Cytokine-based therapies and methods, including monospecific and bispecific non-blocking anti-cytokine antibodies

Abstract

The present disclosure relates to a method for redirecting the active form of an endogenous cytokine to a desired target cell or tissue in a biological system or subject by administering to the biological system or a subject in need thereof a multispecific binding molecule that includes (a) a binding domain that specifically binds to the active form of the cytokine and (b) a binding domain that specifically binds to an epitope on a molecule that is a marker on the target cell or tissue, wherein the multispecific binding molecule, when bound to the cytokine, does not block, or only partially blocks, the ability of the cytokine to bind to and stimulate its cognate receptor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 488,145, filed March 2, 2023, which is incorporated by reference in its entirety. [Background technology]

[0002] Cytokines have a wide range of effects on immune cell behavior. However, several challenges, including their pleiotropic effects and systemic toxicity, significantly limit their therapeutic use (Li & Lim (2020) Science 370, 1034). Systemic toxicity limits the usefulness of various cytokines (e.g., IL-15, IL-2, and IL-4). Administering rhIL-15 as an intravenous bolus dose has proven extremely difficult due to clinical toxicity caused by intense cytokine secretion following administration (Waldmann et al. (2020) Front Immunol 11, 10.3389 / fimmu.2020.00868). Recombinant human IL-2 (rhIL-2) is currently rarely used to treat cancer patients due to its severe toxicity (Schwartz et al. (2002) Oncology 16, 11). A clinical trial of rhIL-4 in oral squamous cell carcinoma was stopped after limited toxicity was observed, manifesting as local pain at the injection site (Werkmeister et al. (2005) Oncology Reports 13, 449).

[0003] Recognizing the limitations of the therapeutic use of recombinant cytokines, others in the art have designed potential solutions, but unfortunately, new problems have arisen. Pegylation was introduced to address half-life issues, but this solution instead resulted in reduced activity, heterogeneous products, manufacturing challenges, PEG accumulation in the liver, and limited half-life extension (see clinical research products by Nektar and Ascendis Pharma). Fc fusion was attempted to address the half-life issue, but other challenges arose, including altered activity, dose-limiting toxicity, manufacturing challenges, increased immunogenicity, and limited half-life extension (see clinical research products by ImmunityBio and Xencor). Site-specifically modified cytokines, called muteins, were attempted to overcome pleiotropic effects, but as a result, increased immunogenicity hindered the clinical application of new biologies.

[0004] To spatially restrict cytokine delivery, attempts have been made to genetically fuse cytokines to antibodies, which function to localize the cytokine to the desired target. Xu et al. described the design and use of a PD1-targeting antibody genetically fused to a modified IL-15 mutein (Xu et al. (2021) Cancer Immunology Research 9, 1141). Martomo et al. described the development of an anti-PD-L1 antibody genetically linked to the sushi domain of the human IL-15 / IL-15 receptor α complex (Martomo et al. (2021) Molecular Cancer Therapeutics 20, 347). Anaveon AG's group described ANV600, a fusion protein containing a PD1-binding moiety and the cytokine IL-2 fused to an anti-IL-2 protein. Thus, all of these solutions involve genetically fusing cytokines to other antibodies, and such modified cytokines pose the development risks and challenges discussed above. See the review article by Santollani and Wittrup (Santollani et al. (2023) Immunological Reviews 320, 10), which lists several approaches adopted by the community to solve the challenges and problems related to the development of cytokine therapies. All of these solutions involve complex engineered cytokines, cytokine receptors, and / or their fusion molecules (WO 2022 / 036079). Thus, there is a need for new compositions and differentiated methods that can amplify the action of natural cytokines, particularly in tumors or tissues, and that overcome the administration challenges and systemic toxicity of currently available approaches. In particular, it would be desirable to develop methods to redirect and localize the effects of cytokines without the need to design and administer fusion proteins that contain cytokines or their receptors in modified forms. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2022 / 036079 [Non-patent literature]

[0006] [Non-Patent Document 1] Li & Lim(2020)Science 370,1034 [Non-patent document 2] Waldmann et al. (2020) Front Immunol 11,10.3389 / fimmu.2020.00868 [Non-patent document 3] Schwartz et al. (2002) Oncology 16,11 [Non-patent document 4] Werkmeister et al. (2005) Oncology Reports 13,449 [Non-patent document 5] Xu et al. (2021) Cancer Immunology Research 9,1141 [Non-patent document 6] Martomo et al. (2021) Molecular Cancer Therapeutics 20,347 [Non-Patent Document 7] Santollani et al.(2023) Immunological Reviews 320,10 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates to novel cytokine-based therapeutics and methods that amplify the action of natural cytokines specifically in desired tumors or tissues to overcome administration challenges and systemic toxicity.

[0008] In one aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a desired target cell or tissue in a biological system, the biological system comprising (i) an active form of the cytokine, (ii) a cell bearing on its surface a receptor for the active form of the cytokine, and (ii) one or more desired target cells or tissues to which the cytokine is redirected; the method comprises exposing the biological system to a multispecific binding molecule comprising (a) at least one first binding domain (cytokine-binding domain) that specifically binds to an epitope on the active form of the cytokine, and (b) at least one second binding domain (target-binding domain) that specifically binds to an epitope on a molecule that is not the corresponding receptor on the target cell or tissue, wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its corresponding receptor, thereby redirecting the active form of the cytokine to the target cell or tissue. The biological system can be an in vitro culture, an animal model, or a human subject.

[0009] In another aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a desired target cell or tissue in a subject, the method comprising administering to the subject a sufficient amount of a multispecific binding molecule comprising (a) a binding domain that specifically binds to the active form of the cytokine (cytokine binding domain) and (b) a binding domain that specifically binds to a molecule that is a marker on the target cell or tissue (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor, thereby redirecting the active form of the cytokine to the target cell or tissue. Administration of the multispecific binding molecule to a subject can extend the half-life of the cytokine in the subject. Administration of the multispecific binding molecule to a subject can increase the amount of the cytokine in the subject's serum.

[0010] In some embodiments, the multispecific binding molecule causes cytokines to accumulate in or around target cells or tissues.

[0011] In one aspect of the disclosure, the cytokine is endogenous to the system.

[0012] In another aspect of the disclosure, the cytokine is exogenous to the system obtained by introducing a recombinant form of the cytokine into the system.

[0013] In another aspect of the present disclosure, cytokines can be either endogenous or exogenous to the system, or a mixture of both.

[0014] In some embodiments, the active form of a cytokine, when redirected, exerts an agonistic effect on cells bearing its corresponding receptor.

[0015] In some embodiments, the multispecific binding molecule, when bound to a cytokine, reduces, but does not completely block, the ability of the cytokine to bind to and / or stimulate its cognate receptor.

[0016] In some embodiments, the multispecific binding molecule, when bound to a cytokine, attenuates the ability of the cytokine to bind to and / or stimulate its cognate receptor, hi some embodiments, the multispecific binding molecule, when bound to a cytokine, alters the clearance characteristics of the cytokine mediated by its cognate receptor.

[0017] In another aspect, the disclosure relates to a method for redirecting an active form of a cytokine to a target cell or tissue of interest, the method comprising: (a) selecting a cytokine of interest; (b) selecting a target molecule that is a marker on the target cell or in the target tissue of interest; (c) generating a panel of binding domains that bind to the cytokine; (d) generating a panel of binding domains that bind to the target molecule; and (e) detecting the cytokine binding domains using an assay that measures the ability of the cytokine, when complexed with the cytokine binding domain, to bind to and / or stimulate its cognate receptor compared to the ability of the unbound cytokine to bind to and / or stimulate its cognate receptor. (f) screening the target binding domains for binding to an appropriate epitope on the target molecule, (g) selecting cytokine binding domains that do not block or only partially block the ability of the cytokine to bind to and / or stimulate its cognate receptor, (h) generating a panel of multispecific binding molecules comprising one or more of the selected cytokine binding domains and one or more selected target binding domains, and (i) screening the multispecific binding molecules in an in vitro cell-based assay that measures their ability to bind to and stimulate the cytokine's cognate receptor in the presence of varying amounts of the multispecific binding molecule. The method may also include screening the multispecific binding molecules in an in vivo assay in a non-human subject that measures their ability to bind to and stimulate the cytokine's cognate receptor when administered to the subject. The method may further comprise performing an epitope binning assay in conjunction with a cytokine binding domain screening step to identify a region or regions on the cytokine that, when bound to a cytokine binding domain within the multispecific binding molecule, retain or partially retain the ability of the cytokine to bind to and stimulate its cognate receptor.

[0018] In some embodiments, the multispecific binding molecule comprises (a) one cytokine-binding domain and one target-binding domain, (b) one cytokine-binding domain and two identical or non-identical target-binding domains, (c) two identical or non-identical cytokine-binding domains and one target-binding domain, or (d) two identical or non-identical cytokine-binding domains and two identical or non-identical target-binding domains.

[0019] In some embodiments, the multispecific binding molecule comprises a cytokine binding domain that specifically binds to IL-15 or IL-15 complexed with IL-15 receptor alpha. The cytokine binding domain may bind to IL-15 with an affinity of less than 100 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM as measured by surface plasmon resonance (SPR).

[0020] In one embodiment of the multispecific molecule, the specific binding of the cytokine binding domain to the cytokine is non-covalent in nature.

[0021] In some embodiments, the multispecific binding molecule comprises a target binding domain that specifically binds to a protein expressed in the tumor microenvironment (TME).

[0022] In some embodiments, the multispecific binding molecule comprises a target binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a tumor cell, and the agonistic action of the cytokine is redirected to the location of the tumor cell.

[0023] In some embodiments, the multispecific binding molecule comprises a target binding domain that specifically binds to a receptor on an immune cell, optionally a T cell, macrophage, dendritic cell, or NK cell.

[0024] In some embodiments, the multispecific binding molecule comprises a cytokine-binding domain and a target-binding domain that bind to epitopes on the same cell (cis binding).

[0025] In some embodiments, the multispecific binding molecule comprises a cytokine-binding domain and a target-binding domain that bind directly or indirectly (trans-binding) to receptors on different cells.

[0026] In some embodiments, the multispecific binding molecule comprises a scaffold, optionally an albumin-based scaffold, a fibronectin-based scaffold, or an immunoglobulin-based scaffold. The immunoglobulin-based scaffold may be derived from IgG1, IgG2, IgG4, IgM, or IgA. The albumin-based scaffold or immunoglobulin-based scaffold may be capable of binding to neonatal Fc receptor (FcRn).

[0027] In some embodiments, a multispecific binding molecule comprises a scaffold, optionally an albumin-based scaffold, a fibronectin-based scaffold, or an immunoglobulin-based scaffold, to which a cytokine-binding domain and a target-binding domain are fused. The multispecific binding molecule may comprise a cytokine-binding domain, a target-binding domain, and a scaffold. The immunoglobulin-based scaffold may be derived from IgG1, IgG2, IgG4, IgM, or IgA. The albumin-based scaffold or immunoglobulin-based scaffold may be capable of binding to neonatal Fc receptor (FcRn).

[0028] In some embodiments, the multispecific binding molecule comprises a first cytokine binding domain, a second target binding domain, and a third Fc domain capable of interacting with an Fcγ receptor.

[0029] In some embodiments, the multispecific binding molecule is a bispecific antibody comprising a binding domain that specifically binds to an epitope on the active form of a cytokine and a binding domain that specifically binds to an epitope on a receptor molecule on a target cell or tissue.

[0030] In another aspect, the present disclosure relates to a method for redirecting the agonistic action of an active form of an endogenous cytokine using a multispecific molecule, wherein the specificity of the multispecific molecule is for the active form of the endogenous cytokine, the association of the multispecific molecule with the active form of the endogenous cytokine is non-blocking, allowing the endogenous cytokine to retain its agonistic action, at least one other specificity of the multispecific molecule is for a non-cytokine molecule, and the multispecific molecule captures the endogenous cytokine and redirects its agonistic action by binding to a cell surface receptor molecule. The endogenous cytokine can be in a soluble or cell surface form. The cell surface receptor molecule can also be a molecule within the extracellular matrix of the target cell. The active form of the endogenous cytokine can be a cytokine, a cytokine complex, or a cytokine isoform.

[0031] In another aspect, the present disclosure relates to a method for redirecting the agonistic action of an active form of a cytokine using a multispecific molecule, wherein the specificity of the multispecific molecule is for the active form of the cytokine, the association of the multispecific molecule with the active form of the cytokine is non-blocking, allowing the cytokine to retain its agonistic action, at least one other specificity of the multispecific molecule is for a non-cytokine molecule, and the multispecific molecule captures the cytokine and redirects its agonistic action by binding to a cell surface receptor molecule. The cytokine can be in a soluble or cell surface form. The cell surface receptor molecule can also be a molecule within the extracellular matrix of the target cell. The active form of the cytokine can be a cytokine, a cytokine complex, or a cytokine isoform.

[0032] In some embodiments, the cell surface receptor molecule targeted by the multispecific molecules of the invention may be a molecule within a lymph node, a tumor-draining lymph node, or the spleen.

[0033] In some embodiments, agonist action is redirected to a desired tissue or cell surface, which may be an immune cell, a tumor cell, a stromal cell, a cell in the tumor microenvironment, a cell in the bone marrow, a cell in a lymph node, an epithelial cell, an endothelial cell, a blood cell, a skin cell, a stem cell, a bone cell, a nerve cell, an adipocyte, or a muscle cell.

[0034] In some embodiments, the cell surface receptor molecule targeted by the multispecific molecules of the invention can be a molecule in tissues associated with an autoimmune disease state.

[0035] In some embodiments, agonist action is redirected to desired tissues to allow cis or trans presentation of endogenous cytokines within the targeted environment.

[0036] In some embodiments, agonist action is redirected to desired tissues to allow for cis or trans presentation of cytokines within the targeted environment.

[0037] In some embodiments, the agonistic action of endogenous cytokines is preferentially localized to desired tissues to allow for cis or trans presentation within the targeted environment.

[0038] In some embodiments, the agonistic action of the cytokine is preferentially localized to a desired tissue to allow for cis or trans presentation within the targeted environment.

[0039] In another aspect, the present disclosure relates to a method for developing a non-blocking multispecific binding molecule, the method comprising selecting an immune signaling molecule, selecting a target molecule, separately testing the multispecific binding molecule for binding to either the immune signaling molecule or the target molecule, testing the multispecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor, and testing the multispecific binding molecule for non-blocking binding to the immune signaling molecule that enables immune signaling agonist activity. Optionally, the method may further comprise modeling the complex between an endogenous cytokine receptor and the immune signaling molecule to define an epitope on the immune signaling molecule that maintains the endogenous cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking multispecific binding molecule that binds to the immune signaling molecule and the target molecule. The multispecific binding molecule may be a bispecific binding molecule.

[0040] In another aspect, the present disclosure relates to a method for developing a non-blocking multispecific binding molecule, the method comprising selecting an immune signaling molecule, selecting a target molecule, separately testing the multispecific binding molecule for binding to either the immune signaling molecule or the target molecule, testing the multispecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor, and testing the multispecific binding molecule for non-blocking binding to the immune signaling molecule that allows immune signaling agonist activity. Optionally, the method may further comprise modeling the complex between the endogenous cytokine receptor and the immune signaling molecule to define an epitope on the immune signaling molecule that maintains the endogenous cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, and using this information to design a cytokine binding domain that binds to the defined epitope, thereby developing a non-blocking multispecific binding molecule that binds to the immune signaling molecule and the target molecule. The multispecific binding molecule may be a bispecific binding molecule.

[0041] In another aspect, the present disclosure relates to a method for developing a non-blocking multispecific binding molecule, the method comprising selecting an immune signaling molecule, selecting a target molecule, separately testing the multispecific binding molecule for binding to either the immune signaling molecule or the target molecule, testing the multispecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor, and testing the multispecific binding molecule for non-blocking binding to the immune signaling molecule that allows immune signaling agonist activity. Optionally, the method may further comprise modeling the complex between the cytokine receptor and the immune signaling molecule to define an epitope on the immune signaling molecule that maintains cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, and using this information to design a cytokine binding domain that binds to the defined epitope, thereby developing a non-blocking multispecific binding molecule that binds to the immune signaling molecule and the target molecule. The multispecific binding molecule may be a bispecific binding molecule.

[0042] In one aspect of the disclosure, the multispecific binding molecule-bound cytokine exhibits agonist activity only upon engagement with its receptor target by the multispecific binding molecule. In another aspect of the disclosure, the multispecific binding molecule-bound cytokine exhibits stronger agonist activity upon engagement with its receptor target by the multispecific binding molecule compared to a monospecific cytokine binding domain that is unable to associate with its receptor target.

[0043] In another aspect, the present disclosure relates to a method for developing non-blocking multispecific binding molecules, the method comprising: selecting an immune signaling molecule; selecting a target molecule; separately testing monospecific binding molecules for binding to either the immune signaling molecule or the target molecule; testing the monospecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor; testing the monospecific binding molecule for non-blocking binding to the immune signaling molecule that enables immune signaling agonist activity; and designing a non-blocking multispecific binding molecule that includes a monospecific binding molecule of the immune signaling molecule and a monospecific binding molecule that binds to the target molecule. The immune signaling molecule can be an endogenous cytokine, chemokine, growth factor, or hormone. The method can optionally include modeling the pharmacological properties of the endogenous cytokine or endogenous cytokine complex. The method can optionally include modeling the pharmacological properties of the target molecule. The method can optionally include identifying a competitive binding profile of the monospecific binding molecule. Identifying competitive binding profiles can be done by epitope binning.

[0044] In another aspect, the present disclosure relates to a method for developing non-blocking multispecific binding molecules, the method comprising: selecting an immune signaling molecule; selecting a target molecule; separately testing monospecific binding molecules for binding to either the immune signaling molecule or the target molecule; testing the monospecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor; testing the monospecific binding molecule for non-blocking binding to the immune signaling molecule that enables immune signaling agonist activity; and designing a non-blocking multispecific binding molecule that comprises a monospecific binding molecule of the immune signaling molecule and a monospecific binding molecule that binds to the target molecule. The immune signaling molecule can be a cytokine (e.g., an endogenous cytokine), a chemokine, a growth factor, or a hormone. The method can optionally include modeling the pharmacological properties of the cytokine (e.g., an endogenous cytokine) or cytokine complex. The method can optionally include modeling the pharmacological properties of the target molecule. The method can optionally include identifying a competitive binding profile of the monospecific binding molecule. Identifying competitive binding profiles can be done by epitope binning.

[0045] In another aspect, the present disclosure relates to a method for developing non-blocking multispecific binding molecules, the method comprising: selecting an endogenous cytokine or endogenous cytokine complex and further modeling the pharmacological properties of the endogenous cytokine or endogenous cytokine complex; selecting a target molecule and further modeling the pharmacological properties of the target molecule; separately testing monospecific binding molecules for binding to either the endogenous cytokine, the endogenous cytokine complex, or the target molecule; and testing the monospecific binding molecules for non-blocking binding to the endogenous cytokine or the endogenous cytokine complex and further performing epitope binning. identifying a competitive binding profile of the monospecific binding molecule by modeling the complex between the cytokine receptor and the endogenous cytokine or endogenous cytokine complex to define an epitope on the endogenous cytokine or endogenous cytokine complex that maintains endogenous cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking bispecific binding molecule that binds to the endogenous cytokine and the target molecule; and designing a multispecific binding molecule that comprises a monospecific binding molecule for the endogenous cytokine and a monospecific binding molecule for the target molecule. Optionally, the method may further include validating the non-blocking multispecific binding molecule for binding to both the endogenous cytokine and the target molecule by in vitro cell-based receptor signaling screening for cytokine activity and target molecule specificity. The method may further include evaluating the efficacy of the non-blocking multispecific binding molecule in vivo. The method may also include evaluating the pharmacokinetic and pharmacodynamic properties of the non-blocking multispecific binding molecule in vivo. Selecting an endogenous cytokine of the method may include determining the endogenous expression level of the endogenous cytokine in the subject, determining the amount of the endogenous cytokine that is present in an active state in the circulation or in a tissue of interest in the subject, determining the distribution profile of endogenous cytokine receptors in the subject, and determining the clearance and metabolic mechanisms of the endogenous cytokine in the subject.Selecting a target molecule can include examining the expression level, tissue specificity, cell surface localization, molecular internalization kinetics, and / or molecular recycling kinetics of the target molecule in a subject. Modeling the pharmacological properties of an endogenous cytokine or endogenous cytokine complex can identify a desirable affinity range for the interaction between a non-blocking bispecific binding molecule and an endogenous cytokine or endogenous cytokine complex, and predict differences in the pharmacokinetics and biodistribution of free endogenous cytokines or endogenous cytokine complexes and / or predict differences in the pharmacokinetics and biodistribution of endogenous cytokines or endogenous cytokine complexes bound to a non-blocking bispecific binding molecule. Modeling the pharmacological properties of a target molecule can identify a desirable affinity range for the interaction between a non-blocking bispecific binding molecule and a target molecule, and / or predict differences in the biodistribution of a target molecule bound to and unbound by a non-blocking bispecific binding molecule. In vitro sandwich assays are available to bridge monospecific binding molecules to endogenous cytokine receptors via binding of endogenous cytokines or cytokine complexes. Modeling can determine the geometry of non-blocking bispecific binding molecule scaffolds that maintain endogenous cytokine receptor specificity and / or signaling properties while binding target molecules.

