Compositions and methods for selective activation of cytokine signaling pathways - Patents.com

Abstract

The present disclosure provides compositions of bispecific antibodies useful in selective cytokine activation on immune cells. The present disclosure also provides compositions of bispecific antibodies useful in the treatment of cancers expressing tumor-associated antigens.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Patent Application No. 63 / 302,514, filed January 24, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing The sequence listing XML associated with this application is provided electronically in XML file format and is incorporated herein by reference. The name of the XML file containing the sequence listing XML is NOVI_049_001WO_SeqList_ST26.xml. The size of the XML file is 158,062 bytes and was created on January 24, 2023. [Background technology]

[0003] 2. Background of the Invention Interleukin-2 (IL-2) is a 15 kDa cytokine essential for the balance between activation and inhibition of the immune response. Under normal conditions, it is secreted by CD4 + Helper T cells are the major producers of this cytokine. During immune activation, CD4 + and CD8 + Both B-lymphocyte-induced and T-cells can be induced to produce IL-2, and this induction is controlled through a negative feedback loop involving B-lymphocyte-induced maturation protein 1 (BLIMP1).

[0004] The IL-2 receptor (IL-2R) has three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Of the three IL-2R subunits, only IL-2Rα is unique to the IL-2R. IL-2Rγ (also called the common γ chain) is shared with the IL-4, IL-7, IL-9, IL-15, and IL-21 receptor complexes. IL-2Rβ is shared with the interleukin-15 (IL-15) receptor complex. IL-15 is a receptor that activates effector NK cells and CD8 +It is a cytokine for the development, proliferation, and activation of memory T cells. IL-15 binds to the IL-15 receptor α (IL-15Rα) and is presented in trans to the IL-2Rβ-IL2Rγ complex on effector cells. IL-15 and IL-2 share binding to the IL-2Rβ-IL2Rγ complex and signal through the STAT3 and STATS pathways. However, unlike IL-2, IL-15 does not upregulate CD4 + CD25 + FoxP3 + Does not support the maintenance of regulatory T (Treg) cells or activates CD8 + In addition, IL-15 does not induce T cell death. + It is the only cytokine known to confer anti-apoptotic signaling in T cells. IL-15 signaling exhibits potent anti-tumor activity against well-established solid tumors in experimental animal models.

[0005] IL-2 signaling is mediated by a dimeric intermediate affinity receptor composed of IL-2Rβ and IL-2Rγ (10 -9 IL-2Rα can be transduced through the dimeric receptor IL-2 (10 -8 M) and does not mediate signaling by itself. However, in combination with CD122 and CD132, it enhances the binding affinity for IL-2 by 100-fold, mediating the binding of high affinity (100-fold) CD4+Foxp3+ T cells expressed on regulatory T cells (Tregs), activated effector T cells, and endothelial cells. -11 M) forms a trimeric receptor. Because Tregs express the highest levels of IL-2Rα under resting conditions, constitutively low levels of IL-2 selectively induce IL-2 signaling on Treg cells and maintain immune suppression. During immune activation, elevated levels of IL-2 also signal through the dimeric receptor to maintain T and NK cell activation and proliferation.

[0006] Due to its role in promoting T-cell and NK-cell activation and proliferation, IL-2 has been explored as a promising therapeutic target in cancer immunotherapy. High-dose aldesleukin (recombinant human IL-2 (hIL-2)) has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma, with objective response rates ranging from 17 to 20%, with complete responses lasting up to 91 months. However, the inconvenient short half-life of aldesleukin inherent to its molecular nature as a cytokine and the severe toxic effects associated with systemic activation of IL-2 signaling have limited the clinical use of aldesleukin. The most severe toxicity associated with aldesleukin is vascular leak syndrome (VLS), which results in multi-organ edema and damage requiring intensive inpatient monitoring and care. Preclinical studies suggest that VLS is caused by IL-2 signaling in vascular endothelial cells.

[0007] To overcome these limitations and fully exploit the potential of IL-2 targeted therapy, different novel approaches such as hIL-2 / mAb conjugates, IL-2 variants with reduced binding to IL-2Rα, PEGylated IL-2 variants, IL-2 / IL-2Rα fusion proteins, and IL-2Rβ / IL-2Rγ agonistic bispecific antibodies (bsAbs) have been applied to develop molecules with a longer half-life in the tumor microenvironment (TME), reduced preferential activation of Tregs, and / or more selective IL-2 signaling activation.

