- terminus” (or "amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C- terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N- terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C- terminus, respectively. “Immediately N-terminal” refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the N-terminus of the polypeptide. “Immediately C-terminal” refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the C-terminus of the polypeptide. As uned herein in the context of nucleic acids, the “5 ’-terminus” (or “five-prime terminus”) and “3’- terminus” (or “carboxyl terminus”) refer to the extreme ends of the nucleic acid sequence, respectively, while the terms “5”’ and “3”’ refer to relative positions in the nucleic acid sequence of the polypeptide toward the 5’-terminus and the 3’-terminus, respectively, and can include the residues at the 5’-terminus and 3’-terminus, respectively.
[0157] Therapeutically Effective Amount: As used herein to the phrase “therapeutically effective amount” refers to the quantity of an agent when administered to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a treatment regimen comprises multiple doses, provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition. A therapeutically effective amount can be ascertained by measuring one or more relevant physiological effects, and it may be adjusted in connection with a dosing regimen and in response to diagnostic analy sis of the subject’s condition. The parameters for evaluation to determine a therapeutically effective amount of an agent are determined by the physician using art accepted diagnostic criteria including but not limited to indicia such as age, weight, sex, general health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computerized tomography, X-ray, and the like. Alternatively, or in addition, other parameters commonly assessed in the clinical setting may be monitored to determine if a therapeutically effective amount of an agent has been administered to the subject such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of the disease, disorder or condition, biomarkers (such as inflammatory cytokines, IFN-y, granzyme, and the like), reduction in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), improvement in Immune-Related Response Criteria (irRC), increase in duration of survival, extended duration of progression free survival, extension of the time to progression, increased time to treatment failure, extended duration of event free survival, extension of time to next treatment, improvement objective response rate, improvement in the duration of response, reduction of tumor burden, complete response, partial response, stable disease, and the like that that are relied upon by clinicians in the field for the assessment of an improvement in the condition of the subject in response to administration of an agent. In one embodiment, a therapeutically effective amount is an amount of an agent when used alone or in combination with another agent provides an provides a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder or condition and does not result in non-reversible serious adverse events in the course of administration of the agent to the mammalian subject.
[0158] Treat: The terms “treat”, “treating”, treatment” and the like refer to a course of action (such as administering to the subject a pharmaceutical composition comprising a hIL-12M in combination with a pharmaceutical composition comprising a hIL2M, optionally in combination with one or more supplementary agents) that is initiated with respect to a subject in response to a diagnosis that the subject is suffering from a disease, disorder or condition, or a symptom thereof, the course of action being initiated so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of: (a) the underlying causes of such disease, disorder, or condition afflicting a subject; and/or (b) at least one of the symptoms associated with such disease, disorder, or condition. In some embodiments, treating includes a course of action taken with respect to a subject suffering from a disease where the course of action results in the inhibition (e.g., arrests the development of the disease, disorder, or condition), prevents the recurrence of disease or ameliorates one or more symptoms associated with the presence of the disease in the subject.
[0159] Variant: The terms “variant”, "protein variant" or "variant protein" or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide by virtue of at least one amino acid modification, substitution, or deletion. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide or may be a modified version of a WT polypeptide. In some embodiments, the variant polypeptide comprises from about one to about ten, alternatively about one to about eight, alternatively about one to about seven, alternatively about one to about five, alternatively about one to about four, alternatively from about one to about three alternatively from one to two amino acid modifications, substitutions, or deletions, or alternatively a single amino acid amino acid modification, substitution, or deletion compared to the parent polypeptide. A variant may be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which the variant is derived.
[0160] Wild Type: By "wild type" or "WT" or "native" when used in reference to a polypeptide or nucleic acid sequence herein is meant to refer to a polypeptide having amino acid sequence or a nucleotide sequence, respectively, that is found in nature, including allelic variations. A wild- type protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been modified by the hand of man.
[0161] It will be understood that individual embodiments, which are separately described herein for clarity and brevity, can be combined without limitation. Thus, the present disclosure includes one or more, or all, combinations of the embodiments described herein as if each and every combination was individually and explicitly disclosed. This also applies to any and all sub- combinations of the embodiments disclosed herein, such that the present disclosure includes one or more, or all, sub-combinations of the embodiments described herein as if each and every sub- combination was individually and explicitly disclosed.
Description of the Embodiments of the Disclosure:
[0162] The present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of:
(a) administering to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M), the hIL12M comprising a P35 subunit (hIL 12P35) and P40M subunit (hIL12P40M) wherein:
• the HL12P35 has at least 95% sequence identity to mature wild type human ML12P35 (SEQ ID NO:2); and
• the hIL12P40M has at least 95% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the hIL12P40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12P40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12P40; and
(b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutein (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (hIL2, SEQ ID NO: 182) that result in reduced binding affinity of the hIL2M to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182).
Human IL 12
[0163] Wild type human IL 12 (wt hIL12) is a covalently disulfide linked heterodimeric protein comprising two wild type subunits, hP40 and hIL12P35. The naturally occurring form of hIL12 comprises an interchain disulfide linkage between residue C96 of P35 (numbered in accordance with SEQ ID NO:1) and residue C199 of P40 (numbered in accordance with SEQ ID NO:3).
Wild Type Human P35
[0164] The wild type human P35 monomer (hIL12P35) is expressed as a 219 amino acid pro- protein (SEQ ID NO: 1) comprising a 22 amino acid signal sequence which is post-translationally removed to render a 197 amino acid mature protein (SEQ ID NO:2). Wild type hIL12P35 (wt HL12P35) contains two intrachain disulfide linkages, the first between residues C64 and C196 and the second between residues C85 and C123 (numbered in accordance with SEQ ID NO: 1). The canonical amino acid sequence of the human pro- hIL12P35 protein (UniProt Reference No. P29459) with the signal sequence (underlined) is:
MCPARSLLLVATLVLLDHLSLARNL PVAT PDPGMFPCLHHSQNLL RAVS NML QK ARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETS FITNG SCLASRKTS FMMALCLS S IYEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAV IDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYL NAS ( SEQ ID NO : 1 ) .
[0165] The mature form of the wild-type human P35 (hIL12P35) less the 22 amino acid signal sequence is expressed as a 197 amino acid mature protein having the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITK DKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMMALCLSS IYED LKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLE EPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS ( SEQ ID NO : 2 ) .
Wild Type Human P40
[0166] Wild type human P40 (hIL12P40) is expressed as a 328 amino acid pro-protein (SEQ ID NO:3) comprising a22 amino acid signal sequence which is post-translationally removed to render a 306 amino acid mature protein (SEQ ID NO: 4). hIL12P40 contains four intrachain disulfides between residues C50 and C90, C131 and C142, C170 and C193, and C300 and C327 (numbered in accordance with SEQ ID NO:3). The canonical amino acid sequence of the hIL12P40 pro- protein (UniProt Reference No. P29460) with the signal sequence (underlined) is:
MCHQQLVI SWFSLVFLAS PLVAIWELKKDVYWELDWYPDAPGEMWLTCDTPE EDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKE DGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTI STDLTFSVKS SRGS SDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVH KLKYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFS LT FCVQVQGKSKREKKDRVFT DKTS ATVI CRKNAS I SVRAQDRY YS S S WS EWAS VPCS ( SEQ ID N0 : 3 )
[0167] The mature form of the wild-type human hIL12P40 less the 22 amino acid signal sequence (hIL12P40) is expressed as a 306 amino acid mature protein (SEQ ID NO:4)
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 4 )
IL12 Receptor
[0168] The IL12 receptor comprises the IL12R(31 and IL12R02 subunits. IL12 receptor activation results from the binding of IL 12 cytokine ligand to both IL 12RJ31 and IL12R02. The binding of the IL 12 cytokine ligand to the IL 12 receptor complex activates the Janus tyrosine kinases, Tyk2 and Jak2, associated with IL12Rβ1 and IL12R02, respectively, to phosphorylate the cytoplasmic tails of the receptors. This results in the recruitment of signal transducer and activator of transcription 4 (STAT4). Homodimerization of STAT4 results in its release from the receptor and translocation of the phosphorylated STAT4 homodimer into the nucleus, where it binds to STAT4-binding elements of the IFN-y gene to produce IFN-y. hIL12 Muteins
[0169] In one embodiment of the method of the present disclosure, the hIL12M comprises: (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) e HL12P40M subunit of the hIL12M having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the hIL12P40M subunit further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL 12.
[0170] In another embodiment of the method of the present disclosure, the hIL12M comprises: (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) e HL12P40M subunit of the hIL12M having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P40 (SEQ ID NO:4), the HL12P40M subunit further comprising one or more amino acid substitutions that reduce the binding affinity of the HL12P40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40.
[0171] In some embodiments, the IL12p40M and IL12P35 are brought into functional, non- cov al ent association wherein the IL12p40M and IL12P35 are covalently attached to a first and second additional molecule (e.g., a polypeptide) wherein the first and second additional molecules provide stable non-covalent association. Polypeptide domains which exhibit stable non-covalent formation of dimeric polypeptide pairs are well known in the art (e.g. leucine zipper motifs, heterodimenc leucine zipper motifs (Moll, et al (2008) Protein Science 10:649-655), immunoglobulin Fc domains and immunoglobulin Fc domains modified to promote heterodimenzation as discussed in more detail below). In some embodiments, the hIL12M comprises a IL12p40M subunit (hIL12P40M) and IL12P35 subunit (IL12P35) wherein the ML12P40M is covalently linked the ML12P35 via at least one disulfide bond alone or in addition the presence of polypeptide domains that facilitate the non-covalent association of the IL12p40M and IL12P35.
[0172] In one embodiment of the disclosure, the hIL12M comprises (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human HL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising one or more amino acid substitutions at a position corresponding to an amino acid residue selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 numbered in accordance with the human IL12p40 precursor (SEQ ID NO: 3), wherein the HL12P40M is covalently linked the ML12P35 via at least one disulfide bond.
[0173] In one embodiment of the disclosure, the hIL12M comprises (a) an HL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising one or more amino acid substitutions at a position corresponding to an amino acid residue selected from the group consisting of P39, D40, E81, F82, KI 06, K217, and K219 of numbered in accordance with SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions at residues W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 is independently selected from the group consisting of an alanine (A) substitution, an arginine (R) substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, a lysine (K) substitution, a phenylalanine (F) substitution, a lysine substitution, a glutamine (Q) substitution, a glutamic acid (E) substitution, a serine (S) substitution, and a threonine (T) substitution, and combinations of any thereof.
[0174] In one embodiment of the disclosure, the hIL12M comprises (a) an hIL12P35 subunit having at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity to mature wild type human hIL12P35 (SEQ ID NO:2); and (b) and an having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human HL12P40 (SEQ ID NO: 4), the IL12p40M further comprising an amino acid substitution(s) selected from the group consisting of (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), tryptophan (W), and tyrosine (Y (e) F82 is selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), histidine (H), lysine (K), phenylalanine (F) proline (P), tryptophan (W), and tyrosine (Y) (I) K106A, (g) D109A, (h) K217A, (i) K219A, (j) E81A/F82A, (k) W37A/E81A/F82A, (1) E81A/F82A/K106A, (m) E81A/F82A/K106A/K219A, (n) E81A/F82A/K106A/K217A, (o) 81A/F82A/K106A/E108A/D115A, (p) E81F/F82A, (q) E81K/F82A, (r) E81L/F82A, (s) E81H/F82A, (t) E81S/F82A, (u) E81A/F82A/K106N, (v) E81A/F82A/K106Q, (w) E81A/F82A/K106T, (x) E81A/F82A/K106R or (y) P39A/D40A/E81A/F82A. In some embodiments, the recombinant polypeptides of the disclosure include an amino acid sequence selected from the group consisting of SEQ ID NOS: 6, 8 and 10.
[0175] In one embodiment of the disclosure, the IL12M comprises an IL12p40M subunit (IL12p40M), the IL12p40M having an amino acid having an amino acid sequence at least 95%, alternatively at least 96% alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, alternatively 100% sequence identity mature human hIL12P40 (SEQ ID NO: 4) , the IL12p40M further comprising an amino acid substitution(s) selected from the group consisting of (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), tryptophan (W), and tyrosine (Y (e) F82 is selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), histidine (H), lysine (K), phenylalanine (F) proline (P), tryptophan (W), and tyrosine (Y) (f) K106A, (g) D109A, (h) K217A, (i) K219A, (j) E81A/F82A, (k) W37A/E81A/F82A, (1) E81A/F82A/K106A, (m) E81A/F82A/K106A/K219A, (n) E81A/F82A/K106A/K217A, (o) 81A/F82A/K106A/E108A/D115A, (p) E81F/F82A, (q) E81K/F82A, (r) E81L/F82A, (s) E81H/F82A, (t) E81S/F82A, (u) E81A/F82A/K106N, (v) E81A/F82A/K106Q, (w) E81A/F82A/K106T, (x) E81A/F82A/K106R or (y) P39A/D40A/E81A/F82A.
[0176] In one embodiment of the disclosure, the IL12p40M the comprises the amino acid substitutions of E81A/F82A/K106A (numbered in accordance with SEQ ID NO:3). In one embodiment of the disclosure, the IL12p40M comprises an amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKA AGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTI STDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAC PAAEESLPIEVMVDAVHALKYENYTSS FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEY PDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSS SWSEWASVPCS ( SEQ ID NO : 10 ) .
[0177] In some embodiments, The IL12p40M comprising one or more amino acid substitutions selected from the group consisting of the amino acid substitutions: (a) W37A; (b) P39A, (c) D40A, (d) E81 is selected from the group consisting of alanine (A); asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), proline (P), tryptophan (W), and tyrosine (Y (e) F82 is selected from the group consisting of alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), histidine (H), lysine (K), phenylalanine (F) proline (P), tryptophan (W), and tyrosine (Y) (f) K106A, (g) D109A, (h) K217A, (i) K219A, (j) E81A/F82A, (k) W37A/E81A/F82A, (1) E81A/F82A/K106A, (m) E81A/F82A/K106A/K219A, (n) E81A/F82A/K106A/K217A, (o) 81A/F82A/K106A/E108A/D115A, (p) E81F/F82A, (q) E81K/F82A, (r) E81L/F82A, (s) E81H/F82A, (t) E81S/F82A, (u) E81A/F82A/K106N, (v) E81A/F82A/K106Q, (w) E81A/F82A/K106T, (x) E81A/F82A/K106R or (y)
P39A/D40A/E81A/F82A , upon association with hIL12P35, forms a dimer (hIL12M) that (i) induces IL-12 signaling in CD8+ T cells; and (ii) has decreased IL-12 signaling in NK cells compared to an hIL12 molecule comprising a wildtype hIL-12p40 polypeptide.
[0178] In some embodiments, the hIL12Ms described herein provide cell-type biased signaling of the downstream signal transduction mediated through the IL 12 receptor compared to a reference polypeptide (e.g., wild type hIL12). In some embodiments, the reduced binding affinity the hP40 mutein of the hIL12M to I L 12RJ31 results in a reduction in STAT4-mediated signaling compared to a reference polypeptide (wt hIL12). In some embodiments, heterodimeric hIL12M-Fc muteins of the present disclosure are partial agonists. In some embodiments, the heterodimeric hIL12M- Fc muteins described herein are partial agonists of STAT3 -mediated signaling (“STAT3 signaling”) and/or STAT4 mediated signaling (“STAT4 signaling”). In some embodiments, the hIL12M has reduced STAT3-mediated signaling compared to a reference polypeptide (wthIL12). In some embodiments, the STAT3 signaling and/or STAT4 signaling is determined by an assay selected from the group consisting of by a gene expression assay, a phospho-flow signaling assay, and an enzyme-linked immunosorbent assay (ELISA).
[0179] A hIL12M comprising the hP40 mutems described herein provide selective activation of certain cell types which provides beneficial properties, such as anti-inflammatory properties, and/or have reduced undesirable properties, such as pro-inflammatory side effects compared to wt hIL12. In some embodiments, the heterodimeric hIL12M-Fc muteins comprising the hP40 muteins described herein provide cell-type biased signaling of the downstream signal transduction mediated through the IL 12 receptor compared to a reference polypeptide (e.g., wild type hIL12). For example, an hIL12M of the present disclosure retain the property of wild-type hIL12 to stimulate or activate IL12 signaling in CD8+ T cells but exhibit a reduction of IFNy and/or STAT4- mediated signaling in natural killer (NK) cells. In some embodiments, the cell-type biased signaling of the hIL12M comprising the hP40 muteins described herein of the present disclosure includes the ability to provide substantial IL12 signaling (e.g., at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%) of the activity of wt hIL12 in CD8+ T cells. In some embodiments, the heterodimeric hIL12M-Fc mutems described herein exhibit increased STAT4 signaling in CD8+T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater and decrease STAT4 signaling in NK cells, for example, at least about a 10%, 20%, 30%, 40%, 50%, 60%, or 70% decrease, as compared to a reference polypeptide (wt hIL12). In some embodiments, the heterodimeric hIL12M-Fc muteins described herein activate interferon gamma (IFNy) in CD8+ T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% and decreased IFNy signaling in NKT cells by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to a reference polypeptide (wt hIL12). Thus, an hIL12M comprising the hP40 muteins described herein exhibit reduced activation of NK cells while retaining the ability to stimulate CD8+ T cells.
Heterodimeric hIL12M-Fc muteins
[0180] In some embodiments, the hIL12M is a hIL12M-Fc. In some embodiments the hIL12M-
Fc is a heterodimeric hIL12M-Fc. [0181] In some embodiments, the present disclosure provides heterodimeric hIL12M-Fc muteins comprising P40 muteins which have improved pharmacological or therapeutic properties, and methods of using such compositions.
[0182] The present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula #1: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein: a) ML12P35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99% or alternatively 100% sequence identity to SEQ ID NO:2; b) hIL12P40M is an human P40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with wild-type pre-human P40 (SEQ ID NO:3), and optionally otherwise identical to SEQ ID NO:4, or is at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99% sequence identity to SEQ ID NO:4; c) LI and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present); d) UH1 and UH2 are each an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); e) Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fc2;
I) Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fcl, wherein the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.
[0183] In some embodiments, the polypeptide of formula [1] is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 or any hIL12P40M-Fc sequence in the informal sequence listing.
[0184] In some embodiments, the polypeptide of formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124 or any hIL12P35-Fc sequence in the informal sequence listing.
[0185] In some embodiments, LI and L2 are independently selected from the group consisting SEQ ID NOS: 27-79. In some embodiments, LI and L2 are independently selected from the group consisting SEQ ID NOS: 36, 37 and 65.
[0186] In some embodiments, UH1 and UH2 are selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
[0187] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a first poly peptide selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 and a second polypeptide selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
[0188] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 80 and a polypeptide of SEQ ID NO: 81.
[0189] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 121 and a polypeptide of SEQ ID NO: 124.
[0190] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 83 and a polypeptide of SEQ ID NO: 82.
[0191] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 141 and a polypeptide of SEQ ID NO: 124.
[0192] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 144 and a polypeptide of SEQ ID NO: 124.
[0193] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 129 and a polypeptide of SEQ ID NO: 124.
[0194] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 147 and a polypeptide of SEQ ID NO: 82. [0195] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 150 and a polypeptide of SEQ ID NO: 82.
[0196] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 153 and a polypeptide of SEQ ID NO: 82.
[0197] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 135 and a polypeptide of SEQ ID NO: 124.
[0198] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein comprising a polypeptide of SEQ ID NO: 138 and a polypeptide of SEQ ID NO: 124.
[0199] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a a polypeptide of SEQ ID NO: 80 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 81.
[0200] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a a polypeptide of SEQ ID NO: 121 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
[0201] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 83 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
[0202] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 141 and a polypeptide of SEQ ID NO: 124.
[0203] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 144 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
[0204] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
[0205] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 147 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82. [0206] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 150 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
[0207] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 153 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 82.
[02081 In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 135 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
[0209] In some embodiments the present disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: 138 and a second nucleic acid sequence encoding polypeptide of SEQ ID NO: 124.
Human P40 Muteins
[0210] The hIL12M muteins of the present disclosure comprise a modified human P40 polypeptide or “hP40 mutein” (also abbreviated “P40M” or “hIL12P40M”) comprising one or more amino acid substitutions, modifications and/or deletions at the interface with the extracellular domain of IL 12R|31 which result in a reduction of the binding affinity of hIL12P40M to IL12R.β1 relative to the mature form of wt hP40 (SEQ ID NO: 4). In some embodiments, the binding affinity of the hP40 mutein for the extracellular domain of hIL12Rβ1 is reduced by about 10%, alternatively by about 20%, alternatively by about 30%, alternatively by about 40%, alternatively by about 50%, alternatively by about 60%, alternatively by about 60%, alternatively by about 70%, alternatively by about 80%, alternatively by about 900%, alternatively to about 100% compared to binding affinity of a reference polypeptide (wt hP40) as determined by surface plasmon resonance (SPR) spectroscopy. In some embodiments, the hIL12P40M is a modified wild type human hp40 polypeptide having at least 70% sequence identity to SEQ ID NO:4 (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4). In some embodiments, the hIL12P40M comprises one or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3. In some embodiments, the hIL12P40M comprises one or more amino acid substitutions at residues selected from the group consisting of E81, F82, KI 06, and K217 numbered in accordance with SEQ ID NO:3. In some embodiments the one or more amino acid substitutions at positions W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 are selected from the group consisting of P39A, D40A, E81A, F82A, K106A, D109A, K217A, K219A. In some embodiments, the hP40 mutein comprises two or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3. In some embodiments, the hIL12P40M comprises two or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217 numbered in accordance with SEQ ID NO:3. In some embodiments, the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3. In some embodiments wherein the hIL12P40M comprises two or more ammo acid substitutions at W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219, the two or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: E81A/F82A, E81K/F82A, E81L/F82A, E81H/F82A and E81S/F82A. In some embodiments, the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of E81, F82, KI 06, and K217 numbered in accordance with SEQ ID NO:3. In some embodiments, the hIL12P40M comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, DI 15, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3. In some embodiments wherein the hP40 mutein comprises three or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, the three or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: W37A/E81A/F82A; E81A/F82A/K106A; E81A/F82A/K106A/K219A, E81A/F82A/K106N, E81A/F82A/K106Q, E81A/F82A/K106T, and E81A/F82A/K106R. In some embodiments, the hP40 mutein comprises four or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 numbered in accordance with SEQ ID NO:3. In some embodiments wherein the hP40 mutein comprises four or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, the four or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the sets of amino acid substitutions: E81A/F82A/K106A/K217A, 81A/F82A/K106A/E108A/D115A and
P39A/D40A/E81A/F82A.