[0046] In another aspect, the present disclosure relates to a method for developing non-blocking multispecific binding molecules, the method comprising the steps of selecting a cytokine or cytokine complex and further modeling the pharmacological properties of the cytokine or cytokine complex; selecting a target molecule and further modeling the pharmacological properties of the target molecule; separately testing the monospecific binding molecules for binding to either the cytokine, cytokine complex, or target molecule; testing the monospecific binding molecules for non-blocking binding to the cytokine or cytokine complex and further identifying competitive binding profiles of the monospecific binding molecules by epitope binning; modeling the complex between the cytokine receptor and the cytokine or cytokine complex to define epitopes on the cytokine or cytokine complex that maintain cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking bispecific binding molecule that binds to the cytokine and the target molecule; and designing a multispecific binding molecule to include a monospecific binding molecule to the cytokine and a monospecific binding molecule to the target molecule. Optionally, the method may further include validating the non-blocking multispecific binding molecule for binding to both the cytokine and the target molecule by in vitro cell-based receptor signaling screening for cytokine activity and target molecule specificity. The method may further include evaluating the efficacy of the non-blocking multispecific binding molecule in vivo. The method may also include evaluating the pharmacokinetic and pharmacodynamic properties of the non-blocking multispecific binding molecule in vivo. Selecting a cytokine for the method may include determining the expression level of the cytokine in the subject, determining the amount of exogenous cytokine to be introduced into the subject, determining the amount of cytokine present in an active state in the circulation or in a tissue of interest in the subject, determining the distribution profile of cytokine receptors in the subject, and determining cytokine clearance and metabolic mechanisms in the subject.Selecting a target molecule can include examining the expression level, tissue specificity, cell surface localization, molecular internalization kinetics, and / or molecular recycling kinetics of the target molecule in a subject. Modeling the pharmacological properties of a cytokine or cytokine complex can identify a desirable affinity range for the interaction between a non-blocking bispecific binding molecule and the cytokine or cytokine complex, predict the amount of exogenous cytokine that can be introduced into a system or subject to increase the total amount of cytokine, predict differences in the pharmacokinetics and biodistribution of free cytokine or cytokine complex, and / or predict differences in the pharmacokinetics and biodistribution of a non-blocking bispecific binding molecule-bound cytokine or cytokine complex. Modeling the pharmacological properties of a target molecule can identify a desirable affinity range for the interaction between a non-blocking bispecific binding molecule and the target molecule, and / or predict differences in the biodistribution of a target molecule bound to a non-blocking bispecific binding molecule and an unbound target molecule. In vitro sandwich assays can be used to crosslink a monospecific binding molecule to a cytokine receptor via binding of the cytokine or cytokine complex. Structural modeling can determine the geometry of non-blocking bispecific binding molecule scaffolds that bind to target molecules while maintaining cytokine receptor specificity and / or signaling properties.

[0047] In some embodiments, the method further comprises performing a competition assay between the monospecific binding molecules for endogenous cytokines or endogenous cytokine complexes binding to endogenous cytokine receptors.

[0048] In some embodiments, the method further comprises performing a competition assay between the monospecific binding molecule, when bound to a cytokine receptor, for a cytokine or cytokine complex bound to the cytokine or cytokine complex and for a free cytokine or cytokine complex.

[0049] In some embodiments, the endogenous cytokine complex is an IL-15SA complex comprising IL-15 and IL-15 receptor α. The non-blocking bispecific binding molecule is capable of binding to an epitope on IL-15 receptor α. The non-blocking bispecific binding molecule is capable of binding to an epitope on IL-15. The endogenous cytokine can be IL-15 or IL-2. The non-blocking bispecific binding molecule is capable of binding to IL-15 with higher affinity than IL-15 receptor α. The non-blocking bispecific binding molecule has an affinity for IL-15 that is at least about 10-fold higher than the affinity of the non-specific binding molecule for IL-15. In another embodiment, the non-blocking bispecific binding molecule is capable of binding to IL-15 with lower affinity than IL-15 receptor α. In another embodiment, the non-blocking bispecific binding molecule is capable of binding to IL-15 with an affinity comparable to that of IL-15 receptor α.

[0050] In some embodiments, the target molecule is programmed cell death protein 1 (PD1), CD33, CD16, programmed death-ligand 1 (PD-L1), integrin, disialoganglioside (GD2), CD20, fibroblast activation protein (FAP), carcinoembryonic antigen receptor (CEAR), or carcinoembryonic antigen (CEA).

[0051] In some embodiments, the non-blocking multispecific binding molecule comprises a bispecific molecular scaffold selected from the group consisting of a homodimeric Fc, a heterodimeric Fc, an albumin-based bispecific scaffold, an affibody, an asymmetric antibody, a bispecific T cell engaging antibody (BiTE), a diabody, a dual affinity retargeting molecule (DART), an immunoglobulin domain crossover (CrossMAb), a minibody, a tandem diabody (TandAb), a fibronectin-based scaffold, or an FynomAb, an antibody fusion construct, or an albumin fusion construct. In some embodiments, the Fc may be derived from an antibody isoform found in nature, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM isoform, or may have several site-directed mutations.

[0052] In some embodiments, non-blocking multispecific binding molecules are engineered fusion proteins that include one or more monospecific binding molecule(s) for binding to an immune signaling molecule and another monospecific binding molecule(s) for binding to a target molecule. The monospecific binding molecule can be an antibody, a bivalent antibody fragment, an antigen-binding fragment (Fab) region, a minibody, a monovalent antibody, a single-chain variable fragment (scFv), a reduced immunoglobulin, a disulfide-stabilized variable fragment, a Fab fragment, a nanobody, an immunoglobulin domain antibody, a variable light chain (VL) construct, a variable heavy chain (VH) construct, a phenomer, or a darpin.

[0053] In another aspect, the present disclosure relates to a method for developing a non-blocking multispecific binding molecule, comprising the steps of selecting an endogenous cytokine of interest to be amplified, obtaining data on the system-level characteristics of the endogenous cytokine, obtaining data on the target receptor, modeling and simulating the endogenous cytokine in its native state when associated with an antibody, modeling and simulating receptor targeting of the endogenous cytokine, identifying binders to the endogenous cytokine and the target receptor, performing binding screening and / or competition assays, performing epitope binning of the antibody, defining desirable epitopes on the endogenous cytokine, and performing mixed cell-based receptor signaling screening, wherein the non-blocking multispecific binding molecule associates with the endogenous cytokine and retains the cytokine receptor binding and signaling properties of the endogenous cytokine.

[0054] In another aspect, the disclosure relates to a method for developing non-blocking multispecific binding molecules comprising the steps of selecting a cytokine of interest to be amplified, obtaining data on the system-level properties of the cytokine, obtaining data on the target receptor, modeling and simulating the cytokine in its native state when associated with an antibody, modeling and simulating receptor targeting of the cytokine, identifying binders to the cytokine and target receptor, performing binding screening and / or competition assays, performing epitope binning of the antibody, defining desirable epitopes on the cytokine, and performing mixed cell-based receptor signaling screening, wherein the non-blocking multispecific binding molecule associates with the cytokine and retains the cytokine receptor binding and signaling properties of the cytokine.

[0055] In another aspect, the present disclosure relates to multispecific binding molecules comprising (a) a binding domain that specifically binds to the active form of a cytokine and (b) a binding domain that specifically binds to an epitope on a molecule that is a marker on a target cell or tissue, wherein the multispecific binding molecule, when bound to the cytokine, does not block or only partially blocks the ability of the cytokine to bind to and stimulate its cognate receptor.

[0056] In another aspect, the present disclosure relates to a non-blocking antibody conjugate comprising an antibody and a cytokine, wherein the antibody is capable of presenting the cytokine to its cognate receptor. The antibody conjugate may be multispecific. The antibody may be capable of localizing the cytokine to a desired tissue or target cell surface receptor. The half-life of the conjugate may be the same as the half-life of the antibody. In another aspect, the half-life of the conjugate may be shorter than the half-life of the unconjugated antibody.

[0057] In another aspect, the present disclosure relates to a method of multispecific targeting comprising the steps of generating a non-blocking multispecific binding molecule using the methods described herein and administering the non-blocking multispecific binding molecule to a subject in need thereof, wherein the multispecific binding molecule targets endogenous cytokines in the serum of the subject and activates and / or expands tumor-specific effector cells, thereby inducing tumor cell killing.

[0058] In another aspect, the present disclosure relates to a method of multispecific targeting comprising the steps of generating a non-blocking multispecific binding molecule using the methods described herein and administering the non-blocking multispecific binding molecule to a subject in need thereof, wherein the multispecific binding molecule targets cytokines in the serum of the subject and activates and / or expands tumor-specific effector cells, thereby inducing tumor cell killing.

[0059] In another aspect, the present disclosure relates to a method for redirecting an active form of an endogenous cytokine to a desired target cell or tissue in a biological system, wherein the biological system comprises (i) an active form of the cytokine, (ii) a cell having on its surface a receptor corresponding to the active form of the cytokine, and (iii) one or more desired target cells or tissues to which the cytokine is redirected; the method comprises exposing the biological system to a multispecific binding molecule comprising (a) a binding domain that specifically binds to an epitope on the active form of the cytokine (cytokine binding domain), where the cytokine retains the ability to bind to and stimulate its receptor when complexed with the multispecific binding molecule, and (b) a binding domain that specifically binds to an epitope on a molecule that is not a cytokine receptor on the target cell or tissue (target binding domain), where the cytokine retains the ability to bind to and stimulate its receptor when complexed with the multispecific binding molecule, thereby redirecting the active form of the cytokine to the target cell or tissue.

[0060] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, the multispecific binding molecule comprising one binding domain specific for PD-1 and a second binding domain specific for IL-15, wherein (a) the binding domain specific for PD-1 comprises CDR H1 (SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72), CDR H2 (SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75), CDR H3 (SEQ ID NO:76, SEQ ID NO:77, or SEQ ID NO:78), CDR L1 (SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:81), CDR L2 (SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84), and L3 (SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87); and (b) the binding domain specific for IL-15 comprises CDR H1 (SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54), CDR H2 (SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57), CDR H3 (SEQ ID NO:58, SEQ ID NO:59, or SEQ ID NO:60), CDR L1 (SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63), CDR L2 (SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66), and L3 (SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69).

[0061] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain is specific for PD-1 and comprises a variable fragment light chain (VL) selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0062] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for IL-15 comprises a variable fragment light chain (VL) selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0063] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for PD-1 comprises a VL selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and the binding domain specific for IL-15 comprises a VL selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0064] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for PD-1 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO:19 and SEQ ID NO:20.

[0065] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for IL-15 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18.

[0066] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein (a) the binding domain specific for PD-1 comprises a VH selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20, and (b) the binding domain specific for IL-15 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18.

[0067] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for PD-1 comprises a constant fragment light chain (CL) selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.

[0068] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding domain specific for IL-15 comprises a CL selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4.

[0069] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the multispecific binding molecule comprises a constant fragment heavy chain 1 (CH1) selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, a constant fragment heavy chain 2 (CH2) selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and a constant fragment heavy chain 3 (CH3) selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

[0070] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding molecule is an antibody in a Fab-Fab format. In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the multispecific binding molecule further comprises at least one scFv fused to the C-terminus of the heavy chain.

[0071] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein one Fab is specific for PD-1 and an scFv is specific for IL-15.

[0072] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein one Fab is specific for IL-15 and an scFv is specific for PD-1.

[0073] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding molecule is an antibody in a dual variable domain (DVD) format.

[0074] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding molecule is an antibody in a dual variable domain (DVD) format, and in a further aspect, a VH domain with specificity for PD-1 is fused to the N-terminus of a VH domain with specificity for IL-15, and a VL domain with specificity for PD-1 is fused to the N-terminus of a VL domain with specificity for IL-15.

[0075] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding molecule is an antibody in a dual variable domain (DVD) format, wherein a VH domain with specificity for PD-1 is fused to the N-terminus of a VH domain with specificity for IL-15, and a VL domain with specificity for PD-1 is fused to the N-terminus of a VL domain with specificity for IL-15, and wherein the outermost Fab is specific for PD-1 and the inner Fab is specific for IL-15.

[0076] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein the binding molecule is an antibody in a dual variable domain (DVD) format, wherein a VH domain with specificity for IL-15 is fused to the N-terminus of a VH domain with specificity for PD-1, and a VL domain with specificity for IL-15 is fused to the N-terminus of a VL domain with specificity for PD-1, and wherein the outermost Fab is specific for IL-15 and the inner Fab is specific for PD-1.

[0077] In another aspect, the disclosure relates to a multispecific binding molecule that binds PD-1 and IL-15, wherein the binding molecule is an antibody in a dual variable domain (DVD) format, wherein the DVD comprises a linker connecting a VH domain comprising specificity for PD-1 to a VH domain comprising specificity for IL-15, and a linker connecting a VL domain comprising specificity for PD-1 to a VL domain comprising specificity for IL-15. In a further aspect, the linker is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. In yet another aspect, the linker is selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41.

[0078] In another aspect, the disclosure relates to a multispecific binding molecule that binds PD-1 and IL-15, wherein the binding molecule is a fusion protein. In another aspect, the disclosure relates to a multispecific binding molecule that binds PD-1 and IL-15, wherein the binding molecule comprises two or more PD-1-binding domains, two or more IL-15-binding domains, or two or more PD-1-binding domains and two or more IL-15-binding domains.

[0079] In another aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a desired target cell or tissue in a subject, the method comprising administering to the subject a sufficient amount of a multispecific binding molecule comprising (a) a binding domain that specifically binds to the active form of the cytokine (cytokine binding domain) and (b) a binding domain that specifically binds to a molecule that is a marker on the target cell or tissue (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor, thereby redirecting the active form of the cytokine to the target cell or tissue. In another aspect, the active form of the cytokine is endogenous to the system or subject. In another aspect, the active form of the cytokine is an exogenous cytokine, wherein the exogenous cytokine is added to a biological system or administered to a subject. In another aspect, the active form of the cytokine is a mixture of endogenous and exogenous cytokines.

[0080] In another aspect, the present disclosure relates to a method for redirecting the agonistic action of an active form of an endogenous cytokine using a multispecific molecule, wherein the specificity of the multispecific molecule is for the active form of the endogenous cytokine, the association of the multispecific molecule with the active form of the endogenous cytokine is non-blocking, allowing the endogenous cytokine to retain its agonistic action, at least one other specificity of the multispecific molecule is for a non-cytokine molecule, and the multispecific molecule captures the endogenous cytokine and redirects its agonistic action by binding to a cell surface receptor molecule. The endogenous cytokine can be in soluble or cell surface form. In another aspect, the non-blocking multispecific molecule binds to IL-15 with a lower affinity than IL-15Rα, the affinity being at least about 10-fold, about 100-fold, or less. In another aspect, the non-blocking antibody complex is shorter than the antibody but longer than the free cytokine. [Brief explanation of the drawings]

[0081] For a better understanding of the present disclosure, the same is now described by way of example with reference to the accompanying drawings which illustrate embodiments of the disclosure and, together with the following description, explain the principles of the disclosure. [Figure 1] This is a schematic diagram of the action of a cytokine-binding antibody (labeled Amplifier antibody in the diagram). The antibody captures the active form of a cytokine molecule in the serum of a biological system. Unlike unbound cytokines, which typically have a short half-life in serum, antibody-bound cytokines are protected from different modes of elimination, potentially resulting in a longer half-life in the biological system. The antibody-bound cytokine stimulates its corresponding receptor. The association of the antibody with the cytokine is non-blocking, i.e., the antibody-bound cytokine is in an active form and can be presented to its corresponding receptor on the surface of a cell, such as an immune cell, to induce signal transduction. [Figure 2]1 is a schematic diagram of a bispecific antibody (labeled Bispecific Amplifier antibody in the figure) comprising a cytokine-binding domain and a target-binding domain, and its action. A bispecific amplification antibody is an example of a multispecific binding molecule of the present disclosure. A bispecific amplification antibody has two specificities, with one arm containing specificity for an epitope on the activated form of the cytokine and another arm containing specificity for an epitope on a target cell surface receptor. Localization of the cytokine-binding bispecific antibody to the designated target cell surface receptor presents the cytokine for interaction with its cognate receptor in the local environment of the target cell. [Figure 3] 2A and 2B are schematic diagrams of two modes of bispecific assembly by bispecific amplification antibodies described in FIG. 2. In the cis assembly mode (A), the cytokine-responsive receptor and the target receptor are on the same cell surface. In the trans assembly mode (B), the cytokine-responsive receptor and the target receptor are on two different cells. [Figure 4] 1 is a schematic diagram of an IL-15 bispecific binding (amplifying) antibody and its mechanism of action. The bispecific amplifying antibody captures the active form of IL-15 in serum and redirects it to the tumor microenvironment (TME). The target receptor within the TME can be a cell surface receptor on immune cells, such as PD1, or a tumor cell surface receptor, such as HER2, or a molecule within the tumor stroma, such as collagen. This localized and redirected IL-15 engagement can selectively localize the activation and proliferation of tumor-specific effector cells for targeted tumor cell killing. [Figure 5]Figures A-C show model structures and predicted model structures of antibody Fab and IL-15 complexed with their corresponding receptor chains. Figure A shows the quaternary structure of IL-15 bound to its corresponding receptor chains, IL-15Rα, IL-15Rβ, and the common gamma chain (model based on crystal structure pdb id 4gs7). Figure B shows the model structure of antibody Fab (shown as a ribbon) bound to the cytokine IL-15 (shown as a spatial entity) based on crystal structure pdb id 2xqb. Figure C shows the predicted model structure of Fab (shown as a ribbon) bound to IL-15 (shown as a spatial entity) bound to its corresponding receptor chains, IL-15Rβ, and the common gamma chain. The models are based on pdb id structures 2xqb and 4gs7. [Figure 6] A-E show exemplary IgG1-based formats containing the PD1 antigen-binding domain from pembrolizumab and the IL-15 binding domain from DISC0280. The black heavy chain and gray light chain are specific for IL-15, while the hatched heavy chain and corresponding unshaded light chain are specific for PD1. A depicts the structure of a bispecific antibody in a Fab-Fab format with one Fab arm specific for PD1 and the other Fab arm specific for IL-15. B and C depict the structures of possible types of bispecific antibodies in a Fab-Fab format with one or two scFvs fused to the C-terminus of the heavy chain. In this embodiment, the Fab is specific for one antigen, while the scFv is specific for the other antigen. D depicts the structure of a bispecific antibody in dual variable domain (DVD) format, in which a VH domain with specificity for one antigen is fused to the N-terminus of a VH domain with specificity for another antigen, and a VL domain with specificity for one antigen is fused to the N-terminus of a VL domain with specificity for another antigen. E depicts the structure of a bispecific antibody in dual Fab domain format, in which the outermost Fab is specific for one antigen and the inner Fab is specific for the other antigen. [Figure 7] Panels A and B show digital gel images of reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) of bispecific antibodies in Fab-Fab format with one anti-PD1 scFv (RV17, RV18, RV19, RV20) or two anti-PD1 scFvs (RV23, RV24, RV25, RV26) fused to the C-terminus of the heavy chain. For RV18, RV20, RV24, and RV26, the anti-PD1 scFv was designed in a VL-linker-VH orientation. For RV17, RV19, RV23, and RV25, the anti-PD1 scFv was designed in a VH-linker-VL orientation. In addition, for RV19, RV20, RV25, and RV26, the anti-PD1 scFv was also designed to have an engineered disulfide. In A, RV18 and RV20 each showed two predominant bands, with the higher molecular weight band representing the intact H chain with scFv binding and the lower molecular weight band representing the L chain. Comparison with RV17 and RV19 also showed two predominant bands, with the lower molecular weight band corresponding to the L chain band of RV18 and RV20, whereas the H chains of RV17 and RV19 showed lower molecular weight bands than RV18 and RV20, consistent with the loss of scFv. In B, RV24 and RV26 each showed three predominant bands, with the highest molecular weight band representing the H chain with scFv binding (H + scFv), the middle band representing the H chain, and the lower molecular weight band representing the L chain. In RV23 and RV25, only bands corresponding to the H chain and L chain were observed. Overall, for variants containing anti-PD1 scFvs designed in a VL-linker-VH orientation (RV20 and RV26), the engineered disulfides appear to stabilize the scFvs, as evidenced by stronger H+ scFv bands than variants lacking the engineered disulfides (RV18, RV24). [Figure 8A] A representative UPLC-SEC profile of a bispecific antibody (RV15) in Fab-Fab format is shown. [Figure 8B]A representative UPLC-SEC profile of a bispecific antibody (RV26) in Fab-Fab format with one scFv fused to the HC is shown. [Figure 8C] A representative UPLC-SEC profile of a bispecific antibody (RV29) in DVD format is shown. [Figure 8D] A representative UPLC-SEC profile of a bispecific antibody (RV32) in dual Fab domain format is shown. [Figure 8E] A representative UPLC-SEC profile of a bispecific antibody (RV34) in Fab-Fab format with two scFvs fused to the HC is shown. [Figure 9A] 1 shows LC-MS analysis of the purity of an exemplary bispecific antibody. 2 shows the results of LC-MS analysis of a bispecific antibody (RV15) in Fab-Fab format. [Figure 9B] 1 shows LC-MS analysis of the purity of an exemplary bispecific antibody. 2 shows the results of LC-MS analysis of a bispecific antibody (RV26) in Fab-Fab format with one scFv fused to the HC. [Figure 9C] 1 shows LC-MS analysis of the purity of an exemplary bispecific antibody. 2 shows the results of LC-MS analysis of a bispecific antibody (RV29) in DVD format. [Figure 9D] 1 shows LC-MS analysis of the purity of an exemplary bispecific antibody. 2 shows the results of LC-MS analysis of a bispecific antibody (RV32) in dual-Fab domain format. [Figure 9E] 1 shows LC-MS analysis of the purity of an exemplary bispecific antibody. 2 shows the results of LC-MS analysis of a bispecific antibody (RV34) in Fab-Fab format with two scFvs fused to the HC. [Figure 10] Non-reducing CE-SDS digital gel images of selected variants before (pre-3xFT) and after (post-3xFT) three cycles of freeze-thaw testing are shown. All variants retained their structural integrity, except for RV36, which showed partial cleavage by CE-SDS, likely due to the deletion of the outer Fv domain of anti-IL-15. [Figure 11A]

[0023] Figure 1 shows the thermal unfolding of an exemplary bispecific antibody as measured by differential scanning calorimetry (DSC). Results are shown using a Fab-Fab format (RV15). [Figure 11B] 1 shows the thermal unfolding of an exemplary bispecific antibody as measured by differential scanning calorimetry (DSC). Results are shown using a bispecific antibody (RV32) in a dual Fab domain format. [Figure 11C] 1 shows the thermal unfolding of an exemplary bispecific antibody as measured by differential scanning calorimetry (DSC). Results are shown using a bispecific antibody (RV29) in DVD format. [Figure 11D]

