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COMBINATION THERAPIES COMPRISING CD137/HER2 BISPECIFIC AGENTS AND PD-1 AXIS INHIBITORS AND USES THEREOF

20210246219 · 2021-08-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The disclosure provides compositions and methods for treating previously treated specific HER2-positive advanced or metastatic solid tumors. The disclosure provides compositions and methods for enhancing immune response in an individual having HER2-positive advanced or metastatic solid tumors. The method comprises administering a PD-laxis inhibitor and a bispecific agent that targets CD137 and HER2.

    Claims

    1. A method of treating cancer in a subject, comprising administering to the subject: (a) a CD137/HER2 bispecific agent comprising (i) CDR1, CDR2, CDR3 domains of the heavy chain variable region having the sequence set forth in SEQ ID NO: 64, (ii) CDR1, CDR2, CDR3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 65, and (iii) a lipocalin mutein with the sequence set forth in any one of SEQ ID NOs: 21-39, and (b) a PD-1 axis inhibitor.

    2. A method of treating cancer in a subject, comprising: (a) administering to the subject a CD137/HER2 bispecific agent, wherein the subject is also receiving a PD-1 axis inhibitor, so that the subject receives therapy in both, or (b) administering to the subject a PD-1 axis inhibitor, wherein the subject is also receiving a CD137/HER2 bispecific agent, so that the subject receives therapy in both, wherein the CD137/HER2 bispecific agent comprises (i) CDR1, CDR2, CDR3 domains of the heavy chain variable region having the sequence set forth in SEQ ID NO: 64, (ii) CDR1, CDR2, CDR3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 65, and (iii) a lipocalin mutein with the sequence set forth in any one of SEQ ID NOs: 21-39.

    3. The method of claim 1 or 2, wherein the cancer is HER-2 positive and/or PD-L1 positive.

    4. The method of any one of claims 1-3, wherein the method is capable of providing an enhanced anti-tumor effect.

    5. The method of any one of claims 1-4, wherein the method is capable of providing an additive an-tumor effect as compared to the CD137/HER2 bispecific agent or the PD-1 axis inhibitor alone.

    6. The method of any one of claims 1-4, wherein the method is capable of providing an synergistic an-tumor effect as compared to the CD137/HER2 bispecific agent or the PD-1 axis inhibitor alone.

    7. The method of any one of claims 4-6, wherein the anti-tumor effect is selected from the group consisting of: (a) stimulation of tumor-related immune response; (b) increased IL-2 secretion; (c) increased IL-2 secretion in a tumor microenvironment; (d) increased IFN-gamma secretion; (e) increased IFN-gamma secretion in a tumor microenvironment; (f) expansion of CD4.sup.+ T cells; (g) expansion of CD4.sup.+ T cells in a tumor microenvironment; (h) expansion of CD8.sup.+ T cells; (i) expansion of CD8.sup.+ T cells in a tumor microenvironment; (j) expansion of tumor-infiltrating lymphocytes; (k) activation of NK cells and increased antibody-dependent cell-mediated cytotoxicity (ADCC); (l) activation of NK cells and increased ADCC in a tumor microenvironment; (m) increased level of CD4 in a tumor microenvironment; (n) decreased level of CD4 in a tumor microenvironment; (o) increased level of CD8 in a tumor microenvironment; (p) decreased level of CD8 in a tumor microenvironment; (q) increased level of PD-L1 in a tumor microenvironment; (r) decreased level of PD-L1 in a tumor microenvironment; (s) increased level of Ki67 in a tumor microenvironment; (t) decreased level of Ki67 in a tumor microenvironment; (u) increased level of CD137 in a tumor microenvironment; (v) decreased level of CD137 in a tumor microenvironment; (w) increased level of HER2 in a tumor microenvironment; (x) decreased level of HER2 in a tumor microenvironment; (y) increased level of IL-8 in a tumor microenvironment; (z) decreased level of IL-8 in a tumor microenvironment; (aa) increased level of FoxP3 in a tumor microenvironment; (ab) decreased level of FoxP3 in a tumor microenvironment; (ac) reduction of tumor size; (ad) suppression of tumor growth; (ae) suppression of tumor metastasis; (af) delay of recurrence; or (ag) improved overall survival.

    8. The method of any one of claims 1-7, wherein the method comprises at least one administration cycle, wherein the cycle is a period of about three weeks, wherein for each of the at least one cycles at least one dose of the CD137/HER2 bispecific agent is administered and at least one dose of the PD-1 axis inhibitor is administered.

    9. The method of any one of claims 1-8, wherein the CD137/HER2 bispecific agent and the PD-1 axis inhibitor are administered sequentially or concurrently.

    10. The method of any one of claims 1-9, wherein the CD137/HER2 bispecific agent is administered at a dose of about 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 8.0 mg/kg.

    11. The method of any one of claims 1-10, wherein the PD-1 axis inhibitor is an anti-PD-L1 antibody.

    12. The method of claim 11, wherein the anti-PD-L1 antibody has the sequences set forth in SEQ ID NOs: 76 and 77.

    13. The method of claim 11 or 12, wherein the anti-PD-L1 antibody is administered at a dose of about 1200 mg.

    14. The method of any one of claims 1-10, wherein the PD-1 axis inhibitor is an anti-PD-1 antibody.

    15. The method of any one of claims 1-10, wherein the PD-1 axis inhibitor is an antibody having the sequences set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75.

    16. The method of any one of claims 1-10, wherein the PD-1 axis inhibitor is atezolizumab, durvalulmab, avelumab, nivolumab, pembrolizumab, tislelizumab, or cemiplimab.

    17. The method of any one of claims 1-16, wherein the CD137/HER2 bispecific agent and the PD-1 axis inhibitor are administered at the following doses: (a) about 0.05 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor, (b) about 0.15 mg/kg CD137/HR2 bispecific agent and about 1200 mg PD-1 axis inhibitor, (c) about 0.5 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor, (d) about 1.0 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor, (e) about 2.5 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor, (f) about 5.0 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor, or (g) about 8.0 mg/kg CD137/HER2 bispecific agent and about 1200 mg PD-1 axis inhibitor

    18. The method of any one of claims 1-17, wherein the method comprises at least one administration cycle, such as 2, 3, 4, 5, 10, 15, 20, 25, and 30 cycles, wherein the cycle is a period of about three weeks.

    19. The method of any one of claims 1-18, wherein the method comprises administering one dose of the CD137/HER2 bispecific agent and one dose of the PD-1 axis inhibitor, sequentially with respect to each other, on days 1 of each three-week cycle.

    20. The method of any one of claims 1-19, wherein the method comprises administering one dose of the CD137/HER2 bispecific agent and one dose of the PD-1 axis inhibitor, sequentially with respect to each other, on days 1 of each cycle for 30 three-week cycles.

    21. The method of any one of claims 1-20, wherein the CD137/HER2 bispecific agent comprises a lipocalin mutein specific for CD137 fused at the N-terminus via a linker to the C-terminus of each heavy chain of anti-HER2 antibody.

    22. The method of any one of claims 21, wherein the lipocalin mutein specific for CD137 has the sequence set forth in SEQ ID NO: 22.

    23. The method of any one of claims 22, wherein the anti-HER2 antibody has the sequence set forth in SEQ ID NOs: 79 and 80.

    24. The method of any one of claims 1-23, wherein the CD137/HER2 bispecific agent has the sequences set forth in SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, or SEQ ID NOs: 79 and 84.

    25. The method of any one of claims 1-24, wherein the cancer is HER-2 positive advanced or metastatic solid tumors.

    26. The method of any one of claims 1-25, wherein the subject is previously treated.

    27. The method of any one of claims 1-26, wherein the subject is previously treated with an anti-HER2 therapy.

    28. The method of any one of claims 1-27, wherein the subject is previously treated with a PD-1 axis inhibitor therapy.

    29. The method of any one of claims 1-28, wherein the treatment produces at least one effect chosen from stimulation of tumor-specific immune responses, reduction in tumor size, suppression of the growth of tumor cells, suppression of the metastasis, complete remission, partial remission, stabilization of disease, extension of the term before recurrence, extension of survival time, complete response, and partial response.

    30. A combination comprising an CD137/HER2 bispecific agent antibody comprising (i) CDR1, CDR2, CDR3 domains of the heavy chain variable region having the sequence set forth in SEQ ID NO: 64, (ii) CDR1, CDR2, CDR3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 65, and (iii) a lipocalin mutein with the sequence set forth in any one of SEQ ID NOs: 21-39, and a PD-1 axis inhibitor, wherein the combination is suitable to be administered to a subject in at least one cycle, wherein for each cycle the CD137/HER2 bispecific agent is administered at a dose of about 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 8.0 mg/kg and the anti-PD-L1 axis inhibitor is administered at a dose of about 1200 mg.

    31. The combination of claim 30, wherein the CD137/HER2 bispecific agent and the anti-PD-L1 antibody are administered sequentially or concurrently.

    32. The combination of claim 30 or 31, wherein the combination may be used for treating HER2-positive and/or PD-L1 positive tumors.

    33. The combination of any one of claims 30-32, wherein the combination may produce enhanced anti-tumor effects.

    34. The combination of any one of claims 30-33, wherein the combination may produce additive anti-tumor effects as compared to the CD137/HER2 bispecific agent or the PD-1 axis inhibitor alone.

    35. The combination of any one of claims 30-33, wherein the combination may produce synergistic anti-tumor effects as compared to the CD137/HER2 bispecific agent or the PD-1 axis inhibitor alone.

    36. A kit of parts comprising (a) a pharmaceutical composition comprising an CD137/HER2 bispecific agent antibody comprising (i) CDR1, CDR2, CDR3 domains of the heavy chain variable region having the sequence set forth in SEQ ID NO: 64, (ii) CDR1, CD2, CDR3 domains of the light chain variable region having the sequence set forth in SEQ ID NO: 65, and (iii) a lipocalin mutein with the sequence set forth in any one of SEQ ID NOs: 21-39; and (b) a pharmaceutical composition comprising a PD-1 axis inhibitor.

    37. The kit of parts of claim 36, wherein the CD137/HER2 bispecific agent is at a unit dosage of about 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 8.0 mg/kg and the anti-PD-L1 axis inhibitor is at a unit dosage of about 1200 mg.

    38. The kit of parts of claim 36 or 37, wherein the pharmaceutical composition comprising an CD137/HER2 bispecific agent and the pharmaceutical composition comprising a PD-1 axis inhibitor are capable of producing additive or synergistic anti-tumor effect.