[0008] The most clinically advanced IL-2 targeting molecule is pegaldesleukin, a PEGylated IL-2 variant in phase III clinical trials (NCT03635983 and NCT03729245) that has shown promising results in which PEGylation of IL-2 extends its half-life and reduces systemic exposure to high levels of active IL-2. However, bempegaldesleukin still has the potential to induce systemic IL-2 signaling and therefore carries the risk of causing systemic toxicity. hIL-2 / mAb conjugates ligate recombinant hIL-2 to a tumor-targeting mAb. The introduction of the tumor-targeting mAb extends the half-life of the molecule and increases tumor specificity. However, the hIL-2 portion of the molecule is still systemically active and therefore still carries the risk of systemic toxicity. Similarly, IL-2Rβ / IL-2Rγ agonistic bsAb has the potential to extend half-life and reduce Treg-preferential IL-2 activation. Nevertheless, they still carry the risk of systemic IL-2 activation and associated toxicity.There is a need for compositions and methods for the selective activation of IL-2. Summary of the Invention

[0009] The present disclosure provides a method for inhibiting tumor growth, below: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A composition comprising Provide for the use of.

[0010] The present disclosure provides a method for the treatment of cancer, below: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A composition comprising Provide for the use of.

[0011] The present disclosure provides a method for enhancing T cell-mediated cell killing, below: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A composition comprising Provide for the use of.

[0012] The present disclosure provides a composition for use in a method of T cell activation, comprising: below: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A composition comprising:

[0013] The present disclosure relates to a) a first composition comprising a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second composition comprising a bispecific antibody having an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A composition comprising:

[0014] In some embodiments, the first tumor associated antigen and the second tumor associated antigen are different tumor associated antigens. In some embodiments, the first tumor associated antigen and the second tumor associated antigen are the same tumor associated antigen. In some embodiments, the first tumor associated antigen and the second tumor associated antigen are expressed on the surface of the same tumor cell.

[0015] In some embodiments, the first tumor associated antigen and / or the second tumor associated antigen is human epidermal growth factor receptor 2 (HER2). In some embodiments, the first tumor associated antigen and / or the second tumor associated antigen is mesothelin (MSLN).

[0016] In some embodiments, the antigen-binding domain that binds to a first tumor-associated antigen and the antigen-binding domain that binds to a second tumor-associated antigen bind to two different epitopes of the same tumor-associated antigen. In some embodiments, the antigen-binding domain that binds to the first tumor-associated antigen and the antigen-binding domain that binds to the second tumor-associated antigen are identical.

[0017] In some embodiments, the first subunit of the cytokine receptor and the second subunit of the cytokine receptor are expressed on the surface of the same immune cell. In some embodiments, the immune cell is a T cell.

[0018] In some embodiments, the cytokine receptor binds IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-12, IL-23, IFNα, IFNβ, IFNε, IFNk, IFNo, IFNδ, IFNτ, IFNω, IFNζ, IFNγ, or IFNλ.

[0019] In some embodiments, the first subunit of the cytokine receptor or the second subunit of the cytokine receptor is IL-2Rγ. In some embodiments, the first subunit of the cytokine receptor is IL-2Rβ and the second subunit of the cytokine receptor is IL-2Rγ.

[0020] In some embodiments, the composition comprises: c) a third bispecific antibody comprising an antigen-binding domain that binds to a third tumor-associated antigen and an antigen-binding domain that binds to an antigen expressed on T cells. Further includes:

[0021] In some embodiments, the third tumor associated antigen is different from the first tumor associated antigen and the second tumor associated antigen. In some embodiments, the antigen expressed on T cells is CD3.

[0022] In some embodiments, the first bispecific antibody, the second bispecific antibody, and / or the third bispecific antibody have an IgG isotype. In some embodiments, the first bispecific antibody, the second bispecific antibody, and / or the third bispecific antibody are chimeric, humanized, or human antibodies.

[0023] In some embodiments, the composition allows antigen-dependent activation of cytokine receptors. In some embodiments, the composition allows antigen-dependent activation of IL-2 receptor signaling in immune cells expressing IL-2Rγ and IL-2Rβ. In some embodiments, the composition allows antigen-dependent activation of IL-15 receptor signaling in immune cells expressing IL-2Rγ and IL-2Rβ.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used to implement the present invention, but suitable methods and materials are described below.All publications, patent applications, patents, and other references described herein are expressly incorporated by reference in their entirety.In case of discrepancy, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0025] Other features and advantages of the present invention will be apparent from, and are encompassed by, the following detailed description and claims. [Brief description of the drawings]