[0023] Figure 1 shows thermal unfolding measured by differential scanning calorimetry (DSC) of an exemplary bispecific antibody. Results are shown using a bispecific antibody (RV26) in Fab-Fab format with one scFv fused to the HC. [Figure 12] Panels A and B show representative adjusted SPR sensorgrams of RV:PD1:IL-15:IL-15 βγ receptor complex formation using RV30. Panel A shows an SPR sensorgram using an anti-PD1 capture surface. Panel B shows an SPR sensorgram using an anti-human Fc capture surface. For both panels, the baseline was set to zero at the beginning of the RV capture step. The arrows indicate the time of injection of either the ligand or analyte. The lower curve shows the response to injection of the ligand alone; a final injection of PBST instead of IL-15 βγ receptor resulted in the formation of the RV30:PD1:IL-15 complex. The upper curve shows the response to injection of the ligand and analyte; the formation of the RV30:PD1:IL-15:IL-15 βγ receptor complex. Subsequent injection of higher concentrations of IL-15 βγ receptor resulted in the response corresponding to the upper curve, which indicated increased formation of the RV30:PD1:IL-15:IL-15 βγ receptor complex. [Figure 13]Figure 1 shows significant in vivo expansion of CD8+ T cells, NK cells, and NKT cells in the peripheral blood of C57BL / 6 mice 4 days after treatment with 100 mg of anti-IL-15 antibody (RV1) and 10 mg of human IL-15, versus 100 mg of isotype control antibody (anti-RSV) and 10 mg of human IL-15. [Figure 14] Figure 1 shows a significant increase in the ratio of CD8+ T cells:CD4+ T cells in the spleens of C57BL / 6 mice 7 days after treatment with 100 mg of anti-IL-15 antibody (RV1) and 10 mg of human IL-15, or 100 mg of isotype control antibody (anti-RSV) and 10 mg of human IL-15. [Figure 15] A and B show the levels of GM-CSF (A) and TNF-α (B) in the supernatants of human PBMCs after 4 days of ex vivo culture with RV1, RV2, or isotope control (anti-RSV) antibody in the presence of human IL-15 (50 pM or 1 nM) or in the absence of IL-15. [Figure 16A] IL-2 levels in the supernatant of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment are shown. [Figure 16B] Figure 1 shows the levels of IFN-γ in the supernatants of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment. * indicates that the interpolated sample concentration was above the upper limit of detection in the MSD assay. [Figure 16C] 1 shows the levels of granzyme A in the supernatant of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment. [Figure 16D]Figure 1 shows the levels of granzyme B in the supernatants of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 precomplexed with IL-15, RV1 with free IL-15, IL-15Rα precomplexed with IL-15, IL-15 alone, or no treatment. * indicates that the interpolated sample concentration was above the upper limit of detection in the MSD assay. [Figure 16E] 1 shows the levels of perforin in the supernatant of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment. [Figure 16F] 1 shows the levels of GM-CSF in the supernatant of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment. [Figure 16G] 1 shows the levels of TNF-α in the supernatant of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM RV1 pre-complexed with IL-15, RV1 with free IL-15, IL-15Rα pre-complexed with IL-15, IL-15 alone, or no treatment. [Figure 17] Panels A–C show the abundance of CD3+CD8+ cells (A), CD3+CD4+ cells (B), and CD56+ NK cells (C) in human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 18]Panels A–C show the abundance of Ki-67-positive CD3+CD8+ cells (A), CD3+CD4+ cells (B), and CD56+ NK cells (C) in human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 19] Panels A and B show the abundance of Ki-67-positive CD56++ NK cells (A) and CD56+CD16+ NK cells (B) in human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 20A] IL-2 levels in the supernatant of human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment are shown. [Figure 20B] Figure 1 shows the levels of IFN-γ in the supernatants of human PBMCs after 4 days of ex vivo culture with 100 nM, 10 nM, or 1 nM of IL-15 precomplexed with either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. * indicates that the interpolated sample concentration was above the upper limit of detection in the MSD assay. [Figure 20C] 1 shows the levels of granzyme A in the supernatant of human PBMCs after 4 days of in vitro culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 20D]Figure 1 shows the levels of granzyme B in the supernatants of human PBMCs after 4 days of in vitro culture with 100 nM, 10 nM, or 1 nM of IL-15 precomplexed with either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. * indicates that the interpolated sample concentration was above the upper limit of detection in the MSD assay. [Figure 20E] Shown are the levels of perforin in the supernatants of human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 20F] 1 shows the levels of GM-CSF in the supernatant of human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 20G] Shown are levels of TNF-α in the supernatant of human PBMCs after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 21] Panels A and B show the abundance of Ki-67-positive CD3+CD8+ cells (A) and CD3+CD4+ cells (B) among ex vivo activated human T cells after 4 days of ex vivo culture with IL-15 pre-complexed with 100 nM, 10 nM, or 1 nM of either RV1, RV2, RV29, RV32, or IL-15Rα-Fc, IL-15 alone, or no treatment. [Figure 22]A-D show the induction of STAT5 phosphorylation in CD4+ T cells (A), CD8+ T cells (B), CD56-strong NK cells (C), and CD56+CD16+ NK cells (D) by IL-15 alone, IL-15 complexed with IL-15Rα, Fc, RV1, RV3, or isotype control (anti-RSV) antibodies. [Figure 23] Figures A-C show stimulation outputs predicting changes in the concentration of IL-15 (ligand) bound to an IL-15 capture antibody under different treatment conditions. Figure A shows a simulation based on the assumption of an antibody with an affinity (Kd) of 0.1 nM for IL-15 and a half-life of the antibody-cytokine complex of 1 day. A scan of predicted changes in serum IL-15 concentrations with different doses of antibody treatment is shown. Figure B shows a simulation based on the assumption of an antibody with an affinity (Kd) of 0.1 nM for IL-15 and a half-life of the antibody-cytokine complex of 3 days. A scan of predicted changes in serum IL-15 concentrations with different doses of antibody treatment is shown. Figure C shows a simulation based on the assumption of an antibody with an affinity (Kd) of 1 nM for IL-15 and a half-life of the antibody-cytokine complex of 3 days. A scan of predicted changes in serum IL-15 concentrations with different doses of antibody treatment is shown. DETAILED DESCRIPTION OF THE INVENTION

[0082] The present disclosure relates to systems and methods for amplifying the effects of natural cytokines, particularly in tumors or tissues, and overcoming administration challenges and systemic toxicity. The technology disclosed herein overcomes the limitations of existing cytokine therapies.

[0083] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0084] An "antibody fragment," as defined herein, generally comprises a portion of an intact antibody comprising the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. In certain embodiments, an antibody fragment retains at least a portion of the hinge region and optionally the CH1 region of an IgG heavy chain. In some embodiments, an antibody fragment retains the entire constant region of an IgG heavy chain and includes an IgG light chain.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. In certain embodiments, monoclonal antibodies used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In some embodiments, the "monoclonal antibodies" are isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0086] Monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0087] The term "binding domain" refers to the region of a polypeptide that binds to another molecule.

[0088] The term "bispecific" is intended to include any agent having two antigen-binding moieties (e.g., antigen-binding polypeptide constructs) each with a unique binding specificity, e.g., a first antigen-binding moiety binds to an epitope on a first antigen and a second antigen-binding moiety binds to an epitope on a second antigen.

[0089] The term "cytokine" as used herein refers to a collective term for proteins released by one cell population and acting on another cell as intercellular mediators. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factors-α and -β; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-β; platelet growth factors; TGF-α and TGF-β; and the like. transforming growth factors (TGFs); insulin-like growth factors I and I; erythropoietin (EPO); osteogenic factors; interferons such as interferon-α, β, and γ; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, and the like; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0090] As used herein, an "endogenous cytokine" includes a cytokine, cytokine complex, or cytokine isoform produced inside an organism or cell. Endogenous cytokines can be in soluble or cell surface (membrane-anchored) form.

[0091] As used herein, an "exogenous cytokine" includes a cytokine, cytokine complex, or cytokine isoform, or a recombinantly produced modified variant of a wild-type cytokine. An exogenous cytokine can be introduced into a system or organism as part of a therapy.

[0092] As used herein, "IgG" refers to a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3. "Immunoglobulin (Ig)" herein refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have several structural forms, including, but not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains. "Immunoglobulin (Ig) domain" herein refers to a region of an immunoglobulin that exists as a distinct structural entity in the protein structure, as recognized by those skilled in the art. Typically, Ig domains have a characteristic rectangular sandwich-fold shape. The known Ig domains in the IgG class of antibodies are VH, Cγ1, Cγ2, Cγ3, VL, and CL.

[0093] The term "immune signaling molecule," as used herein, refers to an endogenous cytokine, chemokine, growth factor, or hormone.

[0094] "Specifically binds" or "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of a binding molecule to bind to a specific antigenic determinant can be measured via enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding construct to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen-binding construct that binds to an antigen, or an antigen-binding molecule comprising an antigen-binding portion thereof, has a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 M or less, e.g., 10 M to 10 M, e.g., 10 M to 10 M).

[0095] As used herein, the terms "target," "target antigen," or "target receptor" refer to a molecule that is specifically bound by the variable region of a given antibody. The target antigen or target receptor can be a protein, carbohydrate, lipid, or other chemical. The molecule can be, for example, programmed cell death protein 1 (PD1), CD33, CD16, programmed death ligand 1 (PD-L1), integrin, disialoganglioside (GD2), CD20, fibroblast activation protein (FAP), carcinoembryonic antigen receptor (CEAR), and carcinoembryonic antigen (CEA).

[0096] The term "target cell" as used herein means a cell that expresses a target antigen.

[0097] Multispecific binding molecules Multispecific binding molecules disclosed herein can comprise one cytokine-binding domain and one target-binding domain; one cytokine-binding domain and two identical or non-identical target-binding domains; two identical or non-identical cytokine-binding domains and one target-binding domain; or two identical or non-identical cytokine-binding domains and two identical or non-identical target-binding domains.

[0098] In one embodiment, a multispecific binding molecule may comprise a binding domain that specifically binds to the active form of a cytokine and a binding domain that specifically binds to an epitope on a molecule that is a marker on a target cell or tissue, where the multispecific binding molecule, when bound to the cytokine, does not block, or only partially blocks, the ability of the cytokine to bind to and stimulate its cognate receptor.

[0099] In some embodiments, the multispecific binding molecule is a bispecific antibody. A bispecific antibody may contain a binding domain that specifically binds to an epitope on an activated form of a cytokine and a binding domain that specifically binds to an epitope on a molecule that is not a cytokine receptor on a target cell or tissue. For example, a bispecific antibody may bind to the epitope IL-15, and the second specificity may be for an epitope on the checkpoint receptor PD1.

[0100] The cytokine binding domain may specifically bind to any cytokine, including, but not limited to, IL-2, IL-15, or IL-15 complexed with IL-15 receptor alpha. In certain embodiments, the cytokine binding domain binds to the IL-15-IL:15 receptor alpha complex with an affinity of less than 100 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM, as measured by surface plasmon resonance (SPR). In certain embodiments, the cytokine binding domain binds to the IL-2-IL:2R complex with an affinity of less than 100 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM, as measured by SPR.

[0101] A multispecific (e.g., bispecific) binding molecule can have an affinity for IL-15 that is at least about 10-fold greater than the affinity of a non-specific binding molecule for IL-15, which affinity can be, for example, at least about 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the affinity of a non-specific binding molecule for IL-15.

[0102] Multispecific binding molecules may comprise target binding domains that specifically bind to proteins expressed in the tumor microenvironment (TME), tumor-associated antigens (TAA) expressed on the surface of tumor cells (where the agonistic effects of cytokines are redirected), and / or receptors on immune cells (e.g., T cells, macrophages, NK cells). In some embodiments, the target is selected from the group consisting of programmed cell death protein 1 (PD1), CD33, CD16, programmed death-ligand 1 (PD-L1), integrins, disialoganglioside (GD2), CD20, fibroblast activation protein (FAP), carcinoembryonic antigen receptor (CEAR), and carcinoembryonic antigen (CEA).

[0103] Multispecific binding molecules may contain cytokine-binding domains and target-binding domains that bind to cytokine and target receptors on the same cell (cis-binding) or to receptors on different cells (trans-binding).

[0104] The multispecific binding molecule may include a scaffold (e.g., an albumin-based scaffold, a fibronectin-based scaffold, or an immunoglobulin-based scaffold). The scaffold may be, for example, an albumin-based bispecific scaffold, an affibody, an asymmetric antibody, a bispecific T cell-triggering antibody (BiTE), a diabody, an immunoglobulin domain crossover (CrossMAb), a minibody, a tandem diabody (TandAb), a fibronectin-based scaffold, a dual variable domain (DVD) antibody, or an FynomAb, an antibody fusion construct, or an albumin fusion construct. The scaffold may be, for example, a dual affinity retargeting molecule (DART). The scaffold may be, for example, an albumin-based scaffold, a fibronectin-based scaffold, or an immunoglobulin-based scaffold. The immunoglobulin-based scaffold may be derived from IgG1, IgG2, IgG4, IgM, or IgA. The albumin-based scaffold or immunoglobulin-based scaffold can be capable of binding to the neonatal Fc receptor (FcRn), which is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2-microglobulin.

[0105] A multispecific binding molecule can be a fusion protein comprising a monospecific binding molecule for binding to an immune signaling molecule and another monospecific binding molecule for binding to a target molecule. Examples of monospecific binding molecules include, but are not limited to, antibodies, bivalent antibody fragments, antigen-binding fragment regions, minibodies, monovalent antibodies, single-chain variable fragments (scFv), reduced immunoglobulins and disulfide-stabilized variable fragments, Fab fragments, nanobodies, immunoglobulin domain antibodies, phenomers, and DARPins. In another aspect of the present disclosure, the non-blocking cytokine binding domain is not the native receptor for the cytokine, such as IL-15Rα for IL-15 or IL-2Rα for IL-2.

[0106] In another aspect, the present disclosure relates to a non-blocking antibody conjugate comprising an antibody and a cytokine, wherein the antibody is capable of presenting the cytokine to its corresponding receptor. The antibody conjugate may be multispecific, for example, the antibody conjugate may be bispecific, trispecific, or specific for at least 4, 5, 6, 7, 8, 9, 10, or more targets. The antibody may be capable of localizing the cytokine to a desired tissue or target cell surface receptor. The half-life of the non-blocking antibody conjugate may be the same as the half-life of the antibody. The half-life of the non-blocking antibody conjugate may be longer or shorter than the half-life of the antibody alone.

[0107] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, the multispecific binding molecule comprising one binding domain specific for PD-1 and a second binding domain specific for IL-14, wherein the binding domain specific for PD-1 comprises CDR H1 (SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72), CDR H2 (SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75), CDR H3 (SEQ ID NO:76, SEQ ID NO:77, or SEQ ID NO:78), CDR L1 (SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:81), CDR L2 (SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84), and CDR L3 (SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87), and the binding domain specific for IL-15 comprises CDR H1 (SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54), CDR H2 (SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57), CDR H3 (SEQ ID NO:58, SEQ ID NO:59, or SEQ ID NO:60), CDR L1 (SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63), CDR L2 (SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66), and CDR L3 (SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69).

[0108] In another embodiment, the multispecific binding molecule that binds PD-1 and IL-15 comprises a binding domain specific for PD-1 comprising a variable fragment light chain (VL) selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0109] In another embodiment, the multispecific binding molecule that binds PD-1 and IL-15 comprises a binding domain specific for PD-1 comprising a heavy chain (VH) selected from the group consisting of SEQ ID NO:19 and SEQ ID NO:20.

[0110] In another embodiment, the multispecific binding molecule that binds to PD-1 and IL-15 comprises a binding domain specific for IL-15 comprising a variable light chain (VL) selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0111] In another embodiment, the multispecific binding molecule that binds PD-1 and IL-15 comprises a binding domain specific for IL-15 comprising a heavy chain (VH) selected from the group consisting of SEQ ID NO:17 and SEQ ID NO:18.

[0112] In another embodiment, a multispecific binding molecule that binds PD-1 and IL-15 comprises a binding domain specific for PD-1 comprising a VL selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and a Fab arm specific for IL-15 comprising a VL selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

[0113] In another embodiment, a multispecific binding molecule that binds PD-1 and IL-15 comprises a binding domain specific for PD-1 comprising a VH selected from the group consisting of SEQ ID NO:19 and SEQ ID NO:20, and a binding domain specific for IL-15 comprising a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO:17 and SEQ ID NO:18.

[0114] In some embodiments, the multispecific binding molecule that binds PD-1 and IL-15 is in a Fab-Fab format and can comprise a light chain variable region. In other embodiments, the multispecific binding molecule that binds PD-1 and IL-15 comprises a binding molecule that further comprises at least one scFv fused to the C-terminus of the heavy chain.

[0115] In another aspect, the disclosure relates to a multispecific binding molecule that binds to PD-1 and IL-15, wherein one binding domain is specific for PD-1 and comprises CDR H1 which may comprise the amino acid sequence of SEQ ID NO: 70, or a sequence at least about 90% identical thereto; CDR H1 which may comprise the amino acid sequence of SEQ ID NO: 71, or a sequence at least about 90% identical thereto; CDR H1 which may comprise the amino acid sequence of SEQ ID NO: 72, or a sequence at least about 90% identical thereto; CDR H2 which may comprise the amino acid sequence of SEQ ID NO: 73, or a sequence at least about 90% identical thereto; CDR H2 which may comprise the amino acid sequence of SEQ ID NO: 74, or a sequence at least about 90% identical thereto; and CDR H3 which may comprise the amino acid sequence of SEQ ID NO: 76, or a sequence at least about 90% identical thereto; CDR H3, which may comprise the amino acid sequence of SEQ ID NO: 78, or a sequence at least about 90% identical thereto; CDR L1, which may comprise the amino acid sequence of SEQ ID NO: 79, or a sequence at least about 90% identical thereto; CDR L1, which may comprise the amino acid sequence of SEQ ID NO: 80, or a sequence at least about 90% identical thereto; CDR L1, which may comprise the amino acid sequence of SEQ ID NO: 81, or a sequence at least about 90% identical thereto; CDR L2, which may comprise the amino acid sequence of SEQ ID NO: 82, or a sequence at least about 90% identical thereto; CDR L2, which may comprise the amino acid sequence of SEQ ID NO: 83, or a sequence at least about 90% identical thereto; L2, and a CDR-L3 which may comprise the amino acid sequence of SEQ ID NO: 85, or a sequence at least about 90% identical thereto, a CDR-L3 which may comprise the amino acid sequence of SEQ ID NO: 86, or a sequence at least about 90% identical thereto, or a CDR-L3 which may comprise the amino acid sequence of SEQ ID NO: 87, or a sequence at least about 90% identical thereto;and the other binding domain is specific for IL-15 and may comprise the amino acid sequence of SEQ ID NO: 52, or a sequence at least about 90% identical thereto; CDR H1 may comprise the amino acid sequence of SEQ ID NO: 53, or a sequence at least about 90% identical thereto; CDR H1 may comprise the amino acid sequence of SEQ ID NO: 54, or a sequence at least about 90% identical thereto; CDR H2 may comprise the amino acid sequence of SEQ ID NO: 55, or a sequence at least about 90% identical thereto; CDR H2 may comprise the amino acid sequence of SEQ ID NO: 56, or a sequence at least about 90% identical thereto; CDR H2 may comprise the amino acid sequence of SEQ ID NO: 57, or a sequence at least about 90% identical thereto; and CDR H3 may comprise the amino acid sequence of SEQ ID NO: 58, or a sequence at least about 90% identical thereto; CDR H3 may comprise the amino acid sequence of SEQ ID NO: 59, or a sequence at least about 90% identical thereto; or CDR H4 may comprise the amino acid sequence of SEQ ID NO: 60, or a sequence at least about 90% identical thereto. H3, CDR L1 which may comprise the amino acid sequence of SEQ ID NO: 61 or a sequence at least about 90% identical thereto, CDR L1 which may comprise the amino acid sequence of SEQ ID NO: 62 or a sequence at least about 90% identical thereto, or CDR L1 which may comprise the amino acid sequence of SEQ ID NO: 63 or a sequence at least about 90% identical thereto, CDR L2 which may comprise the amino acid sequence of SEQ ID NO: 64 or a sequence at least about 90% identical thereto, CDR L2 which may comprise the amino acid sequence of SEQ ID NO: 65 or a sequence at least about 90% identical thereto, or CDR L2 which may comprise the amino acid sequence of SEQ ID NO: 66 or a sequence at least about 90% identical thereto, and CDR L3 which may comprise the amino acid sequence of SEQ ID NO: 67 or a sequence at least about 90% identical thereto, CDR L3 which may comprise the amino acid sequence of SEQ ID NO: 68 or a sequence at least about 90% identical thereto, or CDR L3 which may comprise the amino acid sequence of SEQ ID NO: 69 or a sequence at least about 90% identical thereto;

[0116] In another embodiment, the multispecific binding molecule is in a dual variable domain (DVD) format.

[0117] In another aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a target, wherein the active form of the cytokine is endogenous to a system or subject.

[0118] In another aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a target, wherein the active form of the cytokine is an exogenous cytokine, and the exogenous cytokine is added to a biological system or administered to a subject.

[0119] In another aspect, the present disclosure relates to a method for redirecting an active form of a cytokine to a target, wherein the active form of the cytokine is a mixture of endogenous and exogenous cytokines in a biological system or subject.

[0120] In another aspect, the disclosure relates to a method for redirecting an active form of a cytokine to a target, wherein a non-blocking bispecific binding molecule binds to IL-15 with a lower affinity than IL-15Rα, the affinity being at least about 10-fold, about 100-fold, or less.

[0121] Methods for redirecting active forms of cytokines to target cells or tissues In one aspect, the present disclosure relates to a method for redirecting an active form of a cytokine (e.g., an endogenous cytokine, an exogenous cytokine) to a target cell or tissue of interest.

[0122] In one embodiment, a method redirects an active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) to a target cell or tissue of interest in a biological system (e.g., in vitro culture, animal model, human subject). The biological system may include (i) the active form of the cytokine, (ii) a cell bearing on its surface a receptor for the active form of the cytokine, and / or (iii) one or more target cells or tissues of interest to which the cytokine is redirected. The method includes exposing the biological system to a multispecific binding molecule that includes (a) a binding domain that specifically binds to an epitope on the active form of the cytokine (cytokine binding domain) and (b) a binding domain that specifically binds to an epitope on a molecule that is not a cytokine receptor on the target cell or tissue (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its receptor, thereby redirecting the active form of the cytokine to the target cell or tissue.