    Description

    III. DESCRIPTIONS OF FIGURES

    [0059] FIG. 1: shows the ability of the CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 in combination with the PD-1 antibody shown in SEQ ID NOs: 72 and 73 to costimulate T-cell activation. In the experiment, tumor cell line NCI-N87 was cultured on a plate overnight. The next day, human peripheral blood mononuclear cells (PBMCs) were added to the plate and incubated with the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) alone, the PD-1 antibody (SEQ ID NOs: 72 and 73) alone, or a combination of the two, at the molar ratio of 1:10 in the presence of 0.05 ng/mL staphylococcal enterotoxin B (SEB). Levels of secreted interleukin 2 (IL-2), as marker of T-cell activation, were determined by an electrochemiluminescence-based assay and normalized to the levels of corresponding IgG4 isotype control antibody (SEQ ID NOs: 15 and 16), as described in Example 1. Results from two tested donors are depicted in FIG. 1A and 1B, respectively. The combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-1 antibody (SEQ ID NOs: 72 and 73) was able to induce a dose-dependent IL-2 secretion, with an improved (lower) EC.sub.50, as compared to the CD137/HER2 bispecific agent or the PD-1 antibody alone.

    [0060] FIG. 2: shows the ability of the CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 in combination with a PD-1 antibody shown in SEQ ID NOs: 72 and 73 (FIG. 2A) or in SEQ ID NOs: 74 and 75 (FIG. 2B) to costimulate T-cell activation. In the experiment, tumor cell line NCI-N87 was cultured on a plate overnight. The next day, human PBMCs were added to the plate and incubated with the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) at various concentrations in combination with the PD-1 antibody shown in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75 at the fixed concentration of 10 nM or 100 nM, in the presence of 1 ng/mL of SEB. The CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-1 antibody (SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75) were also tested alone for comparison. Levels of secreted IL-2 were determined by an electrochemiluminescence-based assay as readout for T-cell activation and normalized to the levels of corresponding IgG4 isotype control antibody (SEQ ID NOs: 15 and 16), as described in Example 2. Representative results of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) tested at 10 nM and 3.33 nM are depicted. The combination of the CD137/HER2 bispecific agent (SEQ ID NOs 81 and 80:) and the PD-1 antibody shown in SEQ ID NOs: 72 and 73 and the combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-1 antibody shown in SEQ ID NOs: 74 and 75 both induced higher IL-2 secretion and act synergistically or additively. When combined with SEQ ID NOs: 72 and 73, the induced IL-2 secretion levels were higher when 100 nM antibody was used than when 10 nM antibody was used. On the other hand, the concentration (at 100 nM or 10 nM) of SEQ ID NOs: 74 and 75 did not affect the IL-2 secretion when used in combination with the CD137/HER2 bispecific agent.

    [0061] FIG. 3: shows the results of representative experiments in which the ability of the combination of a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 and a PD-L1 antibody (SEQ ID NOs: 76 and 77) to induce T-cell activation was investigated. In the experiment, tumor cell line NCI-N87 (HER2 high, PD-L1 low), JIMT-1 (HER2 moderate, PD-L1 moderate) or MDA-MB-231 (HER2 low, PD-L1 moderate) was cultured on a plate overnight. The next day, human PBMCs were added to the plate and incubated with the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) at various concentrations, alone or in combination with the PD-L1 antibody (SEQ ID NOs: 76 and 77) or IgG4 isotype control antibody (SEQ ID NOs: 15 and 16) at 100 nM, in the presence of 0.05 ng/mL SEB. Levels of secreted IL-2 were determined as described in Example 3. Exemplary data of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) tested at the concentration of 2.5 nM and 0.16 nM are shown in FIG. 3. The functional activity of the CD137/HER2 bispecific agent in combination with the PD-L1 antibody, measured by the ability to activate T cells or increase IL-2 secretion, is synergistic/additive in the presence of tumor cells expressing high or moderate level of HER2 (NCI-N87 and JIMT-1).

    [0062] FIG. 4: describes the overall study design of a Phase 1b study to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) and assess the safety, efficacy, and PK of a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 administered in combination with a PD-L1 antibody shown in SEQ ID NOs: 76 and 77 in specific advanced or metastatic HER-2 positive solid tumors (e.g., bladder, breast and gastrointestinal). The study includes a dose escalation period followed by an expansion period.

    [0063] FIG. 5. provides an overview over the design of representative CD137/HER2 bispecific fusion proteins as described herein. Representative CD137/HER2 bispecific fusion proteins were made based on an antibody specific for HER2 (e.g., an antibody shown in SEQ ID NOs: 79 and 80) and one or more a lipocalin muteins specific for CD137 (e.g., the lipocalin shown in any one sequence of SEQ ID NOs: 21-39). One or more lipocalin muteins were genetically fused to the C- and/or the N-terminus of either the heavy chain or the light chain of a HER2 specific antibody as depicted in FIG. 1A-1D, resulting in the fusion proteins, e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, and SEQ ID NOs: 79 and 84. An engineered IgG4 backbone with the mutations S228P, F234A, and L235A was used for the HER2 specific antibody as included in the fusion proteins.

    IV. DETAILED DESCRIPTION OF THE DISCLOSURE

    [0064] CD137 is a costimulatory immune checkpoint and member of the tumor necrosis factor receptor (TNFR) family. It is primarily expressed on activated CD4.sup.+ and CD8.sup.+ T cells, activated B cells, and natural killer (NK) cells, and plays an important role in the regulation of the immune response. The clustering of CD137 leads to activation of the receptor and downstream signaling (Yao et al., Nat Rev Drug Discov, 2013, Snell et al., Immunol Rev, 2011). In a T cell pre-stimulated by the T cell receptor (TCR) binding to a cognate major histocompatibility complex (MHC) target, costimulation via CD137 leads to enhanced activation, survival, and proliferation, as well as the production of pro-inflammatory cytokines and an improved capacity to kill.

    [0065] In line with the mode of CD137 activation, which requires receptor clustering, a monospecific CD137-targeting agent, such as an anti-CD137 antibody, may not be efficient by itself to cluster CD137 and lead to efficient activation. Besides, recent work around TNFR family members illustrates the mechanisms of anti-TNFR antibodies whereby the antibodies interact via their Fc regions with Fc-gamma receptors, engaging activating Fc-gamma receptor-expressing immune cells, and facilitate the subsequent anti-tumor activity (Bulliard et al., Immunol Cell Biol, 2014, Bulliard et al., J Exp Med, 2013). This, therefore, suggests an anti-CD137 antibody may trigger CD137 clustering depending on the abundance of Fc-gamma receptor-positive cells, which are not selectively tumor-localized but distributed throughout the body. Accordingly, the efficacy and target-specificity of anti-CD137 monotherapy may be of concerns. In fact, some anti-CD137 therapeutics under clinical studies, such as urelumab and utomilumab, show disappointing efficacy results with low-dose and/or reveal toxicity at high-dose or effective-dose (Bulliard et al., Immunol Cell Biol, 2014, Bulliard et al., J Exp Med, 2013).

    [0066] Therefore, there is an unmet need for CD137-targeting therapeutics that are both effective and safe. An ideal CD137-targeting agent should lead to clustering of CD137, and do so in a tumor localized fashion on tumor-infiltrating lymphocytes. As described herein, to obtain such a CD137-targeting agent, bispecific agents may be designed to target CD137 on one end and a differentially expressed tumor target on the other end.

    [0067] In this respect, HER2 is a clinically-validated target across a broad spectrum of tumor types. Amplification of the HER2 gene and overexpression of its product have been shown to play an important role in the development and progression of various types of cancer including breast, bladder, gastric, gastroesophageal, colorectal, and biliary tract cancer. CD137/HER2 bispecific agents as provided herein are therefore envisioned to promote CD137 clustering by bridging T cells with HER2-positive tumor cells and deliver a costimulatory signal to tumor antigen-specific T cells, providing localized immune activation and leading to tumor destruction.

    [0068] Furthermore, the combination of checkpoint immunotherapies and immunotherapy and tumor-targeted therapy have been demonstrated to show superiority over monotherapies in some instances (Karachaliou et al., Ann Transl Med, 2017, Ott et al., J Immunother Cancer, 2017). CD137-targeting agents are being explored in combination with other therapies. Although preclinical studies with mouse xenograft models have shown that the anti-CD137 therapy may benefit from combining with checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies (Dai et al., Clin Cancer Res, 2015, Wei et al., Oncoimmunology, 2014, Chen et al., Cancer Immunol Res, 2015, Kohrt et al., J Clin Invest, 2014, Kohrt et al., J Clin Invest, 2012, Morales-Kastresana et al., J Immunother Cancer, 2013), there remains a need to balance the improvement in efficacy and associated risks including non-targeted immune activation and toxicitiy for real life applications of CD137-targeting combination therapy. Clinically, agonist anti-CD137 antibody urelumab has been studied in combination with nivolumab (anti-PD-1 mAb) presenting discouraging efficacy results and limited clinical activity (Massarelli, 31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer, 2016). Urelumab was also examined in a neoadjuvant trial of cetuximab (anti-EGFR mAb) and revealed the potential to strengthen anti-tumor immunity, albeit only in the presence of NK cells already activated by cetuximab (Srivastava et al., Clin Cancer Res, 2017). Another anti-CD137 antibody, utomilumab is being studied with another PD-1 inhibitor, pembrolizumab. While the phase lb data showed clinical benefit and revealed certain trends in biomarkers, the combination was not sufficient to distinguish between the benefits of utomilumab and the effects of pembrolizumab monotherapy (Tolcher et al., Clin Cancer Res, 2017).

    [0069] Despite the ongoing developments, there are still needs to identify biomarkers associated with clinical benefit to CD137-targeting therapeutics and optimal combination therapies with suitable modes of actions.

    [0070] Therefore, in some embodiments, the present disclosure provides combinations comprising a CD137-targeting agent and a PD-1 axis inhibitor. A provided CD137-targeting agent is preferably a CD137/HER2 bispecific agent and accordingly a provided combination will comprise a CD137/HER2 bispecific agent and a PD-1 axis inhibitor.

    [0071] The present disclosure also provides methods of making one or more combinations comprising a CD137/HER2 bispecific agent with a PD-1 axis inhibitor as well as compositions comprising such combination(s). In some embodiments, the present disclosure includes pharmaceutical compositions, as well as pharmaceutical kits, comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor. Provided pharmaceutical compositions may be in solid, liquid, sustained release such as transdermal, transnasal, or depot dosage units and may further include a suitable pharmaceutical carrier. In some embodiments, the present disclosure provides methods of using a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, for the treatment of specific HER2-positive advanced or metastatic solid tumors. The present disclosure also provides methods of using a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, to enhance immune response in an individual having HER2-positive advanced or metastatic solid tumors.

    A. Exemplary Combinations of the Disclosure and Uses and Applications Thereof.

    [0072] In some embodiments, a provided combination comprises at least a CD137/HER2 bispecific agent and a PD-1 axis inhibitor. In some embodiments, a provided combination comprises at least a CD137/HER2 bispecific agent and a PD-L1 inhibitor. In some embodiments, a provided combination comprises at least a CD137/HER2 bispecific agent that is a CD137/HER2 bispecific fusion protein set forth in SEQ ID NOs: 81 and 80 and a PD-L1 inhibitor that is a PD-L1 antibody set forth in SEQ ID NOs: 76 and 77. In some embodiments, a provided combination comprises at least a CD137/HER2 bispecific agent that is a CD137/HER2 bispecific fusion protein set forth in SEQ ID NOs: 81 and 80 and a PD-1 inhibitor that is a PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75.