[0026] [Figure 1] Schematic diagram of activation of IL-2 signaling by the combination of agonistic anti-IL-2Rβ×anti-TAA bispecific antibody (bsAb) and agonistic anti-IL-2Rγ×anti-TAA bsAb. We describe a novel IL-2 agonist approach using a combination of two bsAbs. The idea behind this concept is to use a combination of IL-2Rβ×TAA bsAb and IL-2Rγ×TAA bsAb to activate IL-2 signaling by selectively bringing IL-2Rβ and IL-2Rγ into mechanical proximity with each other in tumor microenvironments containing TAA-positive tumor cells. In healthy tissues (i.e., in the absence of cells expressing TAA), the pair of bsAbs can bind to IL-2Rβ and IL-2Rγ on T cells or NK cells, respectively, but cannot bring the two receptors into close proximity, and IL-2 signaling is not activated (A). In the presence of TAA-positive tumor cells (B and C), binding of the bsAb pair to the TAA brings the bsAb pair into close proximity. Subsequent binding of the bsAb pair to IL-2Rβ and IL-2Rγ on T cells or NK cells brings the two receptors into close proximity to each other, triggering activation of the IL-2R signaling pathway. The TAA targeting arms of the bsAb pair can target the same TAA epitope (B) or two different TAA epitopes (C). This novel strategy of IL-2 signaling activation limits the agonist effect to the tumor microenvironment containing TAA-positive tumor cells, avoiding systemic toxicity. Avoiding preferential activation of CD25-expressing Treg cells is another advantage of this novel approach. [Diagram 2]Binding properties of IL-2Rβ×TAA and IL-2Rγ×TAA bsAbs to their respective targets as assessed by ELISA. Concentration-dependent binding properties of IL-2Rβ×HER2 KiH bsAbs (P2C4×Trastuzumab and P2C4×Pertuzumab), IL-2Rγ×HER2 KiH bsAbs (P1A3×Trastuzumab and P1A3×Pertuzumab), and IL-2Rβ×IL-2Rγ KiH bsAbs to their targets IL-2Rβ (A), IL-2Rγ (B), HER2 (C) and an unrelated protein (D) as assessed by ELISA. For κλ-body generation, anti-IL-2Rβ arms (AL1-AL5) were combined with membrane-distal (domain 1) HER2-binding arms (N2-28) and IL-2Rγ arms (AM1-AM11) were combined with membrane-proximal (domain 4) HER2 arms (N2-19). IL-2Rβ arms (AL4) and IL-2Rγ arms (AM5) were combined with different MSLN arms targeting different epitopes (O30, O35, O38 and O41) in another set of κλ-bodies. Binding properties of κλ-bodies to IL-2Rβ (E) and IL-2Rγ (F). Binding properties of IL-2Rβ-bsAb and IL-2Rγ-bsAb to HER2 (G left and right panels, respectively) and MSLN (H and right panels, respectively) are shown. hIgG1 was used as an isotype control. [Diagram 3] Figure 1 shows simultaneous co-engagement of two target antigens by IL-2R x HER2 bsAbs assessed by Octet. Co-engagement was assessed using Protein A biosensors loaded with different bsAbs in the presence of individual targets or the two targets simultaneously with the corresponding bsAbs. Co-engagement of two targets is shown for P1A3 x Pertuzumab (A), P2C4 x Pertuzumab (B) and P2C4 x Trastuzumab (C). [Figure 4]Figure 1 shows the lack of interference of IL-2RxTAA bsAbs with IL-2 and IL15 signaling. IL-2 reporter cell lines (expressing dimeric IL-2R) were incubated with recombinant hIL-2 (fixed concentration of 0.2 or 1 nM) or hIL-15 (fixed concentration of 0.003 nM). The effect of a dose range of IL-2Rβ-κλ bodies (A, B) or IL-2Rγ-κλ (C, D) on IL-2 or IL-15 signaling was assessed. hIgG1 is used as an irrelevant and non-blocking control antibody. Data were normalized to an isotype control fixed at 100% (no neutralization of IL-2 / IL-15 signaling, dashed line). Neutralizing IL-2 or IL-15 antibodies were included as controls. [Figure 5-1] Figure 5 shows activation of IL-2 signaling by combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of soluble HER2. Schematic of the assay format (A). IL-2 reporter cell lines were incubated with combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 bsAbs targeting either the same (B, C) or two different (D) HER2 epitopes in the presence of different concentrations of soluble HER2. Activation of IL-2 signaling was represented by an increase in reporter gene expression. An anti-IL-2Rβ×anti-IL-2Rγ bsAb, P1A3×P2C4, was used as a positive control. [Figure 5-2] See description of Figure 5-1. [Figure 6-1]Figure 6 shows activation of IL-2 signaling by combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of HER2-coated microparticles. Schematic of the assay format (A). IL-2 reporter cell lines were incubated with combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 bsAbs targeting either different (B,C) or the same (D,E) HER2 epitopes in the presence of different numbers of HER2-coated streptavidin microparticles. Activation of IL-2 signaling was represented by an increase in reporter gene expression. Human IL-2 and the anti-IL-2Rβ×anti-IL-2Rγ bsAb, P1A3×P2C4, were used as positive controls. [Figure 6-2] See description of Figure 6-1. [Figure 7]Figure 1 shows activation of IL-2 signaling by combinations of IL-2Rβ×TAA and IL-2Rγ×TAAκλ-body in the presence of TAA-coated microparticles. IL-2 reporter cell lines were incubated with combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2κλ-body targeting two different HER2 epitopes (N2-28 and N2-19 arms) in the presence of HER2-coated streptavidin microparticles (beads-reporter cell ratio, 6:1). Different combinations are shown, where IL-2Rγ×HER2κλ-body (AM1N2-19 to AM5N2-19) was mixed with AL1N2-28 (A), AL2N2-28 (B), AL3N2-28 (C), AL4N2-28 (D) or AL5N2-28 (E). IL-2 reporter cell lines were also incubated with various combinations of IL-2Rβ×MSLN and IL-2Rγ×MSLNκλ-bodies targeting two different MSLN epitopes (F-I) in the presence of MSLN-coated streptavidin microparticles. Different combinations are shown, where IL-2Rγ×MSLNκλ-bodies (AM5O30 / N, AM5O35 / N, AM5O41 / N) were mixed with AL4O38 / N (F), AL4O35 / N (G), AL4O30 / N (H) or AL4O41 / N (I). Activation of IL-2 signaling was represented by an increase in reporter gene expression. Human IL-2 antibody and irrelevant hIgG1 antibody were used as positive and negative controls, respectively. [Figure 8]Figure 1. Activation of IL-2 signaling in the presence of MSLN-coated microparticles. IL-2 reporter cell lines were incubated with