[0123] In another embodiment, the method redirects the agonistic action of an activated form of a cytokine using a multispecific molecule, wherein the specificity of the multispecific molecule is for the activated form of the cytokine, wherein association of the activated form of the cytokine by the multispecific molecule is non-blocking and allows the cytokine to retain its agonistic action, wherein at least one other specificity of the multispecific molecule is for a non-cytokine molecule, and wherein the multispecific molecule captures the cytokine and redirects its agonistic action by binding to the non-cytokine cell surface target receptor molecule.

[0124] Agonist action can be redirected to a desired tissue or cell surface, for example, to a desired stroma to enable cis or trans presentation of endogenous cytokines within the targeted environment.

[0125] Cytokines (eg, endogenous cytokines, exogenous cytokines) can be in soluble or cell surface form.

[0126] The non-cytokine target molecule may be a cell surface receptor molecule.

[0127] Target tissues (e.g., desired tissues) or cell surfaces include, for example, immune cells, tumor cells, stromal cells, cells within the tumor microenvironment, cells within the bone marrow, cells within lymph nodes, epithelial cells, endothelial cells, blood cells, skin cells, stem cells, bone cells, nerve cells, adipocytes, and muscle cells.

[0128] The target cell can be any cell that expresses a target antigen. Virtually any antigen can be targeted by the proteins and polypeptides described herein, including, but not limited to, proteins, subunits, domains, motifs, and epitopes belonging to the following proteins: CD2; CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon α, interferon β, interferon γ; TNF-α, TNFβ2, TNFc, TNFαβ, TNF -RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, A1 adenosine receptor, lymphotoxin β receptor, TALI, BAFF-R, EPO;LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β1, integrin β2, integrin α4 / β7, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin αv, αVβ3 integrin, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, eotaxin 1, BLyS (B-lymphocyte stimulatory substance), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), tissue factor, VEGF, VEGFR, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, T-cell receptor α / β, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucins MUC1, MUC18, heparanase 1, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA 125, prostate-specific membrane antigen (PSMA), high-molecular-weight melanoma-associated antigen (HMW-MAA), carcinoma-associated antigen, and geoprotein. IIb / IIIa (GPIIb / IIIa), tumor-associated antigens expressing Lewis Y-related carbohydrate chains, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, VNR integrin, IL-8, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein, and Clostridium perfringens toxin. Those skilled in the art will understand that the targets listed above refer not only to specific proteins and biomolecules but also to the biochemical pathway(s) that include them.

[0129] The "active form" of a cytokine (e.g., endogenous cytokine, exogenous cytokine) can be a complex. The active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) is a state in which, when reoriented, it can exert an agonistic effect on a cell bearing its corresponding receptor. In some embodiments, the active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) is a single-chain polypeptide. In other embodiments, the active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) can be more complex, including two or more chains, homodimers, heterodimers, or multimers. Some cytokine chains can associate with other protein chains to form complexes; for example, IL-15 can associate with IL-15Ra to form a complex called the IL-15 superagonist complex (IL-15SA). The active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) can be glycosylated, aglycoslated, or have other forms of post-translational modification. The active form of a cytokine (eg, endogenous cytokine, exogenous cytokine) can be an isoform of the cytokine or a variant of the cytokine.

[0130] In one embodiment, cytokine (e.g., endogenous cytokine, exogenous cytokine) refers to a broad family of soluble proteins available in biological systems that have natural functions, including interacting with corresponding receptors and participating in cell signaling functions. The family of soluble proteins can be signaling proteins and belong to the classes of cytokines, chemokines, growth factors, enzymes, endogenous regulatory peptides and proteins, or other biologically active proteins that exist in soluble form.

[0131] In another embodiment, the active form of the cytokine (e.g., endogenous cytokine, exogenous cytokine) is directly tethered to the cell surface. In one embodiment, the active form of the endogenous cytokine is indirectly tethered to the cell surface by binding to another protein that is tethered to the cell surface. In another embodiment, the active form of the exogenous cytokine is indirectly tethered to the cell surface by binding to another protein that is tethered to the cell surface.

[0132] In another embodiment, a method involves redirecting the active form of an endogenous cytokine to a desired target cell or tissue in a subject by administering to the subject a sufficient amount of a multispecific binding molecule comprising: (a) a binding domain that specifically binds to the active form of the cytokine (cytokine binding domain), and (b) a binding domain that specifically binds to a molecule that is a marker on the target cell or tissue (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor, thereby redirecting the active form of the cytokine to the target cell or tissue. Administration of the multispecific binding molecule to a subject can extend the half-life of the cytokine in the subject. For example, the half-life of the cytokine can be extended by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 100-fold, 1000-fold over the native half-life of the cytokine.

[0133] In another embodiment, a method involves redirecting the active form of an exogenous cytokine to a desired target cell or tissue in a subject by administering to the subject a sufficient amount of a multispecific binding molecule comprising: (a) a binding domain that specifically binds to the active form of the cytokine (cytokine-binding domain), and (b) a binding domain that specifically binds to a molecule that is a marker on the target cell or tissue (target-binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor, thereby redirecting the active form of the cytokine to the target cell or tissue. Administration of the multispecific binding molecule to a subject can extend the half-life of the cytokine in the subject. For example, the half-life of the cytokine can be extended by at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 100-fold, 1000-fold over the native half-life of the cytokine.

[0134] Administration of a multispecific binding molecule to a subject can increase the amount of cytokines (e.g., endogenous cytokines, exogenous cytokines) in the subject's serum. The amount of cytokine in the subject's serum can be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In other embodiments, the amount of cytokine in the subject can be increased 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, or 50-fold compared to the cytokine level before administration of the multispecific binding molecule.

[0135] The multispecific binding molecules can cause endogenous cytokines to accumulate in or around target cells or tissues.

[0136] The multispecific binding molecules can cause exogenous cytokines to accumulate in or around target cells or tissues.

[0137] The multispecific binding molecule, when bound to a cytokine, reduces (e.g., reduces the ability of the cytokine to bind to and / or stimulate its cognate receptor by about 0-90%, 0-75%, 0-60%, 0-50%, 0-40%, 0-30%, 0-20%), but does not completely block, the ability of the cytokine to bind to and / or stimulate its cognate receptor. The ability of the cytokine to bind to and / or stimulate its cognate receptor may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0138] A multispecific binding molecule according to paragraph 0015, which restores or enhances the ability of a cytokine to bind to its cognate receptor when the multispecific binding molecule also associates with a target molecule using the target binding domain.

[0139] In one embodiment, the cytokine-binding molecule of the multispecific binding molecule of the invention is an antibody that was developed against a cytokine with the intent of neutralizing the cytokine, but which for various reasons was ultimately unable to be developed as a neutralizing antibody. In a specific embodiment, the antibody increases the serum concentration of the cytokine upon treatment of a biological system. In another embodiment, the antibody induces agonistic effects of the cytokine upon treatment of a biological system.

[0140] In another aspect, a method relates to redirecting an active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) to a target cell or tissue of interest, the method comprising: (a) selecting a cytokine of interest; (b) selecting a target molecule that is a marker on the target cell or in the target tissue of interest; (c) generating a panel of binding domains that bind to the cytokine; (d) generating a panel of binding domains that bind to the target molecules; and (e) using an assay that measures the ability of the cytokine, when complexed with the cytokine binding domain, to bind to and / or stimulate its cognate receptor compared to the ability of the unbound cytokine to bind to and / or stimulate its cognate receptor. (f) screening the cytokine-binding domains; (g) selecting cytokine-binding domains that do not block or partially block the ability of the cytokine to bind to and / or stimulate its cognate receptor; (h) generating a panel of multispecific binding molecules comprising one or more of the selected cytokine-binding domains and one or more selected target-binding domains; and (i) screening the multispecific binding molecules in an in vitro cell-based assay that measures their ability to bind to and stimulate the cytokine's cognate receptor in the presence of varying amounts of the multispecific binding molecule. Optionally, the method may further include screening the multispecific binding molecules in an in vivo assay in a non-human subject that measures their ability to bind to and stimulate the cytokine's cognate receptor when administered to the subject. Additionally or alternatively, the method may include performing an epitope binning assay in conjunction with a cytokine binding domain screening step to identify a region or regions on the cytokine that, when bound to the cytokine binding domain, retain or partially retain the ability of the cytokine to bind to and stimulate its cognate receptor.

[0141] Any suitable assay can be used for screening, including, but not limited to, label-based approaches such as ELISA or label-free approaches such as surface plasmon resonance (SPR). Flow cytometry-based techniques can be used to measure the binding of multispecific molecules and cytokines to their cytokine-responsive receptors or cell surface targets. Cross-linking or sandwich binding screens can be used to evaluate the mutual association of cytokine-responsive receptors and cell surface targets. Western blot or alternative techniques can be used to evaluate signaling effects such as phosphorylation induced by cytokine-responsive receptors. Various RNASeq-based techniques or protein expression tracking approaches can also be used to observe the effects of cytokine-responsive receptor signaling. Histochemical approaches can also be used to observe the effects of cytokine-responsive receptor signaling.

[0142] Methods for multispecific targeting In one aspect, the present disclosure relates to methods of multispecific targeting. In certain aspects, the methods of multispecific targeting comprise the steps of generating a non-blocking multispecific binding molecule using the methods disclosed herein below and administering the non-blocking multispecific binding molecule to a subject in need thereof, wherein the multispecific binding molecule targets cytokines (e.g., endogenous cytokines, exogenous cytokines) in the serum of the subject and activates and / or proliferates tumor-specific effector cells, thereby inducing tumor cell killing.

[0143] The present disclosure encompasses the administration of non-blocking multispecific binding molecules (e.g., bispecific antibodies) to animals, particularly mammals, and particularly humans, to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection.

[0144] In one embodiment, the non-blocking multispecific binding molecules described herein are used for the treatment or prevention of diseases or disorders where altered efficacy of effector cell function (e.g., ADCC, CDC) is desired. Non-blocking multispecific binding molecules and compositions thereof are particularly useful for the treatment or prevention of primary or metastatic neoplastic disease (i.e., cancer) and infectious diseases. The molecules of the invention can be provided in pharmaceutically acceptable compositions known in the art or as described herein. As described in more detail below, the molecules of the invention can be used in methods for treating or preventing cancer, autoimmune diseases, inflammatory disorders, or infectious diseases.

[0145] The non-blocking multispecific binding molecules described herein may also be advantageously utilized in combination with other therapeutic agents known in the art for the treatment or prevention of cancer, autoimmune diseases, inflammatory disorders, or infectious diseases. The non-blocking multispecific binding molecules disclosed herein may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection, such as an anti-cancer agent, an anti-inflammatory agent, or an anti-viral agent.

[0146] Thus, the present disclosure provides methods for preventing, treating, or alleviating one or more symptoms associated with cancer and related conditions by administering one or more non-blocking multispecific binding molecules. Without intending to be bound by any mechanism of action, non-blocking multispecific binding molecules that bind with higher affinity than comparable molecules result in the selective targeting and efficient destruction of cancer cells.

[0147] The present disclosure further encompasses administering one or more non-blocking multispecific binding molecules in combination with other therapies known to those of skill in the art, including, but not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery, for the treatment or prevention of cancer. In some embodiments, the molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more anti-cancer agents, therapeutic antibodies, or other agents known to those of skill in the art for the treatment and / or prevention of cancer. Examples of dosing regimens and therapies that can be used in combination with the non-blocking multispecific binding molecules disclosed herein are well known in the art.

[0148] Cancers and related disorders treatable or preventable by the methods and compositions of the present invention include, but are not limited to, leukemia, lymphoma, multiple myeloma, bone and connective tissue sarcoma, brain cancer, breast cancer, adrenal gland cancer, thyroid cancer, pancreatic cancer, pituitary cancer, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, bile duct cancer, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, salivary gland cancer, pharynx cancer, skin cancer, kidney cancer, and bladder cancer.

[0149] In certain embodiments, the molecules of the invention (e.g., bispecific antibodies) inhibit or reduce primary tumor growth or metastasis of cancer cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% compared to primary tumor growth or metastasis in the absence of the molecules disclosed herein.

[0150] In certain embodiments, cytokines in the presence of the multispecific molecules of the invention induce proliferation of various immune effector cells, including NK cells and T cells, by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold compared to the absence of the multispecific molecules.

[0151] In one embodiment, the cytokines in the presence of the multispecific binding molecule induce the selective expansion of a subset of T cells or NK cells. In a particular embodiment, there is the selective expansion of CD8+ T cells.

[0152] The present disclosure encompasses the use of one or more non-blocking multispecific binding molecules disclosed herein to prevent, treat, or manage one or more symptoms associated with inflammatory disorders in a subject. The present disclosure further encompasses administering a non-blocking multispecific binding molecule in combination with a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents. The present disclosure also provides a method for preventing, treating, or managing one or more symptoms associated with an autoimmune disease, the method further comprising administering to the subject a non-blocking multispecific binding molecule in combination with a therapeutically or prophylactically effective amount of one or more immunomodulatory agents. Examples of autoimmune disorders that can be treated by administering the non-blocking polyspecific binding molecules of the invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic alveolar fibrosis, idiopathic thrombocytopenia, and the like. ITP, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, These include, but are not limited to, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-body syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpeticum vasculitis, vitiligo, and Wegener's granulomatosis.Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infection. Some autoimmune disorders are associated with inflammatory conditions, and therefore there is overlap between what is considered an autoimmune disorder and an inflammatory disorder. Thus, some autoimmune disorders may also be characterized as inflammatory disorders. Examples of inflammatory disorders that can be prevented, treated, or managed according to the methods of the present invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infection.

[0153] The non-blocking multispecific binding molecules of the invention can also be used to reduce inflammation experienced by animals, particularly mammals, suffering from an inflammatory injury. In certain embodiments, the non-blocking multispecific binding molecules disclosed herein reduce inflammation in a subject by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% compared to inflammation in a subject to which the molecule is not administered.

[0154] The present disclosure provides methods and pharmaceutical compositions comprising non-blocking multispecific binding molecules (e.g., bispecific antibodies). The present disclosure also provides methods for treating, preventing, and ameliorating one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of at least one non-blocking multispecific binding molecule or a pharmaceutical composition comprising at least one non-blocking multispecific binding molecule. In certain embodiments, the subject is an animal, such as a mammal, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys, such as cynomolgus monkeys, and humans). In certain embodiments, the subject is a human. In yet another specific embodiment, the non-blocking multispecific binding molecule is derived from the same species as the subject.

[0155] The present disclosure provides methods and pharmaceutical compositions comprising non-blocking multispecific binding molecules (e.g., bispecific antibodies). The present disclosure also provides approaches for developing complementary diagnostic methods to help recognize patients who may be most suitable for treatment with multispecific binding molecules. In certain embodiments, the complementary diagnostic approach involves screening the levels of endogenous cytokines or other relevant factors in the patient to be treated. In another specific embodiment, the diagnostic information can be used in modeling and planning administration strategies for the multispecific binding molecules as drugs.

[0156] The route of administration of the composition depends on the condition being treated. For example, intravenous injection may be preferred for the treatment of systemic disorders such as metastatic lymphoma or tumors. Alternatively, subcutaneous injection may be a preferred route of administration. The dosage of the composition to be administered can be determined by one of ordinary skill in the art without undue experimentation, in conjunction with standard dose-response studies. Relevant circumstances to be considered when making such a determination include the condition(s) being treated, the choice of composition to be administered, the age, weight, and response of the individual subject, and the severity of the subject's symptoms. Depending on the condition, the composition can be administered to the subject orally, parenterally, intranasally, intravesically, intravaginally, rectally, lingually, sublingually, bucally, intrabuccally, and / or transdermally.

[0157] The pharmaceutical compositions of the present invention can be administered parenterally, for example, by intravenous, intramuscular, intrathecal, and / or subcutaneous injection. Parenteral administration can be achieved by incorporating the compositions of the present invention into a solution or suspension. Such solutions or suspensions can include sterile diluents such as water for injection, saline, fixed oils, propylene glycol, glycerin, propylene glycol, and / or other synthetic solvents. Parenteral formulations can also include antibacterial agents such as benzyl alcohol and / or methylparabens, antioxidants such as ascorbic acid and / or sodium bisulfite, and chelating agents such as EDTA. Buffers such as acetates, citrates, and phosphates, and agents for adjusting isotonicity such as sodium chloride and dextrose may also be added. Parenteral preparations can be enclosed in ampoules, disposable syringes, and / or glass or plastic multi-dose vials. Rectal administration involves administering the composition to the rectum and / or large intestine. This can be achieved using suppositories and / or enemas. Suppository formulations can be prepared by methods well known in the art. Transdermal administration involves the transdermal absorption of a composition through the skin. Transdermal formulations include patches, ointments, creams, gels, ointments, etc. The compositions of the present invention can be administered intranasally to a patient. As used herein, intranasal administration or intranasal administration refers to administering a composition to the mucous membranes of a patient's nasal passages and / or nasal cavity.

[0158] The pharmaceutical compositions of the present disclosure may be used in accordance with the methods described herein to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection. The pharmaceutical compositions of the present invention are intended to be sterile and in a form suitable for administration to a subject.

[0159] The present invention also encompasses protocols for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection, in which a non-blocking multispecific binding molecule is used in combination with a therapeutic agent (e.g., a prophylactic or therapeutic agent) other than the non-blocking multispecific binding molecule. The present invention is based, in part, on the recognition that a non-blocking multispecific binding molecule can augment, synergize with, enhance the efficacy, improve the tolerability, and / or reduce the side effects of other cancer treatments, including current standard and experimental chemotherapies. The combination therapies of the present invention have additive efficacy, additive therapeutic effect, or synergistic effect. The combination therapies of the present invention allow for reduced dosages of the therapeutic agents (e.g., prophylactic or therapeutic agents) utilized in combination with a non-blocking multispecific binding molecule to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection, and / or reduce the frequency of administration of such prophylactic or therapeutic agents to a subject suffering from a disease, disorder, or infection, thereby improving the quality of life of the subject and / or achieving a prophylactic or therapeutic effect. Furthermore, the combination therapies of the present invention reduce or avoid undesirable or adverse side effects associated with the administration of current monotherapies and / or existing combination therapies, thereby improving patient compliance with treatment protocols. Numerous molecules that can be used in combination with the non-blocking multispecific binding molecules of the present disclosure are known in the art.

[0160] Methods for developing non-blocking polyspecific binding molecules In one aspect, the present disclosure relates to a method for developing a non-blocking multispecific binding molecule as disclosed herein. In one embodiment, the method for developing a non-blocking multispecific binding molecule comprises the steps of: (a) selecting an immune signaling molecule; (b) selecting a target molecule; (c) separately testing the multispecific binding molecule for binding to either the immune signaling molecule or the target molecule; (d) testing the multispecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor; and (e) testing the multispecific binding molecule for non-blocking binding to the immune signaling molecule that enables immune signaling agonist activity. Optionally, the method may further comprise the steps of: (f) modeling the complex between a cytokine (e.g., endogenous cytokine, exogenous cytokine) receptor and the immune signaling molecule to define an epitope on the immune signaling molecule that maintains cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule-binding cytokine (e.g., endogenous cytokine, exogenous cytokine), thereby developing a non-blocking multispecific binding molecule that binds to the immune signaling molecule and the target molecule. The multispecific binding molecule can be, for example, a bispecific antibody.

[0161] In another embodiment, a method for developing a non-blocking multispecific binding molecule comprises the steps of: (a) selecting an immune signaling molecule; (b) selecting a target molecule; (c) separately testing the monospecific binding molecules for binding to either the immune signaling molecule or the target molecule; (d) testing the monospecific binding molecule for binding to the immune signaling molecule and stimulation of the corresponding receptor; (e) testing the monospecific binding molecule for non-blocking binding to the immune signaling molecule, which allows immune signaling agonist activity; and (f) designing a non-blocking multispecific binding molecule that includes a monospecific binding molecule of the immune signaling molecule and a monospecific binding molecule that binds to the target molecule. The active form of a cytokine (e.g., endogenous cytokine, exogenous cytokine) can be, for example, a cytokine, cytokine complex, or cytokine isoform. The immune signaling molecule can be, for example, a cytokine (e.g., endogenous cytokine, exogenous cytokine), chemokine, growth factor, or hormone. The immune signaling molecule can be, for example, a signaling peptide.

[0162] The methods disclosed herein may also include modeling the pharmacological properties of a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex, modeling the pharmacological properties of a target molecule, or identifying competitive binding profiles of monospecific binding molecules. In certain embodiments, parameters such as the steady-state level of the active form of the cytokine, the clearance rate of the cytokine from different excretion pathways, the expression level of the target molecule within the system, the expression patterns of the cytokine and target receptor in different compartments of the biological system, and the affinity of the multispecific molecule for the cytokine and target can be used to simulate scenarios. This modeling and simulation can be used to predict the range of desirable characteristics of the multispecific molecule, such as affinity for the cytokine or the degree of blockade desired, and the cytokine exposure achievable in the biological system. Such simulations can be used to model various pharmacodynamic and pharmacokinetic characteristics of the multispecific molecule in the biological system of interest. In one embodiment, the simulation is based on solving partial differential equations. Epitope binning can be used to identify competitive binding profiles. In one embodiment, this information can be used in conjunction with structural models of the protein and its complexes, or other experimental site-directed mutagenesis approaches, to engineer multispecific molecules for optimal characteristics.

[0163] In another embodiment, a method for developing non-blocking multispecific binding molecules includes: (a) selecting a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex and modeling the pharmacological properties of the cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex; (b) selecting a target molecule and modeling the pharmacological properties of the target molecule; (c) separately testing monospecific binding molecules for binding to either the cytokine (e.g., endogenous cytokine, exogenous cytokine), cytokine (e.g., endogenous cytokine, exogenous cytokine) complex, or target molecule; and (d) testing the monospecific binding molecules for non-blocking binding to the cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex. (e) modeling the complex between the cytokine receptor and the cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex to define epitopes on the cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex that maintain cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking bispecific binding molecule that binds to the cytokine (e.g., endogenous cytokine, exogenous cytokine) and the target molecule; and (f) designing a multispecific binding molecule that includes a monospecific binding molecule for the cytokine (e.g., endogenous cytokine, exogenous cytokine) and a monospecific binding molecule for the target molecule.The method may also optionally include validating the non-blocking multispecific binding molecule for binding to both the cytokine (e.g., endogenous cytokine, exogenous cytokine) and the target molecule by in vitro cell-based receptor signaling screening for cytokine (e.g., endogenous cytokine, exogenous cytokine) activity and target molecule specificity. The method may also include assessing the efficacy of the non-blocking multispecific binding molecule and / or assessing the pharmacokinetic and pharmacodynamic properties of the non-blocking multispecific binding molecule in vivo.