    [0073] In some embodiments, a provided combination is the combination of a CD137/HER2 bispecific agent and a PD-1 axis inhibitor. In some embodiments, a provided combination is the combination of a CD137/HER2 bispecific agent and a PD-L1 inhibitor. In some embodiments, a provided combination is the combination of a CD137/HER2 bispecific agent and a PD-1 inhibitor. In some embodiments, a provided combination is the combination of a CD137/HER2 bispecific fusion protein set forth in SEQ ID NOs: 81 and 80 and a PD-L1 antibody set forth in SEQ ID NOs: 76 and 77. In some embodiments, a provided combination is the combination a CD137/HER2 bispecific fusion protein set forth in SEQ ID NOs: 81 and 80 and a PD-1 antibody set forth in SEQ ID NOs: 72 and 73. In some embodiments, a provided combination is the combination of a CD137/HER2 bispecific fusion protein set forth in SEQ ID NOs: 81 and 80 and a PD-1 antibody set forth in SEQ ID NOs: 74 and 75.

    [0074] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to activate T cells or stimulate T-cell responses. In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to synergistically or additively activate T cells or stimulate T-cell responses. In some embodiments, a provided combination leads to T-cell activation with a comparable or better potency and/or efficacy as compared to a CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, and SEQ ID NOs: 79 and 84) and/or a PD-1 axis inhibitor (e.g. SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, and SEQ ID NOs: 76 and 77) comprised in a particular combination. The stimulated T-cell response or T-cell activation may be measured, for example, in a functional T-cell activation assay as essentially described in Examples 1-3.

    [0075] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to induce increased IL-2 secretion relative to the bispecific agent or inhibitor alone. In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to synergistically or additively induce increased IL-2 secretion. In some preferred embodiments, provided combinations may be able to induce a concentration-dependent IL-2 secretion. In some embodiments, provided combinations may lead to increased IL-2 secretion with a comparable or better efficiency as compared to to a CD137/HER2 bispecific agent (e.g. SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, and SEQ ID NOs: 79 and 84) and/or a PD-1 axis inhibitor (e.g. SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, and SEQ ID NOs: 76 and 77) alone. IL-2 secretion may be measured, for example, in a functional T-cell activation assay as essentially described in Examples 1-3.

    [0076] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to stimulate T-cell responses in the presence of tumor cells and/or in a tumor microenvironment. In some particular embodiments, a provided fusion protein may be able to stimulate T-cell responses in the presence of HER2-positive tumor cells. The T-cell activation by provided combinations in the presence of tumor cells and/or in a tumor microenvironment may be assessed, for example, in a functional T-cell activation assay essentially described in Examples 1-3.

    [0077] In some embodiments, a provided combination may be able to activate T cells or stimulate T-cell responses in a HER2 dependent manner. In some embodiments, provided combinations may lead to local induction of the IL-2 production by T-cells in the microenvironment in HER2-positive cancer. The HER2 dependent activation of T-cell by provided combinations may be determined, for example, in a functional T-cell activation assay essentially described in Example 3, in which a combination of a CD137/HER2 bispecific agent and a PD-1 axis inhibitor was tested in the presence of tumor cells expressing different levels of HER2 and demonstrated the ability to activate T cells differently depending on the HER2 expression level.

    [0078] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, exerts additive anti-tumor effects. In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, exerts synergistic anti-tumor effects. Provided combinations, thereof, in some embodiments, when administered, allow lower doses with improved efficacy and a wider safety window, as compared to a CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, SEQ ID NOs: 79 and 84) alone and/or a PD-1 axis inhibitor (e.g. SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, SEQ ID NOs: 76 and 77) alone.

    [0079] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to induce increased secretion of IL-2. In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to induce increased secretion of IFN-gamma.

    [0080] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to stimulate CD4.sup.+ T cell proliferation and/or activation, preferably in a tumor microenvironment.

    [0081] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to stimulate CD8.sup.+ T cell proliferation and/or activation, preferably in a tumor microenvironment.

    [0082] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to induce the expansion of tumor-infiltrating lymphocytes.

    [0083] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to activate NK cells and increase ADCC, preferably in a tumor microenvironment.

    [0084] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to induce changes in biomarker levels in a subject. In some embodiments, a provided combination may decrease the level of a biomarker in a subject. In some embodiments, a provided combination may increase the level of a biomarker in a subject. The biomarker may be, for example, CD4, CD8, PD-L1, Ki67, CD137, HER2, IL-8, and FoxP3.

    [0085] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to reduce the tumor size in a subject.

    [0086] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to suppress tumor growth in a subject.

    [0087] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to suppress tumor metastasis in a subject.

    [0088] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to delay tumor recurrence in a subject.

    [0089] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, in combination with the other, may be able to improve overall survival for a subject.

    [0090] In some embodiments, provided combinations may be administered to a subject, e.g., a mammal such as a human. In some embodiments, a subject administered with provided combinations may be confirmed diagnosis of previously treated advanced and/or metastatic HER2-positive tumor.

    [0091] In some embodiments, components of a provided combination may be administered simultaneously or sequentially with respect to any other component of the combination. For example, a CD137/HER2 bispecific agent and a PD-1 axis inhibitor may be administered simultaneously with one another or sequentially with respect to each other.

    [0092] In some embodiments, components of a provided combination may be administered as adjuvant to any other component of the combination, i.e., one component of a provided combination may be administered after a subject receives the treatment of another component. For example, a CD137/HER2 bispecific agent may be administered after a subject receives the treatment of a PD-1 axis inhibitor or a PD-1 axis inhibitor may be administered after a subject receives the treatment of a CD137/HER2 bispecific agent.

    [0093] In some embodiments, when the components of a provided combination are administered simultaneously, they can be administered in a single formulation or in distinct formulations. In some embodiments, when the components of a provided combination are administered as distinct formulations, whether simultaneously or sequentially, the components may be administered at a single site or at separate sites and using the same route or different routes. In some embodiments, when the components of a provided combination are administered sequentially, the time between administration of the components may be determined using certain factors such as the length of time a particular component persists, either systemically or at the administration site; or the length of time that the cellular effects of the component persist, either systemically or at the administration site.

    [0094] In some embodiments, combinations of the disclosure, or compositions comprising provided combinations are envisaged to be used as anti-tumor agents, anti-infection agents, and/or immune modulators. For example, in some embodiments, provided combinations may be used for increasing IL-2 secretion. In some embodiments, provided combinations may be used for increasing INF-gamma secretion. In some embodiments, provided combinations may be used for increasing tumor-infiltrating lymphocytes. In some embodiments, provided combinations may be used for activating NK cells and increasing ADCC. In some embodiments, provided combinations may be used for inducing lymphocyte activation and/or proliferation. In some embodiments, provided combinations may be used for enhancing immune functions. In some embodiments, provided combinations may be used for inducing CD137 clustering and activation on T cells and directing such T cells to tumor cells, preferably HER2-positive tumor cells. In some embodiments, provided combinations may be used for inducing a lymphocyte response in tumor microenvironment. In some embodiments, provided combinations may be used for providing anti-tumor effects. In some embodiments, provided combinations may be used for inducing changes in the level of a biomarker, such as, CD4, CD8, PD-L1, Ki67, CD137, HER2, IL-8, and FoxP3. In some embodiments, provided combinations may be used for treating and/or delaying progression of a cancer.

    [0095] In some embodiments, provided combinations may be used for treating HER2-expressing tumors. In some embodiments, provided combinations may be used for treating HER2-positive tumors. In some embodiments, provided combinations may be used for treating HER2-equivocal tumors. In some embodiments, provided combinations may be used for treating HER2-negative tumors. In some embodiments, provided combinations may be used for treating HER2-expressing tumors. The HER2 expressing status of a tumor may, for example, be analyzed at the protein level by immunohistochemistry (IHC) or other methods known in the art. In some embodiments, provided combinations may be used for treating HER2-positive advanced or metastatic solid tumors.

    [0096] In some embodiments, provided combinations may be used for treating PD-L1-positive tumors.

    [0097] In some embodiments, each component of a provided combination may, by itself, possess anti-tumor, anti-infective, and/or immunostimulatory activity. In some embodiments, combinations of components, such as a combination of a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, may provide greater anti-tumor, anti-infective, and/or immunostimulatory activity any one single component may provide. In some embodiments, combinations of components, may provide additive or synergistic anti-tumor, anti-infective, and/or immunostimulatory activity.

    [0098] In some embodiments, provided combinations may have favorable pharmacokinetic properties to permit a dosing schedule of about twice a week, of about once a week, of about once every ten days, of about once every two weeks, of about once every three weeks, of about once every four weeks, of about once every five weeks, of about once every month, of about once every six weeks, of about once every seven weeks, of about once every eight weeks, or of about once every two months. In some embodiments, one component of a provided combination may, by itself, have favorable pharmacokinetic properties.

    [0099] In some embodiments, one or more components of a provided combination are administered with a frequency of about twice a week, of about once a week, of about once every ten days, of about once every two weeks, of about once every three weeks, of about once every four weeks, of about once every five weeks, of about once every month, of about once every six weeks, of about once every seven weeks, of about once every eight weeks, or of about once every two months.

    [0100] In some embodiments, a combination is provided in the form of a kit of parts. In some embodiments, a kit of part comprises a pharmaceutical composition comprising a CD137/HER2 bispecific agent and a pharmaceutical composition comprising a PD-1 axis inhibitor. In some embodiments, a pharmaceutical composition comprising a CD137/HER2 bispecific agent and a pharmaceutical composition comprising a PD-1 axis inhibitor are provided in at least two separate unit dosage forms. In some embodiments, pharmaceutical compositions comprised in a kit of parts are provided in a unit dosage that corresponds to the dose to be administered to a subject per single administration. As an illustrative example, a CD137/HER2 bispecific agent is administered at a unit dosage of about 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 8.0 mg/kg and a PD-L1 axis inhibitor is administered at a unit dosage of about 1200 mg.

    B. Exemplary CD137/HER2 Bispecific Agents.

    [0101] In some embodiments, a provided combination comprises a CD137/HER2 bispecific agent. In some embodiments, a CD137/HER2 bispecific agent of the disclosure may be a CD137/HER2 bispecific fusion protein containing at least two subunits in any order: (1) a first subunit that comprises a full-length immunoglobulin or an antigen-binding domain thereof specific for HER2, and (2) a second subunit that comprises a lipocalin mutein specific for CD137 (FIG. 5). In some embodiments, a provided CD137/HER2 bispecific agent may be used in a method provided herein.

