WO2019168791A2 - Fusions d'anticorps/il-2 à chaîne unique qui activent sélectivement des lymphocytes t régulateurs - Google Patents

Fusions d'anticorps/il-2 à chaîne unique qui activent sélectivement des lymphocytes t régulateurs Download PDF

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WO2019168791A2
WO2019168791A2 PCT/US2019/019421 US2019019421W WO2019168791A2 WO 2019168791 A2 WO2019168791 A2 WO 2019168791A2 US 2019019421 W US2019019421 W US 2019019421W WO 2019168791 A2 WO2019168791 A2 WO 2019168791A2
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antibody
cytokine
cells
immunocytokine
jes6
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WO2019168791A3 (fr
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Kenan Christopher GARCIA
Jamie SPANGLER
Jeffrey A. Bluestone
Eleanora TROTTA
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The Board Of Trustees Of The Leland Stanford Junior University
The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • lnterleukin-2 is a four-helix bundle cytokine that plays a critical role in immune cell differentiation, growth, and activity.
  • IL-2 signals through formation of either a high-affinity quaternary complex with the interleukin-2 receptor-a (IL-2Ra, CD25), I L-2R , and IL-2Ry (common gamma, y c ) chains (K d ⁇ 10 pM), or an intermediate-affinity ternary complex (K d ⁇ 1 nM) with only the IL-2R and IL-2Ry chains. Consequently, expression of the non-signaling IL-2 Ret subunit regulates cytokine sensitivity.
  • IL-2Ra interleukin-2 receptor-a
  • I L-2R interleukin-2 receptor-a
  • IL-2Ry common gamma, y c
  • K d ⁇ 10 pM common gamma, y c
  • IL-2Ra is robustly expressed on regulatory T (T Reg ) cells but is virtually absent from naive effector cells such as memory-phenotype (MP) CD8 + T cells and natural killer (NK) cells, resulting in differential responsiveness of these immune cell subsets to IL-2.
  • T Reg regulatory T
  • MP memory-phenotype
  • NK natural killer
  • IL-2 complex formation intracellular Janus kinase (JAK) proteins constitutively associated with IL-2R and IL-2Ry phosphorylate tyrosine residues in the receptor intracellular domains, which recruit and activate signal transducer and activator of transcription (STAT)-5 to coordinate immune-related gene expression programs.
  • the IL-2 complex also signals secondarily through the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3- kinase (PI3K) pathways.
  • MAPK mitogen-activated protein kinase
  • PI3K phosphatidyl
  • Stimulation with IL-2 is crucial for the maintenance of regulatory T (T Reg ) cells and for the differentiation of CD4 + T cells into defined effector T cell subsets following antigen-mediated activation.
  • T Reg regulatory T
  • IL-2 signaling optimizes both effector T cell generation and differentiation into memory cells.
  • IL-2 also promotes the activation and expansion of natural killer (NK) cells.
  • IL-2 thus exerts paradoxical effects on immune cell homeostasis, promoting activation and proliferation of both immunostimulatory effector cells (i.e. CD4 + and CD8 + T cells and NK cells) and immunosuppressive T Reg cells.
  • immunostimulatory effector cells i.e. CD4 + and CD8 + T cells and NK cells
  • immunosuppressive T Reg cells Its vital role in immune regulation has made IL-2 an attractive therapeutic target in a range of immune-linked diseases, both to promote the immune response, as in cancer and infectious disease, and to repress the immune response, as in autoimmune disorders and graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • the clinical performance of IL-2 has been limited by the multifarious nature of its activities, which thwart efficacy and lead to toxicity or harmful off-target effects. It would therefore be of tremendous therapeutic value to decouple the immunostimulatory and immunosuppressive activities of IL-2 to cater to particular disease applications.
  • cytokine-directed antibodies that bias activity toward specific T cell subsets.
  • Co-administration of antibodies with IL-2 offers important therapeutic advantages such as prolonged in vivo half-life due to neonatal Fc receptor interactions.
  • Boyman and colleagues established that immunocomplexes formed by pre-association of two mouse IL-2 (mlL-2)-directed antibodies with the cytokine elicit contrasting effects: mlL-2:JES6-1 immunocomplexes actively induce proliferation of IL-2Ra hi cells, preferentially expanding Tp eg cells over effector cells, whereas mlL-2:S4B6 immunocomplexes stimulate proliferation of all immune cells, but particularly favor effector cells.
  • JES6-1 immunocomplexes promote graft tolerance and show efficacy in preclinical models of diabetes and S4B6 immunocomplexes exhibit potent anti-tumor activity without inducing toxicity.
  • S4B6 immunocomplexes exhibit potent anti-tumor activity without inducing toxicity.
  • JES6-1 sterically obstructs IL-2 interaction with the IL-2Rp and y c subunits to block signaling on IL-2Ra'° effector cells, but also undergoes a unique allosteric exchange mechanism with the IL-2Ra subunit, wherein surface-expressed IL-2Ra displaces the JES6-1 antibody and liberates the cytokine to signal through the high-affinity heterotrimeric receptor on IL-2Ra hi Tr Q9 cells.
  • This phenomenon occurs because key residues in the IL-2 AB interhelical loop engage the JES6-1 antibody and the IL-2Ra subunit in distinct orientations; thus, IL-2-antibody and IL-2-receptor binding are mutually exclusive, leading to bidirectional exchange.
  • Activation of the IL-2 signaling pathway on IL-2Ra hi cells further upregulates IL-2Ra expression to create a positive feedback loop that extraordinarly favors T ⁇ 3 ⁇ 49 expansion.
  • Antibody-IL-2 fusion proteins are provided that selectively activate IL-2Ra hi cells. In vivo, administration of an effective dose of such a fusion protein skews the immune response to favor signaling and expansion of Tp eg cells over effector cells.
  • the JES6-1 antibody exerts complex steric and allosteric effects on IL-2 to differentially activate immune cell subsets based on their IL-2 receptor surface expression profiles and this biased activation is further propagated through transcriptional feedback.
  • the fusion protein specifically binds to IL-2 and potentiates IL-2 activity in IL-2Ra hi cells.
  • the fusion protein may be specific for mouse IL-2 and mlL-2 receptors; or may be utilized as a paradigm for development of a human counterpart agent.
  • the fusion proteins may selectively potentiate IL-2 activity by (a) extending the cytokine half-life in vivo to achieve increased stability of circulating cytokine; (b) sterically blocking binding of IL-2 to IL-2Rp and yc and allosterically impeding binding of IL-2 to IL-2Ra; and (c) upregulating IL- 2 Ra expression to create a positive feedback loop that perpetuates proliferation of IL-2Ra hi cells.
  • the affinity of the antibody for IL-2 is from about 5-fold to about 10 3 — fold higher than the affinity of IL-2 for IL-2Rp, and in some embodiments the affinity is from about 50 to about 200-fold higher.
  • the antibody agent has an affinity 3 to 10-fold higher than the affinity of IL-2 for IL-2Ra.
  • fusion proteins referred to as immunocytokines (ICs), which‘shield’ the cytokine from non-specifically engaging immune cells and instead target it preferentially to TReg cells based on surface receptor expression levels.
  • ICs immunocytokines
  • the fusion proteins provided herein utilize antibody engineering to achieve the effect, thus obviating the need for cytokine manipulation to keep both cytokine-receptor affinity and cytokine activity intact. Furthermore, off- target effects are obviated by fully sequestering the cytokine rather than simply lowering its receptor interaction affinity.
  • the structure-based design principles used to engineer an effective single-agent cytokine-antibody fusion are readily extended to other ligand-antibody interactions for exclusive targeting of soluble factors to specific cell subsets of interest.
  • the window of antibody-cytokine affinity‘tunability’ for optimization of preferential TReg cell expansion is provided.
  • the location of interface disruption with respect to chain can also affect IC activity.
  • VL mutants exhibit uniformly stronger signaling activity than VH mutants, which may be impacted by the topology of the fusion itself, as the greater proximity to tethered IL-2 for VL compared to VH may render the VL interface more robust against affinity disruption.
  • the IC format has clear advantages over mixed cytokine/antibody complex administration as it eliminates dosing ratio considerations and concerns about the free cytokine inducing off-target effects or undergoing rapid clearance from the bloodstream.
  • the engineered IC JY3 elicited greater IL-2Ra hi cell expansion in an adoptive T cell transfer model and prevented DSS-induced colitis more effectively than the mixed complex.
  • the enhanced behavior of JY3 IC over the mixed complex provides an immediately useful reagent for expanding TReg cells to combat autoimmune disease, and the structure-guided engineering strategy used to develop this construct is applied to the design of other mechanism-driven therapeutic immunocytokines.