[0164] A cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex can be selected by determining the expression level of the cytokine in a subject, determining the amount of cytokine (e.g., endogenous cytokine, exogenous cytokine) that is present in an active state in the circulation or in a tissue of interest in a subject, determining the distribution profile of cytokine receptors in a subject, determining the effect of administering additional cytokines (e.g., endogenous cytokine, exogenous cytokine) to a subject, and / or determining the clearance and metabolic mechanisms of the cytokine in a subject.

[0165] A target molecule can be selected by examining the expression level, tissue specificity, cell surface localization, molecular internalization kinetics, and / or molecular recycling kinetics of the target molecule in a subject.

[0166] The pharmacological properties of a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex can be modeled by identifying a desired affinity range for interaction with a non-blocking bispecific binding molecule cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex, predicting the differences in pharmacokinetics and biodistribution of the free cytokine or cytokine complex, and / or predicting the differences in pharmacokinetics and biodistribution of a non-blocking bispecific binding molecule-bound cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex.

[0167] By modeling the pharmacological properties of a target molecule, one can identify a desirable affinity range for the interaction between a non-blocked bispecific binding molecule and the target molecule and / or predict differences in the biodistribution of a target molecule bound and unbound to a non-blocked bispecific binding molecule.

[0168] Monospecific binding molecules can be tested for non-blocking binding to a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex, for example, by an in vitro sandwich assay that crosslinks the monospecific binding molecule to a cytokine receptor via binding of the cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex.

[0169] The methods disclosed herein may further include performing a competition assay between monospecific binding molecules for a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex bound to a cytokine receptor.

[0170] Modeling of the relationship between a cytokine receptor and a cytokine (e.g., endogenous cytokine, exogenous cytokine) or cytokine (e.g., endogenous cytokine, exogenous cytokine) complex, e.g., structural modeling, can determine the geometry of a non-blocking bispecific binding molecule scaffold that maintains cytokine receptor specificity and / or signaling properties while binding to a target molecule.

[0171] In another aspect, a method for developing a non-blocking multispecific binding molecule includes: (a) selecting a cytokine of interest (e.g., endogenous cytokine, exogenous cytokine) whose effect needs to be amplified in a system for a desired biological or therapeutic effect; (b) obtaining data on the system-level characteristics of the cytokine (e.g., endogenous cytokine, exogenous cytokine); (c) obtaining data on the target receptor; (d) modeling and simulating the cytokine (e.g., endogenous cytokine, exogenous cytokine) in its native state when associated with an antibody; (e) modeling and simulating receptor targeting of the cytokine (e.g., endogenous cytokine, exogenous cytokine); and (f) (i) identifying binders to a cytokine (e.g., endogenous cytokine, exogenous cytokine) and target receptor; (g) performing binding screening and / or competition assays; (h) performing epitope binning of antibodies; (i) defining desirable epitopes on the cytokine (e.g., endogenous cytokine, exogenous cytokine); and (j) performing mixed cell-based receptor signaling screening, wherein the non-blocking multispecific binding molecule associates with the cytokine (e.g., endogenous cytokine, exogenous cytokine) and retains the cytokine receptor binding and signaling properties of the cytokine (e.g., endogenous cytokine, exogenous cytokine).

[0172] Methods for generating and characterizing antibodies that function as cytokine capture binding domains within amplified antibodies A schematic diagram of the cytokine capture antibody mechanism utilized in the non-blocking bispecific binding antibody molecules of the present invention is shown in Figure 1. Methods utilized in generating and characterizing antibodies having binding domains that target the cytokine IL-15 in such a manner are described below.

[0173] Antibody discovery strategies are carried out using either display techniques such as phage display or immunization of live animals, such as mice or rabbits, with human IL-15 or a fragment thereof, followed by selection to generate a panel of antibodies capable of binding to human IL-15. Such antibody discovery techniques are well established in the art. Those skilled in the art can use one of such discovery strategies or any other discovery approach to discover a panel of antibodies or polypeptides capable of binding to the cytokine IL-15. Antibodies described in the literature that are capable of binding to IL-15, such as DISC0280 (Finch et al. Brit J Pharma (2011) 162, 480), B-E29, MOB-1254Z, PABZ-081, MOB-0784CT, HPAB-0238-YC, HPAB-0359-WJ, MOM-18387, etc., have been discovered using such approaches and have found utility in relation to the multispecific antibodies described in the present invention.

[0174] The binding affinity of an antibody to IL-15 can be estimated using techniques such as ELISA or label-free approaches such as surface plasmon resonance. The binding characteristics of the IL-15:antibody complex to its cognate receptor, such as IL-15Rβγ, can be determined to estimate the level of blocking or non-blocking nature of the antibody association. Antibodies that do not block or only partially block receptor association compared to the binding of free IL-15 can be applied in the amplification antibody described herein. The anti-IL-15 antibodies listed above can be evaluated in SPR assays for their ability to bind to IL-15 but not block or only partially block IL-15 receptor association (IL-15Rβγ) compared to the binding of free IL-15.

[0175] Receptor signaling-based assays are available to determine the effect of IL-15:antibody complexes on the ability of IL-15 to bind to its receptor and induce functionally relevant effects in target cells expressing the receptor on their cell surface. Assays can be based on assessing the phosphorylation of relevant intracellular proteins, such as STAT5. Alternatively, target cell proliferation, observed as a change in cell number or an intracellular marker of cell proliferation, such as Ki-67, can be assessed. Induction of cytokine release after treatment with IL-15:antibody complexes is another alternative functional indicator. IL-15 is mixed with an IL-15 antibody at different ratios (e.g., 1:1, 2:1, 1:10, 10:1, 1:100, 100:1, or other ratios), and the complex mixture is screened for binding to cells expressing one or more of the IL-15 receptor chains, i.e., IL-15R alpha (IL-15Rα), IL-15 / 2R beta (IL-15Rβ), and common gamma chain (γ). Examples of such cells include CTLL-2, KIT225, or M-07e. Alternatively, binding can be assessed using engineered cell lines such as Discoverx's U2OS kit number 93-0998c3. Alternatively, PBMCs, or T cells or NK cells isolated from human blood samples can be used to assess functional signaling effects. Antibodies that cannot bind to IL-15, such as the RSV-targeting antibody palivizumab, can be used as a control to quantify the effect of free IL-15 binding on cells expressing these receptors.

[0176] Using a panel of antibodies, antibody-bound IL-15 can induce signaling at its cognate receptor at various levels, ranging from complete non-blocking to blocking. Antibodies that induce receptor signaling equivalent to free IL-15 are referred to as non-blocking antibodies. For some antibodies, antibody-bound IL-15 exhibits reduced signaling upon receptor association, and these are referred to as partial blocking antibodies. A third class of blocking antibodies appears to completely block IL-15 interaction and signaling through its cognate receptor.

[0177] Furthermore, the blocking, partial blocking, or non-blocking properties of cytokine-capture antibodies can be assessed in vivo in living animals, such as mice or non-human primates. Upon treatment of an animal with human IL-15 cytokine and an antibody capable of binding and associating with the cytokine, the effect of the cytokine-antibody complex can be observed in vivo, focusing on the proliferation of specific immune cells in the animal. Changes in the levels of other cytokines, such as IFNγ (interferon-γ), can also be observed in the animal due to the in vivo action of the cytokine:capture antibody complex. In some embodiments, the IL-15:antibody complex may exhibit the ability to associate with the receptor complex (IL-15Rβγ) in certain assay formats, thus appearing as a non-blocking complex, while in other assays it may limit receptor complex-mediated activity and act as a blocking complex. In other embodiments, the IL-15:capture antibody complex may exhibit non-blocking activity with certain cell types, but appear as a blocking complex with other cells. In one embodiment, the blocking or partially blocking behavior of this complex can be induced in relation to the composition of the bispecific antibody to function as a non-blocking complex.

[0178] Bispecific antibodies comprising an IL-15-binding domain and a target-binding domain A schematic representation of the mechanism of action of bispecific antibodies (amplifying antibodies) targeting a cytokine and a second receptor target is shown in Figure 2, and a schematic representation of IL-15 redirection is shown in Figure 4. Methods utilized in generating and characterizing antibodies having a binding domain for the cytokine IL-15 and a second binding domain for a receptor target are described below.

[0179] The amplifying antibodies described herein are a new class of antibodies that can associate with a cytokine, either endogenous to the system or exogenous cytokines administered to the system, and also bind to a second target, a cell surface receptor. As a result of this dual association, the antibody increases the local concentration of the cytokine at the site presenting the second target, thereby spatially modulating the effect of the cytokine. Such antibodies associate with the cytokine and amplify its effect compared to the action of the cytokine alone. Amplifying antibodies can be designed as bispecific antibodies containing a cytokine capture arm and a second target binding arm, where the second target is typically a cell surface receptor or part of the extracellular matrix. Bispecific antibodies can contain one or more valencies for cytokine capture and similarly can have one or more valencies for target binding. A single valency allows capture of a single copy of a cytokine molecule, while designs using more than one valency can allow capture of up to an equivalent number of cytokine copies or result in stronger avidity of the cytokine molecule.

[0180] Various technologies, including hybridoma, surface display, and B cell cloning approaches, have enabled the discovery of novel antibodies against desired target antigens in a turnkey approach (Banik, Kushnir, Doranz, and Chambers (2023) Mabs 15(1), 2273018). Various such techniques are applicable to anti-cytokine antibody discovery to obtain antibodies that associate with different epitopes on cytokines. Some anti-cytokine antibodies bind to cytokines and completely block the association of the cytokine with its cognate receptor, exhibiting true antagonistic activity. Other anti-cytokine antibodies can bind to cytokines at epitopes while still allowing the antibody-bound cytokine to interact with its cognate receptor in the same way as the cytokine alone, i.e., agonist antibodies. Other antibodies can associate with cytokines in a way that modulates the cytokine's interaction with its native receptor, i.e., partial / modified agonist antibodies. These second two classes of antibodies associate with cytokines and retain full or partial / modified agonistic activity of the cytokine. Cytokine capture antibodies used in the Amplifier design include full agonist antibodies or partial / modified agonist antibodies.

[0181] Antibodies in either the agonist or partial agonist category are investigated in relation to bispecific antibody molecules. Bispecific molecules are engineered protein designs that contain one or more binding domains (target-binding domains) capable of specifically recognizing a second target other than IL-15 and fused to the above-mentioned IL-15-targeting antibody. Several bispecific molecule formats are known in the literature (Brinkmann U & Kontermann RE (2017) MAbs 9, 182-212). A particular symmetric bispecific design of interest includes an scFv that specifically targets a second receptor target and is fused to the C-terminus of the heavy chain of an anti-IL-15 antibody. An alternative bispecific design involves heterodimerizing heavy chain mutations, such as knob-into-hole mutations, to achieve asymmetric antibodies with one arm that associates with IL-15 and a second arm that associates with a second target (Ridgway JBB, Presta LG, Carter P (1996) Prot Engg Des Sel 9, 617-621; von Kreudenstein TS et al (2013) MAbs 5, 646-654).

[0182] In some embodiments, the second arm of the bispecific molecule comprises one or more binding domains capable of targeting a receptor of interest, such as PD1, CTLA4, PD-L1, CD25, GITR, CD11b, CSF-1R, CD40, CD44, SIRPa, TIM3, TIGIT, KIR, NKG2D, NKG2A, LAG3, CD8, Vg9Vd2, etc., typically expressed on various immune cell subtypes. Some of these second receptors allow for cis association, i.e., mutual association of the second receptor and IL-15 receptor on the same cell (see Figure 3A). Others allow for trans association, i.e., the second receptor is associated on a different cell than the cell to which the IL-15 receptor is associated.

[0183] In some embodiments, the second arm of the bispecific molecule may comprise one or more binding domains capable of targeting a receptor of interest typically expressed on various tumor cells, tumor-associated stromal cells, or the extracellular matrix within the tumor microenvironment (see Figure 3B). Some receptors of interest are cell surface receptors such as PD-L1, CD47, VEGFR, PDGFR, HER2, EGFR, EGFRVIII, IGF1R, PSCA, PSMA, CEA, claudin 18.2, mesothelin, MUC1, ROR1, AXL, GPC3, CD133, CD147, folate receptor, MUC16, CA-IX, CD44, CD49d, and ICAM1. Other targets associated with hematological malignancies include CD20, CD19, CD22, CD52, CD38, SLAMF7, CD37, CD98, DKK-1, CD157, CCR4, CXCR4, BAFF-R, CD123, CECAM5, Dyadherin, and tenascin-C.

[0184] Many alternatives, modifications, and variations are possible with this disclosure. While particular embodiments have been shown and described in detail to illustrate the application of the principles of the present disclosure, it will be understood that the invention may be embodied in other ways without departing from such principles. Accordingly, applicant intends to embrace all such alternatives, modifications, equivalents, and variations that are within the spirit and scope of the present invention.

[0185] All publications and patent documents cited in this disclosure are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification shall supersede any such material. The citation of any reference herein is not an admission that such reference is prior art to the present disclosure. Various terms relating to aspects of this specification are used throughout the specification and claims. Such terms have their ordinary meaning in the art unless otherwise specified. Other terms that are specifically defined should be construed as consistent with the definition set forth herein.

[0186] The articles "a" and "an" as used herein refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0187] The term "about" when referring to a measurable value, such as an amount, time period, or the like, is meant to encompass a variation of no more than ±20%, or in some cases no more than ±15%, or in some cases no more than ±10%, or in some cases no more than ±5%, or in some cases no more than ±1%, or in some cases no more than ±0.1% from the particular value, where such a variation is appropriate.

[0188] The phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, e.g., elements conjunctively expressed in some cases and disjunctively expressed in other cases. Multiple elements listed with "and / or" should similarly be construed, e.g., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the specifically identified elements or not. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with an open-ended term such as "comprising," may refer in one embodiment to A only (optionally including elements other than B), in another embodiment to B only (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc. [Example]

[0189] Below are examples of certain specific embodiments for making and using the cytokine-binding polypeptide constructs described herein. The examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0190] The constructs and methods described herein, unless otherwise indicated, may be prepared and carried out utilizing conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current supplement); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992).

[0191] Example 1: Methods for generating and characterizing bispecific antibodies Bispecific antibodies can be produced in a variety of shapes, also called formats (see reference Brinkmann and Kontermann (2017), MABS 9(2), 182-212, which describes several such formats). Designing and investigating the activity of bispecific molecules in several different formats can potentially result in molecules with slightly different functional and biophysical characteristics. Here, we describe the preparation of a bispecific antibody containing binding domains derived from the anti-PD1 antibody pembrolizumab and the anti-IL-15 antibody DISC0280. The antibodies and controls were prepared in different formats, and representations of exemplary bispecific formats are shown in Figures 6A-6E. All of the formats shown in Figures 6A-6E are based on the use of antibody scaffolds. Furthermore, the heavy chains in black and the light chains in gray are representative of those specific for IL-15, while the heavy chains in diagonal lines and the corresponding light chains in white are representative of those specific for PD1. Figure 6A depicts the structure of a bispecific antibody in a Fab-Fab format, in which one Fab arm is specific for PD1 and the other Fab arm is specific for IL-15. Figures 6B and 6C depict the structures of possible versions of a bispecific antibody in a Fab-Fab format, with one or two scFvs fused to the C-terminus of the heavy chain. In these versions, the Fab is specific for one antigen, while the scFv is specific for the other antigen. Figure 6D depicts the structure of a bispecific antibody in a dual variable domain (DVD) format, in which a VH domain with specificity for one antigen is fused to the N-terminus of a VH domain with specificity for the other antigen, and a VL domain with specificity for one antigen is fused to the N-terminus of a VL domain with specificity for the other antigen. Figure 6E depicts the structure of a bispecific antibody in a dual-Fab domain format, in which the outermost Fab is specific for one antigen and the inner Fab is specific for the other antigen.

[0192] The sequences of the following variants are provided in Appendix A following the Examples. CDR regions were identified using IMGT, Kabat, and Chothia methods. Regions may differ slightly depending on the method used for identification. The pembrolizumab Fab sequence was generated from the IMGT 2D structure database (IMGT / 2Dstructure-DB card for INN 9798). The DISC0280 Fab sequence was generated from PDB ID 2XQB, except that the missing N-terminal residues of the light chain (L) were replaced with the missing N-terminal residues of IMGT:IGLV1-47. * The VH and L domains were added from 01 V-LAMBDA (Z73663). Alternatively, a bispecific antibody containing the anti-mouse PD1 sequence F12.3 obtained from patent US2019 / 0263877A1, specifically sequences 12 and 30, respectively, may be used. The constant region is an IgG1 isotype obtained from Uniprot P0DOX5, which also incorporates the LALAPG Fc null mutation. A glycine-serine linker was used to fuse the scFv to the heavy chain (H) of the bispecific antibody in Fab-Fab format (Figures 6B and 6C), the outer variable domain to the inner variable domain of the DVD bispecific antibody (Figure 6D), and to link the VH and VL domains within the scFv. For the bispecific antibody in dual-Fab domain format (Figure 6E), an inter-Fab hinge obtained / derived from SEQ ID NO: 3 in patent publication WO 2018 / 178101 A1 was used. For bispecific antibodies in Fab-Fab format (Figure 5A), sequence substitutions within the CH3 domain from patent publication US20130195849A1 were used for Fc heterodimerization (Table A2). For Fab-preferred H:L pairs (Figures 6A, 6E), sequence substitutions within the CH1, hinge, and CL domains from US20190338048A1 were used.

[0193] Exemplary bispecific antibodies with different molecular formats were cloned, expressed, and purified as follows. Genes encoding the antibody heavy and light chains were constructed synthetically using codons optimized for human / mammalian expression. The final gene products were subcloned into the mammalian expression vector PTT5 (NRC-BRI, Canada) and expressed in CHO cells. CHO cells were maintained in aerated Erlenmeyer flasks at 120 rpm, 37°C, 5% CO2, and standard humidified conditions in a proprietary medium supplemented with 4 mM glutamine (HyClone, catalog no. CSH0034.01) and 0.1% Pluronic® F-68 (Gibco, Life Technologies, catalog no. 24040-032). For protein production, CHO cells were seeded 2 days before transfection. On the day of transfection, cells were grown at 5-6 x 10 6The cells were diluted to a density of 1000 cells / ml. Cells were transfected with PEI MAX 40 kDa (PEIMAX®, Polyscience, catalog no. 24765) at 1.4 μg of DNA per mL of cells at a DNA:PEI ratio of 1:7.1 (w / w). The transfected DNA was a mixture of a plasmid encoding the recombinant protein of interest (in a pTT vector), GFP DNA (in a pTT vector), and pSV40-Bcl-XL DNA at a w / w ratio of 5.7:1. 0.075% dimethylacetamide (Alta Aesar, catalog no. A10924) and 1x antibiotic / antimycotic (HyClone, catalog no. SV30079.01) were added to the transfected cells and returned to the incubator at 120 rpm, 37°C, and 5% CO. Twenty-four hours after transfection, cultures were supplemented with anti-aggregant (Irvine Scientific, Catalog No. 91150), transferred to a humidified incubator (120 rpm, 5% CO) at 32°C, and harvested after 6 days. For protein production, cells were fed Feed 4 (Irvine Scientific, Catalog No. 94134) supplemented with Kolliphor P188 (Sigma-Aldrich, Catalog No. K4894) and sodium bicarbonate (Sigma-Aldrich, Catalog No. S3817), and glucose (Sigma-Aldrich, Catalog No. G7021) was added as needed.

[0194] Fusions of two scFvs to the H chain of a diabody in Fab-Fab format (Figure 6B) and a DVD diabody (Figure 6D) were transfected at an H:L ratio of 1:2. For diabody in Fab-Fab format (Figure 6A), fusions of one scFv to the H chain of a Fab-Fab format (Figure 6C), and dual Fab domain format (Figure 6E), DNA was transfected at an optimal DNA ratio to allow heterodimer formation (e.g., fusions of one scFv to the H chain of a Fab-Fab format at an H1 / H2 / L1 ratio of 22:8:70 (RV26)). Transfected cells were harvested after 7 days by centrifugation at 3800 rpm and collecting the culture medium, followed by clarification using a 0.2 μm filter. The clarified culture medium was loaded onto a MabSelect SuRe (Cytiva) Protein A column, and the antibody was eluted with 100 mM citrate pH 3.0 or a combination of 100 mM citrate and L-arginine (100 mM citrate pH 3.6 + 200 mM L-arginine, followed by 100 mM citrate pH 3.0 + 200 mM L-arginine). The pooled antibody-containing fractions were neutralized with 10% (v / v) 1 M HEPES. For variants with post-pA amounts greater than 1 mg, the Protein A antibody eluate was then buffer exchanged or further purified by gel filtration (SEC) using DPBS (Cytiva (Hyclone) DPBS / Modified-Calcium-Magnesium) or DPBS + 200 mM L-arginine. For gel filtration, the protein was concentrated using a Vivaspin turbo device and a 30 kDa membrane, then loaded onto a Sephadex 200 HiLoad 16 / 600 200 pg column (Cytiva) via an AKTA system at a flow rate of 1 mL / min. DPBS, pH 7.1, or DPBS + 200 mM L-arginine buffer was used at a flow rate of 1 mL / min, and fractions corresponding to the purified bispecific antibody were collected. Protein was quantified based on absorbance measured at 280 nm.Endotoxin levels were then assessed using FDA-approved LAL test cartridges and an Endosafe® PTS™ or MCS™ reader (Charles River Laboratories).