    [0102] In some embodiments, a provided CD137/HER2 bispecific fusion protein also may contain at least one additional subunit, for example, a third subunit. For instance, a fusion protein may contain a third subunit specific for CD137. In some embodiments, a provided CD137/HER2 bispecific fusion protein may comprise one or more additional subunits (e.g., a fourth, fifth, or sixth subunit).

    [0103] In some embodiments, at least one subunit may be fused at its N-terminus and/or its C-terminus to another subunit.

    [0104] In some embodiments, at least one subunit can be fused to another subunit via a linker. In some further embodiments, a linker is a peptide linker, for example, an unstructured glycine-serine (GS) linker, a glycosylated GS linker, or a proline-alanine-serine polymer (PAS) linker. In some embodiments, a GS linker is a (Gly.sub.4Ser).sub.3 linker ((G.sub.4S).sub.3) as shown in SEQ ID NO: 4. Other exemplary linkers are shown in SEQ ID NOs: 5-14. In some embodiments, a peptide linker may have from 1 to 50 amino acids, such as 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids. For example, in some embodiments, when a first subunit comprises a full-length immunoglobulin, a second subunit may be linked via a peptide linker between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said immunoglobulin. In some further embodiments, a third subunit may be linked via a peptide linker between the N-terminus of the third subunit and the C-terminus of a light chain constant region (CL) of said immunoglobulin.

    [0105] In some embodiments, a lipocalin mutein subunit may be fused to an immunoglobulin subunit of a provided CD137/HER2 bispecific fusion protein. In some embodiments, a lipocalin mutein subunit may be fused at its N-terminus and/or its C-terminus to an immunoglobulin subunit at the C-terminus of the immunoglobulin heavy chain domain (HC), the N-terminus of the HC, the C-terminus of the immunoglobulin light chain (LC), and/or the N-terminus of the LC (FIG. 5). For example, in some embodiments, a lipocalin mutein may be linked, via a peptide linker, at its N-terminus to each of the HC of an immunoglobulin (FIG. 5D).

    [0106] In some embodiments, with respect to a CD137/HER2 bispecific fusion protein of the disclosure, wherein at least one subunit may be or comprise a full-length immunoglobulin, the Fc function of the Fc region of the full-length immunoglobulin to Fc receptor-positive cell may be preserved at the same time while the fusion protein is simultaneously engaging CD137 and HER2.

    [0107] In some embodiments, wherein at least one subunit of a provided CD137/HER2 bispecific fusion protein may be or comprise a full-length immunoglobulin, the Fc function of the Fc region of the full-length immunoglobulin to Fc receptor-positive cell may be reduced or fully suppressed by protein engineering while the fusion protein is simultaneously engaging CD137 and PD-L1. In some embodiments, this may be achieved, for example, by switching from the IgG1 backbone to IgG4, as IgG4 is known to display reduced Fc-gamma receptor interactions compared to IgG1. In some embodiments, to further reduce the residual binding to Fc-gamma receptors, mutations may be introduced into the IgG4 backbone such as F234A and L235A. In some embodiments, an S228P mutation may also be introduced into the IgG4 backbone to minimize the exchange of IgG4 half-antibody (Silva et al., J Biol Chem, 2015). In some embodiments, F234A and L235A mutations may be introduced for decreased ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010) and/or M428L and N434S mutations or M252Y, S254T, and T256E mutations for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the immunoglobulin heavy chain of a provided CD137/HER2 bispecific fusion protein in order to remove the natural glycosylation motif.

    [0108] In some embodiments, the Fc portion of an immunoglobulin included in a CD137/HER2 bispecific fusion protein of the disclosure may contribute to maintaining the serum levels of the fusion protein. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein may become internalized and recycled back to the bloodstream, enhancing its half-life within the body.

    [0109] In some embodiments, with respect to a provided CD137/HER2 bispecific fusion protein, a first subunit may be or comprise a full-length immunoglobulin or an antigen-binding domain thereof specific for HER2. In some embodiments, an immunoglobulin is a monoclonal antibody against HER2.

    [0110] In some embodiments, a provided HER2 antibody or antigen-binding domain thereof may comprise the three heavy chain CDRs of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, and/or the three light chain CDRs of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45.

    [0111] In some embodiment, a provided HER2 antibody or antigen-binding domain thereof may have a heavy chain variable region (HCVR) shown in SEQ ID NO: 64, and/or a light chain variable region (LCVR) shown in SEQ ID NO: 65.

    [0112] In some embodiments, a provided HER2 antibody or antigen-binding domain thereof may have a heavy chain that is any one of SEQ ID NO: 78 or 79, and/or a light chain shown in SEQ ID NO: 80.

    [0113] In some embodiments, a provided HER2 antibody or antigen-binding domain thereof may have a HCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in SEQ ID NO: 64, and/or a LCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in SEQ ID NO: 65. In other embodiments, a provided HER2 antibody or antigen-binding domain thereof may have a heavy chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-79, and/or a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence of SEQ ID NO: 80.

    [0114] In some embodiments, a provided HER2 antibody is trastuzumab. In some embodiments, a provided HER2 antibody is trastuzumab with an IgG4 backbone.

    [0115] In some embodiments, with respect to a provided CD137/HER2 bispecific fusion protein, a second subunit may be or comprise a lipocalin mutein specific for CD137. In some embodiments, a provided lipocalin mutein may be or comprise a mutein of mature human neutrophil gelatinase-associated lipocalin (hNGAL). A mutein of mature hNGAL may be designated herein as an “hNGAL mutein”.

    [0116] In some embodiments, a provided CD137-binding hNGAL mutein may bind human CD137 with high affinity and be capable of costimulating human T cells when immobilized on a plastic dish together with an anti-CD3 antibody. In some embodiments, a provided CD137-binding hNGAL mutein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-39 or of a fragment or variant thereof. In some embodiments, a provided CD137-binding hNGAL mutein may comprise the amino acid sequence of SEQ ID NOs: 22 or of a fragment or variant thereof. In some embodiments, the amino acid sequence of a provided CD137-binding hNGAL mutein may have a high sequence identity, such as at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or higher identity, to a sequence selected from the group consisting of SEQ ID NOs: 21-39. In some embodiments, the amino acid sequence of a provided CD137-binding hNGAL mutein may have a high sequence identity, such as at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or higher identity, to the sequence of SEQ ID NOs: 22.

    [0117] In some embodiments, a provided CD137/HER2 bispecific fusion protein is generated by genetic fusion of a CD137-specific hNGAL mutein to a trastuzumab IgG4 variant, connected by a flexible, non-immunogenic linker.

    [0118] In some embodiments, a provided CD137/HER2 bispecific fusion protein is capable of engaging HER2 and CD137 simultaneously. In some embodiments, a provided fusion protein may be able to activate CD137 signaling in a HER2-dependent manner. In some embodiments, a provided fusion protein may be able to activate CD137 signaling in HER2-positive tumor microenvironment. In some embodiments, a provided fusion protein may be able to activate costimulate T cell responses and/or enhance T cell function in HER2-positive tumor microenvironment.

    [0119] In some embodiments, a provided CD137/HER2 bispecific fusion protein may comprise the amino acid sequences selected from the group consisting of SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, SEQ ID NOs: 79 and 84.

    [0120] In some embodiments, a provided CD137/HER2 bispecific fusion protein may comprise the amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequences shown in SEQ ID NOs:81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, SEQ ID NOs: 79 and 84. In some embodiments, where the bispecific fusion protein comprise more than one amino acid chain, a given value for the sequence identity relates to the average sequence identity normalized by the number of amino acid residues in both amino acid chains. For example, if a bispecific fusion protein consists of amino acid chain A having 100 amino acids and amino acid chain B having 50 amino acids, and another bispecific fusion protein consists of amino acid chain A′ having 100 amino acids 80% sequence identity to amino acid chain A and amino acid chain B′ having 50 amino acids and 95% sequence identity to amino acid chain B′, the average sequence identity between both fusion proteins will be (100/(100+50))×80%+(50/(100+50))×95%=85% sequence identity. In some preferred embodiments, where the bispecific fusion protein comprise more than one amino acid chain, a given value for the sequence identity means that a protein of interest comprises an amino acid sequence that has at least the given value of sequence identity to one chain of the bispecific fusion protein and comprises an amino acid sequence that has at least the given value of sequence identity to the other chain of the bispecific fusion protein.

    C. Exemplary PD-1 Axis Inhibitors.

    [0121] In some embodiments, a provided combination comprises a component of a PD-1 axis inhibitor. In some embodiments, a PD-1 axis inhibitor of the disclosure may be a PD-1 antagonist, a PD-L1 antagonist, or a PD-L2 antagonist. In some embodiments, a provided PD-1 axis inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, or an anti-PD-L2 antibody. In some embodiments, a provided PD-1 axis inhibitor may be used in a method provided herein.

    [0122] Various PD-1 axis inhibitors are known in the art. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475, and KEYTRUDA®, is a humanized IgG4 monoclonal antibody that binds to and blocks PD-1. It is used via intravenous infusion to treat inoperable or metastatic melanoma, metastatic non-small cell lung cancer (NSCLC) in certain situations, as a second-line treatment for head and neck squamous cell carcinoma (HNSCC), after platinum-based chemotherapy, and for the treatment of adult and pediatric patients with refractory classic Hodgkin's lymphoma (cHL). Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is a humanized IgG4 anti-PD-1 monoclonal antibody which blocks PD-L1 from binding to PD-1. It is used as a first line treatment for inoperable or metastatic melanoma in combination with ipilimumab if the cancer does not have a mutation in BRAF, as a second-line treatment following treatment with ipilimumab and if the cancer has a mutation in BRAF, with a BRAF inhibitor, as a second-line treatment for squamous non-small cell lung cancer, and as a second-line treatment for renal cell carcinoma. Atezolizumab, also known as MPDL3280A or Tecentriq®, is a human IgG1 monoclonal antibody engineered to eliminate Fc-effector function. It targets human PD-L1 and inhibits its interaction with PD-1. Atezolizumab is indicated for the treatment of urothelial carcinoma, a common type of bladder cancer, and is under investigation in other tumor types.

    C-1. Exemplary Anti-PD-L1 Antibodies as PD-1 Axis Inhibitors

    [0123] In some embodiments, a provided PD-1 axis inhibitor is an anti-PD-L1 antibody or antigen-binding domain thereof. In some embodiments, a provided combination comprises an anti-PD-L1 antibody or antigen-binding domain thereof.