a combination of IL-2Rβ×MSLN and IL-2Rγ×MSLN κλ-bodies targeting the same epitope in MSLN using either the O35 (A), O41 (B) or O30 (C) arms. Activation of IL-2 signaling was represented by an increase in reporter gene expression. Human IL-2 antibody and an irrelevant hIgG1 antibody were used as positive and negative controls, respectively. [Figure 9-1] Figure 9 shows activation of IL-2 signaling by combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of HER2+ tumor cells. IL-2 reporter cell lines were incubated with combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs targeting two different HER2 epitopes. Schematic of the assay format (A). Combinations of P1A3×Trastuzumab+P2C4×Pertuzumab (B) or P1A3×Pertuzumab+P2C4×Trastuzumab (C) were added to HER2+BT-474, SK-BR-3 and NCI-N87 tumor cells. Activation of IL-2 signaling was represented by an increase in reporter gene expression. Human IL-2 was used as a positive control. [Figure 9-2] See description of Figure 9-1. [Figure 10-1]Figure 10 shows pSTAT5 induction by IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAb combinations in the presence of HER2+ tumor cells. Schematic of the assay format (A). NK-92 cell line was incubated with IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAb combinations targeting either two different HER2 epitopes (B, C) or the same HER2 epitope (D, E) in the presence of HER2+ BT-474, SK-BR-3 and NCI-N87 tumor cells. IL-2 agonist properties were also tested by combining all four bsAbs (P1A3×Pertuzumab, P2C4×Pertuzumab, P1A3×Trastuzumab and P2C4×Trastuzumab) (F). IL-2 signaling activation in NK-92 cells was analyzed by measuring the levels of pSTAT5 by flow cytometry. Data were presented as the percentage of pSTAT5 positive cells. Human IL-2 was used as a positive control. [Figure 10-2] See description of Figure 10-1. [Figure 11] Figure 1 shows the induction of pSTAT5 by combinations of IL-2R×HER2 or IL-2R×MSLN-κλ-body in the presence of TAA+ tumor cells. NK-92 cell lines were incubated with combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 κλ-body (A, B) or IL-2Rβ×MSLN and IL-2Rγ×MSLN κλ-body (C-E) in the presence of HER2+BT-474 and NCI-N87 tumor cells (A, B, respectively) or MSLN+NCI-H226, OVCAR-3 and NCI-N87 tumor cells (C, D, E, respectively). IL-2 signaling activation in NK-92 cells was analyzed by measuring the levels of pSTAT5 by flow cytometry. Human IL-2 was used as a positive control. Single treatments with hIgG1 and an irrelevant antibody (wavy line) and IL-2Rβ×MSLN or IL-2Rγ×MSLNκλ-bodies were used as negative controls. [Figure 12-1]FIG. 12 shows activation of IL-2 signaling in human primary T cells by a combination of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of HER2+ tumor cells. Schematic of the assay format (A). PBMCs were incubated with a combination of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of two different HER2+ tumor cell lines (B), BT-474 or NCI-N87. IL-2 signaling activation in PBMC data is presented as the percentage of pSTAT5 positive cells in different T cell subsets gated by FACS: Treg, CD8+ and CD4+ T cells. Human IL-2 was used as a positive control. [Figure 12-2] See description of Figure 12-1. [Figure 13-1]Figure 13 shows T cell retargeting killing / lysis of tumor cells by combinations of CD3xTAA bsAb pairs and IL-2RxTAA bsAb pairs. Combinations of CD3xTAA bsAb and IL-2RxTAA bsAb pairs were tested for their tumoricidal activity when combined all together at the same time (A, B) or sequentially, with T cells first prestimulated with IL-2RxTAA bsAb pairs and then stimulated with CD3xTAA bsAb (C, D). Schematic diagram of each TDCC assay format used (A, C). NCI-N87 tumor cell line was used as a source of HER2 and MSLN. T cell retargeted killing / lysis of NCI-N87 cells by two dose ranges of IL-2RxMSLN bsAb pairs (AL4O35 / N+AM5O30 / N or AL4O38 / N+AM5O30 / N) when combined simultaneously with a fixed dose of CD3xHER2 bsAb (B). Sequential T cell retargeted killing / lysis of NCI-N87 cells by a dose range of IL-2 or IL-2RxHER2 bsAb pairs (P1A3xtrastuzumab+P2C4xpertuzumab) followed by a fixed dose of CD3xMSLN bsAb (D). A single CD3xTAA treatment was used as a control (dotted line). In some experiments, the combination of IL-2 and CD3xTAA bsAb was used as a positive control. IL-2Rβ×IL-2Rγ (P1A3×P2C4) or a single treatment with IL-2 or IL-2R×TAA bsAb were used as negative controls. Data presented using a representative PBMC donor as a source of effector cells. [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 13-4] See description of Figure 13-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention The present invention is based in part on a bispecific antibody platform that can selectively activate cytokines in the tumor microenvironment. Specifically, the present invention is based on the activation of cytokines whose signaling requires the interaction of two or more cytokine receptor subunits. By combining bispecific antibodies, each bispecific has one antigen-binding arm specific for a cytokine receptor subunit and one binding arm specific for a cell surface antigen subunit. Binding of the bispecific antibody to a cell surface antigen brings the different subunits of the cytokine receptor expressed on the cell surface (e.g., T cells, and / or NK cells) into close proximity with each other, resulting in activation of cytokine signaling. This bispecific antibody (bsAb) platform optimally exploits the antitumor potential of cytokine activation by combining the favorable pharmacokinetic properties of bsAbs (i.e., significantly longer half-life compared to natural or recombinant cytokines) with selective cytokine activation in the tumor microenvironment (TME). Furthermore, the bsAb platform of the present invention provides benefits in terms of tumor specificity and reduced extratumoral systemic toxicity when compared to cytokine receptor activation using natural or recombinant cytokines. Furthermore, the present invention also avoids preferential activation of cells expressing the trimeric form of the receptor, such as Tregs (e.g., IL-2R trimer highly expressed on Tregs), by targeting only the dimeric form of the receptor.