[0195] The structures and a brief description of the bispecific variants produced (RV15, RV17, RV18, RV19, RV20, RV21, RV22, RV23, RV24, RV25, RV26, RV29, RV30, RV31, RV32, RV33, RV34, RV35, RV36, and RV37) and the monospecific parent antibodies produced (RV1, RV2, RV3, RV9, and RV10) are presented in Table 1. The clones used to generate each bispecific variant are listed in Table 2. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 2-1] [Table 2-2]

[0196] Overall, biophysical characterization of all variants yielded intact proteins, except for RV17, RV19, RV21, RV22, RV23, and RV25, which exhibited significant instability issues. The RV17, RV19, RV23, and RV25 bispecific antibodies in Fab-Fab format, designed with an anti-PD1 scFv (VH-linker-VL orientation) fused to the C-terminus of the heavy chain, exhibited complete loss of scFv as observed by CE-SDS (Figures 7A and 7B). In comparison, the RV18, RV20, RV24, and RV26 bispecific antibodies in Fab-Fab format, designed with an anti-PD1 scFv (VL-linker-VH orientation), exhibited intact constructs as observed by CE-SDS (Figures 7A and 7B). Similarly, the VH-linker-VL orientation of the scFv appears to be unstable, as intact constructs were observed for the format variants (RV18, RV20, RV24, and RV26) in which the anti-PD1 scFv was in the VL-linker-VH orientation. Furthermore, for variants containing the anti-PD1 scFv designed in the VL-linker-VH orientation (RV20 and RV26), the engineered disulfide appears to stabilize the scFv, as evidenced by a stronger H+ scFv band than for variants without the engineered disulfide (RV18, RV24) (Figure 7). For bispecific antibodies in the Fab-Fab format designed with one anti-IL-15 scFv fused to the C-terminus of each heavy chain (RV21 and RV22, each variant with a different scFv orientation), most of the protein precipitated and was not further characterized. As is understood in the art, not all conversions of Fab VH-VL to ScFv result in stable proteins, and unstable variants may be stabilized by modifying the scFv and / or optimizing the transfection and purification processes.

[0197] Example 2: Analysis of bispecific antibody purity by UPLC-SEC and LC-MS The purity and percent aggregation of exemplary bispecific antibodies were determined by UPLC-SEC. UPLC-SEC analysis was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) attached to a Waters Acquity UPLC H-Class Bio system set at 30°C and equipped with a photodiode array (PDA) detector. The run time was 7 minutes, and the running buffer was 0.2 M KPO4, 0.2 M KCl, pH 7 + 0.02% Tween® 20 at 0.4 ml / min. Elution was monitored by UV absorbance in the range of 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower 3 software with Apex Track™ and shoulder detection functions. Figures 8A-8E show the UPLC-SEC profiles of representative exemplary bispecific antibodies. The results are shown in Table 3.

[0198] The purity and composition of exemplary bispecific antibodies were assessed using mass spectrometry after native deglycosylation. Because the bispecific antibodies contained Fc N-linked glycans, purified samples were deglycosylated with PNGaseF (Millipore Sigma) as follows: 0.1 U PNGaseF per μg of antibody in 50 mM Tris-HCl, pH 7.0, overnight incubation at 37° C., final protein concentration 0.48 mg / mL. Additionally, because RV31 and RV32 contained O-glycosylation (potentially present in the inter-Fab hinge-like linker), the purified samples were further deglycosylated with 1 U OglyZOR O-glycosidase (OglyZOR, Genovis) per μg of antibody and 0.5 U SialEXO sialidase (SialEXO, Genovis) per μg of antibody in 50 mM Tris-HCl, pH 7.0, at a final protein concentration of 0.45 mg / mL overnight at 37°C. The deglycosylated protein samples were analyzed by intact LC-MS using an Ultimate3000 HPLC system coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific) via an Ion Max electrospray ion source (ThermoFisher Scientific). Samples (5 μg) were injected onto a 2.1 × 30 mm Poros R2 reverse-phase column (ThermoFisher Scientific) and separated using the following gradient conditions: 0–3 min: 20% solvent B; 3–6 min: 20–90% solvent B; 6–7 min: 90–20% solvent B; 7–9 min: 20% solvent B. Solvent A was degassed 0.1% aqueous formic acid, and solvent B was degassed acetonitrile. The flow rate was 3 mL / min. After the column, the flow was split, with 100 μL / min directed to the electrospray interface. The column was heated to 82.5 °C, and the solvent was heated to 80 °C before the column to improve protein peak shape. The LTQ-Orbitrap XL was calibrated using ThermoFisher Scientific's LTQ Positive Ion ESI calibration solution (caffeine, MRFA, and Ultramark 1621).The cone voltage (source fragmentation setting) was 40 V, the FT resolution was 7,500, and the scan range was m / z 400–4,000. The LTQ-Orbitrap XL was tuned for optimal detection of larger proteins (>50 kDa) using α-lactalbumin (0.5 mg / mL, Millipore Sigma). LC-MS system performance was assessed prior to sample analysis using in-house standards: a deglycosylated IgG standard (Waters IgG standard), and a mixture of deglycosylated IgG and approximately 80 kDa proteins. For each LC-MS run, mass spectra acquired across the entire antibody peak were summed, and the entire multiply charged ion envelope was deconvoluted into a molecular weight profile using the MaxEnt 1 module in MassLynx data analysis software (Waters) (parameters: peak half-width = 1.0, number of iterations = 10, minimum intensity ratio: left = 60%, right = 60%). The apparent relative amounts of each antibody species in each sample were determined from the peak heights of the resulting molecular weight profiles.

[0199] The results from LC-MS analysis of the bispecific antibody and the component IL-15 cytokine-capturing monospecific antibody and PD1 receptor-targeting monospecific antibody are shown in Table 4, and LC-MS mass spectra of representative variants are shown in Figures 9A-9E. Overall, the data show a partial loss of approximately 195 Da in the H:H and H:L correct pairings with only traces (<2%) of mispairs, which likely corresponds to the deletion of the N-terminal residue QS from the anti-IL-15 binding domain light chain. To address the instability of the anti-IL-15 binding domain light chain, a combination of stability engineering and process optimization can be performed. [Table 3] [Table 4-1] [Table 4-2]

[0200] Example 3: Thermal stability of bispecific antibodies The stability of selected bispecific heterodimeric antibodies and wild-type controls was assessed by freeze-thaw (F / T) cycling and / or differential scanning calorimetry (DSC). For freeze-thaw testing, a small-scale F / T study was performed using three cycles of freezing to -80°C for 30 minutes and thawing to room temperature (RT to 22–25°C) for 30 minutes. Samples were then evaluated for integrity and aggregation before and after the F / T cycle by CE-SDS and UPLC-SEC, respectively. For DSC, after preparative SEC processing, 400 μL of sample at a concentration of 0.4 mg / mL in PBS was used for DSC analysis using a MicroCal VP-Capillary DSC (Malvern Instruments). Five blank injections of buffer were performed at the beginning of each DSC run to stabilize the baseline, and buffer was injected as a reference before each sample injection. Each sample was scanned from 20°C to 100°C at a rate of 60°C / hr with low feedback, an 8 second filter, a 5 minute preTstat, and a nitrogen pressure of 70 psi. The resulting thermograms were referenced and analyzed using Origin 7 software.

[0201] After the FT cycle test, all variants except RV36 retained their structural integrity as measured by the % main peak in UPLC-SEC. For RV36, CE-SDS further revealed partial cleavage, likely due to the deletion of the outer Fv domain of anti-IL-15 (Figure 10). This instability is likely due to the inter-Fv linker. RV29, which is similar to RV36, exhibited a more stable structure due to its shorter inter-Fv linker. Regarding stability assessment via DSC (Table 5), the onset Tm revealed that the most stable bispecific constructs were asymmetric antibodies (RV15, RV33), followed by the dual-Fab domain format antibodies (RV31 and RV32), the DVD antibodies (RV29 and RV36), and then the scFv-containing antibody (RV26). The stability of the VH / VL domains also showed a similar pattern, with the variable domain in the Fab format exhibiting the highest melting temperature (TM), followed by the outer Fv domain in the DVD format, and then the scFv format. Thermograms of exemplary bispecific antibodies (RV15, RV32, RV29, and RV26) are shown in Figures 11A-11D. [Table 5]

[0202] Example 4: Antigen affinity measurements of bispecific antibodies and controls The ability of the bispecific antibodies to present bound cytokines for functionally relevant activity was assessed to determine whether the antibodies could bind antigen with high affinity and form complexes with the IL-15Rβγ receptor chain heterodimer. Antigen binding affinity was measured by surface plasmon resonance (SPR) as follows.

[0203] SPR biosensor assay EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS: N-hydroxysuccinimide; SPR: surface plasmon resonance; EDTA: ethylenediaminetetraacetic acid

[0204] SPR consumables Series S Sensor Chip CM5, Biacore amine coupling kit (NHS, EDC, and 1 M ethanolamine), and 10 mM sodium acetate buffer were purchased from Cytiva Life Sciences (Marlborough, MA). High-purity grade IL-15, IL-15Rβγ protein containing human IL-2 / IL-15Rβ and common gamma (γ) receptor chain heterodimer, and human PD-1 were purchased from Acrobiosystems (Newark, DE). PBS running buffer (PBST) containing 1% Tween 20 was purchased from Teknova Inc. (Hollister, CA). Goat polyclonal anti-human Fc antibody was purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA). EDTA was purchased from Bioshop (Burlington, ON). All SPR assays were performed using a Biacore T200 Surface Plasmon Resonance instrument (Cytiva Life Sciences, Marlborough, MA) with PBST running buffer (to which 0.5 M EDTA stock solution was added to a final concentration of 3.4 mM) at 25°C. Anti-human Fc capture surfaces were generated using a Series S Sensor Chip CM5 with the default parameters set in the Immobilization Wizard of the Biacore T200 control software, targeting 3000 resonance units (RU).

[0205] SPR experiments were performed in two ways: 1) using the variants and / or variants complexed with IL-15 as the ligands, with IL-15 or IL-15Rβγ receptors, respectively, as the analytes, and 2) using PD1 antigen, PD1:variant complexes, and / or PD1:variant:IL-15 as the ligands, with the variants, IL-15, and IL-15Rβγ, respectively, as the analytes. Additionally, SPR experiments were performed using IL-15-binding variants for PD1, and IL-15 and PD1-binding variants for IL-15Rβγ.

[0206] For IL-15 binding, one method involved indirect capture of antibody variants onto an anti-human Fc antibody SPR surface, followed by injection of four concentrations of IL-15 for kinetic analysis using single-cycle kinetics. Variants were captured onto individual anti-hFc surfaces at 2.5 μg / mL for 30 seconds at a flow rate of 10 μL / min. Overall, this resulted in capture of approximately 131–650 RU of variants on the anti-human Fc surface. The first flow cell was left empty to serve as a blank control. Immediately following this capture step, four concentrations of IL-15 (0.104 nM, 0.52 nM, 2.6 nM, and 13 nM) were injected sequentially into all four flow cells at 50 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 seconds at 30 μL / min. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction under analysis. D ) was derived.

[0207] The other method used to assess IL-15 binding involved indirect capture of antibody variants onto a PD1 flow cell surface, followed by injection of four concentrations of IL-15 for kinetic analysis using single-cycle kinetics. PD1 surfaces were prepared by injecting PD1 at 5 μg / mL, pH 4.5, and immobilizing via NHS / EDC using the Immobilization Wizard in the Biacore control software set to 500 RU. Variants were injected at 5 μg / mL into individual flow cells for 30 seconds at a flow rate of 10 μL / min. Generally, this resulted in capture of approximately 500-700 RU on the PD1 surface. Immediately following this capture step, four concentrations of IL-15 (0.104 nM, 0.52 nM, 2.6 nM, and 13 nM) were injected sequentially over all four flow cells at 10 μL / min for 180 seconds, with a dissociation phase of 900 seconds. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, at 10 μL / min for 30 seconds. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction being analyzed. D ) was derived.

[0208] For IL-15Rβγ receptor binding, one method involved indirect capture of antibody variants onto an anti-human Fc antibody flow cell surface. The variants were injected into individual flow cells at 2.5 μg / mL for 30 seconds at a flow rate of 10 μL / min. The first flow cell was left empty to serve as a blank control. Next, 20 nM IL-15 was injected at 10 μL / min for 180 seconds to allow antibody:IL-15 complex formation. Overall, this resulted in approximately 360-690 captures. Immediately following this capture step, four concentrations of IL-15Rβγ receptor (0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected sequentially into all four flow cells at 20 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 seconds at 10 or 30 μL / min. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction under analysis. D ) was derived.

[0209] The other method used to assess IL-15Rβγ receptor binding involved indirect capture of antibody variants onto a PD1 flow cell surface, followed by injection of 20 nM IL-15 and then four concentrations of IL-15Rbg receptor for kinetic analysis using single-cycle kinetics. PD1 surfaces were prepared by injecting PD1 at 5 μg / mL, pH 4.5, and immobilizing via NHS / EDC using a Wizard set to 500 RU. Bispecific variants were injected at 5.0 μg / mL for 30 seconds at a flow rate of 10 μL / min onto individual flow cells. IL-15 was then injected at 20 nM for 180 seconds at a flow rate of 10 μL / min to allow for variant-IL-15 complex formation. Immediately following this capture step, four concentrations of IL-15Rβγ receptor (0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected sequentially over all four flow cells at 20 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, at 10 μL / min for 30 seconds. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction being analyzed. D ) was derived.

[0210] For PD1 binding, one method used involved indirect capture of antibody variants onto the flow cell surface via the Fc region, followed by injection of four concentrations of PD1 for kinetic analysis using single-cycle kinetics. For the protein A (pA) flow cell surface, variants were injected into individual flow cells at 2 μg / mL for 40 seconds at a flow rate of 5 μL / min. Overall, this resulted in capture of approximately 150–935 RU on the pA surface. The first flow cell was left empty to serve as a blank control. Immediately following this capture step, four concentrations of PD1 (0.3125 nM, 1.25 nM, 5 nM, and 20 nM) were injected sequentially into all four flow cells at 50 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The pA surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 seconds at 10 μL / min. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction under analysis. D ) was derived. For the anti-human Fc antibody flow cell surface, variants were injected at 2.5 μg / mL into individual flow cells at a flow rate of 10 μL / min for 30 seconds. Generally, this resulted in capture of approximately 211-261 RU of variant on the anti-human Fc surface. The first flow cell was left empty to serve as a blank control. Immediately after this capture step, four concentrations of PD1 (0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected sequentially into all four flow cells at 20 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, at 30 μL / min for 30 seconds. A buffer injection was performed with each analyte injection used for reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to derive the rate constants and affinity (KD) of the interaction under analysis.

[0211] In the other method used to assess PD1 binding, four concentrations of antibody variants (0.31 nM, 1.25 nM, 5 nM, and 20 nM; or 0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected over the PD1-ligand flow cell surface using single-cycle kinetics. The PD1 surface was prepared by injecting PD1 at 5 μg / ml, pH 4.5, and immobilizing via NHS / EDC using Wizard at 500 RU. The four concentrations of variants were injected sequentially over all four flow cells at 10 μL / min for 150 s, with a dissociation phase of 800 or 900 s. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 s at 10 μL / min. A buffer injection was performed for each analyte injection used as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to determine the rate constants and affinity (K) of the interaction under analysis. D ) was derived.

[0212] For SPR experiments of PD1 binding using IL-15-binding variants, the method involved indirect capture of antibody variants onto the surface of an anti-human Fc antibody flow cell. The variants were injected into individual flow cells at 2.5 μg / mL for 30 seconds at a flow rate of 10 μL / min. The first flow cell was left empty to serve as a blank control. Next, 20 nM IL-15 was injected at 10 μL / min for 180 seconds to allow antibody:IL-15 complex formation. Overall, this resulted in capture of approximately 170-190 RU. Immediately following this capture step, four concentrations of PD1 (0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected sequentially into all four flow cells at 50 μL / min for 150 seconds, with a dissociation phase of 900 seconds. The anti-human Fc surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 seconds at 10 μL / min. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to derive the rate constants and affinity (KD) of the interaction under analysis.

[0213] For SPR experiments of IL-15Rβγ receptor binding using IL-15 and PD1 binding variants, the method involved indirect capture of antibody variants onto an anti-human Fc antibody flow cell surface, where the variants were injected at 2.5 μg / mL into individual flow cells at a flow rate of 10 μL / min for 30 seconds. The first flow cell was left empty to serve as a blank control. Next, 20 nM IL-15 was injected at 10 μL / min for 180 seconds to allow antibody:IL-15 complex formation. Next, 30 nM PD1 was injected at 10 μL / min for 180 seconds to allow antibody:IL-15:PD1 complex formation. Overall, this resulted in capture of approximately 190-235 RU. Immediately following this capture step, four concentrations of IL-15bg receptor (0.24 nM, 1.2 nM, 6 nM, and 30 nM) were injected sequentially over all four flow cells at 50 μL / min for 150 s, with a dissociation phase of 900 s. The antibody capture surface was regenerated twice with 10 mM glycine, pH 1.5, for 30 s at 30 μL / min. A buffer injection was performed with each analyte injection to serve as a reference. The resulting single-cycle kinetic sensorgrams were double-referenced and fitted to a 1:1 binding model using Biacore T200 BiaEvaluation software to derive the rate constants and affinity (KD) of the interaction under analysis.

[0214] Overall, the antigen affinity of the heterodimeric antibodies was assessed with reference to the corresponding wild-type control, RV1 Mab for IL-15 binding, and RV9 Mab for PD1 binding. Overall, the variants bound to PD1 and IL-15 with high affinity similar to that of the wild-type control (see Table 6), with the following caveats / exceptions: 1) some variants exhibited low purity (<95%) as measured by UPLC-SEC peak profiling, resulting in KDs that may be affected by present impurities; 2) impaired accessibility was observed for some variants (e.g., RV18, RV20) due to binding on the chip, resulting in an apparent reduction in affinity for PD1; 3) when variants with two anti-PD1 domains were the analyte, the resulting KDs may reflect KDs indicative of binding activity as well as affinity; and 4) RV17 and RV19 exhibited truncations consistent with deletion of the anti-PD1 scFv (Figures 7A and 7B), thereby resulting in a lack of binding to PD1, as expected.

[0215] Furthermore, the variants are capable of forming desirable IL-15:PD1:IL-15Rβγ receptor complexes, as shown by SPR experiments assessing IL-15Rβγ receptor affinity using a PD1 capture surface (Table 6, Figure 12A) and SPR experiments assessing binding of variants binding to IL-15 and PD1 to the IL-15Rβγ receptor using an anti-human Fc capture surface (Table 7, Figure 12B). On the PD1 capture surface, the RV is first captured by the anti-PD1 binding domain(s), followed by capture of IL-15 by the anti-IL-15 domain(s), and then capture of the IL-15βγ receptor(s) by the IL-15-binding anti-IL-15 domain(s). On the anti-human Fc capture surface, RV is first captured, followed by capture of IL-15 by the anti-IL-15 domain(s), then capture of PD1 by the anti-PD1 domain(s), and then capture of IL-15βγ receptor(s) by the IL-15-binding anti-IL-15 domain(s). For RV30 and RV37, the PD1 binding affinity was similar when the variants were bound to IL-15 (Table 7) or not (Table 6). The IL-15-binding variants also had similar affinities for the IL-15Rβγ receptor when bound to PD1 or not (Tables 6 and 7). [Table 6-1] [Table 6-2] [Table 7]

[0216] Example 5: Anti-IL-15 antibodies induce in vivo proliferation of IL-15-responsive lymphocytes (CD8+ T cells, NK cells, and NKT cells) The in vivo effects of exemplary anti-IL-15 antibodies were evaluated in C57BL / 6 mice. Because anti-IL-15 antibodies do not bind to mouse IL-15, mice were treated intraperitoneally with 10 mg of GMP-grade human IL-15 (Acro Biosystems) (functionally active in mice). Simultaneously, mice were subcutaneously treated with 100 mg of anti-IL-15 antibody (RV1) or an IgG1 isotype control (anti-RSV). Mice were monitored for 7 days. The capture antibody was well tolerated as monitored by body weight and clinical observations. The effects of anti-IL-15 antibodies on immune cell populations were assessed in peripheral blood 4 days after treatment and in spleens at sacrifice on day 7. Lymphocyte subsets were assessed by flow cytometry.

[0217] Compared with the isotype control group, administration of anti-IL-15 antibody resulted in an increase in lymphocyte subsets known to respond to IL-15 on day 4. The numbers of CD8+ T cells, NK cells, and NKT cells were significantly increased in peripheral blood (p = 0.0001, two-way ANNOVA followed by Tukey's multiple comparison test), but not CD4+ T cells, CD4+ Treg cells, or B cells (Figure 13). In addition, analysis of spleens on day 7 showed a significant increase in the proportion of CD3+ T cells that were CD8+ in the presence of anti-IL-15 antibody compared with the isotype control (p = 0.0001, two-way ANNOVA followed by Tukey's multiple comparison test (Figure 14)). These results suggest that anti-IL-15 antibody can bind to cells expressing the IL-15Rβγ receptor in vivo and present IL-15.

[0218] Example 6. Anti-IL-15 antibodies induce cytokine production in ex vivo cultures of PBMCs The effect of an exemplary anti-IL-15 antibody (RV1) on cytokine production was evaluated in human peripheral blood mononuclear cells (PBMCs). Frozen PBMCs were purchased from AllCells Inc. (Alameda, CA), and the cells were thawed immediately before the experiment. The cells were cultured in duplicate in 96-well U-bottom plates (200,000 cells per well) with the indicated concentrations of test samples for 4 days. After 4 days, the levels of cytokines secreted by the cells and present in the culture supernatant were measured by MSD (Meso Scale Dynamics) analysis. Two experiments were performed. In the first experiment, two forms of anti-IL-15 antibody (RV1, Fc-active, and RV2, Fc-null) and an isotype control (anti-RSV, Fc-active) were cultured at 10 mg / ml in duplicate in the absence or presence of human IL-15 (50 pM or 1 nM) for 4 days. GM-CSF was detected in the supernatants of cultures containing anti-IL-15 antibodies with an activating Fc (RV1), but not with an inactive Fc (RV2), both in the presence of exogenously added IL-15 (50 pM and 1 nM) and endogenous IL-15 levels (no IL-15 added to the cultures) (Figure 15A). Similarly, significantly increased levels of TNF-α were observed in cultures containing anti-IL-15 antibodies with an activating Fc and 1 nM IL-15 (p = 0.0001, two-way ANNOVA (Figure 15B)). In addition, 50 pM IL-15 and mixtures with endogenous levels of IL-15 also increased TNF-α levels in RV1 compared with RV2 and the isotype control. Other cytokines (IL-2, IFN-γ, granzyme A / B, and perforin) were not detected above background levels in this assay. These data suggest that the Fc-active form of the anti-IL-15 antibody RV1 can bind to and enhance the activity of endogenous and exogenous IL-15. In a second experiment, the Fc-active IL-15 capture antibody was either precomplexed with IL-15 (at equimolar concentrations by overnight incubation at 4°C in low protein-binding polypropylene 96-well U-bottom plates) or complexed with IL-15 during the assay.Human PBMCs (AllCells Inc., Alameda, CA) were cultured in duplicate in 96-well U-bottom plates for 4 days in the presence of 100 nM, 10 nM, or 1 nM RV1 precomplexed with IL-15, or with RV1 and free IL-15 (equimolar concentrations). This included IL-15 alone, medium alone controls, and IL-15 precomplexed with IL-15Rα-Fc (IL-15Rα-Fc fusion protein, ACROBiosystems). After 4 days, culture supernatants were analyzed by MSD for the presence of IL-2, IFN-γ, granzyme A, granzyme B, perforin, GM-CSF, and TNF-α (Figures 16A–G). When PBMCs were cultured with RV1 precomplexed with IL-15, RV1 with free IL-15, and IL-15Rα-Fc precomplexed with IL-15, a dose-dependent increase in all cytokines was observed. In contrast, IL-15 alone did not elevate TNF-α levels above the negative control, and GM-CSF levels were only slightly elevated. These data suggest that the production of both GM-CSF and TNF-α is enhanced by the active Fc in the RV1 and IL-15Rα-Fc constructs. Furthermore, we demonstrate that binding of RV1 to IL-15 during the assay resulted in functional antibody-IL-15 complex formation and enhanced GM-CSF and TNF-α production, likely due to Fc-mediated activation of monocytes and macrophages by PBMC cultures (typically 10% of PBMCs). For the remaining cytokines, when they were precomplexed with IL-15 or incubated with free IL-15 to form complexes in vitro, the anti-IL-15 antibody was able to stimulate cytokine release by PBMCs in a manner similar to both free IL-15 and IL-15 bound to IL-15Rα-Fc, confirming that the complexes formed between the anti-IL-15 antibody (RV1) and IL-15 were functional.