    [0124] Illustrative examples of anti-PD-L1 antibodies of the disclosure may comprise an antigen-binding regions which cross-blocks or binds to the same epitope as an anti-PD-L1 antibody comprising the VH and VL regions of a previously known antibody, such as atezolizumab (also known as MPDL3280A or RG7446, trade name Tecentrie), avelumab (also known as MSB0010718C, trade name Bavencie), durvalumab (previously known as MED14736, trade name Imfinzi®), BMS-936559 (also known as MDX-1105), and anti-PD-L1 antibodies disclosed in PCT International Application Publication Nos. WO 2010/077634, WO 2007/005874, WO 2010/089411, WO 2011/066389, WO 2013/079174, WO 2015/048520, WO 2016/061142, WO 2016/111645, WO 2015/061668, WO 2016/007235, and WO 2017/148424. A provided anti-PD-L1 antibody also may be or comprises an antigen-binding region or the VH and VL regions or of any one of the above-mentioned anti-PD-L1 antibodies.

    [0125] In some embodiments, a provided anti-PD-L1 antibody or antigen-binding domain thereof may comprise an antigen-binding region, such as any one of the three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) from an antibody selected from the group consisting of atezolizumab, avelumab, durvalumab, and anti-PD-L1 antibodies as described in WO2017148424.

    [0126] In some embodiments, a provided PD-L1 antibody or antigen-binding domain thereof may comprise the three heavy chain CDRs of SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, and/or the three light chain CDRs of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63.

    [0127] In some embodiments, a provided anti-PD-L1 antibody or antigen-binding domain thereof may have a heavy chain variable region (HCVR) shown in SEQ ID NO: 70, and/or a light chain variable region (LCVR) shown in SEQ ID NO: 71.

    [0128] In some embodiments, a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain shown in SEQ ID NO: 76, and/or a light chain shown in SEQ ID NO: 77.

    [0129] In some embodiments, a provided PD-L1 antibody or antigen-binding domain thereof may have a HCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in SEQ ID NO: 70, and/or a LCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in SEQ ID NO: 71. In some embodiments, a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence of SEQ ID NOs: 76, and/or a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence of SEQ ID NO: 77.

    [0130] In some embodiments, an anti-PD-L1 antibody of the disclosure is atezolizumab. In some embodiments, an anti-PD-L1 antibody of the disclosure is durvalumab or avelumab.

    C-2. Exemplary Anti-PD-1 Antibodies as the PD-1 Axis Inhibitor

    [0131] In some embodiments, a provided PD-1 axis inhibitor is an anti-PD-1 antibody or antigen-binding domain thereof. In some embodiments, a provided combination comprises an anti-PD-1 antibody or antigen-binding domain thereof.

    [0132] Illustrative examples of anti-PD-1 antibodies of the disclosure may comprise an antigen-binding region which cross-blocks or binds to the same epitope as an anti-PD-1 antibody comprising the VH and VL regions of a previously known antibody, such as nivolumab (also known as ONO-4538, BMS-936558, or MDX1106, marketed as Opdivo®), pembrolizumab (also referred to as lambrolizumab or MK03475, trade name Keytruda®), PDR001, tislelizumab (BGB-A317), cemiplimab (REGN281), MEDI0680 (formerly AMP-514), pidilizumab (CT-011), ENUM-388D4 (including the D4-1, D4-2 and D4-3 variants), ENUM-244C8 (including each of its variants as well), and anti-PD-1 antibodies disclosed in the U.S. Patent Application Publication Nos. US 2003/0039653, US 2004/0213795, US 2006/0110383, US 2007/0065427, US 2007/0122378, US 2009/0217401, US 2011/0008369, and US2015/0203579 and PCT International Application Publication Nos. WO 2003/099196, WO 2006/121168, WO 2007/005874, WO 2008/156712, WO 2009114335, WO 2010/027423, WO2 011/110604, WO 2012/145493, WO 2013/014668, WO 2014/194302, WO 2015/035606, and WO 2016/106159. A provided anti-PD-1 antibody also may be or comprises an antigen-binding region or the VH and VL regions or of any one of the above-mentioned anti-PD-1 antibodies.

    [0133] In some embodiments, a provided anti-PD-1 antibody or antigen-binding domain thereof may comprise an antigen-binding region, such as any one of the three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the three light chain CDRs (LCDR1, LCDR2 and LCDR3) from an antibody selected from the group consisting of nivolumab, pembrolizumab, PDR001, MEDI0680, pidilizumab, ENUM-388D4, and ENUM-244C8.

    [0134] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof may comprise the three heavy chain CDRs of SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, and/or the three light chain CDRs of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51. In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof may comprise the three heavy chain CDRs of SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, and/or the three light chain CDRs of SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57.

    [0135] In some embodiments, a provided anti-PD-1 antibody or antigen-binding domain thereof may have a HCVR of any one of SEQ ID NOs: 66 and 68, and/or a light chain variable LCVR of any one of SEQ ID NOs: 67 and 69.

    [0136] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof may have a heavy chain of any one of SEQ ID NOs: 72 and 74, and/or a light chain of any one of SEQ ID NOs: 73 and 75.

    [0137] In some embodiments, the heavy chain and light chain pair of a provided PD-1 antibody or antigen-binding domain thereof comprise the HCVR and LCVR, respectively, as follows: SEQ ID NOs: 66 and 67 or SEQ ID NOs: 68 and 69.

    [0138] In some embodiments, the heavy chain and light chain pair of a provided PD-1 antibody are or comprise the amino acid sequences as shown in SEQ ID NOs: 72 and 73 or SEQ ID NO: 74 and 75.

    [0139] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof may have a HCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 66 and 68, and/or a LCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence shown in any one of SEQ ID NOs: 67 and 69. In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof may have a heavy chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence of any one of SEQ ID NOs: 72 and 74, and/or a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence of any one of SEQ ID NO: 73 and 75.

    [0140] In some embodiments, an anti-PD-1 antibody of the disclosure is nivolumab or pembrolizumab. In some embodiments, an anti-PD-1 antibody of the disclosure may be tislelizumab or cemiplimab.

    D. Exemplary Methods of the Disclosure

    [0141] In some embodiments, the present disclosure provides prophylactic and/or therapeutic methods, comprising administering to a subject an effective amount of a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor.

    [0142] In some embodiments, the present disclosure provides methods for increasing IL-2 secretion, preferably in a tumor microenvironment. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to increase IL-2 secretion, preferably in a tumor microenvironment, relative to the bispecific agent or the inhibitor alone. In some embodiments, a provided method may elicit a synergistic increase in IL-2 secretion. In some embodiments, a provided method may elicit an additive increase in IL-2 secretion.

    [0143] In some embodiments, the present disclosure provides methods for increasing IFN-gamma secretion, preferably in a tumor microenvironment. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to increase IFN-gamma secretion, preferably in a tumor microenvironment, relative to the bispecific agent or the inhibitor alone. In some embodiments, a provided method may elicit a synergistic increase in IFN-gamma secretion. In some embodiments, a provided method may elicit an additive increase in IFN-gamma secretion.

    [0144] In some embodiments, the present disclosure provides methods for inducing T lymphocyte activation and/or proliferation. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to induce T lymphocyte activation and/or proliferation. In some embodiments, a provided method may elicit a synergistic T lymphocyte activation and/or proliferation. In some embodiments, a provided method may elicit an additive T lymphocyte activation and/or proliferation.

    [0145] In some embodiments, the present disclosure provides methods for enhancing immune functions. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to induce enhanced immune functions. In some embodiments, a provided method may elicit a synergistic enhancement of immune function. In some embodiments, a provided method may elicit an additive enhancement of immune function.

    [0146] In some embodiments, the present disclosure provides methods for increasing CD4.sup.+ T cells, preferably in a tumor microenvironment. In some embodiments, the present disclosure provides methods for increasing CD8.sup.+ T cells, preferably in a tumor microenvironment. In some embodiments, the present disclosure provides methods for activating NK cells and increasing ADCC, preferably in a tumor microenvironment. In some embodiments, the present disclosure provides methods for monitoring and changing tumor biomarker levels, such as CD4, CD8, PD-L1, Ki67, CD137, HER2, and IL-8 levels. Provided methods may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to induce expansion of CD4.sup.+ T cells preferably in a tumor microenvironment, expansion of CD8.sup.+ T cells preferably in a tumor microenvironment, activation of NK cells and increased ADCC preferably in a tumor microenvironment, and/or changes in tumor biomarker levels.

    [0147] In some embodiments, the present disclosure provides methods for inducing CD137 clustering and activation on T cells and directing such T cells to tumor cells, preferably HER2-positive tumor cells. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to induce CD137 clustering and activation on T cells and directing such T cells to tumor cells, preferably HER2-positive tumor cells.

    [0148] In some embodiments, the present disclosure provides methods for inducing a localized lymphocyte response in the vicinity of tumor cells, preferably HER2-positive tumor cells. The method may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to induce a localized lymphocyte response in the vicinity of tumor cells, preferably HER2-positive tumor cells.

    [0149] In some embodiments, the present disclosure provides methods for treating or delaying cancer progression. In some embodiments, the present disclosure provides methods for providing anti-tumor effects, such as reduction of tumor size, suppression of tumor growth, suppression of metastasis, delay of recurrence, and improved overall survival. Provided methods may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, exerts anti-tumor effects.

    [0150] In some embodiments, the present disclosure provides methods for treating HER2-positive advanced or metastatic solid tumors. In some embodiments, the present disclosure provides methods for treating PD-L1-positive tumors. Provided methods may comprise administering to a subject a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, each in an amount that, when in combination with the other, is effective to exert anti-tumor effects. In some embodiments, a provided method may elicit a synergistic anti-tumor effect. In some embodiments, a provided method may elicit an additive anti-tumor effect.

    [0151] In some embodiments, with respect to provided methods comprising administering to a subject an effective amount of a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, the CD137/HER2 bispecific agent may comprise the amino acid sequences selected from the group consisting of SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, and SEQ ID NOs: 79 and 84, and/or the PD-1 axis inhibitor may comprise the amino acid sequences selected from the group consisting of SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, and SEQ ID NOs: 76 and 77 or may be nivolumab, pembrolizumab, or atezolizumab.

    [0152] In some embodiments, with respect to provided methods comprising administering to a subject an effective amount of a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, the CD137/HER2 bispecific agent may comprise the amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 83 and 80, and SEQ ID NOs: 79 and 84, and/or the PD-1 axis inhibitor may comprise the amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 72 and 73, SEQ ID NOs: 74 and 75, and SEQ ID NOs: 76 and 77, or those of nivolumab, pembrolizumab, or atezolizumab. The meaning of sequence identities to bispecific fusion proteins comprising two amino acid chains as defined above applies mutatis mutandis to antibodies.

    [0153] In some embodiments, with respect to provided methods comprising administering to a subject an effective amount of a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor, the combination is a combination from the group consisting of SEQ ID NOs: 81 and 80 and SEQ ID NOs: 72 and 73, SEQ ID NOs: 81 and 80 and SEQ ID NOs: 74 and 75, SEQ ID NOs: 81 and 80 and SEQ ID NOs: 76 and 77.