[02111 In some embodiments, the hIL12P40M comprises the set of amino acid substitutions E81A/F82A and is referred to herein as “2xAla” (SEQ ID NO:6). In some embodiments, the HL12P40M comprises the set of amino acid substitutions E81A/F82A/K106A and is referred to herein as “3xAla” (SEQ ID NO:8). In some embodiments, the hIL12P40M comprises the set of amino acid substitutions E81A/F82A/K106A/K217A and is referred to herein as “4xAla” (SEQ ID NO: 10).
[O2 I2] In some embodiments, the binding affinity of heterodimeric hIL12M-Fc muteins of the present disclosure comprising one or more, optionally two or more, optionally three or more, or optionally 4 or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, KI 06, E108, DI 15, H216, K217, L218, and K219 (numbered in accordance with SEQ ID NO:3) for the extracellular domain (ECD) of IL12Rβ1 is reduced by at least 5%, optionally by at least 10%, optionally by at least 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, optionally by at least 70%, relative to the binding affinity of wild ty pe hP40 (SEQ ID NO:4) for the extracellular domain (ECD) of IL12R.β1 as determined by surface plasmon resonance.
GSA Linkers:
[0213] In the polypeptides of formulae [1] and [2], Fc domain fusions incorporating a P40 mutein and/or P35 may optionally contain a GSA linker molecule between the P40 mutein and the upper hinge. As used herein the term “GSA linker” refers to a polypeptide having 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19 or 20 amino acids comprised of amino acids selected from the group consisting of glycine, serine and alanine. In some embodiments, the polypeptide linker is a glycine-serine polymer of the structure (GGGGSm)n (SEQ ID NO: 192), (GGGSm)n (SEQ ID NO: 193), (GGGAm)n (SEQ ID NO: 194) and (GGGGAm)n (SEQ ID NO: 195), and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4. In the construction of such polymers, it may be desirable to avoid repeated “GSG” sequences which potentially may provide introduction of a non-naturally occurring glycosylation site. Exemplary glycine-serine linkers include but are not limited to the monomers: GGGGS (referred to as “G4S”) (SEQ ID NO: 65), GGGGA (referred to as “G4A”) (SEQ ID NO: 53), GGGS (referred to as “G3S”) (SEQ ID NO: 50) and GGGA (referred to as “G3A”) (SEQ ID NO: 43), or homopolymers (e.g. “GGGGSGGGGS” also referred to as (G4S)? (SEQ ID NO: 36)) or heteropolymers thereof. Exemplary' GSA linkers are provided in Table 2 below:
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Upper Hinge:
[0214] The heterodimeric hIL12M-Fc muteins of the present disclosure are heterodimers comprising polypeptides of the formulae [1] and [2], which each incorporate an upper hinge region of a human immunoglobulin molecule. The term “upper hinge” or “UH” refers to an amino acid sequence corresponding to amino acid residues 216-220 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the upper hinge region is a naturally occurring upper hinge region of a human immunoglobulin selected from the LH regions of human IgGl, human IgG2, human IgG3 and human IgG4 upper hinge domains. In some embodiments, the upper hinge region is the upper hinge region of a human IgGl immunoglobulin. In some embodiments, the upper hinge region is the upper hinge region of a human IgGl immunoglobulin comprising the pentameric amino acid sequence: EPKSC (SEQ ID NO: 11).
[0215] In some embodiments, the upper hinge region contains an unpaired cysteine residue at position 220 (EU numbering) that typically, in a complete immunoglobulin molecule, binds to a cysteine on a light chain. When only the Fc domain is used comprising the hinge domain, the unpaired cysteine in the hinge domain creates the potential of the formation of improper disulfide bonds. Consequently, in some embodiments the cysteine at position 220 (C220, numbered in accordance with EU numbering) is substituted with an amino acid that does not promote disulfide bonding. In some embodiments, the Fc domain comprises a C220S mutation having the amino acid sequence EPKSS (SEQ ID NO: 12).
Fcl and Fc2:
[0216] |The heterodimeric hIL12M-Fc muteins of the present disclosure are heterodimers comprising polypeptides of the formulae [1] and [2], which each incorporate an Fc region (Fcl and Fc2) of a human immunoglobulin molecule modified to promote heterodimerization.
[0217] As used herein the term “Fc” and “Fc monomer” are used interchangeably herein to designate the monomeric polypeptide subunit of an Fc dimer. An Fc comprises an amino acid sequence (from amino to carboxy terminal) comprising a lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule. In some embodiments, the Fc monomer is a polypeptide comprising the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule domains of human IgGl, human IgG2, human IgG3 and human IgG4 hinge domains. The CH2 domain of hlgGl corresponds to amino acid residues 231-340 (EU numbering) and is provided as SEQ ID NO: 14. The CH3 domain of hlgGl corresponds to amino acid residues 341-447(EU numbering) and is provided as SEQ ID NO: 15.
[02181 The polypeptides of the formulae [1] and [2] each incorporate a lower hinge region of a human immunoglobulin. As used herein, the term “lower hinge” or “LH” refers to an amino acid sequence corresponding to amino acid residues 221-229 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the lower hinge region is a naturally occurring lower hinge region of a human immunoglobulin selected from the LH regions of IgGl , IgG2, IgG3 and IgG4 lower hinge domains. In some embodiments, the lower hinge region is the lower hinge region of a human IgGl immunoglobulin. In some embodiments, the lower hinge region is the lower hinge region of a human IgGl immunoglobulin comprising the decameric amino acid sequence: DKTHTCPPCP (SEQ ID NO: 13).
[0219] In some embodiments, Fcl and Fc2 are derived from a polypeptide corresponding to amino acids 221-447 (EU numbering) of the human IgGl immunoglobulin having the amino acid sequence (EU numbering indicated, SEQ ID NO: 16):
230 240 250 260 270
DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED
280 . 290 . 300 310 320
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RWSVLTVLH QDWLNGKEYK
330 340 350 360 370
CKVSNKALPA PIEKTI SKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK
380 390 400 410 420
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
430 440 447
NVFSCSVMHE ALHNHYTQKS LSLSPGK ( SEQ ID NO : 16)
[0220] As indicated in above sequence, the C-terminal residue of the wild-type form of the IgGl Fc domain is a lysine, referred to as K447 in accordance with EU numbering. The K447 is inconsistently removed by the producer cell during recombinant product. As a result, the population of recombinant Fc monomers may be heterogenous in that some fraction of the recombinantly produced Fc monomers will contain K447 and others will not. Such inconsistent proteolytic processing by producer cells may therefore result in a heterogenous population of hIL12M-Fc . Typically, particularly in the context of human pharmaceutical agents, such heterogeneity of the active pharmaceutical ingredient is to be avoided. Consequently, in addition to modifications to the Fc monomer sequence promote heterodimerization, the present disclosure provides Fc monomers that further comprising a deletion of the C-terminal K447 or a deletion of G446 and K447 and nucleic acid sequences encoding Fc monomers comprising a: (a) a deletion of the lysine residue at position 447 (K447,EU numbering, abbreviated as AK447 or des-K447), or (b) deletion of both the glycine at position 456 (G446 EU numbering, abbreviated as des-G446) and K447 (this double deletion of G446 and K447 being referred to herein as des-G446/des-K447 or AG446/AK447).
[0221] In some embodiments the modifications to the Fc domains of the heterodimeric hIL12M- Fc mutein to promote heterodimerization. In some embodiments the modifications to the Fc domains of the heterodimeric hIL12M-Fc mutein to promote heterodimerization are complementary “knob-into-hole” mutations. In some embodiments, the modifications of the Fc domains to promote heterodimerization of the hIL12P35 and hIL12P40M-Fc domains comprises the amino acid substitution T366W (“knob”) in the first domain and the amino acid substitutions T366S/L368A/Y407V (“hole”) in the second domain.
[0222] In some embodiments the present disclosure provides a heterodimeric hIL12M-Fc mutein wherein the hIL12P35-Fc and hIL12P40M-Fc polypeptides of the heterodimeric hIL12M- Fc mutein are covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds. In some embodiments, the ML12P35 and hIL12P40M-Fc are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C96 of the hP35 domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C199 of the hP40M domain of the hIL12P40M-Fc. In some embodiments, the hIL12P35 and hIL12P40M are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C226 of the lower hinge domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C226 of the lower hinge domain of the hIL12P40M-Fc. In some embodiments, the hIL12P35-Fc and hIL12P40M are covalently linked via a disulfide bond between the sulfhydryl group of amino acid C229 of the lower hinge domain of the hIL12P35-Fc and the sulfhydryl group of amino acid C229 of the lower hinge domain of the hIL12P40M-Fc. In some embodiments, a first Fc domain comprises the amino acid substitution S354C, and the second Fc domain comprises the amino acid substitution Y349C. In some embodiments, the heterodimeric hIL12M-Fc mutein comprises a first Fc domain comprising the amino acid substitution S354C and the second Fc domain comprising the amino acid substitution Y349C and wherein the hIL12P35-Fc and hIL12P40M domains are linked via a disulfide bond between the S354C of the first Fc domain and Y349C of the second Fc domain. In some embodiments, the hIL12P35-Fc andhIL12P40M-Fc of the heterodimeric hIL12M-Fc mutein are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds, optionally four or more disulfide bonds between the side chains of the following cysteine residue pairs: (a) C96 of the hP35 and Cl 99 of the hP40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution.
Modification ofhp40 K282 To Avoid Proteolytic Cleavage
[0223] In some embodiments, the heterodimeric IL12Fc muteins of the present disclosure comprise an amino acid substitution of the lysine (K) residue at position 260 (K260) of the mature form of the human P40 polypeptide (SEQ ID NO: 4 corresponding to position 282 of the human P40 precursor polypeptide SEQ ID NO: 3). As described in Webster, et al (United States Patent No. 7,872,107 issued January 18, 2011), a substitution at position 260 of the mature human P40 polypeptide renders the human P40 polypeptide resistant to proteolytic cleavage. In some embodiments, the human P40 polypeptide of the heterodimeric IL12Fc muteins of the present disclosure comprise a substitution of the lysine at position K282 (numbered in accordance with SEQ ID NO:3) polypeptide with a non-basic amino acid. In some embodiments the non-basic amino acid is selected from the group consisting of alanine, glycine, asparagine or glutamine. In some embodiments, the P40 polypeptide of the heterodimeric IL12Fc muteins of the present disclosure comprise a mutation at position K282 (numbered in accordance with SEQ ID NO: 3) selected from the group consisting of K282G, K282A, K282N, K282Q (numbered in accordance with SEQ ID NO:3.
[0224| In some embodiments, the heterodimeric IL12Fc muteins of the present disclosure comprise the human P40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A/F82A/K106A/K282G, E81A/F82A/K106A/K282A,
E81 A/F82A/K106A/K282N, and E81A/F82A/K106A/K282Q (numbered in accordance with SEQ ID NO:3). In some embodiments, the heterodimeric IL12Fc muteins of the present disclosure comprise the human P40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A/F82A/K282G, E81A/F82A/K282A, E81A/F82A/K282N, and E81 A/F82A/K282Q (numbered in accordance with SEQ ID NO:3).
[0225] In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT
IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 155 ) .
[0226] In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT
IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR
CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRADN
KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP
KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 156 ) .
[0227] In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT
IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR
CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRNDN
KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP
KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 157 ) .
[0228] In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT
IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR
CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRQDN
KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP
KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 158 ) .
[0229] |In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT
IQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR
CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN
KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP
KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 159 ) . [0230| |In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRADN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 160 ) .
[02311 In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRNDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 161 ) .
10232] In one embodiment, the present disclosure provides a heterodimeric IL12Fc mutein comprising a human P40 mutein having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKAAGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRQDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCS ( SEQ ID NO : 162 )
Modifications of Fc Subunits to Promote Heterodimerization
[0233] As provided in formulae [1] and [2] above, the Fcl and Fc2 monomers of the dimeric Fc contain amino acid substitutions that promote heterodimerization between Fcl and Fc2. A variety of techniques are established for the promotion of heterodimerization of Fc domains. See, e.g. Kim, et al., United States Patent No. 11,087,249, issued August 3, 2021. In some embodiments, the modifications to promoter heterodimerization of the Fcl and Fc2 monomers are the HF-TA mutations and the HA-TF mutations as described in Moore, et al (2011) mAbs 3(6):546-557. The HF-TA method employs the S364H/T394F substitutions on one Fc monomer and the Y349T/F405A substitutions on the complementary Fc monomer. The (HA-TF) method employs the S364H/F405A substitutions on one Fc monomer and the Y349T/T394F substitutions on the complementary Fc monomer. Alternatively, the Fcl and Fc2 monomers are modified to promote heterodimerization by the ZW1 heterodimerization method which employs the T350V/L35 IY/F405A/Y407V substitutions on one Fc monomer and the T350V/T366L/K392L/T394W substitutions on the complementary Fc monomer. Von Kreudenstein, et al (2013) mAbs, 5(5):646-654. Alternatively, the Fcl and Fc2 monomers are modified to promote heterodimerization by the EW-RVT heterodimerization method which employs the K360E/K409W substitutions on one Fc monomer and the Q347R/D399V/F405T substitutions on the complementary Fc monomer. Choi , et al (2015) Molecular Immunology 65(2):377-83.
[02341 In one embodiment, Fcl and Fc2 are modified to promote heterodimerization by the employment of the “knob-into-hole” (abbreviated KiH) modification as exemplified herein. The KiH modification comprises one or more amino acid substitutions in a first Fc monomer (e.g. Fcl) that create a bulky “knob” domain on a first Fc and one or more amino acid substitutions on a second Fc monomer (e.g. Fc2) that create a complementary pocket or “hole” to receive the “knob” of the first Fc monomer.
[O235| A variety of amino acid substitutions have been established for the creation of complementary knob and hole Fc monomers. See, e.g. Ridgway, et al (1996) Protein Engineering 9(7):617-921; Atwell, et al (1997) J. Mol. Biol. 270:26-35; Carter, et al. United States Patent No. 5,807,706 issued September 15, 1998; Carter, et al 7,695,936 issued April 13, 2010; Zhao et al. “A new approach to produce IgG4-like bispecific antibodies,” Scientific Reports 11 : 18630 (2021); Cao et al. “Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody,” and Liu et al. "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds". Frontiers in Immunology. 8: 38. doi: 10.3389/fimmu.2017.00038 (2017).
[0236] In some embodiments, the Fc domain comprises two Fc monomers wherein the CH3 domain of a first Fc monomer wherein the threonine at (EU numbering) position 366 is modified with a bulky residue (e.g. a T366W) create a “knob” and the substitution, and a second Fc monomer comprising one or more substitutions in complementary residues of the CH3 domain of the second Fc monomer to create a pocket or “hole” to receive the bulky residue, for example by amino acid substitutions such as T366S, L368A, and/or Y407V.
[0237 | In one embodiment, the Fcl monomer of formula 1 is a “knob” modified Fc monomer comprising the amino acid substitution T366W and the Fc2 monomer of formula 2 is a “hole” modified Fc comprising the set of amino acid substitutions T366S/L368A/Y407V. [0238| Alternatively, the Fcl monomer of formula 1 is a “hole” modified Fc monomer comprising the set of amino acid substitutions T366S/L368A/Y407V and the Fc2 monomer of formula 2 is a “knob” modified Fc monomer comprising the amino acid substitution T366W.
[02391 An example of an engineered Fc heterodimeric pair comprising complementary KiH modifications is provided in Table 3 below:
Figure imgf000070_0001
[0240] As noted, the heterodimeric hIL12M-Fc muteins of the present disclosure are provided as a complementary heterodimeric pair of polypeptides of the formulae [1] and [2] wherein the first and second polypeptide are linked by at least one disulfide bond. In some embodiments, the incorporation of a disulfide bond between the polypeptides of formulae [1] and [2] may be achieved by cysteine substitutions at particular points within the Fcl and Fc2 domains. In one embodiment, the Fcl domain of the polypeptide of formula [1] is derived from the Fc domain of hlgGl comprising an amino acid substitution S354C (EU numbering) and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hlgGl comprising an amino acid substitution Y349C (EU numbering) to provide a disulfide bond between the S354C of Fcl and Y349C of Fc2. Alternatively, the Fcl domain of the polypeptide of formula [1] is derived from the Fc domain of hlgGl comprising an amino acid substitution Y349C (EU numbering) and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hlgGl comprising an amino acid substitution S354C (EU numbering) to provide a disulfide bond between the S354C of Fcl and Y349C of Fc2.
[0241] In some embodiments, the hIE12P40M-Fc and hIL12P40M of the heterodimeric hIL12M-Fc mutein are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds , optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) C96 of the hIL12P35 and C199 of the hIL12P40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution. [0242] Further examples of complementary KiH engineered heterodimeric Fc pairs that may be used in the practice of the present disclosure are provided in Table 4 below.
Figure imgf000071_0002
Figure imgf000071_0001
Figure imgf000071_0003
Modifications to Reduce Effector Functions
[0243] In some embodiments the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to reduce effector function. In some embodiments, the Fc domain may be modified to substantially reduce binding to Fc receptors (FcyR and FcR) which reduces or abolishes antibody directed cytotoxicity (ADCC) effector function. Modification of Fc domains to reduce effector function are well known in the art. See, e.g., Wang, et al. (2018J IgGFc engineering to modulate antibody effector functions, Protein Cell 9(l):63-73. For example, mutation of the lysine residue at position 235 (EU numbering) from leucine (L) to glutamic acid (E) is known to reduce effector function by reducing FcgR and Clq binding. Alegre, et al. (1992) J. Immunology 148:3461-3468. Additionally, substitution of the two leucine (L) residues at positions 234 and 235 (EU numbering) in the IgGl hinge region with alanine (A), i.e., L234A and L235A, results in decreased complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). Hezereh et al., (2001) J. Virol 75(24): 12161-68. Furthermore, mutation of the proline at position 329 (EU numbering) to alanine (P329A) or glycine, (P329G) mitigates effector function and may be combined with the L234A and L235A substitutions. In some embodiments, the Fc domains (Fcl and Fc2) of the compositions of the present invention may comprises the amino acid substitutions L234A/L235A/P329A (EU numbering) referred to as the “LALAPA” substitutions or L234A/L235A/P329G (EU numbering) referred to as the “LALAPG” substitutions. In some embodiments, the Fc domains (Fcl and Fc2) of the compositions of the present disclosure may comprises the amino acid substitutions E233P/L234V/L235A/AG237 (referred to in the scientific literature as the PVAdelG mutation).
[0244| In some embodiments, the Fc domains (Fcl and Fc2) of the compositions of the present disclosure are from hIgG4. In such instances where the Fc domains of the heterodimeric IL12muteins are derived from hIgG4, attenuation of effector function may be achieve by introduction of the S228P and/or the L235E mutations (EU numbering). In some embodiments, the IgG4 Fc incorporates an amino acid substitution at postion 297 (e.g. N297G). Such mutations eliminate a glycosylation site of the IgG4Fc domain that significantly ablates effector function.
[0245] Examples of paired KiH Fc dimeric constructs that may be incorporated into the hIL12 muteins of the present disclosure are provided in Table 5 below:
Figure imgf000073_0001
Fc Sequence Modifications to Extend Duration of Action:
[0246| In some embodiments the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to incorporate amino acid substitutions which extend the duration of action of the molecule and prevent clearance. In some embodiments, such modifications to the Fc monomer include the amino acid substitutions M428L and N434S (EU numbering) referred to as the “LS” modification. The LS modification may optionally be combined with amino acid substitutions to reduce effector function and provide for disulfide bonds between Fcl and Fc2. Table 6 below provides exemplary Fcl and Fcl heterodimeric pairs possessing complementary sequence modifications to promote heterodimerization that may be employed in the design of the Fcl and Fc2 polypeptides of the formulae [1] and [2],
[02471 The following Table 6 provides exemplary Fc heterodimeric pairs which may be used in the preparation of Fcl and Fc2 polypeptides of the heterodimeric hIL12M-Fc muteins of the present disclosure:
Figure imgf000074_0001
[0248] In some embodiments, the Fc domains (Fcl and Fc2) of the compositions of the present disclosure are from hIgG4. In such instances where the Fc domains of the heterodimeric IL12muteins are derived from hIgG4, heterodimerization of the Fcl and Fc2 domains by the introduction of the mutations K.370E, K409W and E357N, D399V, F405T (EU numbering) in the complementary Fc sequences that comprise the heterodimeric Fc domain.
Fc Modifications to Eliminate Glycosylation Sites
[0249] In some embodiments the amino acid sequence of the Fcl and/or Fc2 monomers modified to promote heterodimerization may be further modified to eliminate N-linked or O-linked glycosylation sites. Aglycosylated variants of Fc domains, particularly of the IgG4 subclass are known to be poor mediators of effector function. Jefferies et al. 1998, Immol. Rev., vol. 163, 50- 76). It has been shown that glycosylation at position 297 (EU numbering) contributes to effector function of IgG4 Fc domains. Edelman, et al (1969) PNAS (USA) 63:78-85. In some embodiments, the Fc domains of the compositions of the present disclosure comprise one or modifications to eliminate N- or 0 linked glycosylation sites. Examples of modifications at N297 to eliminate glycosylation sites in the IgG4 Fc domain include the amino acid substitutions N297Q and N297G.
Exemplary heterodimeric hIL12M-Fc muteins:
[0250 | The following table provides a summary of particular compositions of the present disclosure comprising KiH heterodimerization, are provided in Table 7 below
Figure imgf000075_0001
Figure imgf000076_0001
[0251] In some embodiments, the present disclosure provides heterodimeric hIL12M-Fc muteins, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]:
ML12P40M- Lla-UH1— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein the polypeptide of formula [1] is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 141, 144, 147, 150 and 153, and the second polypeptide of the formula [2] is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
[0252] In some embodiments, the hIL12M-Fc muteins of the present disclosure are the heterodimeric hIL12M-Fc muteins provided in Table 8 below.