  • an immunocytokine of the invention comprises a variable region domain comprising specific V H and V L region sequences, including without limitation the V H sequences of JES6-1 (SEQ ID NO:4) or variants provided here, for example with amino acid substitutions at one or more of residues: D55, D58, E60, R62.
  • Exemplary substitutions include substituting any of these residues with, for example, S, G, L, V, I, A; which substitutions specifically include, without limitation, D55A, D58A, E60A, R62A.
  • the VH sequence is paired with the VL sequence of JES6-1 (SEQ ID NO:5) with amino acid substitutions at one or more of residues: S34, Y41 , H100, Y101 , S34+Y41 , S34+Y101 , Y41+Y101 , and S34+Y41+Y101.
  • Exemplary substitutions include substituting any of these residues with, for example, S, G, L, V, I, A; which substitutions specifically include, without limitation, S34A, Y41A, H100A, Y101A, S34A+Y41 A, S34A+Y101 A, Y41A+Y101A, and S34A+Y41A+Y101A.
  • a VH with the sequence set forth in SEQ ID NO:4 is paired with a VL comprising one or more of the amino acid substitutions provided above.
  • variable regions can be provided as a single chain, including without limitation a single-chain variable fragment (scFv) polypeptide, joined to an IL-2 sequence, e.g. hlL-2, mll_-2, etc.
  • the variable region can be joined covalently or non-covalently to an Fc polypeptide, which Fc polypeptide is optionally a human, rat, mouse, etc. sequence.
  • the variable region can also be modified to extend serum half-life via conjugation to polyethylene glycol (PEG) moieties or fused to purification or selective localization tags.
  • the variable regions and the IL-2 sequence may be directly fused, or connected by a linker.
  • Linkers can be proteolytically sensitive or resistant, and may be from about 2 to about 30 amino acids in length, e.g. from about 5 to about 20 amino acids in length.
  • a panel of immunocytokine proteins are provided, or a kit comprising a plurality of immunocytokine proteins, comprising the V H and V H variants disclosed above, and optionally comprising a JES6-1 protein as a reference.
  • the panel of proteins find use in the analysis of IL-2 signaling, particularly in its effect on Treg cells and the selective expansion of IL-2Ra hi cells, such as immunosuppressive Tk b9 cells in the individual.
  • a panel or kit may comprise 2, 3, 4, 5, 6, or more different immunocytokine proteins.
  • a pharmaceutical composition comprising an antibody agent of the invention, and a pharmaceutically acceptable excipient.
  • the pharmaceutical formulation can be provided in a unit dose, e.g. in a dose effective for the selective expansion of IL-2Ra hi cells when administered to an individual.
  • methods are provided for the research, analysis, treatment, etc. of disease, by administering an effective dose and regimen of a pharmaceutical composition of the invention to an individual for the selective expansion of IL-2Ra hi cells.
  • the treatment can preferentially expand immunosuppressive T Reg cells in the individual.
  • in animal model for an autoimmune disease is tested.
  • the animal is tested for an organ transplant, e.g. suffering from an episode of graft rejection.
  • the IC is used as a research tool for the in vitro, ex vivo, or in vivo expansion of regulatory T cells, e.g. in an adoptive transfer regimen.
  • methods are provided for engineering a functionally active immuncytokine comprising a fusion protein of a cytokine and an antibody that binds to the cytokine, by by altering residues selected by structure-guided design based on an immunocomplex of the cytokine, anti-cytokine antibody, and cytokine receptor; and determining the substituted residues that maintain function of the immunocytokine.
  • the residues may be positioned at the cytokine/antibody interface.
  • the molecular structure of the immunocomplex may be determined to guide the selection of sites for mutation.
  • a model for equilibrium dynamics of the cytokine and cytokine receptor may be determined.
  • the affinity of the cytokine/anti-cytokine fusion for the cytokine receptor may be altered to maximize the desired binding kinetics.
  • FIG. 1A-1D Unique antibody-receptor exchange mechanism underlies T Reg bias of mixed IL-2/JES6-1 complex.
  • FIG. 1A Schematic of the mechanistic rationale for IL-2/JES6-1 complex-mediated selective potentiation of Tk b9 cells.
  • the JES6-1 antibody (shown in single chain format) sterically obstructs IL-2 engagement of the IL-2Rp and y c subunits, preventing activation of ll_-2Ra Lo effector cells (left).
  • allosteric exchange between JES6-1 and the IL-2 Rot subunit allows for exclusive signaling on IL-2Ra Hi TR eg s, biasing toward an immunosuppressive response (right).
  • FIG. 1A Schematic of the mechanistic rationale for IL-2/JES6-1 complex-mediated selective potentiation of Tk b9 cells.
  • the JES6-1 antibody (shown in single chain format) sterically obstructs IL-2 engagement of the
  • IL-2 was immobilized and 500 nM IL-2 Rot (top) or 500 nM JES6-1 antibody (bottom) was injected at time 0 min. After 10 minutes, various concentrations of JES6-1 antibody ranging from 31 nM to 2 mM (top) or various concentrations for IL-2 Rot ranging from 0.5 mM to 32 mM (bottom) were added and second-harmonic generation signal change was monitored. Exchange schemes are shown at left. (FIG.
  • Root mean squared displacement (RMSD), dihedral angles, and inter-residue distances for the interhelical loops and flanking residues are plotted for a representative transition between the JES6-1 -bound and IL-2Ra-bound states (bottom).
  • FIG. 1 D Overlay of three representative simulated conformations each from the JES6-1-bound state, the intermediate states, and the IL-2Ra-bound state of IL-2 that form the primary transition path, with interhelical regions colored as in (c).
  • FIG. 2A-2D IL-2/JES6-1 immunocytokine fusion fails to recapitulate TReg-promoting activity of the mixed antibody-cytokine complex.
  • FIG. 2A Schematic of the IL-2/JES6-1 single chain immunocytokine (IC) fusion with the cytokine tethered to the N-terminus of the antibody light chain via a (Gly4Ser)2 flexible linker.
  • FIG. 2B Equilibrium surface plasmon resonance titrations of soluble IL-2 (gray) or JES6-1 IC (blue) binding to immobilized IL-2Ra. Fitted equilibrium dissociation constants (Kd) are indicated.
  • FIG. 2A Schematic of the IL-2/JES6-1 single chain immunocytokine (IC) fusion with the cytokine tethered to the N-terminus of the antibody light chain via a (Gly4Ser)2 flexible linker.
  • FIG. 2B Equilibrium surface plasmon resonance t
  • FIG. 2C STAT5 phosphorylation response (mean ⁇ S.D.) of IL-2Ra + (top) or IL-2Ra (bottom) YT-1 human NK cells stimulated with IL-2, IL-2/JES6-1 complex, or JES6-1 IC.
  • FIG. 2D NOD mice were injected with the indicated IL-2 treatment cohorts for five consecutive days. Mice were sacrificed 72 hours later and their spleens were analyzed for immune cell subset composition via flow cytometry. The ratio of TReg to CD8 + effector T cells is shown for each cohort (mean ⁇ s.d.).
  • FIG. 3A-3F Disruption of antibody-cytokine affinity enhances immunocytokine activity on IL-2Ra+ cells.
  • FIG. 3A Crystallographic structure of the IL-2/JES6-1 interface (PDB ID 4YQX) with interfacial antibody residues that were mutated to alanine highlighted in yellow (heavy chain) or green (light chain).
  • Human IL-2Ra is overlaid from the IL-2 cytokine-receptor quaternary complex structure for reference (PDB ID 2B5I).
  • FIG. 3B Equilibrium surface plasmon resonance titrations of soluble IL-2 binding to immobilized JES6-1 or the indicated antibody variants.
  • FIG. 3C Equilibrium surface plasmon resonance titrations of soluble IL-2, JES6-1 IC, or JES6-1 IC variants binding to immobilized IL-2Ra (top) or IL-2Rp (bottom).
  • FIG. 3D STAT5 phosphorylation response of IL-2Ra+ (top) or IL-2Ra- (bottom) YT-1 human NK cells treated with IL-2, JES6-1 IC, or JES6-1 IC variants. Data represent mean ⁇ s.d.
  • FIG. 3E Comparison of the STAT5 phosphorylation activity of the indicated IC variants (% IL-2-induced signal at 1.2 mM concentration) versus IL-2 affinity of their corresponding antibodies.
  • Activity of the IL-2/JES6-1 complex is indicated by the dashed blue line.