[0028] Without being bound by theory, the present invention includes bsAbs that engage cytokine receptors but do not block or reduce endogenous cytokine signaling. Because therapeutic proteins often induce anti-drug antibodies (ADA), the therapeutic use of either recombinant cytokines, such as IL-2 peptides or modified IL-2 peptides, raises the possibility of inducing such responses in subjects that may also affect endogenous IL-2, resulting in toxicity and adverse effects. Such effects have been observed with erythropoietin (EPO) immunogenicity and may lead to EPO-resistant anemia. An important advantage of the use of bsAbs of the present invention is that any ADA formation limits the exposure and effectiveness of the bsAb, but endogenous cytokines are not affected. For example, compositions comprising bispecific antibodies of the present invention do not block or inhibit endogenous signaling induced by IL-2 or IL-15.

[0029] Without being bound by theory, the composition engages with IL-2Rγ and IL-2Rβ in an antigen-dependent manner and does not preferentially activate Tregs. The composition also allows T cell activation and proliferation, resulting in superior killing of target cells. In some embodiments, the composition may be enhanced using additional T cell redirection with a third bispecific antibody that binds to CD3 on T cells and tumor antigen on target cells.

[0030] In various embodiments, the present invention provides compositions containing a pair of bsAbs. One of the bsAbs targets a first subunit of a multisubunit cytokine receptor and a cell surface protein (i.e., has a specific antigen binding domain). The other bsAb targets a second subunit of a multisubunit cytokine receptor and a cell surface protein expressed on the surface of the same cell that expresses the target antigen of the first bsAb. The cell expressing the cell surface protein binds the two bsAbs, bringing cytokine receptors on T cells or NK cells into close proximity, resulting in cytokine signaling activation.

[0031] Multidomain cytokine receptors include, for example, cytokine receptors that share a common gamma chain, such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, as well as other cytokine receptors that share another common subunit, such as IL-12 and IL-23. Other multidomain cytokine receptors include IFNα, IFNβ, IFNε, IFNk, IFNNo, IFNδ, IFNτ, IFNω, IFNζ, IFNγ, and IFNλ.

[0032] The cell surface receptor is a tumor-associated antigen (TAA).TAAs are well known in the art and include, for example, glioma associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, glypican 3 (GPC3), cMet, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β.-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, CD123, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAXS, SART3, CLL-1, Fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGSS, polysialic acid, PLAC1, RUI, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mut hsp70-2, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-Ia, LMP2, NCAM, Ras mutant, gpIOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, FAP, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, CLDN18.2, TSHR, UPK2, and mesothelin.