Figure imgf000076_0002
Figure imgf000077_0001
1, STK-021 (DR1442M/DR1535M)
[0253] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula #1:
ML12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KTSATVICRKNAS ISVRAQDRYYSSSWSEWASVPCSEPKS SDKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKG QPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG ( SEQ ID NO : 80 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITK DKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMMALCLSS IYED LKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLE EPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSEPKS SDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LAAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLS PG ( SEQ ID NO : 81 )
2, STK-022 (1947/ DR1948M)
[0254] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula 1 : hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hILl 2P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLT IQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLR CEAKNYSGRFTCWWLTTI STDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KTSATVICRKNAS I SVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSEPKSSDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAP
IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQK SLSLSPG ( SEQ ID NO : 121 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 )
3, STK-023 (DR1537M/ DR1536M)
[0255] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKS KREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG ( SEQ ID NO : 83 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 82) .
4, DR2086M/ DR1948M
[02561 In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKS QREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPG (SEQ ID NO: 141) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG (SEQ ID NO: 124) .
5, DR2087M/DR1948M [0257 | In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKSNREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPG ( SEQ ID NO : 144 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 )
6, STK-026 (DR2088M/ DR1948M)
[0258] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS
GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ
KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT
CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKS GREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPG ( SEQ ID NO : 129 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P E VT 0 VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) .
7, DR2090M/DR1536M
[0259] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
MCHQQLVI SWFSLVFLAS PLVAIWELKKDVYWELDWYPDAPGEMWLTC DTPEEDGITWTLDQSSEVLGSGKTLTIQVKAAGDAGQYTCHKGGEVLSHS LLLLHAKEDGIWSTDILKDQKEPKNKT FLRCEAKNYSGRFTCWWLTTIST DLT FSVKS SRGS S DPQGVTCGAATLSAERVRGDNKE YEYSVECQEDSACP AAEESLPIEVMVDAVHKLKYENYTSS FFIRDI IKPDPPKNLQLKPLKNSR QVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSQREKKDRVFTDKTSATVIC RKNAS ISVRAQDRYYSS SWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LAAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDI AVEWESNGQPENNYKTT PPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLS PG ( SEQ ID NO : 147 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG ( SEQ ID NO : 82 )
8, DR2091M/DR1536M
[0260] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKSNREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG ( SEQ ID NO : 150 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG ( SEQ ID NO : 82 )
9, STK-027 (DR2092M/ DR1536M)
[0261] In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]:
ML12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKS GREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPG ( SEQ ID NO : 153 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKTS FMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKS SLEEPDFYKTKIKLCILLHAFRIRAVTI DRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG ( SEQ ID NO : 82 )
10. STK-028 (DR2455M/DR1948M):
In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGS GKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQ KEPKNKTFLRCEAKNYSGRFTCWWLTT ISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKL KYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS
Y FS LT FCVQVQGKS KREKKDRVFTDKT S ATVI CRKNAS I S VRAQDRY YS SSWSEWASVPCSGGGGSGGGGSEPKSS DKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPG ( SEQ ID NO : 135 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG (SEQ ID NO:124)
11. STK-029 (DR2456M/DR1948M):
[02621 In one embodiment, the present disclosure provides a heterodimeric hIL12M-Fc mutein, the heterodimeric hIL12M-Fc mutein comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35-L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSG KTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKE PKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGA ATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYEN YTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLT
FCVQVQGKSGREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVY TLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG (SEQ ID NO: 138) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMM ALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQA LNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNA SGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
S RT P E VT C VWD VS H E D P E VK FN W Y VD GVE VH NAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG (SEQ ID NO:124)
PEGylation [0263 | In some embodiments, the hIL2 and/or hIL12M the present disclosure may be conjugated to one or more polyethylene glycol molecules or “PEGylated.” Although the method or site of PEG attachment to the binding molecule may vary, in certain embodiments the PEGylation does not alter, or only minimally alters, the activity of the binding molecule.
[0264] The present disclosure provides PEGylated heterodimeric hIL12M-Fc muteins. In some embodiments, hIL12P35 polypeptide of the heterodimeric hIL12M-Fc mutein PEGylated. In some embodiments, hIL12P40M-Fc polypeptide of the heterodimeric hIL12M-Fc mutein PEGylated. In some embodiments, both the hIL12P35-Fc and the hIL12P40M-Fc of the heterodimeric hIL12M- Fc mutein are PEGylated.
[0265] In some embodiments, conjugation of the PEG moiety to the hIL2 and/or hIL12M may be accomplished via a sulfhydryl (-SH) group of a cysteine residue. In some embodiments, the PEGylation of the d heterodimeric hIL12M-Fc muteins is provided at one or both of the naturally occurring cysteine residues at position 220 (C220, EU Numbering) of the upper hinge region of the hIL12P35-Fc and/or the hIL12P40M-Fc heterodimeric hIL12M-Fc muteins.
[0266] PEGs suitable for conjugation to a polypeptide sequence of either or both of hIL12M and hIL2M molecules of the present disclosure are generally soluble in water at room temperature, and have the general formula
R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
The PEG can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure.
[0267| PEGylation of hIL2 and/or hIL12M may be facilitated by the incorporation of one or more non-natural amino acids having side chains to facilitate selective PEG conjugation. Specific PEGylation sites can be chosen such that PEGylation of the binding molecule does not affect its binding to the target receptors.
[026S| PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure. [0269 | |A molecular weight of the PEG used in the present disclosure is not restricted to any particular range. The PEG component of the binding molecule can have a molecular mass greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50kDa. In some embodiments, the molecular mass is from about 5kDa to about lOkDa, from about 5kDa to about 15kDa, from about 5kDa to about 20kDa, from about lOkDa to about 15kDa, from about lOkDa to about 20kDa, from about lOkDa to about 25kDa, or from about lOkDa to about 30kDa. Linear or branched PEG molecules having molecular weights from about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons. In one embodiment of the disclosure, the PEG is a 40kD branched PEG comprising two 20 kD arms.
[0270] |The present disclosure also contemplates compositions of conjugates wherein the PEGs have different n values, and thus the various different PEGs are present in specific ratios. For example, some compositions comprise a mixture of conjugates where n=l, 2, 3 and 4. In some compositions, the percentage of conjugates where n=l is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to resolve conjugate fractions, and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
[0271] PEGs suitable fbr conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
[0272| Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15: 100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. US Patent No. 5,650,234), which react preferentially with the side chain of lysine residues to form a carbamate linkage but are also known to react with histidine and tyrosine residues. Use of a PEG-aldehyde linker targets a single site on the N-terminus of a polypeptide through reductive amination. [02731 Pegylation frequently occurs at the a-amino group at the N-terminus of the polypeptide, the epsilon amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant polypeptides possess a single alpha and a number of epsilon amino and imidazole groups, numerous positional isomers can be generated depending on the linker chemistry. General PEGylation strategies known in the art can be applied herein.
[02741 The PEG can be bound to a binding molecule of the present disclosure via a terminal reactive group (a “spacer") which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol. The PEG having the spacer which can be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating succinic acid ester of polyethylene glycol with hi- lly droxysuccinylimide.
[0275 | The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure. Specific embodiments PEGs useful in the practice of the present disclosure include a lOkDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), lOkDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), a 20kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), a 20kDa linear PEG- NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME- 200HS, NOF), a 20kDa 2-arm branched PEG-aldehyde the 20 kDA PEG-aldehyde comprising two lOkDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), a 20kDa 2-arm branched PEG-NHS ester the 20 kDA PEG-NHS ester comprising two lOkDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40kDa 2-arm branched PEG-aldehyde the 40 kDA PEG-aldehyde comprising two 20kDA linear PEG molecules (e.g., Sunbright® GL2- 400AL3), a 40kDa 2-arm branched PEG-NHS ester the 40 kDA PEG-NHS ester comprising two 20kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), a linear 30kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and a linear 30kDa PEG-NHS ester.
[02761 In some embodiments, a linker can used to join the PEG molecule to the heterodimeric hIL12M-Fc mutein . Suitable linkers include “flexible linkers” which are generally of sufficient length to permit some movement between the modified polypeptide sequences and the linked components and molecules. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Suitable linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids. Examples of flexible linkers are described in Section IV. Further, a multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or SO- SO) of these linker sequences may be linked together to provide flexible linkers that may be used to conjugate two molecules. In some embodiments, the linker is a GSA linker as described above. Alternative to a polypeptide linker, the linker can be a chemical linker, e.g., a PEG-aldehyde linker. In some embodiments, the binding molecule is acetylated at the N-terminus by enzymatic reaction with N-terminal acetyltransferase and, for example, acetyl CoA. Alternatively, or in addition to N- terminal acetylation, the binding molecule can be acetylated at one or more lysine residues, e.g., by enzymatic reaction with a lysine acetyltransferase. See, for example Choudhary et al. (2009) Science 325 (5942):834 840.
Biased Activity of Heterodimeric hIL12M-Fc Muteins
[02771 |As discussed above, the heterodimeric hIL12M-Fc muteins of the present disclosure provide cell-type biased signaling of the downstream signal transduction mediated through the IL12 receptor compared to a reference polypeptide (e.g., wild type hIL12). In particular, the heterodimenc hIL12M-Fc muteins of the present disclosure retain significant hIL-12 signaling in CD8+ T cells and have decreased hIL-12 signaling in NK cells compared to a wildtype hIL-12. The selective activation of CD8+ T cells versus NK cells can be evaluated by the activation of interferon gamma (IFNy) while providing a reduction in STAT4 signaling in NK cells.
Enhanced IFNy In CD8+ T Cells v. NK Cells:
[027<S| The heterodimeric hIL12M-Fc muteins of the present disclosure activate interferon gamma (IFNy) in CD8+ T cells and have decreased IFNy signaling in CD8+ T cells compared to the wildtype IL 12. A series of experiments were performed to evaluate the IFNy inducing effects of wt hIL12 and hIL12 proteins comprising P40 subunit E81A/F82A indicated as “2xAla”, E81 A/F82 A/Kl 06 A indicated as “3xAla” and substitution W37A and KiH heterodimeric hIL 12M- Fc comprising the same mutations E81A/F82A indicated as “2xAla Fc”, E81A/F82A/K106A indicated as “3xAla Fc” and the substitution W37A (W37A Fc), on CD8, CD4 and NK cells, respectively. The molecules were produced recombinantly in substantial accordance with the teaching of Example 1 and evaluated for IFNy in substantial accordance with the teaching of Example 3 herein. As illustrated by the data provided in Figure 1, the heterodimeric hIL12M-Fc muteins of the present disclosure activate IFNy in CD8+ T cells and have decreased IFNy signaling in CD8+ T cells compared to the wildtype IL12. Reduced STAT4 Signaling in NK cells:
10279] In some embodiments, the heterodimeric hIL12M-Fc muteins describe herein exhibit substantially full or increased STAT4 signaling in CD8+T cells and decreased STAT4 signaling in NK cells compared to a reference polypeptide (wt hIL12). A series of experiments were performed to evaluate the effect of various IL12 muteins comprising hP40 muteins, both as proteins and as Fc fusions, of STAT4 signaling in CD8+ T cells, CD4+ T cells relative to STAT4 signaling in NK cells. The evaluation of STAT4 was performed in substantial accordance with the teaching of Example 2. The test articles evaluated were wt hIL12 and hIL12 proteins comprising a P40 subunit with the substitutions E81A/F82A indicated as “2xAla”, E81A/F82A/K106A indicated as “3xAla” and KiH heterodimeric hIL12M-Fc constructs comprising wild type hIL12P35 and hP40 (wt Fc), E81A/F82A indicated as “2xAla Fc”, E81 A/F82A/K106A indicated as “3xAla Fc” on CD8, CD4 andNK cells, respectively. The results of these experiments are provided in Figure 2 of the attached drawings. As illustrated by the results provided in Figure 2, the heterodimeric hIL12M-Fc muteins of the present disclosure provide differential STAT4 signaling on CD8+ T cells relative to NK cells. Additionally, the data provided in Figure 2, particularly Panel A and Panel B demonstrate that the heterodimeric hIL12M-Fc muteins of the present disclosure act as IL 12 partial agonists on T cells.
Use in the Treatment of Neoplastic Disease:
10280] The heterodimeric hIL12M-Fc muteins described herein are useful in the treatment of neoplastic disease. To demonstrate the activity of the heterodimeric hIL12M-Fc muteins, surrogate murine IL12Fc muteins containing analogous mutations to the human molecules were generated to evaluate the effects in anMC38 mouse tumor model. A sequence alignment of the naturally occurring human and mouse P40 and P35 polypeptides are provided in Figures 7 and 8, respectively, of the attached drawings. A description of the heterodimeric mIL12Fc test agents used in the MC38 tumor study are summarized in the Tables 9 and 10 below:
Figure imgf000089_0001
Figure imgf000090_0001
MC38 Tumor Study #1:
[O2S11 Briefly, approximately IxlO6 MC38 cells in Matrigel were implanted subcutaneously into 6-8 week old C57BL/6 mice and the tumors permitted to attain an average tumor volume at the initiation of treatment of approximately 100 mm3- 120mm3, The mice were separated into individual treatment groups. The mice were treated by intraperitoneal administration of the various test agents at the doses and dosing schedule indicated in the Table 11 below. In this study bodyweight (BW), an indication of toxicity, and tumor volume (TV) as an indicator of anti-tumor efficacy were measured twice per week.
Figure imgf000090_0002
[02S2] The data arising from the study described above are presented in Figures 3 (tumor volume), Figure 4 (body weight), and Figure 5 (survival) of the attached drawings. Note that the labeling of the panels in Figures 3 and 4 corresponds to the treatment group in Table 11 with tumor volume and body weight on the y-axes respectively and time (study days) is represented on the x- axes in each figure
[0283] Figure 3 provides a spider plot summary of the effect on tumor volume with respect to each animal in each study group. As demonstrated by the data presented, the murine surrogate of the heterodimeric IL12Fc mutein as described herein was effective in the control of tumor growth in this study.
[02S41 Figure 4 provides the average body weight of the animals during the course of the above study. Although the wild type IL 12 Fc test agents evaluated in this study demonstrated an inhibition of tumor growth, the data presented in Figure 4 indicates that such wild type IL12 Fc fusions are associated with significant toxicity as indicated by a significant loss of bodyweight (see, e g. Figure 4, Panels B, C, and E). In contrast, the heterodimeric IL12Fc mutein evaluated in group H comprising the 2xAla mutations in the mP40 domain did not suggest significant toxicity. This is particularly noteworthy as the IL12Fc mutein comprising the 2xAla P40 mutations was administered at a dose at more than 50 fold higher than the other wild type IL12Fc conjugates evaluated. The ability of the IL12Fc mutein comprising the 2xAla mutations to control tumor growth in the absence of significant toxicity is further supported by the data provided in Figure 5 which indicates that all animals treated in with IL12Fc mutein comprising the 2xAla survived while there was a significant negative effect on survival in those test groups administered the wild type IL12Fc conjugates despite the apparent antitumor effect these other molecules demonstrated. Consequently, these data demonstrate that the heterodimeric IL12Fc muteins of the present disclosure are useful in the treatment of neoplastic disease and exhibit significantly lower toxicity than wild type IL12Fc conjugates that do not possess the mutations in the P40 domain of the IL12Fc conjugate.
MC38 Tumor Study 2:
[0285] |A second MC38 tumor study in substantial accordance with the foregoing and the study design is provided in Table 12 was performed. In this study bodyweight (BW), an indication of toxicity, and tumor volume (TV) as an indicator of anti-tumor efficacy were measured twice per week. Mice were bled prior to the start of treatment (0 hours) and four hours, 1 day and 7 days following administration of the test agent. Some animals were taken down at days 2 and 24 post administration of the test agent for immunohistochemical evaluation and FACS analysis.
Table 12.
Figure imgf000092_0001
[0286] The data relating to the effects on tumor growth of these test agents and the study described in Table 12 are provided in Figure 6 of the attached drawings. The data relating to the IL12 proteins (not conjugated to an Fc dimer) are presented in Figure 6, Panel A and the Fc conjugated IL 12 proteins is presented in Figure 6, Panel B. A comparison of the data in Figure 6 Panels A and B demonstrate that the IL 12 molecules when conjugated to a dimeric Fc domain provide significantly improved anti -tumor efficacy relative to their non-Fc conjugated counterparts.
[0287| The blood samples obtained at 0 hours and 4 hours, 1 day and 7 days following administration of the test agents in treatment groups A-H were evaluated for the concentration of murine interferon gamma (mIFNg) in serum as determined by enzyme-linked immunosorbent assay (ELISA). The data obtained is presented in Figure 9 of the attached drawings. Panel A of Figure 9 indicates the results from treatment groups A-E (i.e., the IL12 molecules not conjugated to the Fc) and Panel B indicates the results from treatment groups F, G, and H (i.e., the IL12 molecules conjugated to the Fc). As can be seen from the data presented in Figure 9, the Fc conjugated heterodimeric mIL12-Fc molecules comprising the amino acid substitutions (i.e., "2x Ala" and “3xAla”) demonstrated a significant delay in the induction of interferon gamma in relation to the other treatment groups, in particular in relation to the IL 12 Fc comprising the wild type P40 sequence. This delay in the induction of IFNg results in a decrease in the acutue toxicity associated with IL 12 treatment.
[02881 As noted, samples from this study were subjected to FACS analysis and the NK cells sorted from spleen and tumor tissues. The percentage of lymphocytes in each tissue in response to each of the treatment groups of Table 12 is provided in Figure 10 of the attached drawings. As indicated the Fc conjugated molecules resulted in lower induction of NK cells relatve to the non- Fc conjugated molecules in each tissue type. However, as shown in Figure 10, Panel B and C (expanded view of NK frequence in tumor tissue), the Fc conjugated heterodimeric mIL12-Fc molecules comprising the P40 amino acid substitutions (i.e., “IL12 2xAla Fc” and “IL12 3xAla Fc”) demonstrated a lower frequency of intratumoral NK cells relative to the heterodimeric mIL12 Fc comprising the wild type P40 sequence (“IL 12 WT Fc”).
[02891 Furthermore, the phenotype of NK cells from spleen in the above study were evaluated for T-bet relative to intracellular Granzyme B. T-bet is required for NK cell effector function and NK cell cytolyitic activity. The results of this analysis is provided in Figure 11 of the attached drawings with T-bet on the vertical axis and granzyme B on the horizontal axis. As can be seen from the data presented in Figure 10, there is a loss of Tbet + NK cells with heterodimeric mIL12 Fc comprising the wild type P40 sequence (“IL 12 WT Fc”) but not with the Fc conjugated heterodimeric mIL12-Fc molecules comprising the P40 amino acid substitutions (i.e., “IL 122xAla Fc” and ;‘IL12 3xAla Fc”).
Dose Titration of mIL12 Fc Constructs in CT26 Model:
[029()| The effect of dosing at various levels of mIL12 Fc heterodimers was evaluated in a CT 26 tumor model. Briefly 6-8 week old BALB/c miles were subcutaneously implanted with approximately 0.3 x 106 CT26 mouse tumor cells in Matrigel. Mice were randomized into groups when the tumor reached an average volume of 118 mm3. Treatment groups and the study design are summarized in Table 13. Body weight and tumor volume were measured twice per week. Some mice were sacrified on Day 8 and day 29 of the study for FACS, IHC and serum analys. Serum PK was measured at Days 1, 8, 15, 22 and 28 of the study.
Figure imgf000093_0001
Figure imgf000094_0001
[0291] The data relating to effect on tumor growth in the above study is provided in Figures 12, 13, and 14 of the attached drawings. As shown in Figure 12, the wild type IL-12WT Fc demonstrates a potent anti-tumor activity in CT26 model at 0.8ug (0.5ug IL-12)/dose. The IL-12 3xAla Fc leads to tumor regression with a 5-7 days delay compared to IL-12WT Fc. CT26 model appears to be more sensitive to IL-12 treatment than our in-house MC38. In Figure 13, neither IL-12WT Fc nor IL-12 3xAla Fc lead to weight loss. In view of the toxicity observed with the test agents in the MC38 study conducted in C57BL/6 mice, the BALB/c mice appeared to be more resistant to IL-12WT Fc toxicity. Figure 14 provides FACS analysis which indicates that wild type IL 12 Fc results in NK cell degranulation but that 3xAla IL 12 Fc does not substantially induce NK cell degranulation.
Evaluation of Toxicity in Combination withNK Cell Depletion Study #1 S6-21-005
J 0292 ] A study was conducted in mice to evaluate the effect on NK cell depletion in combination with the IL12 Fc test agents. NK cells were depleted using an the NK 1.1 antibody 6-8 week old C57BL/6 treated with an NK cell depleting antibody (aNKl. l/IL12) followed by IL-12 administration. PBS or aNKl.l was administered on days -3, 0, 3 and 7. IL12 was administered on days 0, 4, and 8 (see Table 14). Moribund mice were taken down for serum analysis and IHC. BW was measured every day. Some mice were bled and evaluated for the presence of absence of NK cells to confirm that the anti=NKl .1 antibody was depleting the NK cells. These evaluations confirmed by FACS that the anti-body was indeed depleting the NK cells. The remaining surviving of the mice were taken down on day 13.
Figure imgf000094_0002
[0293| PBS/aNKl.l was administered on days -3, 0, 3 and 7. IL12 was administered on days 0, 4, and 8. Animals were monited for bodyweight, survivable. Animals were sacrified on day 13. The results of the study are presented in Figure 15. As can be seen from the data presented, NK depletion alleviates toxicity as evaluated by body weight loss and prevents mortality after 1 ,6ug IL-12WT Fc treatment. NK cell depletion it also substantially reduces mortality and morbidity in at the dose of 4.8ug. Additionally, NK cell depletion is transient and NK cells rebound at d6 after the last NK depletion dose.