  • Data represent mean ⁇ s.d.
  • FIG. 3F Comparison of the STAT5 phosphorylation activity of the indicated IC variants (% IL-2- induced signal at 1.2 mM concentration) to their IL-2Ra affinities (representative of their exchanging propensities).
  • Activity of the IL-2/JES6-1 complex is indicated by the dashed blue line.
  • FIG. 4A-4D Engineered double mutant immunocytokine recovers TReg-biased activity of the IL- 2/JES6-1 complex.
  • FIG. 4A Equilibrium surface plasmon resonance titrations of soluble IL-2, JES6-1 IC, or double/triple mutant JES6-1 IC variants binding to immobilized IL- 2Ra (top) or IL-2RP (bottom).
  • FIG. 4B STAT5 phosphorylation response of IL-2Ra+ (top) or IL- 2Ra- (bottom) YT-1 human NK cells treated with IL-2, JES6-1 IC, or double/triple mutant JES6- 1 IC variants.
  • FIG. 4C Equilibrium surface plasmon resonance titrations of soluble IL-2 binding immobilized JES6-1 antibody or the JY3 antibody variant.
  • FIG. 4D C57/BL6 mice were subjected to the indicated treatments for seven consecutive days. Animals were euthanized 24 hours after the final injection and spleen cells were harvested and analyzed via flow cytometry. The percentage of TReg cells within the CD4 + T cell subpopulation is presented (mean ⁇ s.d.).
  • FIG. 5A-5E Engineered immunocytokine selectively potentiates the growth of activated adoptively transferred CD8+ T cells while boosting immunosuppression in the recipient.
  • FIG. 5A Schematic of the adoptive transfer procedure. CD8+ T cells were purified from OT-I/Ly 5.1 mice and adoptively transferred into B6 mice (Ly 5.2), which were then stimulated by SIINFEKL peptide and subjected to the indicated treatments for four consecutive days. Mice were sacrificed 48 hours after the final injection and relative expansion was quantified via flow cytometry for the adoptively transferred (AT) CD8 + T cells (FIG. 5B) and the recipient TReg cells (FIG. 5C), MR CD8 + T cells (FIG. 5D), and NK cells (FIG. 5E). Data represent mean ⁇ s.d.
  • FIG. 6A-6D Engineered immunocytokine reduces disease severity in a mouse model of colitis.
  • FIG. 6A Schematic of the mouse colitis study. BALB/c mice were treated once daily for 7 days with PBS, IL-2 plus a control antibody, IL-2/JES6-1 complex, or JY3 IC. Beginning on day 8, mice were subjected to 3% DSS in their drinking water to induce colitis. Weight loss (FIG. 6B) and disease activity index (FIG. 6C) were assessed on day 15. Mice were sacrificed on day 16 and colon length (FIG. 6D) was measured. Statistical significance by one-way ANOVA + Dunnett’s multiple comparison post-test is indicated. Data represent mean ⁇ s.d.
  • FIG. 7 Design of a single-chain cytokine-antibody fusion linking IL-2 and JES6-1. Crystallographic structure of the IL-2/JES6-1 complex (PDB ID 4YQX) with the distance annotated between the C-terminal residue of IL-2 (red) and the N-terminal residue of the JES6-1 VL domain (green). The JES6-1 antibody is shown as a single-chain variable construct (scFv).
  • FIG. 8A-8B IL-2-receptor and IL-2-JES6-1 antibody affinities dictate exchange mechanism and biased immune cell activation.
  • FIG. 8A Equilibrium surface plasmon resonance titrations of soluble IL-2 binding to immobilized IL-2Ra (cyan), IL-2Rf3 (navy), or JES6-1 (marine blue). Fitted equilibrium dissociation constants (Kd) are indicated.
  • FIG. 8B Hypothetical plot of the TReg to effector cell expansion ratio versus IL-2-antibody affinity in the framework of the JES6-1 allosteric exchange mechanism.
  • the cytokine-antibody affinity is very low, the cytokine will constitutively dissociate from the antibody, resulting in non-specific activation of both TReg and effector immune cells.
  • the cytokine-antibody affinity is very high, the antibody cannot be displaced by IL-2Ra, blocking IL-2 activity on both TReg and effector cells.
  • the affinity of the JES6-1 antibody allows for receptor-antibody exchange to induce biased TReg expansion, whereas the increased affinity of JES6-1 IC precludes its stimulation of TReg proliferation.
  • FIG. 9 Engineered immunocytokine selectively potentiates TReg cell proliferation.
  • C57/BL6 mice were administered the indicated treatments for seven consecutive days. Animals were euthanized 24 hours after the final injection and spleen cells were harvested and analyzed via flow cytometry. Raw data plots of IL-2Ra expression versus CD4 (left) and Foxp3 (right) expression are presented. One representative plot from three replicate mice per condition is shown.
  • FIG. 10A-10B Biased TReg potentiation is highly sensitive to specific binding and structural properties of immunocytokine.
  • FIG. 10A Equilibrium surface plasmon resonance titrations of the soluble IL-2 interaction with immobilized JES6-1 antibody or the S34A+Y101A antibody variant.
  • FIG. 10B C57/BL6 mice were subjected to the indicated treatments for seven consecutive days and sacrificed 24 hours after the final injection. Spleen cells were harvested and analyzed via flow cytometry. The percentage of TReg cells within the CD4+ T cell subpopulation is presented (mean ⁇ s.d ).
  • FIG. 11A-11 B JY3 IC stimulates biased TReg cell expansion and upregulates IL- 2Ra expression in a dose-dependent fashion in C57/BL6 mice.
  • C57/BL6 mice were administered PBS or the indicated concentrations of IL-2/JES6-1 complex or JY3 IC for five consecutive days. Animals were euthanized 72 hours after the final injection and spleen cells were harvested and analyzed via flow cytometry.
  • the ratio of TReg cells to CD8+ effector T cells (FIG. 11 A) and mean fluorescence intensity (MFI) of IL-2Ra (FIG. 11 B) is presented for each cohort. Data represent mean ⁇ s.d.
  • FIG. 12A-12B JY3 IC stimulates biased TReg cell expansion and upregulates IL- 2 Ra expression in a dose-dependent fashion in non-obese diabetic mice.
  • Non-obese diabetic (NOD) mice were administered PBS or the indicated concentrations of IL-2/JES6-1 complex or JY3 IC for five consecutive days. Animals were euthanized 72 hours after the final injection and spleen cells were harvested and analyzed via flow cytometry.
  • the ratio of TReg cells to CD8+ effector T cells (FIG. 12A) and mean fluorescence intensity (MFI) of surface-expressed IL- 2Ra (FIG. 12B) is presented for each cohort. Data represent mean ⁇ s.d. Note that the IL- 2/JES6-1 complex dose was restricted to 5 pg because 3/4 mice that were administered 30 pg of the IL-2/JES6-1 complex died during the course of the experiment.
  • FIG. 13A-13D JY3 IC increases expression of IL-2Ra on immune cells in a model of adoptive CD8+ T cell transfer.
  • CD8+ T cells were purified from OT-I/Ly 5.1 mice and adoptively transferred into B6 mice (Ly 5.2), which were then stimulated by SIINFEKL peptide and subjected to the indicated treatments for four consecutive days. Mice were sacrificed 48 hours after the final injection and mean fluorescence intensity (MFI) of surface-expressed IL-2Ra was quantified via flow cytometry for the adoptively transferred (AT) CD8 + T cells (FIG. 13A) and the recipient TReg cells (FIG. 13B), MP CD8+ T cells (FIG. 13C), and NK cells (FIG. 13D). Data represent mean ⁇ s.d.
  • FIG. 14 Schematic of JES6-1 variable region sequences, SEQ ID NO:4 and SEQ ID NO:
  • FIG. 15A-15B Table 1 provides the Kd values of immunocomplexes.
  • Table 2 provides the Kd values of immunocomplexes.
  • IL-2 is a member of the common gamma cytokine family, each member of which has a four alpha helix bundle; the family also includes IL-4, IL-7, IL-9, IL-15 and IL-21.
  • IL-2 signals through formation of either a high-affinity quaternary complex (K d ⁇ 10 pM) with the interleukin-2 receptor-a (IL-2Ra, CD25), I L-2Rp, and yc chains, or an intermediate-affinity ternary complex (K d ⁇ 1 nM) with only the IL-2Rp and yc chains. Consequently, expression of the non-signaling IL- 2Ra subunit regulates cytokine sensitivity.