    [0154] In some embodiments, provided methods result in a response in a subject. In some embodiments, a response is a partial response. In some embodiments, a response is a complete response. In some embodiments, a response is a sustained response (e.g., a sustained partial response or complete response) in the subject after cessation of a treatment.

    [0155] In some embodiments, a subject of the disclosure may have been treated with a cancer therapy before the treatment of a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor. In some embodiments, a subject of the disclosure may have cancers that are resistant to one or more cancer therapies, where the cancers may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, a subject of the disclosure may have cancers that are resistant to trastuzumab. As described herein, the cancers may be at an early stage or at a late stage.

    [0156] In some embodiments, a subject of the disclosure may suffer from advanced or metastatic breast cancer.

    [0157] In some embodiments, provided methods include administration of an effective amount of an CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84). In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered to the individual at a dose of about 0.05 mg/kg to about 8 mg/kg bodyweight, about 0.15 mg/kg to about 8 mg/kg bodyweight, about 0.5 mg/kg to about 8 mg/kg bodyweight, about 1 mg/kg to about 8 mg/kg bodyweight, about 0.05 mg/kg to about 5 mg/kg bodyweight, about 0.15 mg/kg to about 5 mg/kg bodyweight, about 0.5 mg/kg to about 5 mg/kg bodyweight, or about 1 mg/kg to about 5 mg/kg bodyweight. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered to the individual at a dose of about 0.5 mg/kg to about 8 mg/kg bodyweight, about 1 mg/kg to about 8 mg/kg bodyweight, about 0.5 mg/kg to about 5 mg/kg bodyweight, or about 1 mg/kg to about 5 mg/kg bodyweight. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered to the individual at a dose of about 1 mg/kg to about 8 mg/kg bodyweight. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered to the individual at a dose of about 1 mg/kg to about 5 mg/kg bodyweight. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered at a dose of about 0.05 mg/kg bodyweight, about 0.15 mg/kg bodyweight, about 0.5 mg/kg bodyweight, about 1.0 mg/kg bodyweight, about 2.5 mg/kg bodyweight, about 5.0 mg/kg bodyweight, or about 8.0 mg/kg bodyweight. As a general proposition, the therapeutically effective amount of an CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) administered to a human will be in the range of about 0.01 to about 50 mg/kg, whether by one or more administrations. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) may be administered from about weekly to every about 4 weeks, from about weekly to every about 3 weeks, or from about weekly to every about 2 weeks. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) may be administered about weekly, every about 2 weeks, every about 3 weeks, or every about 4 weeks. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) may be administered on days 1 of each cycle, on days 1 of each 7-day cycle, on days 1 and 8 of each 14-day cycle, on days 1, 8 and 15 of each 21-day cycle, or on days 1, 8, and 15 of each 28-day cycle. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) may be administered every about 3 weeks, on days 1 of each cycle. In some embodiments, at least one cycle, such as 2, 3, 4, 5, 10, 15, 20, 25, 30 or more cycles, are administered. In some embodiments, the provided CD137/HER2 bispecific agent has a sequence as set forth in SEQ ID NOs: 81 and 80.

    [0158] In some embodiments, provided methods of the disclosure include administration of an effective amount of a PD-1 axis inhibitor selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, and a PD-L2 antagonist.

    [0159] In some embodiments, a PD-L1 antagonist is an antibody, such as an antibody that is capable of inhibiting PD-L1 binding to its binding partner(s), such as PD-1 and B7.1. In some embodiments, a PD-L1 antagonist is the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77, which may be administered at a dose of about 800 mg to about 1500 mg from about weekly to every about 4 weeks, from about weekly to every about 3 weeks, from about weekly to every about 2 weeks, such as about weekly, every about 2 weeks, every about 3 weeks, or every about 4 weeks (e.g., about 1000 mg to about 1300 mg, e.g., about 1100 mg to about 1200 mg). In some embodiments, a PD-L1 antagonist is the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77, which may be administered at a dose of about 800 mg to about 1500 mg every about three weeks (e.g., about 1000 mg to about 1300 mg every about three weeks, e.g., about 1100 mg to about 1200 mg every about three weeks). In some embodiments, the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77 may be administered at a dose of about 1200 mg every about three weeks.

    [0160] In some embodiments, a PD-1 antagonist is an antibody, such as an antibody that is capable of inhibiting PD-1 binding to its binding partner(s), such as PD-L1 and PD-L2. In some embodiments, a PD-1 antagonist is the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, which may be administered at a dose of about 100 mg to about 600 mg every about one, two, there, four, five or six weeks (e.g., about 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, or 600 1300 mg every about one, two, there, four, five or six weeks). In some embodiments, the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 may be administered at a dose of about 240 mg every about two weeks. In some embodiments, the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 may be administered at a dose of about 480 mg every about four weeks. In some embodiments, the PD-1 antibody set forth in SEQ ID NOs: 74 and 75 may be administered at a dose of about 200 mg every about three weeks.

    [0161] As a general proposition, the therapeutically effective amount of a PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations. In some embodiments, for example, a provided PD-1 axis inhibitor(e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered in a dose of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered at about 15 mg/kg. In some embodiments, the PD-1 axis inhibitor set forth in SEQ ID NOs: 72 and 73 may be administered at about 3 mg/kg every about two weeks. In some embodiments, the PD-1 axis inhibitor set forth in SEQ ID NOs: 74 and 75 may be administered at about 2 mg/kg every about three weeks. Other dosage regimens may also be useful. In one embodiment, a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered to a human at a flat dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered at a dose of about 1150 mg to about 1250 mg every about three weeks. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered at a dose of about 1200 mg every about three weeks. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73) may be administered at a dose of about 240 mg. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 74 and 75) may be administered at a dose of about 200 mg. In some embodiments, a dose of a provided PD-1 axis inhibitor of the disclosure may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses). In some embodiments, a provided PD-1 axis inhibitor may be administered at a reduced dose, when administered in a combination treatment as compared to a single treatment. In some embodiments, for example, a provided method for treating or delaying progression of cancer in a subject comprises a dosing regimen comprising treatment cycles, wherein a subject is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) at a dose of about 1200 mg, wherein each cycle is about 21 days (i.e., each cycle is repeated every about 21 days). In some embodiments, for example, a provided method for treating or delaying progression of cancer in a subject comprises a dosing regimen comprising treatment cycles, wherein a subject is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73) at a dose of about 240 mg wherein each cycle is about 14 days or at a dose about 480 mg wherein each cycle is about 28 days. In some embodiments, for example, a provided method for treating or delaying progression of cancer in a subject comprises a dosing regimen comprising treatment cycles, wherein a subject is administered, on days 1 of each cycle, a human PD-1 axis binding antagonist (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 74 and 75) at a dose of about 240 mg, wherein each cycle is about 21 days. In some embodiments, at least one cycle, such as 2, 3, 4, 5, 10, 15, 20, 25, 30 or more cycles, are administered.

    [0162] In some embodiments, a CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) and a PD-1 axis inhibitor (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) of the disclosure are administered in a single dosing regimen. The administration of these components may be sequentially (at different times) or concurrently (at the same time) within the context of the dosing regimen. For example, in some embodiments, methods of the disclosure comprises a dosing regimen comprising treatment cycles, wherein a subject is administered, on days 1 of each cycle, an CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) at a dose of about 0.05 mg/kg bodyweight, 0.15 mg/kg bodyweight, 0.5 mg/kg bodyweight, 1.0 mg/kg bodyweight, 2.5 mg/kg bodyweight, 5.0 mg/kg bodyweight, or 8.0 mg/kg bodyweight, and immediately after, a PD-1 axis inhibitor (e.g., a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) is administered at a dose about 1200 mg. In some embodiments, each cycle is about 21 days. In some embodiments, at least one cycle, such as 2, 3, 4, 5, 10, 15, 20, 25, 30 or more cycles, are administered.

    [0163] In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered in a separate composition as a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77). In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) is administered in the same composition as a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77).

    [0164] In some embodiments, a CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) and a PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) of the disclosure are administered by the same route of administration. In some other embodiments, a CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) and a PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) of the disclosure are administered by the same route of administration or by different routes of administration. In some embodiments, a provided CD137/HER2 bispecific agent (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, a provided PD-1 axis inhibitor (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77) may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

    [0165] In some embodiments, provided methods may further comprise an additional therapy. In some embodiments, an additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. Such additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, an additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.).

    E. Pharmaceutical Formulations

    [0166] In some embodiments, molecules of the disclosure including provided combinations and components of combinations, such as CD137/HER2 bispecific agents (e.g., SEQ ID NOs: 81 and 80, SEQ ID NOs: 79 and 82, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 79 and 84) and PD-1 axis inhibitors (e.g., a PD-1 antagonist, such as the PD-1 antibody set forth in SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75, or a PD-L1 antagonist, such as the PD-L1 antibody set forth in SEQ ID NOs: 76 and 77), may be formulated in accordance with standard pharmaceutical practice for use as “active ingredients” of therapeutic compositions. Compositions comprising such molecules may contain one or more pharmaceutically acceptable carrier, glidant, diluent, or excipient, which facilitate administration of the composition and/or facilitate delivery of the composition to the site of action. Suitable carriers, diluents and excipients are known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. Compositions of the disclosure may be in any suitable form, for example tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders, to name just a few non-limiting alternatives. Such compositions (or formulations) may be prepared using methods known in the art, such as conventional dissolution and mixing procedures.

    [0167] In some embodiments, formulations of the disclosure may be prepared for various routes and types of administration in the form of a lyophilized formulation, milled powder, or an aqueous solution.

    [0168] In some embodiments, the CD137/HER2 bispecific agent is formulated as aqueous solution with a target protein concentration of about 25 mg/mL. In some embodiments, a commercial formulation of SEQ ID NOs: 76 and 77 may be used containing SEQ ID NOs: 76 and 77 at about 1200 mg/20 mL (60 mg/mL) in the form of a preservative-free solution for intravenous infusion.

    [0169] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure.

    V. EXAMPLES

    Example 1

    Assessment of T-Cell Activation Induced by the Combination of a CD137/HER2 Bispecific Agent and a PD-1 Antibody

    [0170] A T cell assay was employed to assess the ability of a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80, when used in combination with an anti-PD-1 antibody at fixed molar ratio, to costimulate T-cell responses. For this purpose, the combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and a PD-1 antibody shown in SEQ ID NOs: 72 and 73 were added to staphylococcal enterotoxin B (SEB) stimulated human peripheral blood mononuclear cells (PBMCs), in the presence of the tumor cell line NCI-N87, and incubated for 3 days at 37° C. IL-2 secretion levels, as a demonstration of T-cell activation, were measured in the supernatants.

    [0171] PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1.077 g/mL, Biochrom), following Biochrom's protocols. The purified PBMCs were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down and stored in liquid nitrogen until further use. For the assay, PBMCs were thawed and rested in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies) for 16 h at 37° C. in a humidified 5% CO.sub.2 atmosphere.