[0033] Other antibody domains or tumor target binding proteins (e.g., TCR domains) useful in the present invention include those that bind to the following antigens (note that the cancer indications shown represent non-limiting examples): aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha fetoprotein (carcinoma), CA242 (colorectal cancer), placental alkaline phosphatase (carcinoma), prostate specific antigen (prostate), prostatic acid phosphatase (carcinoma), and the like. Sphatase (prostate), epidermal growth factor (carcinoma), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung, breast, stomach, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma, B-cell neoplasms, autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD5 1 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumors, multiple myeloma), CD66e (carcinoma), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (carcinoma), CD123 (leukemia), mucin (carcinoma), CD221 (solid tumor), CD22? (breast, ovarian cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colorectal cancer, gastric cancer), CEACAMS (CEA, C D66e) (breast, colorectal and lung cancer), DLL4 (A-like-4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD184, bleeding oncology, solid tumors), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate and ovarian cancer), ERBB2 (lung, breast, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), FGFR (carcinoma), GD2 ganglioside (carcinoma), G-28 (cell surface antigen glycolipid, melanoma), GD3 idiotype (carcinoma),Heat shock proteins (carcinomas), HER1 (lung, gastric cancer), HER2 (breast, lung and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinomas), IGF1R (solid tumors, blood cancers), IL-2 receptor (T-cell leukemia and lymphoma), IL-6R (multiple myeloma, RA, Castleman's disease, IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane Transmembrane 4 domain subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 (breast, ovarian, cervical, bronchial and gastrointestinal cancers), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domain subfamily A, small cell lung cancer, NHL), nucleolin, Neu oncogene product (carcinoma), P21 (carcinoma), nectin-4 (carcinoma), anti-(paratope of N-glycolylneuraminic acid, breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROB04, TAG 72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, carcinoma), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers),Not limited to these. Some other tumor-associated antigen targets have been reviewed (Gerber, et al, mAbs 2009 1:247-253; Novellino et al, Cancer Immunol Immunother. 2005 54:187-207; Franke, et al, Cancer Biother Radiopharm. 2000, 15:459-76; Guo, et al., Adv Cancer Res. 2013; 119:421-475; Parmiani et al. J Immunol. 2007 178:1975-9). Examples of these antigens include cluster of differentiation (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD 62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90 , CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, CD 138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD1 84, CDw186, CD195, CD202(a,b), CD209, CD235a, CD271, CD303, CD304), Annexin A1, Nucleolin, Endoglin (CD105), ROB04, Aminopeptidase N-like 4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, Idiotype, MAGE A3, p53 non-mutant, NY-ESO-1,GD2, CEA, MelanA / MART1, Ras mutants, gp100, p53 mutants, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYPIB I, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.

[0034] Two bsAbs can target the same epitope (Figure 1B) or two different epitopes (Figure 1C) on a given cell surface protein or TAA. In the absence of a TAA (i.e., in healthy tissue), the bsAb pair cannot bring cytokine receptor subunits into close proximity and does not induce cytokine activation (Figure 1A). The present invention further provides for the targeting of two different cell surface proteins that are co-expressed on the same cell, in particular two TAAs that are co-expressed on the surface of a tumor cell.

[0035] In a particular embodiment, the present invention uses a combination of two bsAbs to provide selective IL-2 signaling activation on a particular type of cell. One bsAb has one antigen binding domain that binds to IL-2Rβ and a second antigen binding domain that binds to a cell surface protein expressed on the same cell (e.g., T cells or NK cells) that expresses IL-2Rβ. The second bsAb has one antigen binding domain that binds to IL-2Rγ and a second antigen binding domain that binds to the same cell surface protein targeted by the first bsAb. The cell surface protein can be specific to a subset of T cells, NK cells or other IL-2R expressing cells. Binding to the same cell surface protein brings the two bsAbs into close proximity to each other. The IL-2Rβ and IL-2Rγ binding arms can then bind to the two receptors and induce signaling activation in cells that express both IL-2R and the cell surface protein.

[0036] Because the two IL-2 receptors, IL-2Rβ (CD122) and IL-2Rγ (CD132), are shared by both IL-2 and IL-15 for signal transduction, the compositions of the invention can be used to selectively activate both the IL-2 and IL-15 signaling pathways in either the TME or specific subsets of cells.

[0037] bispecific antibody Bispecific antibodies (bsAbs) according to the present invention may be generated de novo or engineered from existing monospecific anti-cytokine receptor subunit and cell surface antigen antibodies.

[0038] The bsAb of the present invention can be based on any of the different antibody formats already described. Furthermore, the two bsAb antibodies forming the pair of the present invention can be based on the same format or on two different formats. Generally, IgG-like formats are preferred as they offer favorable properties such as long half-life and potentially reduced immunogenicity, but any other molecular bispecific format can also be used in the present invention.

[0039] The heavy and light chain amino acid sequences of the antibodies are identified by their United States Adopted Names (USAN available, for example, through the American Medical Association at http: / / _www_ama-assn_org or through the CAS registry).