  • IL-2Ra is robustly expressed on regulatory T (T Reg ) cells but is virtually absent from naive effector cells such as memory-phenotype (MR) CD8 + T cells and natural killer (NK) cells, resulting in differential responsiveness of these immune cell subsets to IL-2.
  • T Reg regulatory T
  • MR memory-phenotype
  • NK natural killer
  • IL-2 refers to the human counterpart protein.
  • Mature human IL-2 occurs as a 133 amino acid sequence (less the signal peptide, consisting of an additional 20 N-terminal amino acids), as described in Fujita, et. al , PNAS USA, 80, 7437-7441 (1983).
  • the amino acid sequence of human IL-2 is found in Genbank under accession locator NP_000577.2.
  • Variants of human IL-2 are known in the art and available clinically, e.g.
  • IL-2 aldesleukin (Proleukin) with the alanine removed from its N-terminus and residue 125 replaced with serine; interking, with a serine at residue 125; teceleukin, with a methionine added at the N-terminus; bioleukin, with a methionine added at the N-terminus and residue 125 replaced with alanine; Super-2 (described by Levin et al. (2012) Nature 484(7395):529-33); and the like.
  • the term IL-2 as used herein refers to both the wild-type human sequence and biologically active variants thereof, as known in the art.
  • IL-2 has key roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T cells. In the thymus, where T cells mature, it prevents autoimmune diseases by promoting the differentiation of certain immature T cells into regulatory T cells, which suppress other T cells that are otherwise primed to attack normal healthy cells in the body. IL-2 also promotes the differentiation of T cells into effector T cells and into memory T cells.
  • IL-2 has a narrow therapeutic window, and the level of dosing usually determines the severity of the side effects. Patients can be given high dosages i.v. for five consecutive days, then allowed approximately 10 days to recover between treatment dosages; and hospitalization/intensive care is required throughout due to the side effects. It can also be delivered subcutaneously at a lower dosage.
  • the use of IL-2 as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response.
  • the major side effect of IL-2 therapy is vascular leak syndrome (VLS), which leads to the accumulation of intravascular fluid in the lungs and liver resulting in pulmonary edema and liver damage.
  • VLS vascular leak syndrome
  • High-affinity IL-2 cytokine/receptor quaternary complexes are typically found on CD4 + T regulatory cells (T Reg s) as well as recently-activated T cells.
  • Intermediate-affinity IL-2 cytokine/receptor complexes are present on naive CD8 + T cells and are prominent on antigen- experienced (memory) and memory- phenotype (MP) CD8 + T cells as well as natural killer (NK) cells. Both MP CD8 + T cells and NK cells express relatively high levels of I ⁇ -2Rb and readily respond to IL-2 injections in vivo.
  • IL-2 Rot is upregulated.
  • IL-2 binds the high-affinity heterotrimeric receptor sequentially, first engaging IL- 2 Rot and then sequentially recruiting the b and y receptor subunits.
  • Antibody refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • intact antibodies as produced in nature are approximately 150 Kd tetrameric agents comprised of two identical heavy chain polypeptides (about 50 Kd each) and two identical light chain polypeptides (about 25 Kd each) that associate with each other into what is commonly referred to as a ⁇ -shaped” structure.
  • Each heavy chain is comprised of at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHi, Chh, and the carboxy-terminal CHs (located at the base of the Y’s stem).
  • VH amino-terminal variable
  • CHi constant domain
  • CHi constant domain
  • Chh constant domain
  • carboxy-terminal CHs located at the base of the Y’s stem
  • a short region known as the“switch” connects the heavy chain variable and constant regions.
  • The“hinge” connects CH 2 and CH 3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody.
  • Each light chain is comprised of two domains: an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”.
  • VL amino-terminal variable
  • CL carboxy-terminal constant
  • Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • the constant regions of antibodies of the invention can be the original species of origin, e.g. rat, mouse, etc., or the constant regions can be substituted with other constant regions of interest, including without limitation the constant regions of any of the known human isotypes.
  • Isotypes include IgG , IgGa, IgGs, lgG 4 , IgAi , IgAa. Included in the constant regions of interest are human lgG4 constant regions with the amino acid substitution S241 P (see, for example, Angal et al. (1993) Mol Immunol. 30(1):105-8.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • Human light chain constant regions include the l and k constant regions, which can be used to substitute for the original constant regions.
  • Antibodies that retain the variable region sequence but have (for example) human constant region sequences are often referred to as“chimeric” antibodies.
  • Each domain in a natural antibody has a structure characterized by an“immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed anti parallel beta barrel.
  • Each variable domain contains three hypervariable loops known as“complementarity determining regions” (CDR1 , CDR2, and CDR3) and four somewhat invariant“framework” regions (FR1 , FR2, FR3, and FR4).
  • the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure
  • the Fc region of naturally occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity.
  • affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation as well as those that have been mutated to enhance serum half-life or effector function (i. e. antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity).
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an“antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody is polyclonal; in some embodiments, an antibody is monoclonal.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art.
  • Humanized antibodies generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies.
  • the entire antibody, except the CDRs is a polypeptide of human origin or is identical to such an antibody except within its CDRs.
  • the full CDRs or a subset of these regions, some or all of which are encoded by nucleic acids originating in a non human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs.
  • Antibodies can be humanized using a variety of techniques including CDR-g rafting, veneering or resurfacing, and chain shuffling.
  • the humanized antibody may comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 969-973. Structure-based methods may be employed for humanization and affinity maturation, for example as described in USSN 11/004,590. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al, 1999, J. Mol. Biol. 294: 151 -162; Baca et al, 1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al. 1996, J. Biol. Chem.
  • an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs, cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular JmmunoPharmaceuticals (“SMIPsTM " ), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, mutants of the tenth type III domain of human fibronectin (monobodies or Ad
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]
  • Specific antibody fragments include, but are not limited to (i) Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) Fd fragment consisting of the VH and CH1 domains, (iii) Fv fragment consisting of the V L and V H domains of a single antibody; (iv) fAb fragment which consists of a single variable region, (v) isolated CDR regions, (vi) F(ab’)2 fragments, (vii) single chain Fv molecules (scFv), wherein a V H domain and a V L domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site, (viii) bispecific single chain Fv and (ix) "diabodies” or "triabodies", multivalent or multispecific fragments constructed by gene fusion.
  • Specific binding or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
  • Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a K d for an antigen or epitope of at least about 10 -4 M, at least about 10 5 M, at least about 10 6 M, at least about 10 7 M, at least about 1Q 8 M, at least about 10 9 M, at least about 10 10 , or greater, where K refers to a dissociation rate of a particular antibody-antigen interaction.
  • an antibody that specifically binds an antigen will have a K d that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope.
  • Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a K d for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where K d refers to an association rate of a particular antibody-antigen interaction.
  • K d refers to an association rate of a particular antibody-antigen interaction.
  • Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, each incorporated herein by reference in their entirety and for all purposes; Huston et al. , Methods in Enzymology, 203: 46- 88, 1991 ; Shu, L. et al., PNAS 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038- 1040, 1988.
  • the present invention further includes compositions immunocytokines, e.g. in a pharmaceutical formulation, including unit dose formulations.
  • antibody agent refers to an agent that specifically binds to a particular antigen.
  • the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • exemplary antibody agents include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies (i.e. , antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular JmmunoPharmaceuticals (“SMIPsTM " ), single chain antibodies, cameloid antibodies, and antibody fragments.
  • SMIPsTM Small Modular JmmunoPharmaceuticals
  • the term“antibody agent” also includes intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g. bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the term encompasses stapled peptides.
  • the term encompasses one or more antibody-like binding peptidomimetics.
  • the term encompasses one or more antibody-like binding scaffold proteins.
  • the term encompasses monobodies or adnectins.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in an antibody described herein. In some embodiments an included CDR is substantially identical to a CDR of the invention in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a CDR of the invention in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the CDR of the invention. In some embodiments an included CDR is substantially identical to a CDR of the invention in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.
  • an included CDR is substantially identical to a CDR of the invention in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the CDR of the invention but the included CDR has an amino acid sequence that is otherwise identical with that of the CDR of the invention. In some embodiments an included CDR is substantially identical to a CDR of the invention in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the CDR of the invention but the included CDR has an amino acid sequence that is otherwise identical to the CDR of the invention.
  • an included CDR is substantially identical to a CDR of the invention in that at least one amino acid within the included CDR is substituted as compared with the CDR of the invention but the included CDR has an amino acid sequence that is otherwise identical with that of the CDR of the invention.
  • an included CDR is substantially identical to a CDR of the invention in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the CDR of the invention but the included CDR has an amino acid sequence that is otherwise identical to the CDR of the invention.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
  • Antibodies of the invention may be isolated or recombinant.