Evaluation of Antitumor Efficacy and Toxicity with NK/CD8 TCell Depletion Study
[0294| A second study was conducted to evaluate the effect on NK and CD8 T-cell depletion in combination with the IL Fc test agents. In this study, 6-8 week old C57BL/6 mice were subcutaneously implanted with IxlO6 MC38 cells in Matrigel. Mice were randomized into groups when the tumors reached the average volume of approximately 85mm3. Again, NK cells were depleted using an the NK 1. 1 antibody. CD8 T cells were depleted. The antibodies and test agents were administered in accordance with the study design provided in Table 15 below. Mice were bled at various timepoints to confirm depletion efficiency. Tumor volume and bodyweight were measured twice weekly and the animals were sacrificed at the conclusion of the study for FACS analysis.
Figure imgf000095_0001
[0295 | The results of the study are presented in Figures 16 and 17. As indicated by the data presented in Figure 16, the depletion of NK cells does mitigate toxicity indicating that the NK cells contribute to the toxicity observed with IL12 agents. However, as shown in Figure 17, the NK cells contribute comparatively minimally to antitumor efficacy. This data indicates that an IL 12 Fc agent that having a biased activation of CD8 T cells and a reduced activation of NK cells (e.g. a heterodimeric hIL12M-Fc compising a hIL12P40M-Fc polypeptide) is efficacious in the treatment of cancers and possesses a substantially reduced toxicity relative to that observed with IL 12 agents comprising a wild-type P40 polypeptide.
Evaluation ofIL12 Agents in B6, RAG2 KO and RAG2/CD132 double KO mice
[0296 J To further evaluate the activity of the hIL12 Fc muteins relative to activation of T cells versus NK cells, an antitumor efficacy study was conducted in B6 mice, RAG 2 knockout mice and RAG2/CD132 double knockout mice with the IL12 and control test agents evaluated above. B6 mice were used as a control group relative to the RAG2 knockoout (KO) mice which lack T and B cells and the RAG2/CD132 double knockout mice which lack T, B and NK cells. Briefly, the approximately IxlO6 MC38 cells were implanted s.c. in Matngel 11 days prior to the initiation of treatment (Day -11) and when the tumor volume reached approximately 120mm3 the mice were randomized into treatment groups as described in Table 16 below. The test articles and contols were administered in accordance with the schedule describe in Table 16 below. Micd were evaluated for weight loss and tumor volume twice weekly.
Figure imgf000096_0002
Figure imgf000096_0001
[0297] The results of this study are presented graphically in Figure 18. As can be observed from the data, the genetic loss of T cells greatly diminishes IL-12-mediated tumor control and genetic loss of T, NK and ILCs renders mice completely resistant to IL-12. When combined with the foregoing data, these studies demonstrate that the antitumor effects of IL 12 are not dependent on the presence of NK cells and that an heterodimeric IL 12 Fc mutein having reduced activation of NK cells retains antitumor efficacy.
Combination Studies:
[0298 | In order to evaluate that activity of the heterodimeric hIL12M-Fc muteins of the present disclosure in combination with supplementary therapeutic agent in the treatment of neoplastic disease, two studies were performed to evaluate the heterodimeric mIL12Fc surrogate muteins in combination with interleukin-2 and an anti-PDl checkpoint inhibitor molecule the MC38 tumor model as previously described herein. The study design is provided in table 17 below
Figure imgf000097_0001
[0299 | The murine IL2 mutem was developed for in vivo studies in mice to correlate activity between the rodent (mouse) and primate (human) environments for human IL2 muteins comprising amino acid substitutions at positions 18, 22 and 126 numbered in accordance with mature wild type hIL2, in particular an hIL2 mutein comprising the amino acid substitutions L18R/Q22E/Q126K. The amino acid sequence of the murine IL2 (mIL2) polypeptide used in this study is:
APTSSSTSSSTAEAQQQQQHLEQLRMDLEELLSRMENYRNLKLPRMLTF KFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRV TVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCHSIISTSPQ (SEQ ID NO: 181) and is N-terminally PEGylated with a 40kD branched chain PEG with a linker and is referred to as PEG-mREH. The results of this study are presented in Figures 19 (PD1) and 20 (mIL2 mutein). As can be seen from the data provided in these Figures, the combination of the heterodimeric Fc P40M mutein provides an enhanced antitumor effect in this model. In particular the combination of the heterodimeric Fc P40M mutein and the anti-PDl antibody desmonstrates a significantly enhanced effect leading to complete response.
[0300] Two additional combination studies in the MC38 tumor model (Combination MC38 Tumor Study #2 and Combination MC38 Tumor Study #3 ) were performed as described in Examples 5, 6, and 7 herein to evaluate toxicity and efficacy of the combination of a murine surrogate of an hIL2M in combination with a murine surrogate of an hIL12 in the treatment of a mammalian subject suffering from a cancer, specifically the mouse MC38 colorectal cancer cell line. The design and structure of the murine surrogate molecules evaluated in these studies are described in Example 6. The study design for Combination MC38 Tumor Study #2 is provided in Table 18 and Example 6 and the data generated from this study are presented in Tables 19 and 20 and Figures 21 and 22 of the attached drawings. The study design for MC38 Tumor Study #3 is provided in Table 21 and Example 7. The data generated of MC38 Tumor Study #3 studies are presented in Figures 23 and 24 of the attached drawings. As demonstrated by the data generated in these studies, the administration of Compound 3, a murine surrogate of an hIL2M mutein of the present disclosure and Compound 2, in combination with a murine surrogate of hIL12M mutein of the present disclosure, generates superior anti-tumor to the administration of either agent alone without unacceptable toxicity.
[0301 ] Although not indicated in Figures, it was observed that the combination heterodimeric wild type IL12 Fc and the anti-PDl antibody was observed to alleviate the toxicity previously observed with the heterodimeric wild type IL 12 Fc in this model. The foregoing data demonstrates that the heterodimeric hIL2 IL12 muteins of the present disclosure are useful in the treatment of neoplastic disease in combination with supplementary therapeutic agents, particularly IL2, IL2 muteins and checkpoint inhibitors such as anti-PDl antibodies.
Synthesis of Polypeptide Domains of hIL12M and hIL2 Molecules
[0302] The hIL2M and hIL12M molecules of the present disclosure comprise polypeptides. However, in some embodiments, the hIL2M and hIL12M of the present disclosure comprise anon- peptidyl components such as a PEG molecule. The process for PEGylation of proteins is discussed elsewhere herein. The following is directed to the synthesis of the polypeptide components of hIL2M and hIL12M of the present disclosure such including the hIL12P40M (or hIL12P40M-Fc) and hIL12P35 (or hIL12P35-Fc) polypeptide subunits as well as the recombinant production of the heterodimeric hIL12M-Fc muteins muteins of the present disclosure.
[0303] The hIL2M and hIL12M polypeptide and/or ML12P40M (hIL12P40M-Fc) and hIL12P35 (hIL12P35-Fc) subunit domains muteins may be produced by conventional methodology for the construction of polypeptides including recombinant or solid phase syntheses as described in more detail below.
Chemical Synthesis
[0304] In some embodiments, polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be prepared by chemical synthesis. The chemical synthesis may proceed via liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and/or peptide/protein backbone modification. Various forms of SPPS are available for synthesizing polypeptide domains of hIL2M and hIL12M molecules of the present disclosure are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J.A. et al., (2005) Protein Pept Lett. 12:723-8). In the course of chemical synthesis, the alpha functions and any reactive side chains may be protected with acid-labile or base-labile groups that are stable under the conditions for linking amide bonds but can readily be cleaved without impairing the peptide chain that has formed.
[0305| In the solid phase synthesis, either the N-terminal or C-terminal amino acid may be coupled to a suitable support material. Suitable support materials are those which are inert towards the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and which do not dissolve in the reaction media being used. Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers; and the like. The successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer.
[0306 | At the end of the solid phase synthesis, the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups. The peptide obtained can be purified by various chromatographic methods including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high pressure liquid chromatography (HPLC) and reversed-phase HPLC.
Recombinant Synthesis
[0307] The polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be produced by recombinant DNA technology'. The techniques and materials for recombinant production of polypeptide in procaryotic and eucaryotic cells are well established and known to those of skill in the art. In the typical practice of recombinant production of polypeptides, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will be accomplish, the nucleic acid sequence being operably linked to one or more expression control sequences encoding by the vector and functional in the target host cell. The recombinant protein may be recovered through disruption of the host cell or from the cell medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide. The recombinant protein may be purified and concentrated for further use including incorporation.
[030S| In some embodiments, polypeptide domains of hIL2M and hIL12M molecules of the present disclosure are produced by recombinant methods using a nucleic acid sequence encoding the polypeptide domains of hIL2M and hIL12M (or fusion proteins thereof). The nucleic acid sequence encoding the desired polypeptide domains of hIL2M and hIL12M molecules can be synthesized by chemical means using an oligonucleotide synthesizer by techniques well known in the art. The nucleic acid molecules encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may contain naturally occurring sequences or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (i.e., either a sense or an antisense strand).
[0309 | In some embodiments, the nucleic acid sequence encoding the wild type human P40 signal peptide and the hIL12P40M polypeptide is selected from the group consisting of SEQ ID NOS: 94, 97, 99, 100, 102, 105, 118, 120, 126, 128, 131, 134, 137, 140, 143, 146, 149, and 152.
In some embodiments, the nucleic acid sequence encoding the wild type human P35 signal peptide and the hIL12P40M polypeptide is selected from the group consisting of SEQ ID NOS:95, 96, 98, 103, 104, 106, and 123. [O3 IO| In general, when expressed in mammalian cells, the the nucleic acid sequence encoding polypeptide domains of hIL12M molecules of the present disclosure is modified to encode a signal peptide to facilitate secretion of the hIL12M. The signal peptide may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector. If a heterologous signal peptide is employed, it is preferably a signal peptide that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In some embodiments, the signal peptide is selected from the group consisting of human serum albumin signal peptide, prolactin albumin signal peptide, the human IL2 signal peptide, human trypsinogen-2, human CD-5, the human immunoglobulin kappa light chain, human azurocidin, Gaussia luciferase and functional derivatives thereof. Particular amino acid substitutions to increase secretion efficiency using signal peptides are described in Stem, et al. (2007) Trends in Cell and Molecular Biology 2: 1-17 and Kober, et al. (2013) Biotechnol Bioeng. 1110(4): 1164-73. Alternatively, the signal peptide may be a synthetic sequence prepared in accordance established principles. See e.g., Nielsen, et al. (1997) Protein Engineering 10(1 ): 1 -6 (Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites)' ,' Bendtsen, et al (2004) J. Mol. Biol 340(4):783-795 (Improved Prediction of Signal Peptides SignalP 3.0),' Petersen, et al (2011) Nature Methods 8:785-796 (Signal P 4.0; discriminating signal peptides from transmembrane regions).
[0311] In some embodiments, the signal peptide the hIL12P40M and hIL12P35 polypeptide subunits of the heterodimeric hIL12M-Fc mutein is the naturally occurring hP40, hP19, and hIL12P35 signal peptide, respectively (i.e. the human hP40 and hIL12P35 signal sequences). In some embodiments, the signal peptide of the hIL12P35 sequence is the naturally occurring wild type human P35 sequence having the amino acid sequence MCPARSLLLVATLVLLDHLSLA (SEQ ID NO: 179). In some embodiments, the signal peptide of the HL12P40M sequence is the naturally occurring wild type human P40 sequence having the amino acid sequence MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 180).
[0312] The inclusion of a signal peptide depends on whether it is desired to secrete the heterodimeric hIL12M-Fc mutein from the recombinant cells in which it is made. If the chosen host cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. When the recombinant host cell is a yeast cell such as Saccharomyces cerevisiae, the alpha mating factor secretion signal peptide may be employed to achieve extracellular secretion as described in Singh, United States Patent No. 7,198,919 Bl.
[0313] The incorporation of a chelating peptide facilitates purification immobilized metal affinity chromatography (IMAC) as described in Smith, et al. United States Patent No. 4,569,794 issued February 11, 1986. Examples of chelating polypeptides useful in the practice of the present disclosure are described in Smith, et al. supra and Dobeli, et al. United States Patent No. 5,320,663 issued May 10, 1995. Particular transition metal chelating polypeptides useful in the practice of the present disclosure binding molecule are polypeptides comprising 3-6 contiguous histidine residues (SEQ ID NO: 197) such as a six-histidine (His)e peptide (SEQ ID NO: 198) and are frequently referred to in the art as “His-tags.” Alternatively, a hemagglutinin tag may be incorporated into the chimeric protein to facilitate purification of protein expressed in eukaryotic cells. By first and second, it should not be understood as limiting to the orientation of the elements of the fusion protein and a heterologous polypeptide can be linked at either the N-terminus and/or C-terminus of the polypeptide domains of heterodimeric hIL12M-Fc mutein . For example, the N-terminus may be linked to a targeting domain and the C-terminus linked to a hexa-histidine tag purification handle (SEQ ID NO: 198).
[0314] In some embodiments, the nucleic acid sequence encoding polypeptide domains of hIL2M and hIL12M molecules of the present disclosure may be “codon optimized” to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast and bacterial host cells, are well known in the and there are online tools to provide for a codon optimized sequences for expression in a variety of host cell types. See e.g., Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David Hacker (Human Press New York). Additionally, there are a variety of web based on-line software packages that are freely available to assist in the preparation of codon optimized nucleic acid sequences.
[0315| The nucleic acid sequences encoding polypeptide domains of hIL2M and hIL12M molecules of the present disclosure prepared may be operably linked to suitable genetic control elements that are capable of effecting expression of the polypeptide in the host cell to be transformed with the expression vector. The specific type of control elements necessary to effect expression will depend upon the cell type to be transformed. In the practice of the present invention, the cell to be transformed is a mammalian T-cell. The term control elements refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation which affect the replication, transcription and translation of the polypeptide coding sequence in a recipient cell. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell. [0316| The nucleic acid sequences encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure is/are operably linked to a promoter sequence. The term "promoter" is used in its conventional sense to refer to a nucleotide sequence at which the initiation and rate of transcription of a coding sequence is controlled. The promoter contains the site at which RNA polymerase binds and also contains sites for the binding of regulatory factors (such as repressors or transcription factors). Promoters can be naturally occurring or synthetic. The promoter can be constitutively active, activated in response to external stimuli (inducible), active in particular cell type or cell state (tissue specific or tumor specific) promoters, and/or regulatable promoters. The term "inducible promoter" refers to promoters that facilitate transcription of the Bioactive polypeptide preferably (or solely) under certain conditions and/or in response to external chemical or other stimuli. Examples of inducible promoters are known in the scientific literature (see, e.g., Yoshida et al., Biochem. Biophys. Res. Comm., 230:426-430 (1997); lida et al., J. Virol., 70(9): 6054-6059 (1996); Hwang et al., J. Virol., 71(9): 7128-7131 (1997); Lee et al., Mol. Cell. Biol., 17(9): 5097-5105 (1997); and Dreher et al., J. Biol. Chem., 272(46): 29364-29371 (1997). Examples of radiation inducible promoters include the EGR-1 promoter. Boothman et al., volume 138, supplement pages S68-S71 (1994).
[0317] When expressing a multi-subunit protein as in the practice of the present invention, each polypeptide subunit may be operably linked to an expression control sequence (monocistronic) or multiple polypeptides may be encoded by a polycistronic construct where multiple polypeptides are expressed under the control of a single expression control sequence. Examples of elements which may be employed to facilitate polycistronic expression internal ribosome entry site (IRES) elements or the foot and mouth disease vims protein 2A (FMVD2A) system. A wide variety of IRES sites are known (see e.g. Doudna JA, Sarnow P. Translation initiation by viral internal ribosome entry sites. In: Translational Control in Biology and Medicine: Mathews et al, Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2007. pp. 129-154; http://www.IRESite.org). Examples of IRES elements include the picomavirus IRES of poliovirus, rhinovirus, encepahlomyocardits virus, the aphthovirus IRES of foot and mouth disease virus, the IRES cricket paralysis virus (CrPV) the hepatitis A IRES of hepatitis A virus, the hepatitis C IRES of hepatitis C virus, the pestivirus IRES of swine fever or bovine diarrhea viruses, the cripavirus IRES, and mammalian IRES elements such as the fibroblast growth factor-1 IRES, the fibroblast growth factor-2 IRES, PDGF IRES, VEGF IRES, IGF-2 IRES. The use of IRES elements typically results in significantly lower expression of the second protein of the polycistronic message. The use of the FMDV2A system results in more efficient production of the downstream proteins as the multiple proteins are first expressed as a fusion protein which contains the autoproteolytic FMDV2A domain which cleaves the polyprotein into functional subunits. Ryan and Drew (1994) EMBO J. 13(4): 928-933. Depending on the construction of the polycistronic coding sequence, especially to facilitate restriction endonuclease sites, the use of the FMDV2A system frequently may in the addition of a small number amino acids to the carboxy terminus of the upstream protein.
[0318] In preparing a bicistronic nucleic acid sequence encoding a heterodimeric hIL12M-Fc mutein, the nucleic acid sequences encoding the hIL12P35-Fc and hIL12P40M-Fc subunits of the heterodimeric hIL12M-Fc muteins of the present disclosure may be provided in a bicistronic expression cassette to provide for co-expression of the subunits in a mammalian host cell. In some embodiments, the present disclosure provides bicistronic nucleic acids arranged as illustrated below:
5’ - hIL12P40M-Fc - P2A - hIL12P35-Fc - 3’
5’ - hIL12P35-Fc - P2A - hIL12P40M-Fc - 3’
5’ - hIL12P40M-Fc - IRES - hIL12P35-Fc - 3’
5’ - hIL12P40M-Fc - T2A - hIL12P35-Fc - 3’ 5’ - hIL12P35-Fc - T2A - hIL12P40M-Fc - 3’ 5’ - hIL12P35-Fc - IRES - hIL12P40M-Fc - 3’ 5’ - hIL12P35-Fc - T2A - hIL12P40M-Fc - 3’
[0319 J Once assembled (by synthesis, site-directed mutagenesis or another method), the nucleic acid sequence encoding the polypeptide domains of hIL2M and hIL12M molecules of the present disclosure will be inserted into an vector. A variety of expression vectors for uses in various host cells are available and are typically selected based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are examples of non-viral vectors. To facilitate efficient expression of the recombinant polypeptide, the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that are functional in the chosen expression host.
[0320[ Expression vectors typically contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
[0321 ] Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
[0322] Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence but is preferably located at a site 5' from the promoter. Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques.
[0323| In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Additional examples of marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B- phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context. Proper assembly of the expression vector can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host.
[0324] In some embodiments of the disclosure, the expression cassete comprising the CMV promoter and nucleic acid sequence encoding the ML12P35 (or hIL12P35-Fc) and ML12P40M (or hIL12P40M-Fc) polypeptides is inserted into a pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA). In some embodiments of the disclosure, the expression cassete comprising the CMV promoter and nucleic acid sequence encoding the ML12P35 and hIL12P40M polypeptides is inserted into the multiple cloning site of the pExSyn2.0 expression vector as prepared in accordance with the teaching of Example 1.
[0325] Host cells are typically selected in accordance with their compatibility with the chosen expression vector, the toxicity of the product coded for by the DNA sequences, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells.
[0326] In some embodiments the hIL12P40M, and hIL12P35 polypeptide subunits of the heterodimeric hIL12M-Fc muteins muteins and biologically active variants and fragments thereof can also be made in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187:195)).
[0327| Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL-2648), Expi293 cells, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA. ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
[032<S] iVector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory' Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non- viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).
[03291 In some embodiments, the nucleic acid sequences encoding the hIL12P35 (or ML12P35- Fc) and hIL12P40M (or IL12P40M-Fc) are each provided a separate expression vectors which are then co-transfected into the host cell. In some embodiments, a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in a mammalian cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M operably linked to a promoter functional in a mammalian cell are co-transfected into a mammalian host cell. In some embodiments, the promoter functional in a mammalian cell of the first and second recombinant expression vectors is the CMV promoter. In some embodiments the first and second recombinant expression vectors are pCDNA3.4 mammalian expression vectors (Life Technologies, Carlsbad, CA). In some embodiments the first and second recombinant expression vectors are pExSyn2.0 expression vectors as prepared in accordance with the teaching of Example 1.
[0330| In some embodiments, the present disclosure provides a recombinant mammalian host cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in the recombinant mammalian host cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a promoter functional in the recombinant mammalian host cell. In some embodiments, the nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) and hIL12P40M (or IL12P40M-Fc) further encodes a signal peptide, in some embodiments. In some embodiments the signal peptide for the hIL12P35 (or HL12P35- Fc) polypeptide is the wild type human P35 signal peptide (SEQ ID NO: 179) . In some embodiments the signal peptide for the hIL12P40M (or IL12P40M-Fc) polypeptide is the wild type human P40 signal peptide (SEQ ID NO: 180). In some embodiments, the nucleic acid sequence encoding the hIL12P35 (or hIL12P35-Fc) and hIL12P40M (or IL12P40M-Fc) signal peptide In some embodiments, recombinant mammalian host cell is selected from the group consisting of CHO and 293 cells. In some embodiments, the present disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding HL12P35 (or hIL12P35-Fc) operably linked to a promoter functional in a CHO cell and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a promoter functional in a CHO cell. In some embodiments, the present disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hIL12P35 (or hIL12P35-Fc) operably linked to a CMV promoter and a second recombinant expression vector comprising a nucleic acid sequence encoding hIL12P40M (or IL12P40M-Fc) operably linked to a CMV. In some embodiments the first and second recombinant expression vectors are pCDNA3.4 mammalian expression vectors (Life Technologies, Carlsbad, CA). In some embodiments the first and second recombinant expression vectors are pExSyn2.0 expression vectors as prepared in accordance with the teaching of Example 1. In some embodiments, the present disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding a hIL12P40M-Fc selected from the group consisting of SEQ ID NOS: 80, 83, 121, 141, 144, 129, 135, 138, 147, 150, and 153 and a second expression vector comprising a nucleic acid sequence encoding a hIL12P35-Fc selected from the group consisting of SEQ ID NOS: 81, 82, and 124. In one embodiment, the cell for production of the hIL12M-Fc is a CHO cell.
[03311 Host cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary' supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.
[03321 Recombinantly-produced polypeptides can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is employed. Alternatively, the recombinant polypeptides can also be recovered from host cell lysates. A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be employed during the recovery phase from cell lysates to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.