[0040] Monospecific anti-cytokine receptor subunits and cell surface antigen binding variable domains may be selected de novo, for example, from a phage display library, where the phage are engineered to express human immunoglobulins or portions thereof, such as Fabs, single chain variable fragments (scFvs), or unpaired or paired antibody variable regions, and then engineered into a bispecific format. Monospecific anti-cytokine receptor subunits and cell surface antigen variable domains may be isolated, for example, from a phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein.

[0041] The antibody library is screened for binding to human cytokine receptor subunit or cell surface antigen extracellular domain, and the obtained positive clones are further characterized, and Fab is isolated from the clone lysate.Such phage display method for isolating human antibody is established in the art.See, for example, U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915 and 6,593,081. The resulting novel variable region combinations are engineered into a bispecific format using methods known in the art and described herein.

[0042] The antibodies of the present invention have two or more antigen-binding sites and are bispecific. Bispecific antibodies of the present invention include antibodies having a full-length antibody structure. Alternatively, bispecific antibodies of the present invention include antibodies that are less than full-length but contain an antigen-binding domain, such as a Fab' fragment.

[0043] As used herein, a "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The full-length antibody heavy chain (HC) consists of the well-known heavy chain variable domain and constant domains VH, CH1, CH2, and CH3. The full-length antibody light chain (LC) consists of the well-known light chain variable domain and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains.

[0044] The term "Fab arm" or "half molecule" refers to one heavy-light chain pair that specifically binds to an antigen.

[0045] The full-length bispecific antibodies of the invention can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules with distinct specificities in an in vitro cell-free environment, or by using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm may be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a distinct epitope.

[0046] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0047] As used herein, "heterodimerization" refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains having non-identical CH3 amino acid sequences.

[0048] A "knob-in-hole" strategy (see, e.g., PCT Publication WO 2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain "hole" with the heavy chain "knob". Exemplary CH3 substitution pairs that form the knob and hole are (represented as modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain) T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0049] Other strategies may be used, such as promoting heavy chain heterodimerization using electrical interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface as described in US Patent Publication No. US2010 / 0015133, US Patent Publication No. US2009 / 0182127, US Patent Publication No. US2010 / 028637, or US Patent Publication No. US2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_T3 as described in US Patent Publication No. US2012 / 0149876 or US Patent Publication No. US2013 / 0195849. 94W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0050] Correct heavy and light chain pairing can be achieved using immunoglobulin domain crossover, a general approach for the generation of bispecific IgG antibodies, as described in WO2015150447A1.

[0051] In addition to the above methods, bispecific antibodies of the invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization by the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability, and the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo disulfide bond isomerization, thereby generating the bispecific antibody by Fab arm exchange.

[0052] Furthermore, bispecific antibodies of the invention can be made using techniques including those disclosed in WO2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces bispecific antibodies that are identical in structure to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain and a second light chain variable region fused to a constant lambda domain. Each combining site exhibits a different antigen specificity with contributions from both the heavy and light chains. The light chain variable region may be of the lambda or kappa family, and is preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide junctions.

[0053] However, it is also possible to obtain the bispecific antibodies of the invention by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are called IgG κλ antibodies or "κλ bodies", a new fully human bispecific IgG format. This κλ body format has characteristics indistinguishable from standard monoclonal antibodies and therefore allows affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, which is preferred compared to previous formats.

[0054] Exemplary anti-IL-2Rβ antibodies that can be used to engineer bispecific antibodies include, for example, P2C4 or a variant of P2C4, P2H7 or a variant of P2H7, P2D12 or a variant of P2D12, P1G11 or a variant of P1G11 (described in WO 2017 / 021540A1, which is incorporated by reference in its entirety).

[0055] P2H7 antibody CDRH1-TYAMH (SEQ ID NO: 155) CDRH2 -WINTGNGNTKYSQNFQG (SEQ ID NO: 156) CDRH3 -DLGQLERLYFW (SEQ ID NO: 157) CDRL1 -RAGQAISSWLA (SEQ ID NO: 158) CDRL2-KASNLES (SEQ ID NO: 159) CDRL3-QQYQSYPYT (SEQ ID NO: 160) VH- (SEQ ID NO: 161) EVQLVQSGTEVKKPGASVKVSCKASGYTFTTYAMHWVRQAPGQSLEWMGWINTGNGNTKYSQNFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLGQLERLYFWGQGTLVTVSS VL- (SEQ ID NO: 162) DIQMTQSPSTLSASVGDRVTLSCRAGQAISSWLAWYQQKPGKAPKLLIYKASNLESGVPSRFSGGGSGAEFTLTISSLQPDDFATYYCQQYQSYPYTFGQGTKLEIR