  • isolated when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step.
  • Variant refers to an entity that shows significant structural identity with a reference entity, specifically human IL-2 or an antibody or fragment thereof provided herein, but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In some embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a“variant” of a reference entity is based on its degree of structural identity with the reference entity.
  • any biological or chemical reference entity has certain characteristic structural elements.
  • a variant by definition, is a distinct chemical entity that shares one or more such characteristic structural elements.
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function.
  • a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc) covalently attached to the polypeptide backbone.
  • a variant polypeptide shows an overall sequence identity with a reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.
  • a polypeptide of interest is considered to be a“variant” of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.
  • a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues.
  • the parent or reference polypeptide is one found in nature. As will be understood by those of ordinary skill in the art, a plurality of variants of a particular polypeptide of interest may commonly be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide.
  • Non-immunogenic, flexible polypeptide linkers are known and used in the art, e.g. linkers comprising glycine and serine residues.
  • a peptide linker has a formula selected from: (GS) n , wherein n is an integer from 6 to 15; (G2S) n , wherein n is an integer from 4 to 10; (G3S) n , wherein n is an integer from 3 to 8; (G4S) n , wherein n is an integer from 2 to 6; (G) n , wherein n is an integer from 12 to 30; and (S) n , wherein n is an integer from 12 to 30.
  • Antigen refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody.
  • an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen).
  • an antigen binds to an antibody and may or may not induce a particular physiological response in an organism.
  • an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer]) etc.
  • an antigen is or comprises a polypeptide.
  • an antigen is or comprises a glycan.
  • an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source).
  • antigens utilized in accordance with the present invention are provided in a crude form.
  • an antigen is a recombinant antigen.
  • administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.
  • administration may involve intermittent dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • antibody therapy is commonly administered parenterally (e.g., by intravenous or subcutaneous injection).
  • biological sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein.
  • a source of interest comprises an organism, such as an animal or human.
  • a biological sample is or comprises biological tissue or fluid.
  • a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell- containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc.
  • a biological sample is or comprises cells obtained from an individual.
  • obtained cells are or include cells from an individual from whom the sample is obtained.
  • a sample is a“primary sample” obtained directly from a source of interest by any appropriate means.
  • a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.
  • sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
  • processing e.g., by removing one or more components of and/or by adding one or more agents to
  • a primary sample For example, filtering using a semi-permeable membrane.
  • Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
  • Combination Therapy refers to those situations in which a subject is simultaneously or concomitantly exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered in overlapping dosing regimens.
  • composition refers to the combination of two or more agents as described herein for co administration or administration as part of the same regimen. It is not required in all embodiments that the combination of agents result in physical admixture, that is, administration as separate co- agents each of the components of the composition is possible; however many patients or practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.
  • composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method.
  • any composition or method described as “comprising” (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method “consisting essentially of (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method.
  • composition or method described herein as “comprising” or “consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method “consisting of” (or “consists of) the named elements or steps to the exclusion of any other unnamed element or step.
  • known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
  • Dosage Form refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject, including a“unit dose”. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • Dosing regimen refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
  • a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • composition as disclosed herein, the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
  • therapeutically effective amount means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • term "therapeutically effective amount” refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse an autoimmune disease process occurring in said individual, or will enhance or increase a suppressive process in said individual.
  • the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a "cure” for autoimmune disease; rather the methods of treatment are directed to the use of the described compositions to "treat" a disease, i.e., to effect a desirable or beneficial change in the health of an individual.
  • Such benefits are recognized by skilled healthcare providers and include, but are not limited to, a stabilization of patient condition, an improvement in vital functions, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof.
  • treatment refers to any administration of an antibody or antibody complex of the invention that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • a patient refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or presence of one or more tumors. In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
  • a patient is a human.
  • a patient is suffering from or susceptible to one or
  • Immunocytokines are provided that selectively activate IL-2Rct hi cells. In vivo, administration of an effective dose of such an antibody, derivative or mimetic thereof biases the immune response to favor signaling and expansion of Tp eg over effector cells.
  • Embodiments of the invention include isolated immunocytokines, pharmaceutical formulations comprising one or more of the antibodies; cell lines that produce these immunocytokines.
  • the immunocytokines comprise a constant region sequence, including without limitation IgG constant regions, e.g. IgG , lgG 2a , lgG 2b , lgG 3 , lgG 4 , etc.
  • Polypeptides of interest also include variable regions sequences that differ by up to one, up to two, up to 3, up to 4, up to 5, up to 6 or more amino acids as compared to the amino acids sequence set forth the variants of JES6-1 provided herein
  • single chain antibodies can be constructed according to the method of U.S. Pat. No. 4,946,778 to Ladner et al, which is incorporated herein by reference in its entirety.
  • Single chain antibodies comprise the variable regions of the light and heavy chains joined by a flexible linker moiety.
  • the antibody fragment known as the single domain antibody, which comprises an isolate V H single domain.
  • the antibodies of the invention can be multispecific antibodies, and notably bispecific antibodies or bispecific scFv chains, also sometimes referred to as "diabodies".
  • Bispecific antibodies and antibody fragments are unimolecular species that bind to two (or more) different antigens, or different epitopes on the same antigen.
  • the antibody is a minibody.
  • Minibodies are minimized antibody-like proteins comprising a scFv joined to a CHS domain. Hu et al, 1996, Cancer Res. 56:3055-3061 , entirely incorporated by reference.
  • the scFv can be joined to the Fc region, and may include some or the entire hinge region.
  • An antibody utilized in accordance with the present invention may also be in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs, cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular jmmunoPharmaceuticals (“SMIPsTM " ), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans-bodies®, Affi bodies®, a TrimerX®, MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®.
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload, e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc., or other pendant group [e.g., poly-ethylene glycol, etc.
  • Exemplary antibody agents include, but are not limited to, Small Modular ImmunoPharmaceuticals (“SMIPsTM " ), single chain antibodies, cameloid antibodies, and antibody fragments.
  • the term encompasses one or more antibody-like binding scaffold proteins. In come embodiments, the term encompasses monobodies or adnectins.
  • immunocytokines of the invention can selectively act on IL-2Ra hi cells, which express sufficient I L-2RT ⁇ to displace the antibody by mass action and initiate an allosteric exchange mechanism that enables IL-2 complex formation and signal activation. Included in the invention are methods of use for these immunocytokines, which may be administered at a dose and regimen sufficient to activate IL-2Ra hi cells.
  • the IL-2Ra hi cells are regulatory T cells, known as T Reg cells.
  • Ti 3 ⁇ 49 cells are a small subset of T-lymphocytes that repress the activity of immune effector cells and therefore have diverse clinical applications in transplantation, allergy, asthma, infectious diseases, graft- versus-host disease (GVHD), and autoimmunity. Ti 3 ⁇ 49 cells can be used to suppress the undesirable immune responses in patients with autoimmune dysregulation.
  • the compositions of the invention can be administered in vivo, e.g. to expand endogenous Tk b9 cells, or can be administered in vitro, e.g. to expand Tr 9 cells from complex lymphocyte populations, to produce therapeutically effective numbers of T ⁇ 3 ⁇ 49 cells, etc.
  • antibodies of the invention can be used in combination with hlL-2 in ex vivo methods for adoptive cell therapy protocols.
  • cells e.g., peripheral blood lymphocytes or purified populations of lymhocytes isolated from a patient and placed or maintained in culture
  • the culture step can include further steps in which the cells are stimulated or treated with other agents, e.g., to stimulate proliferation, or to expand a population of cells that is reactive to an antigen of interest.
  • the cells are then administered to a subject, e.g. in an animal model for autoimmune disease, after they have been treated.
  • Naturally occurring T Reg cells constitute only 1-5% of total CD4 + T cells in blood and they remain largely dormant until activated.
  • the phenotype of human Ti 3 ⁇ 49 cells is recognized in the art to be generally CD4 + CD25 hi (CD25 is also known in the art as IL-2Ra).
  • TR 69 cells can be induced to express forkhead box P3 (FOXP3) protein.
  • T Reg cells can be characterized as positive for the expression of one or more proteins selected from the group consisting of CTLA4, TNFR2, FOXP3, CD62L, Fas, H LA-DR, and CD45RO, and as low or negative for the expression of one or more proteins selected from the group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN- gamma, IL10, and ICOS.
  • the effectiveness of immunocytokines can be monitored by, for example, analyzing samples, including blood samples, for the content of T Reg cells, where exposure to immunocytokines of the invention result in increased numbers and activation of T Reg cells.