[0333] Various purification steps are known in the art and find use, e.g., affinity chromatography. Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural specific binding of one molecular species to separate and purify a second species from a mixture. Antibodies are commonly used in affinity chromatography. Size selection steps may also be used, e.g., gel filtration chromatography (also known as size-exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size. In gel filtration, a protein solution is passed through a column that is packed with semipermeable porous resin. The semipermeable resin has a range of pore sizes that determines the size of proteins that can be separated with the column. In one embodiment, at least one step in the puficiation of the hIL12M- Fc is a column chromatographic purification step employing a Protein A column. Protein A purification of Fc conjugated polypeptides is well known in the art using commercially available equipment and reagents.
[0334] The substantially purified forms of the heterodimeric hIL12M-Fc mutein can be used, e.g., as therapeutic agents, as described herein. The biological activity of the heterodimeric hIL12M-Fc mutein produced in accordance with the foregoing can be confirmed by assay using procedures well known in the art including but not limited to competition ELISA, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fiting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009) with instrumentation commercially available from GE Healthcare Bio-Sciences such as the Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752)); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays). In one embodiment, the activity of an hIL2M of the present disclosure may be assessed by a cell based assay for the induction of intracellular expression of phospho-STAT4 using mammalian T cells such as CD8 T cells or NK cells.
Pharmaceutical Formulations
[0335| In some embodiments, the hIL2M and hIL12M molecules are be incorporated into compositions, including pharmaceutical compositions. The terms “pharmaceutical formulation,” “pharmaceutical composition”, “pharmaceutically acceptable formulation” and “pharmaceutically acceptable composition” are used interchangeably herein. In some embodiments, the pharmaceutical composition comprises the hIL2M and/or hIL12M and at least one pharmaceutically acceptable carrier, buffer, dispersant, preservatives or tonicity agent. In some embodiments, the pharmaceutical composition comprises the hIL2M and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the hIL12M and at least one pharmaceutically acceptable carrier. A pharmaceutical composition comprising the hIL2M and/or hIL12M is formulated to be compatible with its intended route of administration and is compatible with the therapeutic use for which the heterodimenc hIL2M and/or hIL12M is to be administered to the subject in need of treatment or prophyaxis.
[0336] Carriers include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
[0337 J The term buffers includes buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and/or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
[0338] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0339| The pharmaceutical formulations for parenteral administration to a subject should be sterile and should be fluid to facilitate easy syringability. It should be stable under the conditions of manufacture and storage and are preserved against the contamination. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
[0340] In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
Routes of Administration
[03411 In some embodiments of the therapeutic methods of the present disclosure involve the administration of a pharmaceutical formulation comprising a the hIL2M and/or hIL12M molecules to a subject in need of treatment or prophyaxis by a variety of routes of administration, including parenteral administration, oral, topical, or inhalation routes.
[0342] In some embodiments, the methods of the present disclosure involve the parenteral administration of a pharmaceutical formulation comprising a the hIL2M a and/or nd hIL12M molecules to a subject in need of treatment. In some embodiments, the methods of the present disclosure involve the parenteral administration of a pharmaceutical formulation comprising a heterodimeric the hIL2M and a pharmaceutical formulation hIL12M to a subject in need of treatment. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Parenteral formulations comprise solutions or suspensions used for parenteral application can include vehicles the carriers and buffers. Pharmaceutical formulations for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In one embodiment, the formulation is provided in a prefilled syringe presentation.
[0343| In some embodiments of the method of the present disclosure a pharmaceutical formulation comprising a heterodimeric the hIL2M and a pharmaceutical formulation hIL12M is administered to a subject in need of treatment in a formulation to provide extended release. Examples of extended release formulations of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. In one embodiment, the hIL2M and/or hIL12M molecules are formulated with earners that will protect the hIL2M and/or hIL12M against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques known to those of skill in the art of pharmaceutical formulation.
[0344 | In some embodiments of the method of the present disclosure, delivery of the hIL2M and/or hIL12M to a subject in need of treatment is achieved by the administration of a nucleic acid encoding the hIL2M and/or hIL12M. Methods for the administration of a nucleic acids encoding the hIL2M and/or hIL12M to a subject is achieved by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010), or Putnam (Am. J. Health Syst. Pharm. (1996) 53: 151-160 erratum at Am. J. Health Syst. Pharm. (1996) 53:325). In some embodiments, hIL2M and/or hIL12M is administered to a subject by the administration of a pharmaceutically acceptable formulation of recombinant expression vector comprising a nucleic acid sequence encoding the hIL2M and/or hIL12M operably linked to one or more expression control sequences operable in a mammalian subject. In some embodiments, the expression control sequence may be selected that is operable in a limited range of cell types (or single cell type) to facilitate the selective expression of the hIL2M and/or hIL12M in a particular target cell type. In one embodiment, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments the recombinant viral vector is a recombinant adenoassociated virus (rAAV) or recombinant adenovirus (rAd), in particular a replication deficient adenovirus derived from human adenovirus serotypes 3 and/or 5.
[0345] • In some embodiments of the method of the present disclosure, delivery of the hIL2M and/or hIL12M to a subject in need of treatment is achieved by the administration of recombinant host cells modified to express the hIL2M and/or hIL12M, which may be administered in the therapeutic and prophylactic applications described herein. In some embodiments, the recombinant host cells are mammalian cells, e.g., human cells.
Treatment of Neoplastic Disease
10346] The present disclosure provides methods of use of hIL2M in combination with hIL12M of in the treatment of subjects suffering from a neoplastic disease disorder or condition by the administration of a therapeutically effective amount hIL2M and hIL12M, nucleic acids encoding hIL2M and hIL12M, recombinant vector(s) encoding hIL2M and hIL12M or a recombinant cell expressing hIL2M and hIL12M as described herein. In some embodiment, the recombinatnt vector is a recombinant viral vector encoding hIL2M and hIL12M.
[03471 |In one embodiment, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of
(a) administering to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M), the hIL12M comprising a p35 subunit (hIL12p35) and p40M subunit (hIL12p40M) wherein:
• the hIL12p35 has at least 95% sequence identity to mature wild type human hIL12p35 (SEQ ID NO:2); and
• the hIL12p40M has at least 95% sequence identity to mature wild type human hIL12p40 (SEQ ID NO:4), the hIL12p40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12p40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40; and
(b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutem (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182), wherein step (a) is performed in combination with step (b). [034S| In one embodiment, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject, the method comprising the steps of:
(a) administering to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein a = 1 and b = 1, and wherein the polypeptide of formula [1] comprises the ammo acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDT PEEDGITWTLDQSS EVLGSGKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDG IWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTF SVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSA CPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQ LKPLKNS RQVEVS WE Y PDTWST PHS Y FS LT FCVQVQGKS GREKK DRVFTDKTSATVICRKNASI SVRAQDRYYS SSWSEWASVPCSGG GGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL M I S RT P E VT C VWDVS H E D P E VK FN W Y VD G VE VH N AKT K P RE E Q YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKA KGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTT PPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSV LHEALHSHYTQKSLSLS PG ( SEQ I D NO : 129 ) and wherein the polypeptide of formula [2] comprises the amino acid sequence:
RNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTS EEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSC LASRKTS FMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQI F LDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCIL LHAFRIRAVTI DRVMSYLNASGGGGSGGGGSEPKSSDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALAAPIEKTI SKAKGQPREPQVCTLPPSRDELTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGS F FLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) , wherein the polypeptide of formula [1] is linked to the polypeptide of formula [2] by at least one disulfide bond, and
(b) administering to the mammalian subj ect a therapeutically effective amount of an hIL2 mutein (hIL2M) comprising a polypeptide of the amino acid sequence:
PTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS ETTFMCEYADETATIVEFLNRWITFCKSI ISTLT ( SEQ ID NO : 188 ) wherein the N-terminal proline is covalently linked to a40kD branched chain PEG molecule comprising two 20Kd arms. [0349| The compositions and methods of the present disclosure are useful in the treatment of subject suffering from a neoplastic disease characterized by the presence neoplasms, including benign and malignant neoplasms, and neoplastic disease. Examples of benign neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to adenomas, fibromas, hemangiomas, and lipomas. Examples of pre-malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms amenable to treatment using the compositions and methods of the present disclosure include but are not limited to carcinomas (cancers arising from epithelial tissues such as the skin or tissues that line internal organs), leukemias, lymphomas, and sarcomas typically derived from bone fat, muscle, blood vessels or connective tissues). Also included in the term neoplasms are viral induced neoplasms such as warts and EBV induced disease (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular disease including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion and the like.
[0350 | The term “neoplastic disease” includes cancers characterized by solid tumors and non- solid tumors including but not limited to breast cancers; sarcomas (including but not limited to osteosarcomas and angiosarcomas and fibrosarcomas), leukemias, lymphomas, genitourinary cancers (including but not limited to ovarian, urethral, bladder, and prostate cancers); gastrointestinal cancers (including but not limited to colon esophageal and stomach cancers); lung cancers; myelomas; pancreatic cancers; liver cancers; kidney cancers; endocrine cancers; skin cancers; and brain or central and peripheral nervous (CNS) system tumors, malignant or benign, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical carcinoma-in-situ; intestinal polyposes; oral leukoplakias; histiocytoses, hyperprofroliferative scars including keloid scars, hemangiomas; hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis; hyperkeratoses and papulosquamous eruptions including arthritis.
[0351] The term neoplastic disease includes carcinomas. The term "carcinoma" refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The term neoplastic disease includes adenocarcinomas. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. [0352 | As used herein, the term "hematopoietic neoplastic disorders" refers to neoplastic diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
[03531 Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative/myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ambiguous lineage. Exemplary myeloid disorders amenable to treatment in accordance with the present disclosure include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML).
[0354] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin’s Lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Exemplary lymphic disorders amenable to treatment in accordance with the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
[0355] In some instances, the hematopoietic neoplastic disorder arises from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term "hematopoietic neoplastic disorders" refers malignant lymphomas including, but are not limited to, non-Hodgkins lymphoma and variants thereof, peripheral T cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
[0356 | The determination of whether a subject is “suffering from a neoplastic disease” refers to a determination made by a physician with respect to a subject based on the available information accepted in the field for the identification of a disease, disorder or condition including but not limited to X-ray, CT-scans, conventional laboratory diagnostic tests (e.g. blood count, etc ), genomic data, protein expression data, immunohistochemistry, that the subject requires or will benefit from treatment.
Dosing Regimens
[0357 | In one embodiment, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject the method comprising the steps of: (a) administering to the mammalian subject a therapeutically effective amount of a first pharmaceutical composition comprising a hIL12 mutein (hIL12M), the hIL12M comprising a p35 subunit (hIL 12p35) and p40M subunit (hIL12p40M) wherein: (a) the hIL12p35 has at least 95% sequence identity to mature wild type human hIL12p35 (SEQ ID NO:2); and the hIL12p40M has at least 95% sequence identify to mature wild type human hIL12p40 (SEQ ID NON), the hIL12p40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12p40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 wherein the hIL12P35 and hIL12P40M are covalently linked by at least one disulfide bond; and
(b) administering to the mammalian subject a therapeutically effective amount of a second pharmaceutical composition comprising hIL2 mutein (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182), and \ wherein step (a) is performed in combination with step (b).
[035S| In one embodiment, step is performed (a) simultaneously with step (b). In one embodiment, step is performed contemporaneously with step (b). In one embodiment, step (a) is performed sequentially with step (b) and step (b) is performed prior to step (a). In one embodiment, step (a) is performed sequentially with step (b) and step (a) is performed prior to step (b). In one embodiment, step (a) is performed sequentially with step (b) and the hIL12M is provided in advance of the administration of the hIL2M. In one embodiment, step (b) is performed sequentially with step (a) and step (b), the administration of the hIL2M is performed in advance of step (a), the administration of the hIL12M. In one embodiment, step (a) is performed sequentially with step (b) and step (b), the administration of the hIL2M, is performed at least one 1 day, alternatively at least 2 days, alternatively at least 3 days, alternatively at least 4 days, alternatively at least 5 days, alternatively at least 6 days, alternatively at least one week, alternatively at least 10 days, alternatively at least 2 weeks, alternatively at least 3 weeks, alternatively at least 4 w'eeks, in advance of step (a) the administration of the hIL12M.
[03591 In some embodiments, the present disclosure provides a method of treating a neoplastic disease in a mammalian subject, the method comprising the administration of a therapeutically effective amount of a hIL12M combination with a therapeutically effective amount of a hIL2M, wherein the therapeutically effective of amount of the hIL2M is a dose of approximately 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, as a subcutaneous injection and the therapeutically effective of amount of the hIL12M is a dose of approximately 50 pg/kg to approximately 5000 pg/kg, alternatively approximately 100 pg/kg to approximately 2500 pg/kg, alternatively approximately 250 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 1000 pg/kg, administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, as a subcutaneous injection.
[0360 | |In one embodiment, the present disclosure provides a method of treating a neoplastic disease in a human subject, the method comprising the steps of:
(a) administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of hIL12 mutein (hIL12M) consisting of a polypeptide of the formula [1]: hIL 12P40M- L 1 a- UH 1 — Fc 1 [ 1 ] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein the polypeptide of formula [1] has the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDT PEEDGITWTLDQS SEVLGS GKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQ KEPKNKT FLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSS DPQGVT C GAAT L S AE RVRG DN KE Y E Y S VE C QE D S AC P AAE E S L P I E VMVD AVH KL KYENYTS S FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS YFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNAS I SVRAQDRYYS SSWSEWASVPCSGGGGSGGGGSEPKS SDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLS PG ( SEQ ID NO : 129 ) and wherein the polypeptide of formula [2] has the amino acid sequence:
RNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH
EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTS FMM
ALCLSS I YEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVI DELMQA LNFNSETVPQKS SLEE PDFYKTKIKLCILLHAFRIRAVT I DRVMSYLNA SGGGGSGGGGS EPKS S DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P EVT CVWDVS H E D P EVKFNWYVDGVE VHNAKT KP RE EQ Y N S T Y RV VSVLTVLHQDWLNGKEYKCKVSNKALAAPI EKT I SKAKGQPRE PQVCTL PPSRDELTKNQVSLSCAVKGFYPS DIAVEWESNGQPENNYKTT PPVLDS DGS FFLVSKLTVDKSRWQQGNVFSCSVLHEALHS HYTQKSLSLS PG ( S EQ I D NO : 124 ) , wherein the polypeptide of formula [1] is linked to the polypeptide of formula [2] by at least one disulfide bond (STK-026), and
(b) administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of an hIL2 mutein (hIL2M) having the amino acid sequence:
PT S S STKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLT FKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS ETT FMCEYADETATIVEFLNRWIT FCKS I I STLT ( SEQ ID NO : 188 ) wherein the N-terminal proline of SEQ ID NO: 188 is covalently linked by a linker to a 40kD branched chain PEG molecule comprising two 20Kd arms (STK- 012), and wherein step (a) is performed in combination with step (b).
[0361] In one embodiment of the foregoing method, step (b), the administration of the hIL2M, is performed in advance of step (a), the administration of the hIL12M, and the pharmaceutical composition comprising a therapeutically effective amount of the hIL2 mutein (hIL2M) contains a dose of approximately of 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively every two weeks, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (b) is administered by subcutaneous injection, and in step (a), the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M of step a is a pharmaceutical composition contains a dose of approximately 100 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 1000 pg/kg, administered weekly, alternatively bi-weekly, alternatively every' 3 weeks, alternatively every 4 weeks, as a subcutaneous injectionadministered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (a) is administered by subcutaneous injection. Both the hIL12M (STK-026) and hIL2M (STK-012) molecules are modified to provide an extended half- life. Pre-clinical pharmacokinetic studies in primates and clinical experience with STK-012 indicate that the compound persists in vivo in human beings over a period of from 2 to 4 weeks depending on the dose. Pre-clinical pharmacokinetic studies in primates indicate that the STK- 026 molecule persists in vivo for a period of two weeks to more than 4 weeks depending on dose administered. In one embodiment of the method of the present disclosure, the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately 5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every 4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 containing a dose of approximately 5 pg/kg and the hIL12M STK-026 is dosed on schedule of of every 4 weeks, wherein the dosing of the hIL12M STK-026 is initiated two weeks after dosing of the hIL2M STK-012. In one embodiment of the method of the present disclosure, the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately 5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every 4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 contains a dose of approximately 250 pg/kg a to approximately 1000 pg/kg and is dosed on schedule of of every 4 weeks, wherein the dosing of the hIL12M STK- 026 is initiated three weeks after dosing of the hIL2M STK-012.
Combination with Supplementary Anti-Neoplastic Therapeutic Agents:
[0362] In some embodiments, the method of the present disclosure may optionally include, in addition to hIL12M and hIL2M, the administration of one or more active anti -neoplastic agents (“supplementary agents”). Such further anti-neoplastic agents are referred to interchangeably as “supplementary anti-neoplastic combinations” or “supplementary anti-neoplastic combination therapy” and those therapeutic agents that are used in combination with the hIL2M and hIL12M the present disclosure are referred to as “supplementary anti-neoplastic agents.” As used herein, the term “supplementary anti-neoplastic agents” includes anti-neoplastic agents that can be administered or introduced separately, for example, formulated separately for separate administration (e.g., as may be provided in a kit) and/or therapies that can be administered or introduced in combination with the hIL2M and hIL12M molecules.
[0363] In some embodiments, the supplementary anti-neoplastic agent is a chemotherapeutic agent. In some embodiments the supplementary agent is a “cocktail” of multiple chemotherapeutic agents. IN some embodiments the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e g. radiation therapy). The term ‘‘chemotherapeutic agents” includes but is not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chiorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins such as bleomycin A2,, cactinomycin, cahcheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin and derivaties such as demethoxy-daunomycin, 11 -deoxy daunorubicin, 13 -deoxy daunorubicin, detorubicin, 6-diazo-5- oxo-L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, N-methyl mitomycin C; mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate, dideazatetrahydrofolic acid, and folinic acid; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nab-paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes such as cisplatin, oxaplatin and carboplatin; vinblastine; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; taxanes such as paclitaxel, docetaxel, cabazitaxel; carminomycin, adriamycins such as 4'-epiadriamycin, 4- adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate; cholchicine and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0364] The term “chemotherapeutic agents” also includes anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0365] In some embodiments, a supplementary anti-neoplastic agent is one or more chemical or biological agents identified in the art as useful in the treatment of neoplastic disease, including, but not limited to, a cytokines or cytokine antagonists such as INF a, or anti-epidermal growth factor receptor, irinotecan; tetrahydrofolate antimetabolites such as pemetrexed; antibodies against tumor antigens, a complex of a monoclonal antibody and toxin, a T-cell adjuvant, bone marrow transplant, or antigen presenting cells (e.g., dendritic cell therapy), anti- tumor vaccines, replication competent viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or an immunomodulator to achieve additive or synergistic suppression of tumor growth, non-steroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon-β1a (Avonex®), and interferon-β1b (Betaseron®) as well as combinations of one or more of the foreoing as practied in known chemotherapeutic treatment regimens including but not limited to TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others that are readily appreciated by the skilled clinician in the art.
[0366] • In some embodiments, the heterodimeric hIL12M-Fc mutein is administered in combination with BRAF/MEK inhibitors, kinase inhibitors such as sunitinib, PARP inhibitors such as olaparib, EGFR inhibitors such as osimertinib (Ahn, etal. (2016) J Thorac Oncol 11 :S115), IDO inhibitors such as epacadostat, and oncolytic viruses such as talimogene laherparepvec (T- VEC).
[03671 In some embodiments, a “supplementary anti -neoplastic agent” is a therapeutic antibody (including bi-specific and tri-specific antibodies which bind to one or more tumor associated antigens including but not limited to bispecific T cell engagers (BITEs), dual affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs). In some embodiments, the therapeutic antibody is an antibody that binds to at least one tumor antigen selected from the group consisting of HER2 (e.g. trastuzumab, pertuzumab, ado-trastuzumab emtansine), nectin-4 (e.g. enfortumab), CD79 (e.g. polatuzumab vedotin), CTLA4 (e.g. ipilumumab), CD22 (e.g. moxetumomab pasudotox), CCR4 (e.g. magamuizumab), IL23pl9 (e.g. tildrakizumab), PDL1 (e.g. durvalumab, avelumab, atezolizumab), IL17a (e.g. lxekizumab), CD38 (e.g. daratumumab), SLAMF7 (e.g. elotuzumab), CD20 (e.g. rituximab, tositumomab, ibritumomab and ofatumumab), CD30 (e.g. brentuximab vedotin), CD33 (e.g. gemtuzumab ozogamicin), CD52 (e.g. alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate binding protein, GD2 (e.g. dinuntuximab) , GD3, IL6 (e.g. silutxumab) GM2, Ley, VEGF (e.g. bevacizumab), VEGFR, VEGFR2 (e.g. ramucirumab), PDGFRa (e.g. olartumumab), EGFR (e.g. cetuximab, panitumumab and necitumumab), ERBB2 (e.g. trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL Rl, TRAIL R2, RANKL RAP, tenascin, integrin aV|33, and integrin a4|3L
[0368] |In some embodiments, a therapeutic antibody is an immune checkpoint modulator for the treatment and/or prevention neoplastic disease in a subject as well as diseases, disorders or conditions associated with neoplastic disease. The term “immune checkpoint pathway” refers to biological response that is triggered by the binding of a first molecule (e.g. a protein such as PD1) that is expressed on an antigen presenting cell (APC) to a second molecule (e.g. a protein such as PDL1) that is expressed on an immune cell (e.g. a T-cell) which modulates the immune response, either through stimulation (e.g. upregulation of T-cell activity) or inhibition (e.g. downregulation of T-cell activity) of the immune response. The molecules that are involved in the formation of the binding pair that modulate the immune response are commonly referred to as “immune checkpoints.” In one embodiment, the immune checkpoint pathway modulator is an antagonist of a negative immune checkpoint pathway that inhibits the binding of PD1 to PDL1 and/or PDL2 (“PD1 pathway inhibitor). The term PD1 pathway inhibitors includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and/or PDL2. Examples of commercially available PD1 pathway inhibitors useful as supplementary agents in the treatment of neoplastic disease include antibodies that interfere with the binding of PD1 to PDL1 and/or PDL2 including but not limited to nivolumab (Opdivo®, BMS-936558, MDX1106, commercially available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda®MK-3475, lambrolizumab, commercially available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech/Roche, South San Francisco CA). Additional PD1 pathway inhibitors antibodies are in clinical development including but not limited to durvalumab (MEDI4736, Medimmune/ AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), and avelumab (MSB0010718C, Merck Serono/Pfizer) and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in United States Patent No. 8,217,149 (Genentech, Inc) issued July 10, 2012; United States Patent No. 8,168,757 (Merck Sharp and Dohme Corp.) issued May 1, 2012, United States Patent No. 8,008,449 (Medarex) issued August 30, 2011 , United States Patent No. 7,943,743 (Medarex, Inc) issued May 17, 2011.