[0219] Example 7. Anti-IL-15 antibodies stimulate phosphor-STAT5 (pSTAT5) signaling in NK cells The effects of exemplary bispecific and monospecific anti-IL-15 antibodies on T cell and NK cell proliferation in ex vivo cultures of PBMCs were investigated by flow cytometry using the cell proliferation marker Ki-67. The Ki-67 antigen is an established marker for cell proliferation, and in combination with extracellular markers of cell subsets, flow cytometry can be used to identify proliferating cells within mixed cell cultures (Kim and Sederstrom (2015) Curr Protoc Mol Biol 111, 28.6.1). Cryopreserved PBMCs (StemCell Technologies, Vancouver, BC) were thawed and cultured at 200,000 cells per well in 96-well U-bottom plates (low-protein-binding polypropylene plates overnight at 4°C) in the presence of 100 nM, 10 nM, or 1 nM monoclonal anti-IL-15 antibodies (RV1 and RV2), bispecific anti-PD-1 × anti-IL-15 antibodies (RV29 and RV32), or equimolar concentrations of IL-15 precomplexed with IL-15Rα-Fc. IL-15 alone and medium served as positive and negative controls, respectively, and each condition was performed in duplicate. After 4 days of culture, supernatants were collected, and the remaining cells were surface stained for CD3, CD4, CD8, CD16, and CD56 (BioLegend, CA, USA) to identify T cell and NK cell subsets. After surface staining, cells were fixed and permeabilized using a transcription factor staining kit (Thermo Fisher Scientific, MA, USA), and intracellular staining was performed for Ki-67 (BioLegend, CA, USA). Flow cytometry of fixed cells was performed on a CytoFLEX (Beckman Coulter, IN, USA), and the percentage of individual cell populations was determined by analysis using FlowJo (BD BioSciences, NJ, USA). Analysis of surface staining showed a treatment-related dose-dependent increase in the fraction of CD8+ T cells among the CD3+ T cell subset and a corresponding decrease in the abundance of CD4+ T cells in all treatment conditions compared to the negative control (Figures 17A and 17B, respectively), consistent with IL-15-dependent stimulation of CD8 T cells over CD4 T cells.Analysis of the NK cell marker CD56 revealed no dose-dependent changes in NK cell abundance (Figure 17C). Consistent with the increased abundance of CD8 over CD4 T cells, levels of the proliferation marker Ki-67 correlated with IL-15 in a dose-dependent manner across all conditions tested, particularly in CD8+ T cells (Figure 18A). This was more pronounced with anti-IL-15 antibody compared with IL-15 alone or IL-15 complexed with IL-15Rα-Fc. As expected, given our observation that IL-15 preferentially stimulates CD8 cells, the abundance of Ki-67-expressing CD4+ T cells was lower than that observed for CD8+ T cells (Figure 18B), although there was still a treatment dose-dependent increase in Ki-67-positive CD4+ T cells. There were no dose-dependent effects on NK cells with IL-15, IL-15-precomplexed IL-15Rα-Fc, or IL-15-precomplexed RV1, likely due to saturation at the lowest dose of these test samples. Notably, in CD8 cells, dose-dependent effects were observed with IL-15 precomplexed with RV2 (Fc null) and the bispecific anti-PD-1 × anti-IL-15 antibodies RV29 and RV32, although the low levels of PD-1 in primary PBMCs may account for their low potency in this assay. NK cells were further divided into strongly CD56-positive (CD16-) and weakly CD56-positive (CD16+) subsets (Figures 19A and 19B). Again, no dose-dependent Ki-67 staining was observed with IL-15, IL-15-precomplexed IL-15Rα-Fc, or IL-15-precomplexed RV1. There was a dose-dependent effect of the bispecific antibodies (RV29 and RV32) on both NK subsets, and a dose-dependent effect of RV2 on the CD56-diminished CD16+ subset of NK cells. This observation with RV2 is consistent with the increased potency of RV1 and Fc-based presentation of IL-15 by IL-15Rα-Fc via CD16 in the CD56-diminished CD16+ subset of NK cells.In conclusion, this example demonstrates that monoclonal anti-IL-15 antibodies (RV1 or RV2) and bispecific anti-PD-1 x anti-IL-15 antibodies (RV29 and RV32) can form functional complexes with IL-15, resulting in proliferation of both CD8+ T cells and NK cells over CD4+ T cells.

[0220] Cell culture supernatants collected from the cells were analyzed by MSD for the presence of IL-2, IFN-γ, granzyme A, granzyme B, perforin, GM-CSF, and TNF-α (Figures 20A-20G). Despite variability between duplicate samples in some cases, dose-dependent cytokine responses were observed in the majority of tested samples. In particular, IFN-γ, granzymes A and B, perforin, and GM-CSF (Figures 20B-20F) showed dose-dependent increases with IL-15 and IL-15 precomplexed with either IL-15Rα-Fc or RV1, resulting in the highest cytokine levels. The Fc-null anti-IL-15 antibody (RV2) and the bispecific anti-PD-1 × anti-IL-15 antibodies (RV29 and RV32) produced low cytokine levels, consistent with the low levels observed in T cell and NK cell proliferation. As also shown in Figures 15A and 16F, RV1 was the only test sample to stimulate the highest levels of GM-CSF and high levels of TNF-α (Figures 20F and 20G). This confirms previous observations that Fc enhances the production of these cytokines. IL-2 levels were high in negative control cultures of cells alone (Figure 20A), making interpretation of IL-2 difficult. These data demonstrate that anti-IL-15 antibody containing bispecific constructs can bind and present IL-15 in a manner that stimulates both cytokine production and cell proliferation in vitro.

[0221] Example 8: Bispecific anti-IL-15 antibodies stimulate the in vitro proliferation of exhausted T cells expressing PD-1 To test the ability of anti-IL-15 antibody-containing bispecific constructs to bind a second target and present IL-15, exemplary anti-PD-1 x anti-IL-15 bispecific antibodies were evaluated for their ability to induce the proliferation of exhausted primary human T cells expressing PD-1. CD3+ T cells were negatively selected from cryopreserved PBMCs (AllCells Inc, Alameda, CA) using magnetic beads (Miltenyi Biotec, Germany) and cultured in flasks precoated with anti-CD3 antibody (OKT3, 5 mg / ml) and soluble anti-CD28 (2 mg / ml). After 24 hours, 20 IU / ml of recombinant human IL-2 was added. On day 3, cells were diluted to 2.5 million cells / ml and supplemented with fresh IL-2 at 20 IU / ml. On day 6, IL-2 was supplemented at 20 IU / ml. After 7 days, aliquots of cells were stained for CD3, CD4, and PD-1 expression (>90% of CD3+ cells expressed PD-1), and the remaining cells were cryopreserved. Cells were then thawed and cultured in 96-well U-bottom plates (low-protein-binding polypropylene plates overnight at 4°C) in the presence of IL-15 or test samples (IL-15Rα-Fc, RV1, RV2, RV29, and RV32) precomplexed with equimolar concentrations of IL-15. Samples were added in duplicate at concentrations of 100 nM, 10 nM, and 1 nM, and a negative (media only) control was included. After 4 days, cells were surface stained for CD3, CD4, CD8, and PD-1 (BioLegend, CA, USA) to identify PD-1 expression on T cell subsets. After surface staining, cells were fixed and permeabilized using a transcription factor staining kit (Thermo Fisher Scientific, MA, USA), and intracellular staining was performed for Ki-67 (BioLegend, CA, USA). Flow cytometry of fixed cells was performed using a CytoFLEX (Beckman Coulter, IN, USA), and the percentage of individual cell populations was determined by analysis using FlowJo (BD BioSciences, NJ, USA). The majority of CD8+CD3+ and CD4+CD3+ cells expressed Ki-67, indicating expansion of both T cell subsets in all tested samples (Figures 21A and 21B).The abundance of Ki-67-positive CD8 cells was higher than that of CD4 cells, consistent with our previous observation that IL-15 alone or complexed with anti-IL-15 antibodies preferentially stimulates CD8 cells. Additionally, the potency of bispecific anti-PD-1 × anti-IL-15 constructs (RV29 and RV32) was similar to that of monoclonal anti-IL-15 antibodies (RV1 and RV2), particularly in CD8+ T cells, suggesting that bispecific binding to PD-1 enhances the association of IL-15 to the IL-15Rβγ complex, in contrast to observations in PBMCs (Figure 18A). Further evidence that RV29 and RV32 bound to PD-1 is shown in Table 8. The MFI (mean fluorescence intensity) of PD-1 staining in Ki-67-positive cells treated with IL-15 or IL-15 precomplexed with IL-15Rα-Fc, RV1, or RV2 was similar across treatments for CD8+ cells (MFI range = 1693-3224, mean = 2186) and CD4+ cells (MFI range = 4099-6551, mean = 4828). However, a decrease in the MFI of PD-1 staining was detected in Ki-67-positive cells treated with the bispecific constructs RV29 and RV32 for both CD8+ cells (MFI range = 2025-999, mean = 1456) and CD4+ cells (MFI range = 3898-2336, mean = 3092) (Table 8), indicating that the bispecific constructs bound to and blocked PD-1 detection. Additionally, as demonstrated by SPR (Table 6), the MFI of PD-1 was lower in cells treated with RV32 than with RV29, consistent with RV32 having a higher affinity for PD-1. This lack of PD-1 detection was expected due to the epitope overlap between the anti-PD-1 arm of the bispecific and the PD-1 detection antibody (clone EH12.2H7). These data demonstrate that the bispecific anti-PD-1 × anti-IL-15 construct (RV29 and RV32) functionally redirected IL-15 to PD-1-expressing T cells during in vitro activation, resulting in increased potency against PD-1-expressing T cells compared with PBMCs. [Table 8]

[0222] Example 9. Anti-IL-15 antibodies stimulate phosphor-STAT5 (pSTAT5) signaling in NK cells We examined the effect of exemplary anti-IL-15 antibodies on the early stage of cytokine signaling (i.e., phosphorylation of the intracellular signaling molecule STAT5) upon binding of IL-15 to IL-15Rβγ. PBMCs from three independent donors were incubated in duplicate with IL-15, IL-15 complexed with IL-15Rα-Fc, IL-15 complexed with anti-IL-15 antibodies RV1 (IgG1 isotype) and RV3 (IgG4 isotype), or an IgG1 isotype control (anti-RSV). After 20 minutes, cells were stained for CD3, CD4, CD8, CD56, and CD16, followed by intracellular staining for pSTAT5 (Biosciences, CA, USA). Cells were analyzed by flow cytometry (using CytoFLEX, Beckman Coulter, IN, USA), and the mean percentage (and SD) of cells expressing pSTAT5 was measured for CD8+ T cells, CD4+ T cells, CD56-bold NK cells, and CD56+CD16+ NK cells by analysis using FlowJo (BD BioSciences, NJ, USA) (Figures 22A-22D). EC50 values ​​were derived for each treatment and cell subset and are shown in Table 9. IL-15, IL-15 complexed with IL-15Rα-Fc, and isotype control (i.e., free IL-15) resulted in dose-dependent pSTAT5 expression in all four cell subsets. IL-15 complexed with both formats of anti-IL-15 antibody dose-dependently stimulated pSTAT5 in CD56-bold NK cells (Figure 22C) and CD56+CD16+ NK cells (Figure 22D). However, the IgG1 format anti-IL-15 antibody (RV1) was more efficient at inducing pSTAT5 compared with the IgG4 format (RV3), as evidenced by a lower EC50 value, consistent with the observation that the IgG1 Fc of RV1 enhanced binding to CD16 (FcγRIII) on NK cells and subsequently increased pSTAT5 activity compared with the IgG4 isotype (RV3), which did not interact with CD16. The data suggest that secondary presentation (in this case, Fc-based) contributes to increased IL-15 activity upon presentation with an IL-15 capture antibody. [Table 9]

[0223] Example 10. Structural modeling of non-blocking epitopes on the IL-15 cytokine Structural modeling of the quaternary crystal structure of IL-15 bound to its receptors, IL-15Rα, IL-15Rβ, and common gamma chain, was performed using the structure in pdb (id 4gs7). A 2D representation of the structure is shown in Figure 5A. The cytokine is shown as a spatial entity, with the IL-15Rβ and common gamma chain shown as wireframes and the ribbon-like IL-15Rα chain. The IL-15Rβ and common gamma receptor chains, present on effector cells of the immune system, such as T cells and NK cells, transmit activation signals after IL-15 binding. IL-15Rα is responsible for presenting IL-15 to the beta-gamma receptor heterodimer (IL-15Rβγ). Therefore, to achieve a non-blocking or partially blocking association of IL-15 that does not inhibit IL-15 binding to the IL-15Rβγ heterodimer, capturing IL-15 at an epitope close to the interface with the IL-15Rα chain should allow productive association with IL-15Rβγ. Several anti-IL-15 antibodies capable of binding to IL-15 are known in the literature, including DISC0280 (Finch et al. (2011) British Journal of Pharmacology 162, 480), B-E29, MOB-1254Z, PABZ-081, MOB-0784CT, HPAB-0238-YC, HPAB-0359-WJ, MOM-18387, 04H04 (Sestak et al. (2018) Front Immunol 9, 1603), AMG714 / oldesekimab (Wei et al. (2022) Journal of Immunotoxicology 19, 109), and CALY-002 (Vicari et al. (2017) Mabs 9, 927). Some of these antibodies bind to IL-15 and block its interaction with IL-15Rβ and the common gamma chain, whereas DISC0280 binds to an epitope on IL-15 that competes for binding to IL-15Rα. Figure 5B shows a 2D representation of the crystal structure of IL-15 bound to DISC0280, based on pdb id 2xqb, an antibody developed to block IL-15 activity.We modeled the co-complex structure of antibody-bound IL-15 with the IL-15Rβγ receptor heterodimer by fitting the structure of IL-15 to two independent crystal structures, 4gs7 and 2xqb. The model structure is shown in Figure 5C and demonstrates that the antibody potentially binds to IL-15, allowing the bound cytokine to interact with the IL-15Rβγ heterodimer receptor complex. This modeling work provides insight into the nature of the cytokine-binding domain and those epitopes on the cytokine that can potentially stimulate its cognate receptor despite the cytokine being associated by its binding domain.

[0224] Example 11. Modeling the pharmacological effects of IL-15 in the presence of an IL-15 capture antibody The Access tool developed by Applied Biomath (Grant et al., 2023, Mabs, 15, 2192251) was used to exploratory model the effect of antibodies containing cytokine-binding domains on the pharmacological aspects of cytokine exposure in a system. Based on the biophysical characteristics of protein therapeutics, the model mathematically describes key mechanisms, such as drug targeting, distribution, and excretion, and can link the pharmacokinetic and pharmacodynamic aspects of drug action to these properties. Among other features, characteristics such as the affinity of the binding domain for the ligand (cytokine), dosing frequency, drug half-life, ligand half-life, ligand concentration, and blood distribution volume are critical to model performance. The tool accepts exploratory values ​​of specific characteristic parameter values ​​as inputs and can scan other properties as dependent variables. Starting with an IL-15 concentration of 5 pg / ml, i.e., a concentration of approximately 0.0004 nM, and a half-life of 2 hours, Figure 23A shows the modeled change in IL-15 concentration in the presence of an IL-15 capture antibody with an affinity (KD) of 0.1 nM for IL-15, extending the half-life of IL-15 to 1 day (24 hours), and administered every 7 days. The curves show how the persistence, and therefore concentration, of IL-15 changes with different doses of antibody. The legend on the right indicates that the antibody dose range was varied from 0.01 mg / kg to 10 mg / kg. In this setting, the model predicts that the concentration of IL-15 will increase by approximately 10-fold (to approximately 0.004 nM) after weekly administration of 0.316 mg / kg of a cytokine capture antibody containing a cytokine-binding domain. Figure 23B shows the results of a simulation using a similar antibody, but with an extended half-life of 3 days. The results here suggest that the concentration of IL-15 increases approximately 30-fold at the same dose level compared to the simulation in Figure 23A. Figure 23C shows the simulation results when the affinity of the cytokine binding domain is changed to an affinity (KD) of 1 nM. Such models can be further extended to take into account other parameters that may be critical to drug action. Appendix A: Arrays Table A1-1 Table A1-2 Table A1-3 Table A1-4 Table A1-5 Table A1-6 Table A1-7 Table A1-8 Table A2-1 Table A2-2 Table A2-3 Table A2-4 Table A2-5 Table A3-1 Table A3-2 Table 10-1 Table 10-2 Table 11-1 Table 11-2 Table 11-3 Table 11-4 Table 11-5 Table 11-6 Table 11-7 Table 11-8 Table 11-9 Table 11-10 Table 11-11 Table 11-12 Table 11-13 Table 11-14 Table 11-15 Table 11-16 Table 11-17 Table 11-18 Table 11-19 Table 11-20 Table 11-21 Table 11-22 Table 11-23 Table 11-24 Table 11-25 Table 11-26 References 1.Li & Lim(2020)Science 370,1034 2.Waldmann et al. (2020)) Front Immunol 11,10.3389 / fimmu.2020.00868 3.Schwartz et al.(2002)Oncology 16,11 4.Werkmeister et al. (2005) Oncology Reports 13,449 5.Xu et al.(2021)Cancer Immunology Research 9,1141 6. Martomo et al. (2021) Molecular Cancer Therapeutics 20,347 7. Santollani et al. (2023) Immunological Reviews 320,10 8.Kohler et al.,Nature 256:495(1975) 9.Clackson et al.,Nature 352:624-628(1991) 10.Marks et al.,J.Mol.Biol.222:581-597(1991) 11.Morrison et al.,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984) 12.Liljeblad et al,Glyco J 17,323-329(2000) 13.Heeley,Endocr Res 28,217-229(2002) 14.Banik,Kushnir,Doranz,and Chambers (2023) Mabs 15(1),2273018 15.Brinkmann U & Kontermann RE(2017)MAbs 9,182-212 16.Ridgway JBB,Presta LG,Carter P(1996)Prot Engg Des Sel 9,617-621 17.von Kreudenstein TS etal(2013)MAbs 5,646-654 18.E.Creighton,Proteins:Structures and Molecular Properties(W.H.Freeman and Company,1993) 19.A.L.Lehninger,Biochemistry(Worth Publishers,Inc.,current addition) 20.Sambrook,et al,Molecular Cloning:A Laboratory Manual(2nd Edition,1989) 21.Methods In Enzymology(S.Colowick and N.Kaplan eds.,Academic Press,Inc.) 22.Remington’s Pharmaceutical Sciences, 18th Edition (Easton,Pennsylvania:Mack Publishing Company,1990) 23.Carey and Sundberg Advanced Organic Chemistry 3rd Ed.(Plenum Press)Vols A and B(1992) 24.Brinkmann and Kontermann(2017),MABS 9(2),182-212 25.Finch et al.(2011)British Journal of Pharmacology 162,480 26.Sestak et al.(2018)Front Immunol 9,1603 27.Wei et al.(2022)Journal of Immunotoxicology 19,109 28.Vicari et al(2017)Mabs 9,927 29.Grant et al.,2023,Mabs,15,2192251

Claims

1. 1. A method for redirecting an active form of an endogenous cytokine to a desired target cell or tissue in a biological system, the biological system comprising: (i) the active form of the cytokine; (ii) cells having a corresponding receptor on their surface for the active form of the cytokine; and (iii) one or more desired target cells or tissues to which the cytokine is redirected; The method includes subjecting the biological system to (a) a binding domain that specifically binds to an epitope on the activated form of the cytokine (cytokine-binding domain); and (b) exposing the target cell or tissue to a multispecific binding molecule that comprises a binding domain that specifically binds to an epitope on a molecule that is not a cytokine receptor (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor; The method, whereby the active form of the cytokine is redirected to the target cell or tissue.

2. 1. A method for redirecting an active form of an endogenous cytokine to a target cell or tissue of interest in a subject, comprising: (a) a binding domain that specifically binds to the activated form of the cytokine (cytokine-binding domain); and (b) administering to the subject a sufficient amount of a multispecific binding molecule that comprises a binding domain that specifically binds to a molecule that is a marker on the target cell or tissue (target binding domain), wherein the cytokine, when complexed with the multispecific binding molecule, retains the ability to bind to and stimulate its cognate receptor; The method, whereby the active form of the cytokine is redirected to the target cell or tissue.

3. 3. The method of claim 1 or claim 2, wherein the multispecific binding molecule causes the cytokine to accumulate in or around the target cell or tissue.

4. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine, when redirected, exerts an agonistic effect on its corresponding receptor on the target cell.

5. 3. The method of claim 2, wherein administration of the multispecific binding molecule to the subject increases the half-life of the cytokine in the subject.