  • a sample can be obtained, for example, from a blood sample or a bone marrow sample.
  • the invention also includes methods for treating an immunological disorder, e.g. in an animal model of disease, including for example atopic disorders such as allergy, asthma, etc.; autoimmune disease, graft-versus-host disease, transplantation rejection, and the like.
  • an immunological disorder e.g. in an animal model of disease, including for example atopic disorders such as allergy, asthma, etc.; autoimmune disease, graft-versus-host disease, transplantation rejection, and the like.
  • Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, and/or other self-molecules causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical manifestations of the disease.
  • Autoimmune diseases include diseases that affect specific tissues as well as diseases that can affect multiple tissues, which can depend, in part, on whether the responses are directed to an antigen confined to a particular tissue or to an antigen that is widely distributed in the body.
  • Autoimmune disorders that can be assessed by treatment with immunocytokines can be selected from the group consisting of type I diabetes, Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue- Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic Inflammatory Demyelinating Polyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease, Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barre, Hashimoto's Thyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic
  • Asthma is characterized by chronic airway inflammation with intense eosinophil and lymphocyte infiltration, mucus hyperproduction, and airway hyperresponsiveness.
  • Antigen- specific Th2 cells and their cytokines such as IL-4, IL-5, and IL-13 orchestrate these pathognomonic features of asthma, which benefits from administering an effective dose of the compositions of the present invention.
  • CORD chronic obstructive pulmonary disease
  • T cell-mediated immune response is also associated with chronic hepatitis B virus (HBV) infection (see Wu et al. Journal of Gastroenterology and Hepatology 25 (2010) 750-757; Zhang et al. (2010) Hepatology (2010) 51 :81-91).
  • Th17 cells are highly enriched in both peripheral blood and liver of CHB patients, and exhibit a potential to exacerbate liver damage during chronic HBV infection, and such conditions benefit from administering an effective dose of the compositions of the present invention.
  • Atherosclerosis is an inflammatory disease in which interferon (IFN)-y, the signature cytokine of Th1 cells, plays a central role, and such conditions benefit from administering an effective dose of the compositions of the present invention.
  • IFN interferon
  • Methods also include prevention and treatment of transplantation associated conditions, including GVHD and graft rejection.
  • solid organ transplantation is used in accordance with the conventional meaning of the term, where an organ from a donor, which donor may be living or deceased, in placed into the body of a recipient in the appropriate position and cardiovascular connections to be physiologically integrated into the recipient.
  • Solid organs may include kidney, pancreas and including pancreatic islet cells; heart; lungs, intestine, liver, and the like as known in the art.
  • the transplanted organ may be referenced as a“graft”, and the physiological integration of the organ may be referred to as engraftment.
  • Immunosuppression refers to the treatment of a graft recipient with agents, primarily to diminish the immune responses of the host immune system against the graft, although the agents may also diminish GVHD of the donor hematopoietic cells.
  • Immunosuppressive treatment of the transplantation patient begins with the induction phase, perioperatively and immediately after transplantation. Maintenance therapy then continues. Induction and maintenance strategies use different medicines at specific doses or at doses adjusted to achieve target therapeutic levels to give the transplantation patient the best hope for long-term graft survival.
  • Primary immunosuppressive agents include calcineurin inhibitors, which combine with binding proteins to inhibit calcineurin activity, and which include, for example, tacrolimus, cyclosporine A, etc.
  • levels of both cyclosporine and tacrolimus must be carefully monitored. Initially, levels can be kept in the range of 10-20 ng/mL, but, after 3 months, levels may be kept lower (5-10 ng/mL) to reduce the risk of nephrotoxicity.
  • Adjuvant agents are usually combined with a calcineurin inhibitor and include steroids, azathioprine, mycophenolate mofetil, and sirolimus. Protocols of interest include a calcineurin inhibitor with mycophenolate mofetil. The use of adjuvant agents allows clinicians to achieve adequate immunosuppression while decreasing the dose and toxicity of individual agents.
  • Mycophenolate mofetil in kidney transplant recipients has assumed an important role in immunosuppression after several clinical trials have shown a markedly decreased prevalence of acute cellular rejection compared with azathioprine and a reduction in 1-year treatment failures.
  • GVHD is a risk for both HLA- matched and -mismatched transplantations. GVHD can occur even if the donor and recipient are H LA-matched because the immune system still recognizes other differences between their tissues. GVHD is usually mediated by T cells, which react to foreign peptides presented on the MHC of the host.
  • Acute GVHD typically occurs in the first 3 months after transplantation and may involve the skin, intestine, or the liver.
  • High-dose corticosteroids such as prednisone are a standard treatment.
  • Chronic GVHD may also develop after haplotype matched transplant and typically occurs after the first 3 months following transplant. It is the major source of late treatment-related complications, although it less often results in death.
  • chronic GVHD may lead to the development of fibrosis, or scar tissue, similar to scleroderma; it may cause functional disability and require prolonged immunosuppressive therapy.
  • Acute transplant rejection is the rejection by the immune system of a transplanted organ. Acute rejection is characterized by infiltration of the transplanted tissue by immune cells of the recipient, which carry out their effector function and destroy the transplanted tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. “Chronic transplant rejection” generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can typically be described by a range of specific disorders that are characteristic of the particular organ.
  • disorders include fibroproliferative destruction of the airway (bronchiolitis obliterans); in heart transplants or transplants of cardiac tissue, such as valve replacements, such disorders include fibrotic atherosclerosis; in kidney transplants, such disorders include, obstructive nephropathy, nephrosclerorsis, tubulointerstitial nephropathy; and in liver transplants, such disorders include disappearing bile duct syndrome.
  • transplant rejection encompasses both acute and chronic transplant rejection.
  • transplant rejection the transplanted tissue is rejected and destroyed by the recipient’s immune system.
  • Acute rejection may occur to some degree in all transplants, except in the cases of identical twins or during immunosuppression. Acute rejection may begin as soon as one week after transplant and greatest risk for development of acute rejection occurs in the first three months following transplant.
  • Chronic rejection is the long-term loss of function of a transplanted organ.
  • compositions including pharmaceutical compositions.
  • Such compositions typically include the polypeptide(s) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • the compositions are administered through a parenteral route.
  • parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic
  • 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).
  • 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).
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • 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.
  • surfactants e.g., sodium dodecyl sulfate.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable 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.
  • 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.
  • 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.
  • Oral compositions if used, generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PrimogelTM, or corn starch; a lubricant such as magnesium stearate or SterotesTM; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PrimogelTM, or corn starch
  • a lubricant such as magnesium stearate or SterotesTM
  • a glidant such as colloidal silicon dioxide
  • the polypeptide(s) may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • inhalation is performed using pressurized, non-aerosol administration.
  • the polypeptide(s) are administered intranasally by inhalation wherein the inhalation is performed using a pressurized, non-aerosol device.
  • the polypeptide(s) are administered intranasally by inhalation wherein the inhalation is performed using an aerosol device.
  • Systemic administration of the polypeptide(s) can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • polypeptide(s) can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • nucleic acids encoding polypeptides of the invention can be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 41 8:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006- 1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151 - 160, 1996, erratum at Am. J. Health Syst. Pharm. 53 :325, 1996).
  • the polypeptide(s) are prepared with carriers that will protect the polypeptide(s) against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • 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.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Dosage, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDso/EDso.
  • Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso (e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • ICso e.g., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of antibody depends on the polypeptide and antibody selected. For instance, single dose amounts of the antibody may be in the range of approximately 0.001 to 1 mg/kg of patient body weight.
  • the dosage of hlL-2 may be similar to, or less than, that prescribed for PROLEUKIN®.
  • the compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount can include a single treatment or, can include a series of treatments.
  • the compositions are administered every 8 hours for five days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by an additional five days of administration every 8 hours.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • JES6-1 Immunocytokine Heavy Chain is as follows: SEQ ID NO:1 EIQLQQSGPELRRPGSSVKLSCKASGYNITDYLIHWVRHRPEHGLEWIGWIDPEDGETRYAQK FQSKATLTADTSSNAAYMQLSSLTPEDTATYFCARSLDSTYIYPFAYWGQGTLVTVSSAETTAP SVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVT VPSSTWSSQAVTCNVAHPASSTKVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPK VTCVWDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKC KVNSGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMN GQPQENYK
  • SEQ ID NO:1 residues 1-121 JES6-1 VH sequence.
  • SEQ ID NO:1 residues 122-218 Rat lgG2a CH1.