[0369] Examples of antibody therapeutics which are FDA approved and may be used as supplementary agents for use in the treatment of neoplastic disease include atezohzumab, olaratumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrohzumab, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumaxomab, panitumumab, bevacizumab, ramucirumab, siltuximab, enfortumab vedotin, polatuzumab vedotin, [fam] -trastuzumab deruxtecan, cemiplimab, moxetumomab pasudotox, mogamuizumab, tildrakizumab, ibalizumab, durvalumab, inotuzumab, ozogamicin, avelumab, obinutuzumab, ado- trastuzumab emtansine, cetuximab, tositumomab-1131, ibritumomab tiuxetan, gemtuzumab, and ozogamicin.
Physical Methods
[0370] In some embodiments, a supplementary anti-neoplastic agent is one or more non- pharmacological modalities (e.g., localized radiation therapy or total body radiation therapy or surgery). By way of example, the present disclosure contemplates treatment regimens wherein a radiation phase is preceded or followed by treatment with a treatment regimen comprising a hIL 12 mutein and one or more supplementary anti-neoplastic agents. In some embodiments, the present disclosure further contemplates the use of heterodimeric hIL12M-Fc mutein in combination with surgery (e.g. tumor resection). In some embodiments, the present disclosure further contemplates the use of a heterodimeric hIL12M-Fc mutein in combination with bone marrow transplantation, peripheral blood stem cell transplantation or other types of transplantation therapy.
Kits
[0371] Also provided are kits comprising the hIL2M and hIL12M agents of the present disclosure. In some embodiments, the kit comprises a first sterile vial comprising a pharmaceutical formulation of hIL2M and a second sterile vial comprising a pharmaceutical formulation of hIL12M agents and instructions for use. In one embodiment, the kit comprises a first sterile vial comprising a liquid pharmaceutical formulation of hIL2M-PEG and a second sterile vial comprising a liquid pharmaceutical formulation of hIL12M-Fc and instructions for use. In one embodiment, the kit comprises a first sterile vial comprising a liquid pharmaceutical formulation of hIL2M-PEG suitable for use in an autoinjector device and a second sterile vial comprising a liquid pharmaceutical formulation of hIL12M-Fc suitable for use in an autoinjector device and instructions for use. In one embodiment, the kit comprises a first sterile prefilled syringe comprising a liquid pharmaceutical formulation of hIL2M-PEG and a second sterile prefilled syringe comprising a liquid pharmaceutical formulation of hIL12M-Fc and instructions for use. In one embodiment, the kit comprises a sterile vial comprising a lyophilized pharmaceutical formulation of hIL2M-PEG and sterile vial comprising a solution for reconstiution of of the lyophilized hIL2M-PEG formulation, sterile vial comprising a pharmaceutical formulation of hIL12M-Fc and sterile vial comprising a solution for reconstiution of of the lyophilized hIL2M- PEG formulation and instructions for use. In some embodiments, the kit comprises a first sterile vial comprising a pharmaceutical formulation of STK-026 and a second sterile vial comprising a pharmaceutical formulation of STK-
[O372] M agents and instructions for usein some embodiments, the kit may optionally comprise a one or more codes (such as QR codes, passwords, passkeys or similar electronic access codes) which may provide access (or a license) to a remote monitoring system or a telehealth application on a mobile device.
[0373] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a polypeptide is disclosed and discussed and a number of modifications that can be made to the polypeptide are discussed, each and every combination of the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. All patents, patent applications and publications referred to throughout the disclosure herein are incorporated by reference in their entirety
EXAMPLES
Example 1. Recombinant Production of hIL12 Muteins
[0374] The heterodimeric IL12 muteins of the present disclosure are produced in substantial accordance with the following procedure. A pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA) was modified to include additional restriction sites in the Multiple Cloning Cloning Site (MCS) and renamed pExSyn2.0. Nucleic acid sequences encoding the hP40Fc and hIL12P40M polypeptides are cloned into pExSyn2.0 at the EcoRI and BamHI restriction sites, using standard molecular biology cloning techniques. A nucleic acid sequence encoding the wt HL12P35, a Gly-Ser linker and an Hisx8 chelating peptide (SEQ ID NO: 199) were cloned into the pExSyn2.0 vector at the EcoRI and BamHI using standard molecular biology cloning techniques. The vectors were DNA sequenced (MC Lab, South San Francisco, CA) to confirm identity. The vectors encoding the hP40Fc and hIL12P35 are co-transfected into Expi293 Cells in substantial accordance with the manufacturers protocol (Life Technologies, Carlsbad, CA). The His-tagged hIL12 muteins (His-tag on P35 C-termmus) are captured using 0.1 ml Ni Sepharose excel resin (Cytiva, part # GE17371201), equilibrated in Phosphate Buffered Saline (PBS) containing 10 mM Imidazole. The muteins are eluted from the Ni resin with 0.5 ml of PBS containing 250 mM Imidazole and dialyzed into PBS. Concentrations are determined with UV absorbance at 280 nm using extinction coefficients determined from the protein sequence.
[0375| Alternatively, IL12-Fc heterodimer is produced by transfection of two constructs consisting of P35-Fc and P40-Fc monomers. DNA is produced, scaled up, and expi293 or expiCHO cells transfected as described above. The Fc-tagged IL 12 complexes are captured using Protein-A resin equilibrated in PBS, and eluted from the column with 100 mM Sodium Acetate pH2.8. Elutions are neutralized and dialyzed into PBS. Further purification using standard techniques such as Size Exclusion Chromatography and/or Anion Exchange Chromatography are used to produce pure IL12-Fc heterodimer. Concentrations are determined with UV absorbance at 280 nm using extinction coefficients determined from the protein sequence.
Example 2, Human IL12 pSTAT4 Reporter Assay
[0376] To characterize the mutations’ effects on pSTAT4 signaling, the HEK-Blue Human IL 12 pSTAT4 Reporter Assay (Invivogen, San Diego, CA) is performed in substantial accordance with the manufacturers protocol. Example 3, Evaluation of IFN Gamma In Isolated Human Cells:
[0377] The evaluation of IFN gamma activity in isolated human PBMCs is performed in substantial accordance with the following procedure. Isolated human whole PBMCs are removed from storage in liquid nitrogen, thawed, and counted. Cells are divided into two groups from which were isolated either Pan-T Cells or Natural Killer Cells using StemCell negative isolation kits (StemCell Technologies, Cat. #17951, Cat. #19055), per manufacturer’s protocol. Cells are then counted, resuspended in Complete Yssel’s media (IMDM, Gibco, Cat. #122440-053) containing .25% w/v Human Albumin (Sigma, Cat. #A9080), lx ITS-X (human) (Gibco, Cat. #51500056), 30mg/L Transferrin (Roche, Cat. #10652202001), 2mg/L PA BioXtra (Sigma, Cat. #P5585), LA- OA-Albumin (Sigma, Cat. #L9655), IX Penicillin/Streptomycin (Gibco, Cat. #15-140-122), 1% Human Serum (Gemini, Cat. #507533011), and transferred to wells of a 96 well, flat-bottom, tissue-culture treated plate (Fisher Scientific, Cat. #FB012931). The plates used to stimulate Pan- T Cells are coated with 5 ug/mL anti-CD3 antibody (Biolegend, Cat. #300458) in Phosphate Buffered Saline (PBS) (Coming, Cat. #12-031-CV), stored overnight at 4C, and are washed prior to cell isolations. All cells are supplemented with human IL-2 and recombinant human IL-18 (R&D Systems, Cat. #9124-IL-050/CF), final concentrations 100 pM and 50 ng/mL, respectively. Pan-T Cells are additionally supplemented with 10 ug/mL anti-CD28 antibody (Biolegend, Cat. #302934), final concentration 10 ug/mL. IL-12 mutant proteins were titrated in Complete Yssel’s Media at concentrations ranging from 200nM to 2fM, 1: 10 dilutions, and are added to wells in equivalent volume to previously plated cells, the final concentrations typically ranging from lOOnM to HM. Cells are then incubated at 37C, 5% CO2 for 48 hours.
[0378] In the last 4 hours of incubation, cells are treated with 1:1000 Monensin (eBiosciences, Cat. #00-4505-51). After incubation, cells are washed with PBS and stained with Zombie NIR fixable viability dye (Biolegend, Cat. #423105) for 15 minutes at 4C in the dark. Cells are washed twice in pre-made FACS Buffer (BD, 554656) and are fixed in IX Phosflow Fix Buffer I (BD, Cat. #557870), pre-heated to 37C, for 10 minutes at 37C, 5% CO2. Cells are then washed with FACS Buffer twice and permeabilized in Phosflow Perm Buffer III (BD, Cat. #558050), per manufacturer’s recommendation. After permeabilization, cells are washed twice in FACS Buffer, briefly blocked with 1: 10 Human TruStain FcX Fc Block (Biolegend, Cat. #422302) in FACS Buffer and then stained for with anti-IFNy antibody (Biolegend, Cat. #506507), anti-CD4 antibody (BD, Cat. #552838), anti-CD8 antibody (BD, Cat. #563677), and anti-CD56 antibody (Biolegend, Cat. #362504) for 1 hour at room temperature, in the dark. Cells are then washed with FACS Buffer twice and resuspended inFACS Buffer containing 1% PFA (Electron Microscopy Sciences, Cat. #15710) for at least 10 minutes at 4oC in the dark prior to acquisition via flow cytometry. Example 4, Evaluation of pSTAT4 Activity in Human Cells
[0379] The evaluation of STAT4 activity in isolated human PBMCs is performed in substantial accordance with the following procedure. Human Whole PBMCs are isolated from Leukoreduction System Chambers (Stanford Blood Center) using the Erythrocyte Custom Sedimentation Kit (Miltenyi Biotec, Cat. #130-126-357) followed by the Custom Buffy Coat/LRSC PBMC Isolation kit (Miltenyi Biotec, Cat. #130-126-448), per manufacturer’s protocol. These negatively selected PBMCs are washed in warm Complete Yssel’s media (IMDM, Gibco, Cat. #122440-053) containing 0.25% w/v Human Albumin (Sigma, Cat. #A9080), lx ITS- X (human) (Gibco, Cat. #51500056), 30mg/L Transferrin (Roche, Cat. #10652202001), 2mg/L PA BioXtra (Sigma, Cat. #P5585), LA-OA-Albumin (Sigma, Cat. #L9655), IX Penicillin/Streptomycin (Gibco, Cat. #15-140-122), 1% Human Serum (Gemini, Cat. #507533011), are counted, and are transferred to a T175 tissue-culture treated flask (Nunc, Cat. # 159910) at a concentration of 2E06 cells per mL. Media is supplemented with 1 ug/mL anti-CD3 antibody (Biolegend, Cat. #300458) and 1 ug/mL anti-CD28 antibody (Biolegend, Cat. #302934). Cells are incubated for 72 hours at 37C, 5% CO2.
[0380] After incubation, cells are decanted from the flask, washed twice with warm Complete Yssel’s media, and allowed to rest at 37C, 5% CO2 without anti-CD3 or anti-CD28 stimulation. Loosely attached cells are detached with gentle washing and manual agitation prior to washes and rest. After resting, cells are washed with PBS and stained with Zombie NIR fixable viability dye (Biolegend, Cat. #423105) for 15 minutes at 4C in the dark. Cells are washed twice in pre-made Assay Buffer (.5% BSA PBS) and transferred to wells of a 96 well, round-bottom, tissue-culture treated plate (Thermo Scientific, Cat. #163320) and allowed to equilibrate in a 37C, 5% CO2 incubator. After equilibration, cells are treated with an equivalent volume of 2x IL- 12 mutant protein diluted in Assay Buffer at 1 : 10 titrations, final concentrations ranging from luM to lOOfM. Cells are then incubated at 37C, 5% CO2 for 15 minutes.
[0381] | After incubation, cells are fixed in an equivalent volume of pre-warmed, IX Phosflow Lyse/Fix Buffer (BD, Cat. # 558049) for 10 minutes at 37C, 5%CO2. Cells are then washed in Assay Buffer twice and permeabilized in BD Phosflow Perm Buffer III (BD, Cat. #558050), per manufacturer’s recommendation. After permeabilization, cells are washed twice in FACS Buffer, briefly blocked with 1: 10 Human TruStain FcX Fc Block (Biolegend, Cat. #422302) in FACS Buffer and then stained for with anti-pSTAT4 antibody (CD, Cat. #562703), anti-CD4 antibody (BD, Cat. #552838), anti-CD8 antibody (BD, Cat. #563677), anti-CD3 (Biolegend, Cat. #300415), and anti-CD56 antibody (Biolegend, Cat. #362504) for 1 hour at room temperature, in the dark. Cells are then washed with FACS Buffer twice and resuspended in FACS Buffer containing 1% PFA (Electron Microscopy Sciences, Cat. #15710) for at least 10 minutes at 4C in the dark prior to acquisition via flow cytometry.
Example 5: Preparation of Compounds 1. 2. and 3
[0382| To eval uate the efficacy and toxicity of the combination of an hIL2M and hIL 12M of the present disclosure in the treatment of cancer in a mammalian subject, equivalent murine surrogates of the human IL2 and IL 12 muteins were constructed and evaluated in a murine model of colorectal cancer using the MC38 murine colorectal cell line.
[0383] Compound 1 : Wild Type murine IL12 Fc: Compound 1 used in the following efficacy and toxicity studies is a mIL12Fc molecule comprised of a wild-type murine IL 12P35 (mIL12R35, UniProt Ref. P43431) fused to a first murine IgG2 (m!gG2) Fc domain (mIL I 2/G5-Fcl ) using a 15 amino acid GS linker (G4Sx3 (SEQ ID NO: 37)) and a wild-type murine IL12p40 (mIL12p40) fused to a second murine IgG2 (mIgG2) Fc domain (mlLl 2/G5-Fc2) wherein the Fcl and Fc were modified to promote heterodimerization.
[0384] The mIL12P35-Fcl subunit of the mIL12Fc is a polypeptide having the amino acid sequence (mIL12P35 underlined; GS linker italicized):
RVI PVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTS TLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGS IYEDLKMY QTEFQAINAALQNHNHQQI ILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADP YRVKMKLCILLHAFSTRWTINRVMGYLSSAGGGGSGGGGSGGGGSPRG PT I KP CPPCKCPAPNAAGGPSVFI FPPKIKDVLMISLSPIVTCVWDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLGAP IERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNG KTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTK S FSRTPG ( SEQ ID NO : 18 9 )
The mIL12p40-Fc2 subunit of the mIL12Fc is a polypeptide having the amino acid sequence (mIL12p40 underlined)
MWELEKDVYWEVDWTPDAPGETVNLTCDTPEEDDITWTS DQRHGVIGSGKTLT ITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEA PNYSGRFTCSWLVQRNMDLKFNIKSSS SSPDSRAVTCGMASLSAEKVTLDQRDY EKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTS FFIRDI IKPDPPKN LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKG AFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS PRG PT I KPC P P CKCPAPNAAGGPSVFI FPPKIKDVLMI SLS PIVTCVWDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLGAPIER TISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTE LNYKNTEPVLDS DGSYFMYSWLRVEKKNWVERNSYSCSWHEGLHNHHTTKS FS RTPG ( SEQ ID NO : 190 )
[0385] Compound 2: mIL12M-Fc. Compound 2 used in the following efficacy and toxicity studies is a murine mIL12M Fc molecule (mIL12M-Fc) molecule comprised of: (1) a wild-type murine IL12P35 (mIL12P35, UniProt Ref. P43431) fused to a first murine IgG2 (m!gG2) Fc domain (mIL12P35-Fcl) using a 15 amino acid GS linker (G4Sx3 (SEQ ID NO: 37)) as described above with respect to Compound 1 (SEQ ID NO: 189), and (2) a murine IL12p40 modified to reduce binding to the murine IL12Rbl receptor, the mIL12p40 containing the amino acid substitutions E81A, F82A, and K106A numbered in accordance with the mature form of wild type murine IL12p40M (mIL12p40) fused to a second murine IgG2 (mIgG2) Fc domain (mIL12p40M- Fc2) wherein the Fcl and Fc2 domains were modified to promote heterodimerization having the amino acid sequence (mIL12p40M underlined):
MWELEKDVYWEVDWTPDAPGETVNLTCDTPEEDDITWTS DQRHGVIGSGKTLT ITVKA ALDAGQYTCHKGGETLSHSHLLLHAKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCS WLVQRNMDLKFNIKSS5 SS PDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPT AEETLPIELALEARQQNKYENYSTS FFIRDI IKPDPPKNLQMKPLKNSQVEVSWEYPDS WST PHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDR YYNSSCSKWACVPCRVRS PRGPTIKPCPPCKCPAPNAAGGPSVFI FPPKIKDVLMISLS PIVTCVWDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMS GKEFKCKVNNKDLGAPIERTI SKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMP EDI YVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSWHEGLH NHHTTKS FSRTPG ( SEQ ID NO : 191 )
[0386] Compounds 1 and 2 were produced by recombinant expression from a bicistronic vector construct in Expi293 cells and chromatographically purified using a Protein A column in substantial accordance with Examples 1 and standard techniques.
[0387] The activity of the mIL12-Fc (Compound 1) and mIL12M-Fc (Compound 2) murine surrogate compounds were evaluated NK cells and demonstrated activity substantially similar to their human counterparts. Briefly, Bl/6 spleens were processed into single cells suspensions and NK cells were isolated by negative selection. Murine NK cells were treated with mIL2 and hILl 8 for 48 hours and then stimulated with a titration of Compound 1 or Compound 2 for 20 minutes. Cells were fixed, permeabilized, and stained for STAT4 phosphorylation prior to measurement via flow cytometry. The results of this study are presented in Figure 21, Panel A. As illustrated, the mIL12M-Fc molecule (Compound 2) exhibited significantly reduced STAT4 signaling in murine NK cells compared to the wild type mIL12-Fc molecule (Compound 1). For comparison, human hIL12M-Fc comprising a p40 subunit having the amino acid substitutions E81 A/F82A/K106A and wild type hIL12-Fc molecules were evaluate for their ability to stimulate STAT4 siginahng in human NK cells. Briefly, human NK cells were isolated from LRS chamber derived PBMCs and then treated with human IL 12 wild type Fc or human IL12 mutant Fc for 20 minutes. Cells were fixed, permeabilized, and stained for STAT4 phosphorylation prior to measurement via flow cytometry. The results of this experiment are provided in Figure 21, Panel B. As indicated in Figure 21, the m!L12M-Fc and hIL12M-Fc surrogate molecules exhibit similar STAT4 phosphorylation relative to the species matched mIL12-Fc or hIL12-Fc.
[0388] Compound 3: PEGylated murine mIL2M: To evaluate the combination of the IL2M and IL12M molecules, amurine surrogate hIL2M compound comprising: (1) amurine IL2 polypeptide correspond to the amino acid substitutions L18R/Q22E/Q126K of the hIL2M; (2) a 2 amino acid GS linker having the sequence Gly-Ser; and (3) a chelating peptide comprising eight histidine residues (SEQ ID NO: 199), the polypeptide of the mIL2M having the amino acid sequence:
APTSSSTS SSTAEAQQQQQQQQQQQQHLEQLRMDLEELLSRMENYRNLKLPRML TFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTV VKLKGSDNT FECQFDDESATWDFLRRWIAFCHS 11 STS PQGSHHHHHHHH ( SEQ ID NO : 187 ) which is N-terminally linked to a 40 kDa Polyethylene Glycol (PEG) moiety consisting of 2 branched 20kDa arms via an aldehyde linker (NOF) to form Compound 3. Compound 3 was recombinantly expressed in Expi293 cells and chromatographically purified using nickel immobilized metal affinity chromatography in substantial accordance with Example 1 and standard techniques.
Example 6. Combination MC38 Tumor Study #2
[0389] A, Study Design: MC-38 tumor cells (Kerafast, Catalog #ENH204-FP) were thawed and cultured in MC-38 media (DMEM [Gibco, Catalog #11995-065] supplemented with 10% FBS [Coming, 35-016-CV] and Penicillin/Streptomycin [Gibco, Catalog #15140-122] at 37°C and 5% CO2. For passaging, cells were split 1 :4 every 3-4 days (approximately 70% confluence) by dissociation with TrypLE (Gibco, Catalog #12604-013). Cells were passaged 3-4 times before tumor challenge. On the day of tumor challenge, cells were dissociated with TrypLE, harvested, and washed with DPBS (Coming, Catalog #21-031-CV). Cells were spun for 5 minutes at 400g. Supernatants were discarded and cells were resuspended in DMEM to a concentration of 20 x 106 cells/mL. The cell suspension was mixed with an equal part of Matrigel® (Coming, Catalog #356231) on ice resulting in a final cell suspension of 10 x 106 cells/mL. Eight to ten-week-old female C57BL/6J mice (The Jackson Laboratory, Catalog #000664) were subcutaneously injected in one shaved hind flank with 100 pL of cell suspension (1 x 106 cells per mouse). Palpable tumors were measured by length and width, and tumor volumes were calculated with the following formula:
TV = '/2(L x W2) where TV = tumor volume (mm3), L = the longest tumor dimension in mm, W = the shortest tumor dimension in mm.
[0390] When tumors reached an average of -115 mm3 mice were randomized into treatment groups (eight mice per group) on Studay Day -1. Starting the day following randomization, Study Day 0, treatment of the animals commenced in accordance with the following schedule:
Figure imgf000133_0001
Tumor volumes and body weights were measured throughout the study. In those animals where tumor volume exceeded 2000 mm3 were euthanized.