    [0172] The following procedure was performed using triplicates for each experimental condition: tumor cell line NCI-N87 were treated for 30 min at 37° C. with 30 μg/ml mitomycin C (Sigma Aldrich) in order to block proliferation. Mitomycin treated cells were then washed twice in culture medium and plated at 2.5×10.sup.4 cells per well onto a 384 well flat-bottom tissue culture plates to allow adhesion overnight at 37° C. in a humidified 5% CO.sub.2 atmosphere. The target cells had before been grown under standard conditions, detached using Accutase (PAA Laboratories), and resuspended in culture media.

    [0173] On the next days, after washing the plates twice with PBS, 2.5×10.sup.4 PBMCs per well were added to the tumor cells. A dilution series of a combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-1 antibody (SEQ ID NOs: 72 and 73) (1:10 molar ratio), the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) alone, or the PD-1 antibody (SEQ ID NOs: 72 and 73) alone, typically ranging from 0.001 nM to 100 nM, together with 0.05 ng/mL SEB, were added to the respective wells. Plates were covered with a gas permeable seal (4titude) and incubated at 37° C. in a humidified 5% CO.sub.2 atmosphere for 3 days. Subsequently, IL-2 levels in the supernatant were assessed using the human IL-2 DuoSet kit (R&D Systems) as described in the following procedures.

    [0174] 384 well plates were coated for 2 h at room temperature with 1 μg/mL “Human IL-2 Capture Antibody” in PBS. Subsequently, wells were washed 5 times with 80 μl PBS supplemented with 0.05% Tween (PBS-T). After 1 h blocking in PBS-T containing 1% casein (w/w), assay supernatants and a concentration series of an IL-2 standard diluted in culture medium was transferred to respective wells and incubated overnight at 4° C. The next day, a mixture of 100 ng/mL goat anti-hlL-2-Bio detection antibody (R&D Systems) and 1 μg/mL Sulfotag-labelled streptavidin (Mesoscale Discovery) in PBS-T containing 0.5% casein were added and incubated at room temperature for 1 h. After washing, 25 μL reading buffer (Mesoscale Discovery) was added to each well and the resulting electrochemiluminescence (ECL) signal was detected using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

    [0175] The result of representative experiments is depicted in FIG. 1 and the fitted EC.sub.50s for induced IL-2 secretion is summarized in Table 1. An hIgG4 isotype control antibody was tested to set the basal activity. The combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-1 antibody (SEQ ID NOs: 72 and 73) were able to induce a dose-dependent secretion of IL-2, with an improved (lower) EC.sub.50 values as compared to the CD137/HER2 bispecific agent or the PD-1 antibody alone. Meanwhile, IL-2 levels induced by the combination were higher compared to equimolar concentrations of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) or the PD-1 antibody (SEQ ID NOs: 72 and 73) and displayed a synergistic or additive effect.

    TABLE-US-00001 TABLE 1 Efficacy of inducing IL-2 secretion Molecule(s) EC.sub.50 (Donor A) EC.sub.50 (Donor B) SEQ ID NOs: 81 and 80  0.1494 nM  0.1165 nM SEQ ID NOs: 72 and 73  0.4198 nM  0.8538 nM SEQ ID NOs: 81 and 80 + 0.05462 nM 0.05817 nM SEQ ID NOs: 72 and 73

    Example 2

    Assessment of T-Cell Activation Induced by a CD137/HER2 Bispecific Agent in Combination with a PD-1 Antibody

    [0176] Another T cell assay was employed to assess the ability of a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80, when used in combination with a fixed concentration of a PD-1 antibody, to costimulate T-cell responses. For this purpose, the combination of a CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) at various concentrations and a PD-1 antibody shown in SEQ ID NOs: 72 and 73 or in SEQ ID NOs: 74 and 75 at a fixed concentration were added to SEB stimulated human PBMCs, in the presence of the tumor cell line NCI-N87, and incubated for 3 days at 37° C. IL-2 secretion levels, as a demonstration of T-cell activation, were measured in the supernatants.

    [0177] PBMCs from healthy volunteer donors were isolated and treated as described in Example 1. Tumor cell line NCI-N87 were treated and plated at 2.5×10.sup.4 cells per well the day before the assay as described in Example 1.

    [0178] The following procedure was performed using triplicates for each experimental condition: NCI-N87 coated plates were washed twice with PBS, and 2.5×10.sup.4 PBMCs per well were added to the tumor cells. The CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) at various concentrations, ranging from 0.0002 nM to 10 nM, alone or in combination with SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75 at 10 nM or 100 nM were added to the respective wells, together with 1 ng/mL SEB. In the same experiment, the PD-1 antibody (SEQ ID NOs: 72 and 73 or SEQ ID NOs: 74 and 75) was also titrated alone for comparison. Plates were then covered with a gas permeable seal (4titude) and incubated at 37° C. in a humidified 5% CO.sub.2 atmosphere for 3 days. Afterward, IL-2 levels in the supernatant were assessed as described in Example 1.

    [0179] The results of representative experiments are depicted in FIG. 2 (the CD137/HER2 bispecific agent tested at 10 nM and 3.33 nM; data at other concentrations not shown). The combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) with either of the tested PD-1 antibody (SEQ ID NOs: 72 and 73 and SEQ ID NOs: 74 and 75) induced higher IL-2 secretion and act synergistically or additively. When the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) was combined with SEQ ID NOs: 72 and 73, the induced IL-2 secretion levels were higher when 100 nM antibody was used than when 10 nM antibody was used. On the other hand, the concentration (at 100 nM or 10 nM) of SEQ ID NOs: 74 and 75 did not affect the IL-2 secretion when used in combination with the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80).

    Example 3

    Assessment of T-Cell Activation Induced by a CD137/HER2 Bispecific Agent in Combination with a PD-L1 Antibody in the Presence of Tumor Cells Expressing Different Level of HER2 and/or PD-L1

    [0180] To assess the ability of a combination of the CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 and a PD-1 axis inhibitor to costimulate T-cell activation in a HER2 target dependent manner, a further T cell assay was employed. The combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) different concentrations with a PD-L1 antibody shown in SEQ ID NOs: 76 and 77 at a fixed concentration was applied to SEB stimulated T cells, in the presence of a tumor cell line with different HER2 and/or PD-L1 expression levels. Tested tumor cell lines include NCI-N87 (HER2 high, PD-L1 low), JIMT-1 (HER2 moderate, PD-L1 moderate) and MDA-MB-231 (HER2 low, PD-L1 moderate).

    [0181] PBMCs from healthy volunteer donors were isolated and treated as described in Example 1.

    [0182] The following procedure was performed using triplicates for each experimental condition: the tumor cell lines NCI-N87, JIMT-1, and MDA-MB-231 were grown or treated with mitomycin C as described in Example 1 and plated at 8.3×10.sup.3 cells per well in culture medium to allow adhesion overnight at 37° C. in a humidified 5% CO.sub.2 atmosphere. On the next days, after washing the plates coated with tumor cells twice with PBS, 2.5×10.sup.4 PBMCs per well were added to each well. The CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) at various concentrations, ranging from 0.0002 nM to 10 nM, alone or in combination with the PD-L1 antibody (SEQ ID NOs: 76 and 77) or hIgG4 isotype control antibody at 100 nM were added to the respective wells, together with 0.05 ng/mL SEB. In the same experiment, the PD-L1 antibody also titrated alone for comparison. Plates were then covered with a gas permeable seal (4titude) and incubated at 37° C. in a humidified 5% CO.sub.2 atmosphere for 3 days. Afterward, IL-2 levels in the supernatant were assessed as described in Example 1.

    [0183] Exemplary data of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) tested at the concentration of 2.5 nM and 0.16 nM are shown in FIG. 3 (data of the bispecific agent at other concentrations not shown). Co-culturing of T cells with NCI-N87 (HER2 high, PD-L1 low) or JIMT-1 (HER2 moderate, PD-L1 moderate) in presence of the combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-L1 antibody (SEQ ID NOs: 76 and 77) led to a statistically significant increased IL-2 secretion as compared to the bispecific agent or the antibody alone or a combination of the bispecific agent and an hIgG4 isotype control antibody (FIGS. 3A and 3B). Additionally, co-culturing with MDA-MB-231 (HER2 low, PD-L1 moderate) with the combination of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) and the PD-L1 antibody (SEQ ID NOs: 76 and 77) induced higer IL-2 secretion, however, only to the same extend as when the antibody was used alone. This is likely resulting from the Her2 expression on MDA-MB-231 is not sufficient for the CD137/HER2 bispecific agent to have any effects.

    [0184] Overall, the data indicate that the functional activity of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) in combination with the PD-L1 antibody (SEQ ID NOs: 76 and 77), measured by their ability to activate T cells or increase IL-2 secretion, is synergistic or additive and depend on Her2 and PD-L1 expression profile on tumor cells.

    Example 4

    Assessment of Functional In Vivo Activity in a Xenograft Mouse Model Engrafted with Human PBMCs

    [0185] In order to investigate the in vivo activity of a provided combination comprising a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 and a PD-1 axis inhibitor, cell line-derived xenograft mouse model is used. Accordingly, a human cancer cell line is implanted subcutaneously in immune deficient NOG mice, delivered at the age of 4-6 weeks with at least 1 week of quarantine. After the tumors have been reaching volumes of approximately 80-100 mm.sup.3, mice are substituted with human PBMCs. Test molecule(s) are injected at least three times and tumor growth and activity is constantly measured. After reaching study end, mice are sacrificed. Intratumoral infiltration of CD3-, CD4- and CD8-positive cells are assessed via immunohistochemistry. IFN-gamma RNAscope is conducted as further read-out.

    Example 5

    Assessment of Functional In Vivo Activity in a CD137 Humanized Mouse Model

    [0186] In order to investigate the in vivo activity of a provided combination comprising a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 and a PD-1 axis inhibitor, a mouse cell line-derived xenograft mouse model over-expressing human Her2 is used. Accordingly, a mouse cancer cell line is implanted subcutaneously in humanized CD137 C57b mice, delivered at the age of 8-10 weeks with at least 1 week of quarantine. After the tumors have been reaching volumes of approximately 50-80 mm.sup.3, mice are randomized in homogeneous groups. Test molecule(s) are injected at least three times and tumor growth and activity will be constantly measured. After reaching study end, mice are sacrificed. Intratumoral infiltration of CD3-, CD4- and CD8-positive cells are assessed via immunohistochemistry. IFN-gamma RNAscope is conducted as further read-out.

    Example 6

    Clinical Study of CD137/HER2 Bispecific Agent in Combination with PD-1 Axis Inhibitor

    [0187] Data discussed above indicate that a combination comprising a CD137/HER2 bispecific agent and a PD-1 axis inhibitor can act additively or synergistically to stimulate T cell activation in tumor microenvironment. Such a combination could therefore provide clinical benefit in patients with cancer.