  • SEQ ID NO:1 , residues 219-223 is a Rat lgG2a Hinge sequence.
  • SEQ ID NO:1 , residues 224-336 is Rat lgG2a CH2.
  • SEQ ID NO:1 residues 337-443 is Rat lgG2a CH3 sequence.
  • JES6-1 Immunocytokine light Chain is as follows: SEQ ID NO:2 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPK QATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDES ATVVDFLRRWIAFCQSIISTSPQGGGGSGGGGSDIVMTQSPFSLAVSEGEMVTINCKSSQSLLS SGNQKNYLAWYQQKPGQSPKLLIYYASTGQSGVPDRFIGSGSGTDFTLTISDVQAEDLADYYC
  • SEQ ID NO:2 residues 1-149 mouse IL-2 sequence.
  • SEQ ID NO:2 residues 150-159 is a linker.
  • SEQ ID NO:2, residues 160-272 is JES6-1VL sequence.
  • SEQ ID NO:2, residues 273- 379 is Rat kappa CL sequence.
  • the JY3 Immunocytokine Heavy Chain is the same as SEQ ID NO:1.
  • the JY3 immunocytokine light chain sequence comprises the same components as SEQ ID NO:2, with amino acid changes in the JES6-1VL sequence.
  • the complete sequence is as follows, SEQ ID NO:3
  • SEQ ID NO:3 residues 1-149 mouse IL-2 sequence.
  • SEQ ID NO:3 residues 150-159 is a linker.
  • SEQ ID NO:3, residues 160-272 is JY3 sequence with the amino acid substitutions relative to JES6-1 underlined.
  • SEQ ID NO:2, residues 273-379 is Rat kappa CL sequence.
  • variable region of JES6-1 heavy chain (SEQ ID NO:4) is as follows. Exemplary CDR sequences are marked in Figure 14.
  • Immunocytokine variants have been prepared with amino acid substitutions at one or more of residues: D55, D58, E60, R62.
  • Exemplary substitutions include substituting any of these residues with, for example, S, G, L, V, I, A; which substitutions specifically include, without limitation, D55A, D58A, E60A, R62A.
  • variable region of JES6-1 light chain (SEQ ID NO:5) is as follows. Exemplary CDR sequences are marked in Figure 14. DIVMTQSPFSLAVSEGEMVTINCKSSQSLLSSGNQKNYLAWYQQKPGQSPKLLIYYASTGQSG VPDRFIGSGSGTDFTLTISDVQAEDLADYYCLQHYISPPTFGAGTKLELK
  • Immunocytokine variants have been prepared with amino acid substitutions at one or more of residues: S34, Y41 , H100, Y101 , S34+Y41 , S34+Y101 , Y41+Y101 , and S34+Y41+Y101.
  • Exemplary substitutions include substituting any of these residues with, for example, S, G, L, V, I, A; which substitutions specifically include, without limitation, S34A, Y41A, H100A, Y101A, S34A+Y41A, S34A+Y101A, Y41A+Y101A, and S34A+Y41A+Y101A.
  • IL-2 lnterleukin-2
  • IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (comprised of IL-2 receptor-a [IL-2Ra, CD25]), I L-2 Rp , and common y [yc]).
  • IL-2 Rot which is highly expressed on regulatory T (TReg) cells regulates IL-2 sensitivity.
  • TReg regulatory T
  • IL-2 is a pleiotropic cytokine that orchestrates the proliferation, survival, and function of both immune effector cells and regulatory T (TReg) cells to maintain immune homeostasis.
  • TReg regulatory T
  • IL-2 sensitivity is dictated by the non-signaling IL-2Ra chain, which is abundantly expressed on the surface of TReg cells, but virtually absent from naive immune effector cells (i.e. natural killer [NK] cells and memory phenotype [MR] CD8 + T cells).
  • naive immune effector cells i.e. natural killer [NK] cells and memory phenotype [MR] CD8 + T cells.
  • IL-2 cytokine-receptor complex leads to activation of intracellular Janus kinase (JAK) proteins, which are constitutively associated with IL-2Rp and yc. JAK proteins phosphorylate key tyrosine residues in the receptor intracellular domains, leading to recruitment and activation of signal transducer and activator of transcription (STAT)-5 to effect immune- related gene expression and regulate functional outcomes.
  • JAK proteins phosphorylate key tyrosine residues in the receptor intracellular domains, leading to recruitment and activation of signal transducer and activator of transcription (STAT)-5 to effect immune- related gene expression and regulate functional outcomes.
  • STAT signal transducer and activator of transcription Due to its essential role in the differentiation and growth of TReg cells, the IL-2 cytokine has been extensively characterized in pre-clinical models to treat a range of autoimmune diseases, including diabetes and multiple sclerosis.
  • JES6-1 sterically obstructs IL-2 interaction with the IL-2Rp and yc subunits to block signaling on IL-2Ra Lo effector cells, but also undergoes a unique allosteric exchange mechanism with the IL-2Ra subunit, wherein surface- expressed IL- 2Ra displaces the JES6-1 antibody and liberates the cytokine to signal through the high-affinity heterotrimeric receptor on IL-2Ra High TReg cells (Fig. 1a).
  • This phenomenon occurs because key residues in the IL-2 AB interhelical loop engage the JES6-1 antibody and the IL-2Ra subunit in distinct orientations; thus, binding of the antibody and receptor to IL-2 occur in mutually exclusive fashion that manifests as a bidirectional exchange.
  • Activation of the IL-2 signaling pathway on IL-2Ra High cells further upregulates IL-2Ra expression to create a positive feedback loop that extraordinarly favors TReg expansion.
  • IL-2 has been covalently linked to an anti- IL-2 antibody to enhance its in vivo half-life and stability.
  • this approach is incompatible with the allosteric exchange mechanism enacted by the IL- 2/JES6-1 complex as tethering IL-2 to the JES6-1 antibody greatly enhances the apparent antibody-cytokine affinity, obstructing the triggered release that is essential for TReg bias.
  • IL-2 undergoes bidirectional exchange between the JES6- 1 antibody and the IL- 2Ra receptor subunit
  • the allosteric exchange mechanism described previously allows for the displacement of JES6-1 in the cytokine/antibody complex by the surface- bound IL-2Ra receptor subunit (Fig. 1a).
  • This mechanism was supported by structural and surface plasmon resonance (SPR)-based studies.
  • SPR surface plasmon resonance
  • SHG second-harmonic generation
  • IL-2 was labeled with a second-harmonic-active dye and immobilized to a surface.
  • the tethered cytokine was then saturated with either mouse IL-2Ra (Fig. 1 b, top) or JES6-1 (Fig. 1 b, bottom).
  • various concentrations of soluble JES6-1 (Fig. 1 , top) or IL- 2Ra (Fig. 1 b, bottom) were added and changes in SHG signal, indicative of modulations in average tilt angles of the dye particles conjugated to IL-2, were quantified.
  • dose- dependent changes were observed in IL-2 upon adding soluble protein to the immobilized complex, indicative of bidirectional exchange between the antibody and receptor engagement of the cytokine.
  • the equilibrium dynamics captured by the MSM predicted that IL-2 stably adopts a JES6-1 -bound conformation even in the absence of antibody but occasionally relaxes to a distinct metastable state that resembles the IL-2Ra- bound conformation (Fig. 1c).
  • the antibody-bound and receptor-bound states of the cytokine diverge significantly with respect to root mean squared displacement (RMSD), inter-residue distances, and residue-specific dihedral angles in all three interhelical loops (Fig. 1c).
  • the transition from the JES6-1-bound to the IL-2Ra-bound states involves significant conformational rearrangements and, in particular, destabilization of a salt bridge and a hydrogen bond in the AB and BC loops, respectively, that appear to rigidity these regions (Fig. 1c, red and orange). These changes coincide with the loss of a cation-pi interaction between B helix and the beta strand of the CD region, accompanied by increased flexibility of the latter (Fig. 1c, green). Inspection of the primary transition path with higher temporal resolution suggests that loss of loop rigidity occurs in sequential fashion.
  • IL-2-JES6-1 immunocytokine exhibits reduced activation of !L-2Ro i,9h cells and does not promote TReg expansion in vivo.
  • the IL-2 cytokine exhibited over 30-fold more potent activation (as measured by ST AT 5 phosphorylation) on IL-2Ra + cells compared to IL-2Ra cells, as expected due to the higher affinity of the heterotrimeric versus the heterodimeric IL-2 receptor complex.