6. 3. The method of claim 2, wherein administration of the multispecific binding molecule to the subject increases the amount of the cytokine in the serum of the subject.

7. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule, when bound to the cytokine, reduces, but does not completely block, the ability of the cytokine to bind to and / or stimulate its cognate receptor.

8. 1. A method for redirecting an active form of a cytokine to a target cell or tissue of interest, comprising: (a) selecting a cytokine of interest; (b) selecting a target molecule that is a marker on a target cell or in a target tissue of interest; (c) generating a panel of binding domains that bind to said cytokine; (d) generating a panel of binding domains that bind to the target molecule; (e) screening the cytokine binding domain using an assay that measures the ability of the cytokine, when complexed with the cytokine binding domain, to bind to and / or stimulate its cognate receptor compared to the ability of the unbound cytokine to bind to and / or stimulate its cognate receptor; (f) screening the target binding domain for binding to an appropriate epitope on the target molecule; (g) selecting a cytokine binding domain that does not block or only partially blocks the ability of said cytokine to bind to and / or stimulate its cognate receptor; (h) generating a panel of multispecific binding molecules comprising one or more of the selected cytokine binding domains and one or more selected target binding domains; (i) screening the multispecific binding molecules in an in vitro cell-based assay that measures the ability of the cytokine to bind to and stimulate its cognate receptor in the presence of varying amounts of the multispecific binding molecule.

9. 9. The method of claim 8, further comprising screening the multispecific binding molecule in an in vivo assay in a non-human subject that measures the ability of the cytokine to bind to and stimulate its cognate receptor when administered to the subject.

10. 9. The method of claim 8, further comprising performing an epitope binning assay in conjunction with the cytokine binding domain screening step to identify a region or regions on the cytokine that, when bound to the cytokine binding domain, retain or partially retain the ability of the cytokine to bind to and stimulate its cognate receptor.

11. The multispecific binding molecule comprises: (a) one cytokine-binding domain and one target-binding domain; (b) one cytokine-binding domain and two identical or non-identical target-binding domains; (c) two identical or non-identical cytokine-binding domains and one target-binding domain; or 10. The method of any one of the preceding claims, comprising (d) two identical or non-identical cytokine binding domains and two identical or non-identical target binding domains.

12. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule comprises a cytokine binding domain that specifically binds to IL-15 or IL-15 complexed with IL-15 receptor alpha.

13. 13. The method of claim 12, wherein the cytokine binding domain binds to IL-15, or the IL-15-IL:15 receptor alpha complex, with an affinity of less than 100 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM as measured by surface plasmon resonance (SPR).

14. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule comprises a target binding domain that specifically binds to a protein expressed in the tumor microenvironment (TME).

15. 14. The method of any one of claims 1 to 13, wherein the multispecific binding molecule comprises a target binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a tumor cell, and the agonistic action of the cytokine is redirected to the location of the tumor cell.

16. 14. The method of any one of claims 1 to 13, wherein the multispecific binding molecule comprises a target binding domain that specifically binds to a receptor on an immune cell, optionally a T cell, a macrophage, or an NK cell.

17. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule comprises a cytokine-binding domain and a target-binding domain that binds to a cytokine cognate receptor and target on the same cell (cis-binding).

18. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule comprises a cytokine-binding domain and a target-binding domain that binds to a cytokine-cognate receptor and target on different cells (trans-binding).

19. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule comprises a scaffold, optionally an albumin-based scaffold, a fibronectin-based scaffold, or an immunoglobulin-based scaffold.

20. 20. The method of claim 19, wherein the immunoglobulin-based scaffold is derived from IgG1, IgG2, IgG4, IgM, or IgA.

21. 20. The method of claim 18 or 19, wherein the albumin-based scaffold or the immunoglobulin-based scaffold is capable of binding to neonatal Fc receptor (FcRn).

22. 10. The method of any one of the preceding claims, wherein the multispecific binding molecule is a bispecific antibody comprising a binding domain that specifically binds to an epitope on an activated form of the cytokine and a binding domain that specifically binds to an epitope on a molecule that is not a cytokine receptor on the target cell or tissue.

23. (a) a binding domain that specifically binds to the active form of a cytokine; and (b) a multispecific binding molecule comprising a binding domain that specifically binds to an epitope on a molecule that is a marker on a target cell or tissue, The multispecific binding molecule, when bound to the cytokine, does not block or only partially blocks the ability of the cytokine to bind to and stimulate its cognate receptor.

24. 10. The method of claim 1, wherein the biological system is selected from an in vitro culture, an animal model, or a human subject.

25. 1. A method for redirecting the agonistic action of an active form of an endogenous cytokine using a multispecific molecule, comprising: a. the specificity of the multispecific molecule is for an active form of a cytokine; b. the association of the activated form of the cytokine by the multispecific molecule is non-blocking; c. association of the activated form of the cytokine by the multispecific molecule causes the cytokine to retain all or part of its agonistic activity; d. at least one other specificity of the multispecific molecule is for a non-cytokine molecule; and e. The method wherein the multispecific molecule captures the cytokine and redirects its agonistic action by binding to the non-cytokine cell surface receptor molecule.

26. 26. The method of claim 25, wherein the agonist action is redirected to a desired tissue or cell surface.

27. 26. The method of claim 25, wherein the cytokine is an endogenous cytokine.

28. 27. The method of claim 25 or 26, wherein the endogenous cytokine is in soluble form.

29. 27. The method of claim 25 or 26, wherein the endogenous cytokine is a cell surface type.

30. The method of any one of claims 25 to 29, wherein the non-cytokine molecule is a cell surface receptor molecule.

31. 27. The method of claim 26, wherein the desired tissue or cell surface is selected from the group consisting of immune cells, tumor cells, stromal cells, cells within the tumor microenvironment, cells within the bone marrow, cells within the lymph nodes, epithelial cells, endothelial cells, blood cells, skin cells, stem cells, bone cells, nerve cells, adipocytes, and muscle cells.

32. 32. The method of any one of claims 25 to 31, wherein the agonist action is redirected to a desired tissue or cell surface receptor or stroma to enable cis or trans presentation of the endogenous cytokine within the targeted environment.

33. 1. A method for developing non-blocking multispecific binding molecules, comprising: a) selecting an immune signaling molecule; b) selecting a target molecule; c) testing the multispecific binding molecules separately for binding to either the immune signaling molecule or the target molecule; d) testing the multispecific binding molecules for binding to the immune signaling molecules and stimulation of the corresponding receptors; e) testing the multispecific binding molecule for non-blocking or partially blocking binding to the immune signaling molecule that enables immune signaling agonist activity.

34. 34. The method of claim 33, further comprising: f) modeling the complex between an endogenous cytokine receptor and the immune signaling molecule to define epitopes on the immune signaling molecule that maintain endogenous cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking or partially blocking multispecific binding molecule that binds to the immune signaling molecule and the target molecule.

35. The method of any one of claims 25 to 34, wherein the multispecific binding molecule is a bispecific binding molecule.

36. 1. A method for developing non-blocking or partially blocked multispecific binding molecules, comprising: a) selecting an immune signaling molecule; b) selecting a target molecule; c) testing the monospecific binding molecules separately for binding to either said immune signaling molecule or said target molecule; d) testing the monospecific binding molecules for binding to the immune signaling molecules and stimulation of the corresponding receptors; e) testing said monospecific binding molecule for non-blocking or partially blocking binding to said immune signaling molecule that enables immune signaling agonist activity; f) designing the non-blocking multispecific binding molecule to comprise a monospecific binding molecule for the immune signaling molecule and a monospecific binding molecule that binds to the target molecule.

37. 26. The method of claim 25, wherein the active form of the endogenous cytokine is a cytokine, a cytokine complex, or a cytokine isoform.

38. 37. The method of claim 36, wherein the immune signaling molecule is an endogenous cytokine, chemokine, growth factor, hormone, signaling molecule, signaling polypeptide chain, or soluble ligand for its corresponding receptor.

39. 39. The method of any one of claims 33 to 38, further comprising modeling the pharmacological properties of said endogenous cytokine or said endogenous cytokine complex.

40. 40. The method of any one of claims 33 to 39, further comprising modeling the pharmacological properties of the target molecule.

41. 41. The method of any one of claims 33 to 40, further comprising determining a competitive binding profile of said monospecific binding molecule.

42. 42. The method of claim 41, wherein identifying the competitive binding profile comprises epitope binning.

43. 1. A method for developing non-blocking multispecific binding molecules, comprising: a) selecting an endogenous cytokine or endogenous cytokine complex and further modeling the pharmacological properties of said endogenous cytokine or said endogenous cytokine complex; b) selecting a target molecule and further modeling the pharmacological properties of said target molecule; c) testing the monospecific binding molecules separately for binding to either the endogenous cytokine, the endogenous cytokine complex, or the target molecule; d) testing the monospecific binding molecules for non-blocking binding to the endogenous cytokine or endogenous cytokine complex and further identifying competitive binding profiles of the monospecific binding molecules by epitope binning; e) modeling the complex between a cytokine receptor and the endogenous cytokine or the endogenous cytokine complex to define epitopes on the endogenous cytokine or the endogenous cytokine complex that maintain endogenous cytokine receptor specificity and signaling properties upon binding of the monospecific binding molecule, thereby developing a non-blocking bispecific binding molecule that binds to the endogenous cytokine and the target molecule; f) designing a multispecific binding molecule that comprises a monospecific binding molecule for said endogenous cytokine and a monospecific binding molecule for said target molecule.

44. 44. The method of claim 43, further comprising validating the non-blocking multispecific binding molecule for binding to both the endogenous cytokine and the target molecule by in vitro cell-based receptor signaling screening for cytokine activity and target molecule specificity.

45. 45. The method of claim 43 or 44, further comprising assessing the efficacy of the non-blocking multispecific binding molecule in vivo.

46. 47. The method of any one of claims 44 to 46, further comprising assessing the pharmacokinetic and pharmacodynamic properties of the non-blocked multispecific binding molecule in vivo.

47. a) said selecting i) determining the endogenous expression level of said endogenous cytokine in a subject; ii) determining the amount of said endogenous cytokine present in an active state in the circulation or in a tissue of interest in said subject; iii) identifying a distribution profile of the endogenous cytokine receptors in the subject; and iv) identifying clearance and metabolic mechanisms of the endogenous cytokine in the subject.

48. The method of claim 36 or 43, wherein the selecting in b) is determined through examining the expression level, tissue specificity, cell surface localization, molecular internalization kinetics, and / or molecular recycling kinetics of the target molecule in the subject.

49. 44. The method of claim 36 or 43, wherein modeling the pharmacological properties of the endogenous cytokine or endogenous cytokine complex in a) identifies a desired affinity range for the interaction of a non-blocking bispecific binding molecule with an endogenous cytokine or endogenous cytokine complex to predict differences in the pharmacokinetics and biodistribution of a free endogenous cytokine or endogenous cytokine complex and / or predicts differences in the pharmacokinetics and biodistribution of a non-blocking bispecific binding molecule-bound endogenous cytokine or endogenous cytokine complex.

50. 44. The method of claim 36 or 43, wherein said modeling of the pharmacological properties of the target molecule in b) identifies a desired affinity range for the interaction between the non-blocking bispecific binding molecule and the target molecule and / or predicts differences in biodistribution of the target molecule bound and unbound to a non-blocking bispecific binding molecule.

51. 44. The method of any one of claims 33, 34, or 43, wherein the test in d) utilizes an in vitro sandwich assay that crosslinks the monospecific binding molecule with the endogenous cytokine receptor via binding of the endogenous cytokine or the endogenous cytokine complex.

52. 52. The method of any one of claims 33 to 51, further comprising performing a competition assay between said monospecific binding molecules for said endogenous cytokine or endogenous cytokine complex binding to said endogenous cytokine receptor.

53. 53. The method of any one of claims 34 to 52, wherein said modeling in step e) determines the geometry of a non-blocking bispecific binding molecule scaffold that maintains endogenous cytokine receptor specificity and / or signaling properties while binding to said target molecule.

54. The method of any one of claims 25 to 53, wherein the endogenous cytokine complex is an IL-15SA complex comprising IL-15 and IL-15 receptor alpha.

55. 55. The method of claim 54, wherein the non-blocking bispecific binding molecule binds to an epitope of IL-15 receptor alpha.

56. The method of any one of claims 25 to 55, wherein the non-blocking bispecific binding molecule binds to an epitope of IL-15.

57. The method of any one of claims 25 to 56, wherein the endogenous cytokine is IL-15.

58. The method of any one of claims 25 to 56, wherein the endogenous cytokine is IL-2.

59. 58. The method of any one of claims 55 to 57, wherein the non-blocking bispecific binding molecule binds to IL-15 with higher affinity than to IL-15 receptor alpha.

60. 60. The method of claim 59, wherein the non-blocking bispecific binding molecule has an affinity for IL-15 that is at least about 10-fold greater than the affinity of the non-specific binding molecule for IL-15.

61. 61. The method of any one of claims 25 to 60, wherein the target molecule is selected from the group consisting of programmed cell death protein 1 (PD1), CD33, CD16, programmed death-ligand 1 (PD-L1), integrin, disialoganglioside (GD2), CD20, fibroblast activation protein (FAP), carcinoembryonic antigen receptor (CEAR), and carcinoembryonic antigen (CEA).

62. 62. The method of any one of claims 25 to 61, wherein the non-blocking multispecific binding molecule comprises an albumin-based bispecific scaffold, an affibody, an asymmetric antibody, a bispecific T cell engaging antibody (BiTE), a diabody, a dual affinity retargeting molecule (DART), an immunoglobulin domain crossover (CrossMAb), a minibody, a tandem diabody (TandAb), a fibronectin-based scaffold, or a bispecific molecular scaffold selected from the group consisting of FynomAb, an antibody fusion construct, or an albumin fusion construct.

63. 63. The method of any one of claims 25 to 62, wherein the non-blocking multispecific binding molecule is a fusion protein comprising a monospecific binding molecule for binding to the immune signaling molecule and another monospecific binding molecule for binding to the target molecule.

64. 64. The method of any one of claims 34 to 63, wherein the monospecific binding molecule is selected from the group consisting of an antibody, a bivalent antibody fragment, an antigen-binding fragment region, a minibody, a monovalent antibody, a single-chain variable fragment (scFv), a reduced immunoglobulin, and a disulfide-stabilized variable fragment, a Fab fragment, a nanobody, an immunoglobulin domain antibody, a finomer, and a DARPin.

65. 1. A method for developing non-blocking multispecific binding molecules, comprising: a. selecting an endogenous cytokine of interest to be amplified; b. Obtaining data regarding the system level profile of said endogenous cytokines; c. Obtaining data regarding the target receptor; d. Modeling and simulating said endogenous cytokine in its native state when associated with an antibody; e. Modeling and simulating receptor targeting of said endogenous cytokine; f. Identifying binders to said endogenous cytokine and said target receptor; g. performing a binding screen and / or competition assay; h. performing epitope binning of antibodies; i. defining a desired epitope on said endogenous cytokine; j. conducting a mixed cell-based receptor signaling screen; The method, wherein the non-blocking multispecific binding molecule associates with the endogenous cytokine and retains the cytokine receptor binding and signaling properties of the endogenous cytokine.

66. a. an antibody, and b. A non-blocking antibody conjugate containing a cytokine, The non-blocking antibody conjugate, wherein the antibody is capable of presenting the cytokine to its corresponding receptor.

67. 67. The non-blocking antibody conjugate of claim 66, wherein the antibody conjugate is multispecific.

68. 68. The non-blocking antibody conjugate of claim 66 or 67, wherein the antibody is capable of localizing the cytokine to a desired tissue or target cell surface receptor.

69. 69. The non-blocking antibody conjugate of any one of claims 66 to 68, wherein the half-life of the conjugate is the same as the half-life of the antibody.

70. 1. A method of multispecific targeting comprising: a. Producing a non-blocking multispecific binding molecule using the method of any one of claims 25 to 65; b. administering the non-blocking multispecific binding molecule to a subject in need thereof; The method, wherein the multispecific binding molecule targets endogenous cytokines in the serum of the subject and activates and / or expands tumor-specific effector cells, thereby inducing tumor cell killing.

71. 71. The method of claim 70, further comprising administering an exogenous cytokine to the subject.

72. 72. The method of claim 70 or 71, wherein the cytokine is IL-15.

73. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine is endogenous to the system or the subject.

74. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine is a mixture of endogenous and exogenous cytokines in the biological system or the subject.

75. 26. The method of claim 25, wherein the cytokine is an exogenous cytokine.

76. 58. The method of any one of claims 55-57, wherein the non-blocking bispecific binding molecule binds IL-15 with a lower affinity than IL-15 receptor alpha (IL-15Rα), said affinity being at least about 10-fold, 100-fold, or 1000-fold lower.

77. a multispecific binding molecule that binds to PD-1 and IL-15, the multispecific binding molecule comprising one binding domain specific for PD-1 and a second binding domain specific for IL-15; (a) the binding domain specific for PD-1 comprises CDR H1 (SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72), CDR H2 (SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75), CDR H3 (SEQ ID NO:76, SEQ ID NO:77, or SEQ ID NO:78), CDR L1 (SEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:81), CDR L2 (SEQ ID NO:82, SEQ ID NO:83, or SEQ ID NO:84), and CDR L3 (SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87); and (b) the multispecific binding molecule, wherein the binding domain specific for IL-15 comprises CDR H1 (SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54), CDR H2 (SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57), CDR H3 (SEQ ID NO:58, SEQ ID NO:59, or SEQ ID NO:60), CDR L1 (SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63), CDR L2 (SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66), and CDR L3 (SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69).

78. 78. The multispecific binding molecule of claim 77, wherein the binding domain specific for PD-1 comprises a variable fragment light chain (VL) selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

79. 78. The multispecific binding molecule of claim 77, wherein the binding domain specific for IL-15 comprises a VL selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:

2.

80. (a) the binding domain specific for PD-1 comprises a VL selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; and (b) the binding domain specific for IL-15 comprises a VL selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:

2.

81. 81. The multispecific binding molecule of any one of claims 77-80, wherein the binding domain specific for PD-1 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO:

20.

82. 82. The multispecific binding molecule of any one of claims 77 to 81, wherein the binding domain specific for IL-15 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO:

18.

83. (a) the binding domain specific for PD-1 comprises a VH selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20; (b) the binding domain specific for IL-15 comprises a variable fragment heavy chain (VH) selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO:

18.

84. 84. The multispecific binding molecule of any one of claims 77 to 83, wherein the binding domain specific for PD-1 comprises a constant fragment light chain (CL) selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:

10.

85. The multispecific binding molecule of any one of claims 77 to 84, wherein the binding domain specific for IL-15 comprises a CL selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:

4.

86. 86. The multispecific binding molecule of any one of claims 77 to 85, comprising a constant fragment heavy chain 1 (CH1) selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, a constant fragment heavy chain 2 (CH2) selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and a constant fragment heavy chain 3 (CH3) selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:

36.

87. The multispecific binding molecule of any one of claims 77 to 86, wherein the binding molecule is an antibody in Fab-Fab format.

88. 88. The multispecific binding molecule of any one of claims 77 to 87, further comprising at least one scFv fused to the C-terminus of the heavy chain.

89. 89. The multispecific binding molecule of claim 87 or 88, wherein one Fab is specific for PD-1 and the scFv is specific for IL-15.

90. 89. The multispecific binding molecule of claim 87 or 88, wherein one Fab is specific for IL-15 and the scFv is specific for PD-1.

91. 87. The multispecific binding molecule of any one of claims 77 to 86, wherein the binding molecule is a multivalent molecule comprising VH / VL domains or Fab domains.

92. 92. The multispecific binding molecule of claim 91, wherein the VH domain with specificity for PD-1 is fused to the N-terminus of the VH domain with specificity for IL-15, and the VL domain with specificity for PD-1 is fused to the N-terminus of the VL domain with specificity for IL-15.

93. 92. The multispecific binding molecule of claim 91, wherein the VH domain with specificity for IL-15 is fused to the N-terminus of the VH domain with specificity for PD-1, and the VL domain with specificity for IL-15 is fused to the N-terminus of the VL domain with specificity for PD-1.

94. 92. The multispecific binding molecule of claim 91 , wherein the VH domain containing specificity for PD-1 is fused to the N-terminus of the VH domain containing specificity for IL-15, and the VL domain containing specificity for PD-1 is fused to the N-terminus of the VL domain containing specificity for IL-15, and the outermost Fab is specific for PD-1 and the inner Fab is specific for IL-15.

95. 92. The multispecific binding molecule of claim 91 , wherein the VH domain comprising specificity for IL-15 is fused to the N-terminus of the VH domain comprising specificity for PD-1, and the VL domain comprising specificity for IL-15 is fused to the N-terminus of the VL domain comprising specificity for PD-1, and the outermost Fab is specific for IL-15 and the inner Fab is specific for PD-1.

96. 96. The multispecific binding molecule of any one of claims 91 to 95, wherein the DVD comprises a linker connecting the VH domain comprising specificity for PD-1 to the VH domain comprising specificity for IL-15, and a linker connecting the VL domain comprising specificity for PD-1 to the VL domain comprising specificity for IL-15.

97. 97. The multispecific binding molecule of claim 96, wherein the linker is a glycine serine linker.

98. 98. The multispecific binding molecule of any one of claims 91 to 97, wherein the linker is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO:

39.

99. 97. The multispecific binding molecule of any one of claims 91 to 96, wherein the linker is selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO:

41.

100. 87. The multispecific binding molecule of any one of claims 77 to 86, wherein the binding molecule is a fusion protein.

101. 101. The multispecific binding molecule of any one of claims 77-100, wherein the binding molecule comprises two or more PD-1 binding domains, two or more IL-15 binding domains, or two or more PD-1 binding domains and two or more IL-15 binding domains.

102. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine is endogenous to the system or the subject.

103. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine is an exogenous cytokine, and the exogenous cytokine is added to the biological system or administered to the subject.

104. 3. The method of claim 1 or claim 2, wherein the active form of the cytokine is a mixture of endogenous and exogenous cytokines in the biological system or the subject.

105. 58. The method of any one of claims 55-57, wherein the non-blocking bispecific binding molecule binds IL-15 with a lower affinity than IL-15Rα, said affinity being at least about 10-fold, 100-fold, or less.

106. 10. The method of claim 1, wherein the multispecific binding molecule is used for selective expansion of immune cell subsets in a system or a subject.

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