[0391] B. Evaluation of Toxicity: Loss of body weight and survival were evaluated as measures of toxicity. All mice were weighed, and relative body weight (BW) change normalized to the cumulative weight of each animal at the at the initiation of the study (Study day -1) as summarized in Tables 19 and 20 below and the spider plots in Figure 22. The “Mean” is the cumulated body weights of the animals in the group (or surviving animals) normalized to their initial body weights expressed as a percentage of the initial cumulative body weights. Where n is less than 8, this indicates that animals in this treatment did not survive or were euthanized for excess tumor burden. Table 19. Percent Changes in Bodyweight Treatment Groups 1, 3, and 5
Figure imgf000134_0001
Figure imgf000134_0003
Figure imgf000134_0002
[0392] |As indicated by the data provided in Tables 19 and 20 and illustrated graphically in Figure 22, on Study Day 7, mice treated with DPBS control (Group 1), Compound 2 (mIL12M- Fc, Group 5), or Compound 3 (mIL2M, Group 2) indicated a retention of greater than 97% of initial body weights. On Study Day 7, the mice treated with Compound 1 (wild type mIL12Fc, Group 3) showed on average BW reduction of approximately 12% (88% of initial body weight). The addition of Compound 3 (mIL2M) to the treatment regimen with Compounds 1 and 2 further distinguished toxicity differences between Compounds 1 (wild type mIL12Fc) and 2 (mIL12M- Fc). Compound 3 (mIL2M) when administered with Compound 1 (wild type mIL12Fc), treatment Group 4, led to decreases in body weight to an average of approximately 28% (72.1% of initial body weight) on Study Day 7. In treatment Group 4, all but one animal was removed from the study and euthanized due to animal welfare endpoint criteria. In contrast, the combination of Compound 2 with Compound 3 (Group 6) demonstrated average Day 7 body weight reductions of approximately 7% (92.6% of initial body weights) with no animals requiring euthanasia. (Tables
1 and 2). Four animals in treatment Group 1 (PBS) were euthanized due to excess tumor burden.
[0393] C. Anti-Tumor Efficacy: Tumor volume was assessed throughout the study as described in Example 5. The data relating to the tumor volume (y-axis) versus study day (x-axis) is provided with respect to individual animal tumor volumes is provided in Figure 23. For purposes of this study, a complete response (CR) is defined as loss of tumor palpability. As shown in Figure
2 Panels B, C and D, each of Compounds 1, 2, and 3 (Groups 2, 3, and 5 respectively) demonstrated some anti-tumor activity with varying degrees of complete responders (CRs): Group 2 (Panel B) 0 of 8 CRs, Group 3 (Panel B) 1 of 8 CRs and Group 5 (Panel B) 2 of 8 CRs. In contrast, the combination of Compounds 2 and 3 (Group 6) resulted in 5 of 8 CRs and all mice surviving until Study Day 24. the highest number of complete responders (CRs), defined as loss tumor palpability, compared to groups treated with Compound 2 or Compound 3 alone.
[0394] In sum, the foregoing data show that the combination of Compound 3 , a murine surrogate of an hIL2M mutein of the present disclosure and Compound 2, a murine surrogate of hIL12M mutein of the present disclosure, generates superior anti-tumor efficacy (as measured by the number of CRs) to the administration of either agent alone without unacceptable toxicity. In contrast to the combination of a mIL-2M (Compound 3) with a mIL-12M (Compound 2) Group , the combination of an mIL-2M (Compound 3) with a wild type mIL-12 (Compound 1) resulted in body weight loss that required humane euthanization of the majority of animals.
Example 7. Combination MC38 Tumor Study #3
[0395] |A, Study Design: A second MC38 tumor study was conducted to evaluate the test Compounds 1, 2, and 3, alone or in combination, using a weekly schedule. Mice were challenged with MC-38 tumor cells in substantial accordance with the teaching of Example 6. A. When tumors reached an average of ~99 mm3, mice were randomized into treatment groups (n = 7/group. Test Compounds 1, 2, and 3 were prepared in substantial accordance with the teaching of Example 5. The animals were randomized and weighed on Study Day -1. Starting on the following day, Study Day 0, the animals were treated with the amounts of test Compounds at the frequency and via the route of administration as indicated in the following Table 21 :
Figure imgf000136_0002
Figure imgf000136_0005
Figure imgf000136_0001
Figure imgf000136_0003
Figure imgf000136_0004
[0396] B, Body Weights and Tolerability: Loss of body weight and survival were evaluated as measures of toxicity. All mice were weighed, and relative body weight (BW) change normalized to the cumulative weight of each animal at the at the initiation of the study (Study Day -1) as summarized in the spider plots in Figure 24. As illustrated in Figure 24, at Study Day 7 all groups maintained, on average, greater than 99% of their initial body weights. Over the course of the study, no animals were removed from the study due to body weight loss or other clinical symptoms.
[0397] C. Anti-Tumor Efficacy: Tumors were measured throughout the study in substantial accordance with the teaching of Example 5. Figure 25 provides spider plots of tumor volumes (y- axis) observed over the course of the study (x-axis) with a each line representing each individual animal. For purposes of this study, a complete response (CR) is defined as loss of tumor palpability. As shown in Figure 25, all treatment Groups (other than PBS, Group 1, Panel A) exhibited anti-tumor efficacy. As illustrated in Figure 25, Panel F, the greatest anti-tumor effect was in treatment Group 6 receiving a weekly intraperitoneal dose of 48 pg of Compound 2 (mIL12M) in combination with a weekly subcutaneous dose of lOpg of Compound 3 (mIL2M). Taken together, these data show that the combination of Compound 3, a murine surrogate of an hIL2M mutein of the present disclosure and Compound 2, a murine surrogate of hIL12M mutein of the present disclosure, generates a superior outcome in the treatment of cancer in a mammalian subject relative to the administration of either agent alone. Informal Sequence Listing
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
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Figure imgf000173_0001

Claims

CLAIMS I/W e Claim:
1. A method of treating a neoplastic disease in a mammalian subj ect the method comprising the steps of:
(a) administenng to the mammalian subject a therapeutically effective amount of an hIL12 mutein (hIL12M), the hIL12M comprising a p35 subunit (hIL 12p35) and p40M subunit (hIL12p40M) wherein:
• the hIL12p35 has at least 95% sequence identity to mature wild type human hIL12p35 (SEQ ID NO:2); and
• the hIL12p40M has at least 95% sequence identity to mature wild type human hIL12p40 (SEQ ID NO:4), the hIL12p40M further comprising one or more amino acid substitutions that reduce the binding affinity of the hIL12p40M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40; and
(b) administering to the mammalian subject a therapeutically effective amount of an hIL2 mutein (hIL2M), the hIL2M comprising one or more amino acid substitutions relative to the sequence of wild type human IL2 (SEQ ID NO: 182) that result in reduced binding affinity of the hIL2 mutein to the extracellular domain of hCD132 as compared to wild type human IL2 (SEQ ID NO: 182), wherein step (a) is performed in combination with step (b).
2. The method of claim 1 wherein the amino acid substitutions that reduce the binding affinity of the hIL12p40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 are amino acid substitutions at one or more of the residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, K219 and and K282 numbered in accordance with SEQ ID NO:3 (hIL12p40 precursor).
3. The method of claim 2 wherein the amino acid substitutions that reduce the binding affinity of the hIL12p40M subunit of the hIL12M to the extracellular domain of the hILl 2Rbl receptor compared to the wild type human IL12p40 are ammo acid substitutions selected from the group consisting of: W37A; P39A; D40A; E81A; E81N; E81D; E81C; E81Q; E81E; E81P; E81W; E81Y; F82A; F82R; F82E; F82H; F82K; F82P; F82W; F82Y; K106A; K106N; D109A; K217A; K219A; E81A/F82A; W37A/E81A/F82A;
E81A/F82A/K106A; E81A/F82A/K106A/K219A; E81A/F82A/K106A/K217A; 81A/F82A/K106A/E108A/D115A; E81F/F82A; E81K/F82A, E81L/F82A; E81H/F82A; E81S/F82A; E81A/F82A/K106N; E81A/F82A/K106Q; E81A/F82A/K106T;
E81A/F82A/K106R; and P39A/D40A/E81A/F82A.
4. The method of claim 3 wherein the amino acid substitutions that reduce the binding affinity of the hIL12p40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is a set of amino acid substitutions selected from the group consisting of 2xAla = E81 A/F82A; 3xAla = E81A/F82A/K106A; and 4xAla: E81A/F82A/K106A/K217A.
5. The method of claim 3 wherein the amino acid substitutions that reduce the binding affinity of the hIL12p40M subunit of the hIL12M to the extracellular domain of the hIL12Rbl receptor compared to the wild type human IL12p40 is the set of amino acid substitutions E81A/F82A/K106A.
6. The method of any one of claims 1-5 wherein the hIL12M is an hIL12M-Fc heterodimer wherein the ML12P35 and hIL12p40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide of the formula [1]: hIL12P40M- Lla-UH1— Fcl [1] and a second polypeptide of the formula [2] : hIL12P35- L2b-UH2— Fc2 [2] wherein:
• LI and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present);
• UH1 and UH2 are each an upper hinge domain of human immunoglobulin independently selected from the group consisting of the IgGl, IgG2, IgG3 and IgG4 upper hinge, optionally comprising the amino acid substitution C220S (EU numbering); • Fcl is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fc2;
• Fc2 is a polypeptide comprising the lower hinge, CH2 and CH3 domains of a human immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4, comprising one or more amino acid substitutions promote heterodimerization with Fcl; and wherein the polypeptide of formula [1] and the polypeptide of formula [2] are linked by at least one interchain disulfide bond.
7. The method of claim 6 wherein the GSA linker is a polypeptide having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18, 19 or 20 amino acids the polypeptide comprised of amino acids selected from the group consisting of glycine, senne and alanine.
8. The method of claim 6 wherein GSA linker is a glycine-serine polymer of the structure (GGGGSm)n (SEQ ID NO: 192), (GGGSm)n (SEQ ID NO: 193), (GGGAm)n (SEQ ID NO: 194) and (GGGGAm)n (SEQ ID NO: 195), and combinations thereof, where m, n, and o are each independently selected from 1, 2, 3 or 4.
9. The method of claim 6 wherein the GSA linker wherein the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS: 27-79.
10. The method of claim 6 wherein the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOS: 36, 37 and 65.
11. The method of any one of claims of claim 6 wherein Fcl and Fc2 is each a naturally occurring upper hinge region of a human immunoglobulin selected from the UH regions of human IgGl, human IgG2, human IgG3 and human IgG4 upper hinge domains.
12. The method of any one of claims of claim 6 wherein upper hinge region is selected from the group consisting of EPKSC (SEQ ID NO: 1 l)and EPKSS (SEQ ID NO: 12).
13. The method of any one of claims 6-12 wherein Fcl and Fc2 comprise amino acid substitutions that promote heterodimerization between Fcl and Fc2.
14. The method claim 13 where one of Fcl and Fc2 comprises the amino acid substitutions S364H/T394F and the other of Fcl and Fc2 comprises the amino acid substitutions Y349T/F405A.
15. The method claim 13 where one of Fcl and Fc2 comprises amino acid substitutions T350V/L351Y/F405A/Y407V and the other of Fcl and Fc2 comprises the amino acid substitutions T350V/T366L/K392L/T394W.
16. The method claim 13 where one of Fcl and Fc2 comprises amino acid substitutions K360E/K409W and the other of Fcl and Fc2 comprises the amino acid substitutions Q347R/D399V/F405T.
17. The method claim 13 where one of Fcl and Fc2 comprises amino acid substitutions to provide a knob and the other of Fcl and Fc2 comprises amino acid substitutions provide a hole.
18. The method claim 17 where wherein the acid substitution to provide a knob is the T366W and the acid substitutions to provide a hole is the set of amino acid substitutions T366S/L368A/Y407V.
19. The method of any one of claims 6-18 wherein Fcl and Fc2 are covalently linked via one or more, optionally two or more optionally three or more disulfide bonds , optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) C96 of the hP35 and C199 of the hP40M; (b) between C226 of the first Fc monomer and the C226 of the second Fc monomer, (c) between C229 of the first Fc monomer and the C229 of the second Fc monomer; and (d) between S354C of the first Fc domain comprising a S354C amino acid substitution and Y349C of the second Fc domain comprising a Y349C amino acid substitution.
20. The method of any one of claims 6-19 wherein Fcl and Fc2 comprise one or more amino acid substitutions to reduce effector function.
21. The method of claim 20 wherein Fcl and Fc2 are human IgG2 Fc domains and one or both of Fcl and Fc2 comprise amino acid substitutions to reduce effector function selected from the group consisting of: L234E;L234A/L235A; L234A/L235A/P329A; and L234A/L235A/P329G.
22. The method of claim 20 wherein Fcl and Fc2 are human IgG4 Fc domains and one or both of Fcl and Fc2 comprise one or more amino acid substitutions to eliminate N- or 0 linked glycosylation sites.
23. The method of claim 22 wherein the modification to eliminate N- or 0 linked glycosylation sites is selected from the group consisting of N297Q and N297G.
24. The method of any one of claims 6-23 wherein Fcl and Fc2 comprise the amino acid substitutions M428L and N434S.
25. The method of any one of claims 6-24 wherein each of Fcl and Fc2 comprise a deletion of: (a) the lysine residue at position 447 or (b) a deletion of both the glycine at position 446 and the lysine residue at position 447.
26. The method of any one of claims 1-25 wherein the hIL12M is PEGylated.
27. The method of claim 26 wherein the PEG has a molecular mass greater than about 5kDa, greater than about lOkDa, greater than about 15kDa, greater than about 20kDa, greater than about 30kDa, greater than about 40kDa, or greater than about 50kDa.
28. The method of any one of claims 26 or 27 wherein the hIL12 is a hIL12M-Fc heterodimer comprises of polypeptide of formula [1] and polypeptide of formula [2] and wherein and PEG covalently linked to one or both of the C220 residues ( EU Numbering) of the upper hinge regions of the polypeptide of formula [1] and polypeptide of formula [2],
29. The method of any one of claims 1-28 wherein the hIL12P40M further comprises an amino acid substitution selected from the group consisting of K282G, K282A, K282N, K282QG or K282.
30. The method of any one of claims 1-29 where in the hIL12M is recombinantly expressed in a mammalian host cell.
31. The method of claim 30 wherein the mammalian host cell is selected from HEK293 cells and CHO cells.
32. The method of any one of claims l-31wherein the binding affinity of hIL12M for the extracellular domain (ECD) of IL12Rβ1 is reduced by at least 5%, optionally by at least 10%, optionally by at least 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, optionally by at least 70%, relative to the binding affinity of wild type hIL12 for the extracellular domain (ECD) of IL12Rβ1 as determined by surface plasmon resonance.
33. The method of any one of claims l-31wherein the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in signaling inNK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide.
34. The method of any one of claims l-31wherein the hIL12M induces IL-12 signaling in CD8+ T cells and has at least 10%, optionally at least a 20%, optionally at a least 30%, optionally at least a 40%, optionally at least a 50%, optionally at least a 60%, or optionally at least a 70% reduction in in signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide..
35. The method of any one of claims l-31wherein the hIL12M decreased STAT-4 mediated signaling wherein the STAT4 signaling in NK cells is decreased by at least 10%, optionally by at least a 20%, optionally by at least 30%, optionally by at least 40%, optionally by at least 50%, optionally by at least 60%, or optionally by at least 70% signaling in NK cells compared to an hIL12 molecule comprising a wild type hIL-12p40 polypeptide..
36. The method of claim 6 wherein the polypeptide of the formula #1 is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153.
37. The method of claim 6 wherein the polypeptide of the formula #2 is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
38. The method of claim 6 wherein the polypeptide of the formula #1 is selected from the group consisting of SEQ ID NOS: 80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153 and the polypeptide of the formula #2 is selected from the group consisting of SEQ ID NOS: 81, 82, 84, 87, 89, 91, 93, and 124.
39. The method of claim 6 wherein the polypeptide of the formula #1 is a polypeptide having an amino acid sequence:
IWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQV KAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVEC QEDSACPAAEESLPIEVMVDAVHKLKYENYTSS FFIRDI IKPDPPKNLQLKPLKNSR QVEVSWEYPDTWST PHSYFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNAS IS VRAQDRYYS SSWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPCR DELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTV DKSRWQQGNVFSCSVLHEALHSHYTQKSLSLS PG ( SEQ ID NO : 129 ) , and the polypeptide of the formula [2] is a polypeptide having an amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDI TKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTS FMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVP QKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSE PKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF YPSDIAVEWESNGQPENNYKTTPPVLDS DGS FFLVSKLTVDKSRWQQGNVFS CSVLHEALHSHYTQKSLSLSPG ( SEQ ID NO : 124 ) .
40. The method of claim 39 wherein the hIL12M is recombinantly procuced in CHO cells.
41. The method of claim 1 wherein the one or more amino acid substitutions that decrease the binding affinity of the hIL2M to CD 132 are selected from amino acid substitutions at positions 18, 22, and 126 (numbered in accordance with mature human wild type (wt hIL2; SEQ ID NO: 182)
42. The method of claim 41 wherein:
• the amino acid substitution at position 18 selected from the group consisting of L18R, L18G, L18M, L18F, L18E, L18H, L18W, L18K, L18Q, L18S, L18V, LI 81, L18Y, L18H, L18D, L18N and L18T;
• the amino acid substitution at position 22 selected from the group consisting of Q22F, Q22E, Q22G, Q22A, Q22L, Q22M, Q22F, Q22W, Q22K, Q22S, Q22V, Q22I, Q22Y, Q22H, Q22R, Q22N, Q22D, Q22T, and F; and
• the amino acid substitution at position 126 selected from the group consisting of Q126H, Q126M, Q126K, Q126C, Q126D, Q126E, Q126G, Q126I, Q126R, Q126S, or Q126T.
43. The method of claim 42 wherein the amino acid substitutions at positions 18, 22, and 126 are selected from the group consisting of L18R/Q22E/Q126K (“REK”); L18R/Q22E/Q126H (“REH”); L18A/Q22E/Q126H (“AEH”); L18A/Q22E/Q126K (“AEK”); L18D/Q22E/Q126H (“DEH”); L18E/Q22E/Q126H (“EEH”); L18E/Q22E/Q126K (“EEK”); L18F/Q22E/Q126H (“FEH”); L18G/Q22E/Q126H (“GEH”); L18H/Q22E/Q126H (“HEH”); L18H/Q22E/Q126K (“HEK”); L18I/Q22E/Q126H (“IEH”); L18I/Q22E/Q126K (“IEK”); L18K/Q22E/Q126H (“KEH”); L18M/Q22E/Q126H (“MEH”); L18N/Q22E/Q126H (“NEH”); L18Q/Q22E/Q126H (“QEH”); L18R/Q22A/Q126H (“RAH”); L18R/Q22D/Q126H (“RDH”); L18R/Q22E/Q126E (“REE”); L18R/Q22E/Q126M (“REM”); L18R/Q22E/Q126T (“RET”); L18R/Q22E/Q126V (“REV”); L18R/Q22E/Q126L (“REL”); L18R/Q22E/Q126F (“REF”); L18R/Q22E/Q126N (“REN”); L18R/Q22E/Q126R (“RER”); L18R/Q22E/Q126Y (“REY”); L18R/Q22F/Q126H (“RFH”); L18R/Q22G/Q126H (“RGH”); L18R/Q22H/Q126H (“RHH”); L18R/Q22I/Q126H (“RIH”); L18R/Q22K/Q126H (“RKH”); L18R/Q22L/Q126H (“RLH”); L18R/Q22M/Q126H (“RMH”); L18R/Q22N/Q126H (“RNH”);
L18R/Q22R/Q126H (“RRH”); L18R/Q22S/Q126H (“RSH”); L18R/Q22T/Q126H (“RTH”); L18R/Q22T/Q126K (“RTK”); L18R/Q22V/Q126H (“RVH”); L18R/Q22W/Q126H (“RWH”); L18R/Q22Y/Q126H (“RYH”); L18S/Q22E/Q126H (“SEH”); L18T/Q22E/Q126H (“TEH”); L18V/Q22E/Q126H (“VEH”); L18V/Q22E/Q126K (“VEK”); L18W/Q22E/Q126H (“WEH”); and L18Y/Q22E/Q126H (‘YEH”).
44. The method of any one of claims 41-43 wherein the hIL2M further comprises one or more amino acid substitutions selected from the group consisting of T3C, T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P, K35E; R38W, R38G; M39L, M39V; H55Y; V69A; Q74P, Q74N, Q74H, Q74S; M104A; D109C or a non-natural amino acidwith an activated side chain at position 109' AA113, T113N; AA125 is C125A or C125S; S130T, S130G and S130R.
45. The method of any one of claims 41-44 wherein the hIL2M further comprises a deletion of one or more N-terminal amino acid selected from the group or deletions: des- Al; des-Al/ des-P2; des-Al/ des-P2/ des-T3; des-Al/des-P2/des-T3/des-S4; des-Al/des- P2/des-T3/des-S4/des-S5; des-Al/des-P2/des-T3/des-S4/des-S5/des-S6; des-Al/des-P2/des- T3/des-S4/des-S5/des-S6/des-T7.
46. The method of any one of claims 41-45 wherein the wherein the hIL2M exhibits at least a 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of reduction in the binding affinity to CD 132 of wild-type hIL2 as determined by surface plasmon resonance.
47. The method of any one of claims 41-46 wherein the wherein the hIL2M exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90% of the binding affinity to CD25 of wild- type hIL2 as determined by surface plasmon resonance.
48. The method of any one of claims 41-47 wherein the wherein the hIL2M exhibits at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70%, optionally at least 80%, optionally at least 90%, optionally at least 100% of the binding affinity to CD25 of wild-type hIL2 as determined by surface plasmon resonance.
49. The method of any one of claims 41-48 wherein the hIL2M is covalently linked to a carrier molecule that provides for an extended duration of action in a mammalian subject.