    [0188] A Phase Ib, open-label, dose escalation study of a CD137/HER2 bispecific agent shown in SEQ ID NOs: 81 and 80 in combination with a PD-L1 antibody shown in SEQ ID NOs: 76 and 77 in patients with specific HER2-positive advanced or metastatic solid tumors is designed to determine the maximum tolerated dose (MTD) and Phase 2 dose (RP2D). Secondary objectives include assessing efficacy, safety, pharmacokinetics, and immunogenicity of the combination of the CD137/HER2 bispecific agent and the PD-L1 antibody. Exploratory objectives include assessing the preliminary antitumor activity of the combination, the CD137/HER2 bispecific agent exposure-response relationships, and biomarkers. The study includes a dose escalation period followed by an expansion period (FIG. 4A).

    [0189] One treatment cycle is defined as 21 days and consists of intravenous infusions of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 89) given every 3 weeks (Q3W) in combination with the PD-L1 antibody (SEQ ID NOs: 76 and 77) given Q3W (FIG. 4B). Patients receive the CD137/HER2 bispecific agent as a 2-hour intravenous infusion on Day 1 of each cycle, immediately followed by the PD-L1 antibody (1200 mg fixed dose) administered over 60 (±15) minutes. Subsequent doses of the PD-L1 antibody may be administered over 30 (±10) minutes if the first dose is well tolerated.

    [0190] Dosing of the initial two patients at a given dose level is staggered by a minimum of 7 days. A safety review is conducted after the first patient has completed Day 8 visit. If no dose-limiting toxicities (DLTs) are observed for the first patient, staggering may be reduced to 72 hours for the second patient. No staggering is required for subsequent patients if no DLTs are observed in the first two patients. A DLT, as used herein, is defined as an adverse effect (AE) occurring in Cycle 1 for which other causes cannot be identified (and is therefore possibly related to study treatment) and satisfying at least one of the pre-set criteria. Toxicities are graded and documented according to the NCI CTCAE, version 4.03 guidelines. Patients who discontinue prior to completion of cycle 1 are replaced.

    [0191] Dose escalation proceeds using a modified toxicity probability interval (mTPI) adaptive design to make dose recommendations and estimate the maximum tolerated dose (MTD) of the CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) in combination with the approved dose of 1200 mg of the PD-L1 antibody (SEQ ID NOs: 76 and 77). The mTPI design is a model-based approach that has a pre-specified decision matrix that recommends escalating, reducing or maintaining the same dose or stopping dose escalation, based on the number of DLTs observed in the dose level under evaluation. The MTD is defined as the dose level that has an estimated probability of toxicity closest to 0.3. It is expected that 3 to 6 subjects will be enrolled in each dose escalation cohort. Intra-patient dose escalation is not permitted.

    [0192] Dose escalation begins at a dose level of 0.05 mg/kg every 3 weeks (Q3W) in combination with a fixed dose of 1200 mg the PD-L1 antibody Q3W. After a minimum of 3 subjects within this dose have been treated at the target dose level and evaluated through the DLT evaluation period, if the mTPI model recommends escalation, then enrollment may begin for next dose level. The mTPI model's recommendations are based only on DLTs. However, all available safety data as well as emerging PK, persistence and pharmacodynamic data, and DLT modeling recommendations are considered in determining subsequent doses to be tested. Dose escalation continues until the MTD has been reached, until the maximum sample size (30 subjects) has been reached, or until a stopping rule for safety or underdosing takes effect, whichever occurs first. Dose levels of the CD137/HER2 bispecific agent and PD-L1 are tested during the dose escalation period per Table 2. The doses of the CD137/HER2 bispecific agent are adjusted or interrupted as appropriated based on treatment modifications.

    TABLE-US-00002 TABLE 2 Doses of the CD137/HER2 bispecific agent and the PD-L1 antibody evaluated in the dose escalation period CD137/HER2 PD-L1 antibody Minimum bispecific agent (SEQ ID Dose No. of (SEQ ID NOs: 81 and 80) NOs: 76 and 77) Level Patients Dose (mg/kg) Dose (mg) 1 3 0.05 1200 2 3 0.15 1200 3 3 0.5 1200 4 3 1.0 1200 5 3 2.5 1200 6 3 5.0 1200 7 3 8.0 1200

    [0193] Once the MTD has been established, the expansion period of the study investigates the preliminary efficacy and PD effects, in addition to safety and tolerability, of the RP2D established from the dose-escalation period.

    [0194] Dosing continues until criteria for study treatment discontinuation are met (disease progression or withdrawal from the study) or up to 30 cycles. Patients are assessed for tumor response/progression every 6 weeks for the initial 24 weeks of dosing (first 8 cycles) and every 12 weeks after the Week 24 scans.

    [0195] For the dose-finding cohorts, after the last patient in a given cohort has completed at least 1 cycle of study treatment, all safety data are reviewed to determine whether to continue or halt dose escalation, expand individual dose levels to gain additional safety data, determine the MTD and/or RP2D, or explore other dose levels/schedules.

    [0196] Formulations—The CD137/HER2 bispecific agent (SEQ ID NOs: 81 and 80) is provided as aqueous solution in a 20-mL Type I USP/European Pharmacopeia glass vial with a nominal fill of 16 for infusion. The CD137/HER2 bispecific agent is formulated with a target protein concentration of 25 mg/mL in 20 mM Histidine, 250 mM Sorbitol, pH 6.3, 0.01% PS80. The PD-L1 antibody (SEQ ID NOs: 76 and 77) is provided as aqueous solution in a single-use, 20-cc Type 1 USP/European Pharmacopeia glass vial. The vial is designed to deliver 20 mL (1200 mg) of the PD-L1 antibody formulated as 60 mg/mL in 20 mM histidine acetate, 120 mM sucrose, 0.04% polysorbate 20, pH 5.8.

    [0197] Safety assessment—The safety and tolerability of the CD137/HER2 bispecific agent and the PD-L1 antibody are assessed based on AEs (incidence and severity), performance status, physical examinations, 12-lead resting electrocardiograms (ECGs), LVEF assessments and laboratory safety evaluations. Laboratory abnormalities and AEs are graded according to NCI CTCAE v4.03. Management of immune-related (ir) AEs provides detailed guidance for the handling of immune-related (ir) AEs. AEs, ECGs, LVEF assessments and laboratory data are reviewed and summarized on an ongoing basis during the study. All patients who receive at least 1 dose of study treatment are included in the safety analyses.

    [0198] Pharmacokinetic assessment—The single and multiple dose pharmacokinetic profiles of the CD137/HER2 bispecific agent and the PD-L1 antibody are investigated during the course of the study using the following parameters: (1) the integral of the concentration-time curve (area under the concentration-time curve, AUC); (2) maximum plasma, blood, serum, or other body fluid drug concentration (C.sub.max); (3) time to reach Cmax (T.sub.max); (4) terminal half-life (t.sub.1/2); (5) the volume of plasma cleared of the drug per unit time clearance (clearance, CL); (6) the apparent volume of distribution during terminal phase (V.sub.z); and (7) accumulation ratio (AR; AR=C.sub.max (multiple dose)/C.sub.max (single dose)).

    [0199] For the assessment of the pharmacokinetics of the CD137/HER2 bispecific agent, peripheral venous blood (4 mL) is collected from all patients during Cycles 1 and 3 at pre-bispecific agent infusion, and at 5 minutes, 4, 8, 24, 48, 72, 168, and 336 hours after the end of bispecific agent infusion including flush. Samples are also collected pre-bispecific agent infusion on Day 1 of Cycles 2, 4-6, 8, 12, and 16 for quantitation of the bispecific agent.

    [0200] For the assessment of the pharmacokinetics of the PD-L1 antibody, peripheral venous blood (4 mL) is collected from all patients during Cycle 1 at pre-antibody infusion, and at 30 minutes after the end of the PD-L1 antibody infusion including flush. Samples are also collected pre-antibody infusion on Day 1 of Cycles 2, 4, 8, 12, 16 for quantitation of the PD-L1 antibody.

    [0201] Pharmacodynamic assessment—The pharmacodynamic response of the CD137/HER2 bispecific agent and the PD-L1 antibody are assessed by quantifying lymphocyte subtypes or markers in tumor biopsies or peripheral blood and cytokine levels in plasma. Selected pharmacodynamic markers planned for analysis (samples will be taken predose and postdose at pre-specified time points) are presented in Table 3.

    TABLE-US-00003 TABLE 3 Pharmacodynamic markers evaluated Source Marker Biopsy IHC (e.g. CD4, CD8, PDL-1, Ki67, FoxP3, etc.) Genomic analysis Blood Phenotyping (e.g. CD8 T cells, CD4 T cells) Plasma e.g., OD137, HER2, interleukin 8 (IL-8), IFN-gamma

    [0202] Biomarker assessment—Peripheral blood and fresh tumor tissue are collected prior to therapy and at selected time points on treatment (tumor biopsy). Residual sample material available after completion of the designated analyses are used for identification of additional pharmacodynamic or predictive markers or to enhance understanding of disease biology unless prohibited by local laws or regulations. Samples are de-identified to ensure patient privacy.

    [0203] Efficacy assessment—The efficacy of the CD137/HER2 bispecific agent and the PD-L1 antibody is assessed by evaluating tumor response and progression according to RECIST, Version 1.1. Patients are assessed every 6 weeks for the initial 24 weeks of dosing (first 8 cycles). After the Week 24 scans, tumor assessments are conducted every 12 weeks. Tumor assessments would consist of clinical examination and appropriate imaging techniques, e.g., computerized tomography (CT) scans of the chest, abdomen, and pelvis with appropriate slice thickness per RECIST. Other studies such as magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET) scan, and ultrasound are performed as required. The same methods used to detect lesions at baseline are used to follow the same lesions throughout the clinical study.

    [0204] Immunogenicity assessment—The immunogenicity of the CD137/HER2 bispecific agent and the PD-L1 antibody is assessed based on antidrug antibody levels in the venous blood samples. Additionally, the concentration/adverse event—immunogenicity relationship was explored graphically, and tabulated to characterize a relationship between the changes from screening immunogenicity presence and serum concentration of the CD137/HER2 bispecific agent. The potential correlation between immunogenicity and other endpoints (major safety, efficacy and biomarker parameters) is also evaluated.

    [0205] Statistical methods—Tabular summaries of data are descriptive in nature (i.e., number of patients [n], mean, standard deviation, median, minimum and maximum for continuous variables and n and percent for categorical variables). A more detailed description of analysis methods is provided in the statistical analysis plan (SAP) to be completed prior to the clinical database lock.

    [0206] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks, and peer-reviewed publications described herein are hereby incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the following claims.

    [0207] Equivalents: those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

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