  • the mixed IL-2/JES6-1 complex induced weaker activation of both cell lines but, importantly, showed more pronounced obstruction of signaling on IL-2Ra compared to IL-2Ra + cells, rationalizing the complex’s IL-2Ra Hi TReg bias.
  • JES6-1 IC did not activate IL-2Ra cells and induced much weaker activation of IL-2Ra + cells compared to the mixed complex, consistent with its impaired interaction with the IL- 2 Ra subunit (Fig. 2c).
  • Fig. 2c we explored how this differential signaling in vitro would translate into in vivo immune cell subset bias.
  • Administration of IL-2 alone to non-obese diabetic (NOD) mice did not show an increase in TReg relative to CD8 + effector T cell abundance, but treatment with IL- 2/JES6-1 complex doubled the TReg:CD8 + T cell ratio.
  • Affinity mutant immunocytokines demonstrate improved exchange and iL-2Ra + cell activation.
  • VH variable heavy
  • Fig. 3a We formatted each alanine mutant as a full-length antibody and characterized binding to the IL-2 cytokine via SPR titrations. All mutants with the exception of R62A decreased the antibody-cytokine affinity, with a maximum affinity impairment of 89-fold
  • V H mutants generally impair affinity to a greater extent than do V L mutants (Fig. 3b, Table 1).
  • the discrepancy between V L and V H mutant IC constructs is even more apparent when comparing IL-2Ra + cell activity to exchange (as determined by IL-2Ra affinity).
  • activity correlates with exchange within the V L IC mutants
  • the V H IC mutants all elicit weak stimulation of IL-2Ra + cells, independent of their IL-2Ra exchange propensities (Fig. 3F).
  • Multi-site immunocytokine mutants exhibit enhanced IL-2Ra exchange, cellular activation, and in vivo TReg-biased expansion.
  • JES6-1 IC constructs the VL domain mutants S34A, Y41A, and Y101A
  • Fig. 3d we designed three double-alanine mutant and one triple-alanine mutant IC construct to enhance IL- 2RD-exchanging capacity and IL-2Ra + cell-selective signaling.
  • All multi-residue mutant IC constructs potentiated IL-2Ra exchange compared to the parent JES6-1 IC, with the most actively-exchanging mutants (Y41A+Y101A and S34A+Y101A) exhibiting a 2.6-fold IL- 2Ra affinity enhancement relative to the parent JES6-1 IC (Fig. 4a, top, Table 2).
  • JY3 IC Y41A+Y101A IC mutant
  • IC formulation also had benefits in enhancing maximum tolerated dose of the IL-2 cytokine compared to the mixed complex, as administration of a 7.4 , ug dose of IL-2 in mixed complex format was lethal to NOD mice (3/4 mice died), but an equivalent dose of the JY3 IC (30 pg) was well tolerated (0/4 mice died).
  • the IL-2/JES6-1 complex also upregulated IL-2Ra expression on TReg and MR CD8 + T cells, although not on NK cells, and JY3 IC increased surface IL-2Ra levels on TReg and MP CD8 + T cells to a greater extent than the complex and also robustly upregulated IL-2Ra on NK cells (Fig. 13b-d).
  • JY3 IC specifically targets IL-2 activity to IL-2Ra Hi immune cell subsets, and that it promotes more robust expansion and receptor upregulation on these subsets compared to the mixed IL-2/JES6-1 complex.
  • Engineered immunocytokine prevents the development of autoimmune disease in mice.
  • DSS dextran sodium sulfate
  • IL-2/JES6-1 complex-treated mice exhibited significant reductions in disease severity, including attenuated weight loss, increased colon length, and lower disease activity index (Fig. 6b-d), consistent with previous findings.
  • JY3 IC further enhanced autoimmune disease prevention, with more pronounced improvements in weight loss, colon length, and disease activity score compared to the IL-2/JES6-1 complex (Fig. 6b-d).
  • the allosteric receptor-antibody exchange mechanism we describe is specific to the IL-2/JES6-1 system, the structure- based design principles we used to engineer an effective single-agent cytokine-antibody fusion can be extended to other ligand-antibody interactions for exclusive targeting of soluble factors to specific cell subsets of interest.
  • the IC format has clear advantages over mixed complex administration as it eliminates dosing ratio considerations and concerns about the free cytokine inducing off-target effects or undergoing rapid clearance from the bloodstream.
  • our engineered IC elicited greater !L-2Ra Hi9h cell expansion in an adoptive T cell transfer model (Fig. 4) and prevented DSS-induced colitis more effectively than the mixed complex (Fig. 5), even though the two formats induced similar TReg to effector cell expansion ratios (Figs. 4d, 9, 11a, and 12a).
  • the superior phenotypic behavior of the engineered IC could be that it is positioned more optimally on the biphasic TReg to effector cell activity curve based on its altered antibody-cytokine affinity (Fig. 8b).
  • the more extensive IL-2Ra upregulation induced by JY3 IC versus the mixed complex may present an advantage for the immunocytokine by fueling the transcriptional feedback loop that perpetuates IL-2 signaling.
  • the enhanced behavior of JY3 IC over the mixed complex provides an immediately useful reagent for expanding TReg cells to combat autoimmune disease, and the structure-guided engineering strategy used to develop this construct is applied to the design of other mechanism-driven therapeutic immunocytokines.

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Abstract

L'invention concerne des fusions d'anticorps-IL-2 à chaîne unique (immunocytokines) ayant une activité sélective dans l'expansion de sous-ensembles de lymphocytes T. Les procédés d'utilisation comprennent l'administration d'un agent de l'invention pour développer des sous-populations de cellules immunitaires particulières en tant qu'outil de recherche ou pour traiter une maladie ou des modèles de maladie par induction de l'activité IL -2 sélective.
PCT/US2019/019421 2018-02-28 2019-02-25 Fusions d'anticorps/il-2 à chaîne unique qui activent sélectivement des lymphocytes t régulateurs WO2019168791A2 (fr)

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WO2021161287A3 (fr) * 2020-02-16 2021-09-16 Aulos Bioscience, Inc Anticorps anti-il-2 modifiés
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
EP4108683A4 (fr) * 2020-02-21 2024-04-03 Jiangsu Hengrui Pharmaceuticals Co., Ltd. Anticorps anti-il-2 et fragment de liaison à l'antigène de celui-ci et utilisation médicale de ceux-ci

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JP2003527439A (ja) * 2000-03-17 2003-09-16 ミレニアム・ファーマシューティカルズ・インコーポレイテッド 抗ｃｄ１８抗体と抗ｃｃｒ２抗体の混合物を用いる狭窄および再狭窄を抑制する方法
MXPA04005266A (es) * 2001-12-04 2004-10-11 Merck Patent Gmbh Inmunocitocinas con selectividad modulada.
WO2009103157A1 (fr) * 2008-02-22 2009-08-27 University Health Network Mfap4 comme marqueur pour des cellules régulatrices et des cellules anticancéreuses
CN108473569B (zh) * 2016-01-11 2022-11-22 苏黎世大学 针对人白介素-2的免疫刺激性人源化单克隆抗体及其融合蛋白

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Publication number Priority date Publication date Assignee Title
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
WO2021161287A3 (fr) * 2020-02-16 2021-09-16 Aulos Bioscience, Inc Anticorps anti-il-2 modifiés
CN115397852A (zh) * 2020-02-16 2022-11-25 奥罗斯生物科学公司 工程化抗il-2抗体
JP2023506093A (ja) * 2020-02-16 2023-02-14 アウロス バイオサイエンス インコーポレイテッド 操作された抗ｉｌ－２抗体
JP7292526B2 (ja) 2020-02-16 2023-06-16 アウロス バイオサイエンス インコーポレイテッド 操作された抗ｉｌ－２抗体
CN115397852B (zh) * 2020-02-16 2023-08-11 奥罗斯生物科学公司 工程化抗il-2抗体
AU2021221287B2 (en) * 2020-02-16 2023-10-05 Aulos Bioscience, Inc Engineered anti-IL-2 antibodies
IL295210B1 (en) * 2020-02-16 2023-12-01 Aulos Bioscience Inc Transgenic antibodies against 2–IL
US11851485B2 (en) 2020-02-16 2023-12-26 Aulos Bioscience, Inc. Engineered anti-IL-2 antibodies
IL295210B2 (en) * 2020-02-16 2024-04-01 Aulos Bioscience Inc Transgenic antibodies against 2–IL
EP4108683A4 (fr) * 2020-02-21 2024-04-03 Jiangsu Hengrui Pharmaceuticals Co., Ltd. Anticorps anti-il-2 et fragment de liaison à l'antigène de celui-ci et utilisation médicale de ceux-ci

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