50. The method of claim 49 wherein the carrier molecule is selected from the group consisting of Fc polypeptides, hydrophilic polymers (e.g. PEG), hydrophobic polymers (e.g. fatty acid molecules) acylated), human serum albumin.
51. The method of claim 50 wherein the hydrophilic polymer is polyethylene glycol (PEG).
52. The method of claim 1 wherein the hIL2M comprises a polypeptide having the amino acid sequence:
PTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFY MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNI NVIVLELKGSETTFMCEYADETATIVEFLNRWITFCKSIISTLT (SEQ ID NO: 188;
53. The method of claim 52 wherein the hIL2M further comprises a N- terminal 40kD branched chain PEG comprising 2 20Kd arms covalently linked to the N-terminal prolme residue.
54. The method of claim 1 wherein:
(a) the hIL12M is an hIL12M-Fc heterodimer wherein the hIL12P35 and hIL12p40M subunits of the hIL12M are each linked to an Fc polypeptide, the hIL12M-Fc heterodimer comprising a first polypeptide having the amino acid sequence:
IWELKKDVYWELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVL GSGKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDI LKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSS DPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEV MVDAVHKLKYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWE YPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNA SISVRAQDRYYSS SWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALAAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSR WQQGNVFSCSVLHEALHSHYTQKSLSLSPG ( SEQ ID NO : 129 ) , and the polypeptide of the formula [2] is a polypeptide having an amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPC TSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETS FIT NGSCLASRKTSFMMALCLSS IYEDLKMYQVEFKTMNAKLLMD PKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYK TKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSEPK SSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQ PENNYKTT PPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVL HEALHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) , and
(b) the hIL2M comprises a polypeptide having the amino acid sequence: PTSS STKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFK FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL ISNINVIVLELKGSETT FMCEYADETATIVEFLNRWITFCKS IISTLT ( SEQ I D NO : 188 ) herein the N-terminal proline residue is covalently linked to a 40kD branched chain PEG comprising two 20Kd arms.
55. The method of claim 54 wherein the hIL12M is administered sequentially with the hIL2M, the administration being on a weekly dosing schedule wherein the hIL12M is dosed at a level of approximately 100 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 1000 pg/kg, administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every' 4 weeks, as a subcutaneous injection administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (a) is administered by subcutaneous injection and the hIL2M is dosed at a level of between of 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively every two weeks, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (b) is administered by subcutaneous injection.
56. A method of treating a neoplastic disease in a human subject, the method comprising the steps of (a) administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of hIL12 mutein (hIL12M) consisting of a polypeptide of the formula [1]: hIL12P40M- Lla-UHl— Fcl [1] and a second polypeptide of the formula [2]: hIL12P35- L2b-UH2— Fc2 [2] wherein the polypeptide of formula [1] has the amino acid sequence:
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVL GSGKTLTIQVKAAGDAGQYTCHKGGEVLSHSLLLLHAKEDGIWSTDI LKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSS DPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEV MVDAVHKLKYENYTSS FFIRDI IKPDPPKNLQLKPLKNSRQVEVSWE YPDTWSTPHSYFSLTFCVQVQGKSGREKKDRVFTDKTSATVICRKNA SISVRAQDRYYSS SWSEWASVPCSGGGGSGGGGSEPKSSDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALAAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSR WQQGNVFSCSVLHEALHSHYTQKSLSLSPG ( SEQ ID NO : 129 ) and wherein the polypeptide of formula [2] has the amino acid sequence:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEI DHEDITKDKTSTVEACLPLELTKNESCLNSRETS FITNGSCLASRKT SFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVI DELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTID RVMSYLNASGGGGSGGGGSEPKS SDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRT P EVT CVWDVS RED PEVKFNWYVDGVE VHNAKT K PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTIS KAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEA LHSHYTQKSLSLS PG ( SEQ ID NO : 124 ) , wherein the polypeptide of formula [1] is linked to the polypeptide of formula
[2] by at least one disulfide bond (STK-026), and
(b) administering to the human subject a pharmaceutical composition comprising a therapeutically effective amount of an hIL2 mutein (hIL2M) having the amino acid sequence:
PTSS STKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLT FKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLE LKGSETTFMCEYADETATIVEFLNRWITFCKS IISTLT ( SEQ ID NO : 188 ) wherein the N-terminal proline of SEQ ID NO: 188 is covalently linked by a linker to a 40kD branched chain PEG molecule comprising two 20Kd arms (STK-012), and wherein step (a) is performed in combination with step (b), the administration of the hIL2M, is performed in advance of step (a), the administration of the hIL12M, and the pharmaceutical composition comprising a therapeutically effective amount of the hIL2 mutein (hIL2M) contains a dose of approximately of 1 pg/kg to approximately 100 pg/kg, alternatively approximately 5 pg/kg to approximately 80 pg/kg, alternatively approximately 5 pg/kg to approximately 60 pg/kg, alternatively approximately 5 pg/kg to approximately 40 pg/kg, alternatively approximately 5 pg/kg to approximately 30 pg/kg, alternatively approximately 5 pg/kg, alternatively approximately 10 pg/kg, alternatively approximately 20 pg/kg, alternatively approximately 30 pg/kg, alternatively approximately 40 pg/kg, administered weekly, alternatively every two weeks, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (b) is administered by subcutaneous injection, and in step (a), the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M of step a is a pharmaceutical composition contains a dose of approximately 100 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 2000 pg/kg, alternatively approximately 250 pg/kg to approximately 1000 pg/kg, administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, as a subcutaneous injection administered weekly, alternatively bi-weekly, alternatively every 3 weeks, alternatively every 4 weeks, and the pharmaceutical composition of step (a) is administered by subcutaneous injection.
57. The method of claim 56 wherein the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately
5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every
4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 containing a dose of approximately 5 pg/kg and the hIL12M STK-026 is dosed on schedule of every 4 weeks, wherein the dosing of the 11IL12M STK-026 is initiated two weeks after dosing of the hIL2M STK-012.
58. The method of claim 57 wherein the pharmaceutical composition comprising a therapeutically effective amount of the hIL2M STK-012 containing a dose of approximately
5 pg/kg to approximately 40 pg/kg and the hIL2M STK-012 is dosed on a schedule of every 4 weeks in combination with the pharmaceutical composition comprising a therapeutically effective amount of the hIL12M STK-026 contains a dose of approximately 250 pg/kg a to approximately 1000 pg/kg and is dosed on schedule of every 4 weeks, wherein the dosing of the hIL12M STK-026 is initiated three weeks after dosing of the hIL2M STK-012.
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Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4569794A (en) 1984-12-05 1986-02-11 Eli Lilly And Company Process for purifying proteins and compounds useful in such process
US5320663A (en) 1992-07-02 1994-06-14 E. I. Du Pont De Nemours And Company Method of obtaining lead and organolead from contaminated media using metal accumulating plants
US5650234A (en) 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5807706A (en) 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US6683046B1 (en) 1989-12-22 2004-01-27 Hoffmann-La Roche Inc. Purification and characterization of cytotoxic lymphocyte maturation factor and monoclonal antibodies thereto
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US7198919B1 (en) 1983-04-25 2007-04-03 Genentech, Inc. Use of alpha factor sequences in yeast expression systems
US7872107B2 (en) 2005-12-30 2011-01-18 Merck Patent Gmbh Interleukin-12p40 variants with improved stability
US7943743B2 (en) 2005-07-01 2011-05-17 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
US8168757B2 (en) 2008-03-12 2012-05-01 Merck Sharp & Dohme Corp. PD-1 binding proteins
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
WO2015124297A1 (en) * 2014-02-19 2015-08-27 Merck Patent Gmbh Cancer-targeted il-12 immunotherapy
WO2018030806A1 (en) 2016-08-10 2018-02-15 아주대학교산학협력단 Cytokine fused to immunoglobulin fc heterodimer and pharmaceutical composition comprising same
WO2019104092A1 (en) 2017-11-21 2019-05-31 The Board Of Trustees Of The Leland Stanford Junior University Partial agonists of interleukin-2
WO2019149039A1 (en) * 2018-02-01 2019-08-08 北京科诺科服生物科技有限公司 Antitumor protein composition and application thereof
WO2020072821A2 (en) 2018-10-03 2020-04-09 Xencor, Inc. Il-12 heterodimeric fc-fusion proteins
WO2020086758A1 (en) 2018-10-23 2020-04-30 Dragonfly Therapeutics, Inc. Heterodimeric fc-fused proteins
US10696722B2 (en) 2016-08-10 2020-06-30 Ajou University Industry-Academic Cooperation Foundation Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
WO2021067863A2 (en) * 2019-10-03 2021-04-08 Xencor, Inc. Targeted il-12 heterodimeric fc-fusion proteins
WO2021146436A2 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Biased il2 muteins methods and compositions
WO2021146481A1 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Il2 muteins
US11087249B2 (en) 2016-05-24 2021-08-10 Ciambella Ltd. Method and apparatus for triggering execution of a workflow over a network
WO2021216916A1 (en) 2020-04-22 2021-10-28 Dragonfly Therapeutics, Inc. Formulation, dosage regimen, and manufacturing process for heterodimeric fc-fused proteins

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7198919B1 (en) 1983-04-25 2007-04-03 Genentech, Inc. Use of alpha factor sequences in yeast expression systems
US4569794A (en) 1984-12-05 1986-02-11 Eli Lilly And Company Process for purifying proteins and compounds useful in such process
US6683046B1 (en) 1989-12-22 2004-01-27 Hoffmann-La Roche Inc. Purification and characterization of cytotoxic lymphocyte maturation factor and monoclonal antibodies thereto
US5320663A (en) 1992-07-02 1994-06-14 E. I. Du Pont De Nemours And Company Method of obtaining lead and organolead from contaminated media using metal accumulating plants
US5650234A (en) 1994-09-09 1997-07-22 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces
US5807706A (en) 1995-03-01 1998-09-15 Genentech, Inc. Method for making heteromultimeric polypeptides
US7695936B2 (en) 1995-03-01 2010-04-13 Genentech, Inc. Knobs and holes heteromeric polypeptides
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
US7943743B2 (en) 2005-07-01 2011-05-17 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (PD-L1)
US7872107B2 (en) 2005-12-30 2011-01-18 Merck Patent Gmbh Interleukin-12p40 variants with improved stability
US8168757B2 (en) 2008-03-12 2012-05-01 Merck Sharp & Dohme Corp. PD-1 binding proteins
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
WO2015124297A1 (en) * 2014-02-19 2015-08-27 Merck Patent Gmbh Cancer-targeted il-12 immunotherapy
US11087249B2 (en) 2016-05-24 2021-08-10 Ciambella Ltd. Method and apparatus for triggering execution of a workflow over a network
WO2018030806A1 (en) 2016-08-10 2018-02-15 아주대학교산학협력단 Cytokine fused to immunoglobulin fc heterodimer and pharmaceutical composition comprising same
US10696722B2 (en) 2016-08-10 2020-06-30 Ajou University Industry-Academic Cooperation Foundation Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
US11078249B2 (en) 2016-08-10 2021-08-03 Ajou University Industry-Academic Cooperation Foundation Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same
WO2019104092A1 (en) 2017-11-21 2019-05-31 The Board Of Trustees Of The Leland Stanford Junior University Partial agonists of interleukin-2
WO2019149039A1 (en) * 2018-02-01 2019-08-08 北京科诺科服生物科技有限公司 Antitumor protein composition and application thereof
WO2020072821A2 (en) 2018-10-03 2020-04-09 Xencor, Inc. Il-12 heterodimeric fc-fusion proteins
US20200216509A1 (en) 2018-10-03 2020-07-09 Xencor, Inc. Il-12 heterodimeric fc-fusion proteins
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
WO2020086758A1 (en) 2018-10-23 2020-04-30 Dragonfly Therapeutics, Inc. Heterodimeric fc-fused proteins
WO2021067863A2 (en) * 2019-10-03 2021-04-08 Xencor, Inc. Targeted il-12 heterodimeric fc-fusion proteins
WO2021146436A2 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Biased il2 muteins methods and compositions
WO2021146481A1 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Il2 muteins
WO2021216916A1 (en) 2020-04-22 2021-10-28 Dragonfly Therapeutics, Inc. Formulation, dosage regimen, and manufacturing process for heterodimeric fc-fused proteins

Non-Patent Citations (87)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. NP_000197
"Uniprot", Database accession no. Q99665
"UniProtKB", Database accession no. P31785
AHN ET AL., J THORAC ONCOL, vol. 11, 2016, pages 115
ALEGRE ET AL., J. IMMUNOLOGY, vol. 148, 1992, pages 3461 - 3468
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1977, pages 3389 - 3402
ARDOLINO ET AL., J. CLIN. INVEST., vol. 124, 2014, pages 4781 - 4794
ASSIER E ET AL., J IMMUNOL, vol. 172, 2004, pages 7661 - 7668
ATKINS ET AL., CLINICAL CANCER RESEARCH, vol. 3, 1997, pages 409 - 417
ATKINS ET AL., J CLIN ONCOL, vol. 17, no. 7, 1999, pages 2105 - 2116
ATWELL ET AL., J. MOL. BIOL., vol. 270, 1997, pages 26 - 35
BALDARI ET AL., EMBO J., vol. 6, 1987, pages 229 - 234
BENDTSEN ET AL.: "Improved Prediction of Signal Peptides SignalP 3.0", J. MOL. BIOL, vol. 340, no. 4, 2004, pages 783 - 795, XP004516690, DOI: 10.1016/j.jmb.2004.05.028
BIOINFORMATICS, vol. 4
BRUNDA ET AL., J. EXP. MED., vol. 178, 1993, pages 1223 - 1230
BRUNDA ET AL., J. IMMUNOTHER. EMPHASIS TUMOR IMMUNOL., vol. 17, 1995, pages 71 - 77
CAMARERO J.A. ET AL., PROTEIN PEPT LETT., vol. 12, 2005, pages 723 - 8
CAO ET AL., CHARACTERIZATION AND MONITORING OF A NOVEL LIGHT-HEAVY-LIGHT CHAIN MISPAIR IN A THERAPEUTIC BISPECIFIC ANTIBODY
CARSON ET AL., J. IMMUNOL., vol. 162, 1999, pages 4943 - 4951
CHOI ET AL., MOLECULAR IMMUNOLOGY, vol. 65, no. 2, 2015, pages 377 - 83
CHOUDHARY ET AL., SCIENCE, vol. 325, no. 5942, 2009, pages 834 - 840
CLONING IN BACTERIAL CELLS AND DNA MUTAGENESIS, vol. 1
CLONING IN MAMMALIAN CELLS AND YEAST, vol. 2
COHEN, J, SCIENCE, vol. 270, 1995, pages 908
COLIGAN ET AL.: "Current Protocols in Protein Science", 2000, JOHN WILEY AND SONS, INC., pages: 1 - 2
CZAJKOWSKY ET AL., EMBO MOL MED, vol. 4, 2012, pages 1015 - 1028
DELGOFΣE ET AL., CURR OPIN IMMUNOL, vol. 23, no. 5, 2011, pages 632 - 8
DONDELINGER ET AL.: "Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition", FRONTIERS IN IMMUNOLOGY, vol. 9, 2018, XP055572450, DOI: 10.3389/fimmu.2018.02278
DOUDNA JASARNOW P ET AL.: "Translational Control in Biology and Medicine", 2007, COLD SPRING HARBOR LABORATORY PRESS, article "Translation initiation by viral internal ribosome entry sites", pages: 129 - 154
DREHER ET AL., J. BIOL. CHEM., vol. 272, no. 46, 1997, pages 29364 - 29371
DRESCHER ET AL., METHODS MOL BIOL, vol. 493, 2009, pages 323 - 343
DUTCHER ET AL., J IMMUNOTHER CANCER, vol. 2, no. 1, 2014, pages 26
EDELMAN ET AL., PNAS (USA, vol. 63, 1969, pages 78 - 85
FRIDMAN W H ET AL: "The use of cytokines in the treatment of solid tumours", HEMATOLOGY AND CELL THERAPY, SPRINGER-VERLAG, PA, vol. 39, no. 2, 1 April 1997 (1997-04-01), pages 105 - 108, XP019371954, ISSN: 1279-8509 *
GANESAN A, MINI REV. MED. CHEM., vol. 6, 2006, pages 3 - 10
GILLIES ET AL., J. IMMUNOL., vol. 160, 1998, pages 6195 - 6203
GLASSMAN ET AL., CELL, vol. 184, no. 4, 2021, pages 983 - 999
GLYCOCONJUGATES AND PROTEIN EXPRESSION, vol. 3
HENIKOFFHENIKOFF, PNAS(USA, vol. 89, 1989, pages 10915 - 10919
HEZEREH ET AL., J. VIROL, vol. 1-4, no. 24, 2001, pages 12161 - 68
HUMBLET-BARON ET AL., J ALLERGY CLIN IMMUNOL, vol. 138, no. 1, 2016, pages 200 - 209
HWANG ET AL., J. VIROL., vol. 71, no. 9, 1997, pages 7128 - 7131
IHLE ET AL., NATURE, vol. 377, no. 6550, 1995, pages 591 - 4
IIDA ET AL., J. VIROL., vol. 70, no. 9, 1996, pages 6054 - 6059
JEFFERIES ET AL., IMMOL. REV., vol. 163, 1998, pages 50 - 76
JI J ET AL: "Synergistic anti-tumor effect of glycosylphosphatidylinositol-anchored IL-2 and IL-12", THE JOURNAL OF GENE MEDICINE, JOHN WILEY & SONS, INC, US, vol. 6, no. 7, 1 July 2004 (2004-07-01), pages 777 - 785, XP002320355, ISSN: 1099-498X, DOI: 10.1002/JGM.547 *
KALIA ET AL., IMMUNITY, vol. 32, no. 1, 2010, pages 91 - 103
KAUFMAN ET AL., EMBO J, vol. 6, 1987, pages 187 - 195
KOBER ET AL., BIOTECHNOL BIOENG, vol. 1110, no. 4, 2013, pages 1164 - 73
KURJANHERSKOWITZ, CELL, vol. 30, 1982, pages 933 - 943
LASEK ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 63, 2014, pages 419 - 435
LASEK ET AL., CANCER IMMUNOL IMMUNOTHERAPY, vol. 63, no. 5, pages 419 - 435
LEE ET AL., MOL. CELL. BIOL., vol. 17, no. 9, 1997, pages 5097 - 5105
LEONARD ET AL., BLOOD, vol. 90, 1997, pages 2541 - 2548
LEVIN ET AL., NATURE, vol. 484, no. 7395, 2012, pages 529 - 533
LEVYDARNELL, NAT REV MOL CELL BIOL, vol. 3, no. 9, 2002, pages 651 - 62
LI ET AL., NATURE COMMUNICATIONS, vol. 8, no. 1, 2017, pages 1762
LIU ET AL.: "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds", FRONTIERS IN IMMUNOLOGY, vol. 8, 2017, pages 38
LUCKLOWSUMMERS, VIROLOGY, vol. 170, 1989, pages 31 - 39
MCCAFFREY ET AL., NATURE, vol. 418, 2002, pages 6893
MOLL ET AL., PROTEIN SCIENCE, vol. 10, 2008, pages 649 - 655
MOMIN ET AL., SCI. TRANSL. MED., vol. 11, 2019, pages eaaw2614
MOORE ET AL., MABS, vol. 3, no. 6, 2011, pages 546 - 557
MURRAY, J IMMUNOL., vol. 178, no. 5, 2007, pages 2623 - 9
NIELSEN ET AL., PROTEIN ENGINEERING, vol. 10, no. 1, 1997, pages 1 - 6
O'SHEAPLENGE, IMMUNITY, vol. 36, no. 4, 2012, pages 542 - 50
PEACE, D. J.CHEEVER, M. A., J EXP MED, vol. 169, no. 1, 1989, pages 161 - 173
PETERSEN ET AL.: "Signal P 4. D; discriminating signal peptides from transmembrane regions", NATURE METHODS, vol. 8, 2011, pages 785 - 796
PUTNAM, AM. J. HEALTH SYST. PHARM., vol. 53, 1996, pages 325 - 160
RIDGWAY ET AL., PROTEIN ENGINEERING, vol. 9, no. 7, 1996, pages 617 - 921
RYANDREW, EMBO J., vol. 13, no. 4, 1994, pages 928 - 933
SCHORLE ET AL., NATURE, vol. 352, no. 6336, 1991, pages 621 - 624
SCHULTZ, GENE, vol. 54, 1987, pages 113 - 123
SEED, NATURE, vol. 329, 1987, pages 840
SMITH ET AL., MOL. CELL BIOL., vol. 3, 1983, pages 2156 - 2165
STEM ET AL., PROC NATL ACAD SCI USA, vol. 87, 1990, pages 6808 - 6812
STERN ET AL., TRENDS IN CELL AND MOLECULAR BIOLOGY, vol. 2, 2007, pages 1 - 17
TANIA CARMINATE ET AL: "Human IL-2 mutein with higher antitumor efficacy than wild type IL-2", THE JOURNAL OF IMMUNOLOGY, AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 190, no. 12, 15 May 2013 (2013-05-15), pages 6230 - 6238, XP002789660, ISSN: 1550-6606, DOI: 10.4049/JIMMUNOL.1201895 *
VON KREUDENSTEIN ET AL., MABS, vol. 5, no. 5, 2013, pages 646 - 654
WANG ET AL., SCIENCE, vol. 310, 2005, pages 159 - 1163
WANG ET AL.: "IgG Fc engineering to modulate antibody effector functions", PROTEIN CELL, vol. 9, no. 1, 2018, pages 63 - 73, XP055457296, DOI: 10.1007/s13238-017-0473-8
XIA ET AL., NATURE BIOTECHNOL., vol. 20, 2002, pages 1006 - 1010
YOSHIDA ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 230, 1997, pages 426 - 430
ZALEVSKY ET AL., NATURE BIOTECHNOLOGY, vol. 28, 2010, pages 157 - 159
ZALIPSKY ET AL., BIOTEHNOL. APPL. BIOCHEM, vol. 15, 1992, pages 100 - 114
ZHAO ET AL.: "A new approach to produce IgG4-like bispecific antibodies", SCIENTIFIC REPORTS, vol. 11, 2021, pages 18630

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