[0056] P2D12 antibody CDRH1-SYAMS (SEQ ID NO: 163) CDRH2-AISGSGGSTYYADSVKG (SEQ ID NO: 164) CDRH3 -DLGDY (SEQ ID NO: 165) CDRL1 - QASQDIGNYLN (SEQ ID NO: 166) CDRL2-DASNLET (SEQ ID NO: 167) CDRL3-LQLYDYPLT (SEQ ID NO: 168) VH- (SEQ ID NO: 169) HVQLVETGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGDYWGQGTLVTVSS VL- (SEQ ID NO: 170) DIQLTQSPSSLSASVGDRVTITCQASQDIGNYLNWYQLKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCLQLYDYPLTFGGGTKVEIK

[0057] P1G11 antibody CDRH1 -GYYWS (SEQ ID NO: 19) CDRH2-EINHSGSTNYNPSLKS (SEQ ID NO: 20) CDRH3-SSSGDAFD (SEQ ID NO: 171) CDRL1-TRSSGSIASNYVQ (SEQ ID NO: 172) CDRL2-DDNQRPT (SEQ ID NO: 173) CDRL3-QSSHSTAVV (SEQ ID NO: 174) VH- (SEQ ID NO: 175) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYN PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSSSGDAFDIWGQGTMVTVSS VL- (SEQ ID NO: 176) NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIFDDNQRPTGVP DRFSAAIDTSSSSASLTISGLTAEDEADYYCQSSHSTAVVFGGGTKLTVL

[0058] Exemplary anti-IL-2Rβ antibodies that can be used to engineer bispecific antibodies include, for example, Hu-Mikβ1 (National Cancer Institute, Mayo Clinic, University of Chicago Medicine Celiac Disease Center), single domain antibodies IL-2RB_F09, IL-2RB_F17, IL-2RB_F18, IL-2RB_F20 and IL-2RB_F21 (Katherine E. Harris et al., 2021, A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells, Scientific Reports, doi.org / 10.1038 / s41598-021-90096-8). Exemplary anti-IL-2Rβ antibodies can also include, but are not limited to, "AL1", "AL2", "AL3", "AL4" and "AL5" as shown in Table 1.

[0059] Exemplary anti-IL-2Rγ antibodies that can be used to engineer bispecific antibodies include, for example, P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P1A3_B4, P1A3_FW2, P2B9 (described in WO 2017 / 021540A1, which is incorporated by reference in its entirety).

[0060] P1A3_B3, P1A3_E8, P1A3_E9, and P1A3_B4 antibodies CDRH1-GYYWS (SEQ ID NO: 19) CDRH2-EINHSGSTNYNPSLKS (SEQ ID NO: 20) CDRH3-SPGGYSGGYFQH (SEQ ID NO: 21) CDRL1-RSSQSLLHSNGYNYLD (SEQ ID NO: 15) CDRL2-LGSNRDS (SEQ ID NO: 16) CDRL3-MQGTHWPWT (SEQ ID NO: 17)

[0061] P1A3_B3 antibody VH- (SEQ ID NO: 177) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHFGSTNYN PSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCATSPGGYSGGYFQHWGQGTLVTVSS VL- (SEQ ID NO: 178) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRDS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCMQGTHWPWTFGQGTKVEIK

[0062] P1A3_E8 antibody VH- (SEQ ID NO: 179) QVQLQQWGAGMLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHFGSTNYN PSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCATSPGGYSGGYFQHWGQGTLVTVSS VL- (SEQ ID NO: 180) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRDS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCMQGTHWPWTFGQGTKVEIK

[0063] P1A3_E9 antibody VH- (SEQ ID NO: 181) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHFGSTNYN PSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCATSPGGYSGGYFQHWGQGTLVTVSS VL- (SEQ ID NO: 182) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPWTFGQGTKVEIK

[0064] P1A3_B4 antibody VH- (SEQ ID NO: 183) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHFGSTNYN PSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCATSPGGYSGGYFQHWGQGTLVTVSS VL- (SEQ ID NO: 184) DVVMTQSPLSLPVTPGESVSISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPWTFGQGTKVEIK

[0065] Exemplary anti-IL-2Rgamma antibodies may also include, but are not limited to, single domain antibodies IL-2RG_F05, IL-2RG_F16, IL-2RG_F18, IL-2RG_F19 and IL-2RG_F20 (Katherine E. Harris et al., 2021, A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells, Scientific Reports, doi.org / 10.1038 / s41598-021-90096-8). Exemplary anti-IL-2Rgamma antibodies also include, but are not limited to, "AM1", "AM2", "AM3", "AM4", "AM5", "AM6", "AM7", "AM8", "AM9", "AM10" and "AM11" shown in Table 1.

[0066] Exemplary anti-cell surface (e.g., TAA) antibodies that can be used to engineer bispecific molecules include anti-tumor associated antigen (TAA) antibodies known in the art, such as pertuzumab and trastuzumab (HER-2), cetuximab, necitumumab, panitumumab and amivantamab (EGFR), labetuzumab and civisatamab (CEA), amatuximab (mesothelin), codons, and the like. Rituzumab (glypican 3), atezolizumab, avelumab and durvalumab (PD-L1), blinatumomab (CD19), brentuximab vedotin (CD30), daratumumab (CD38), gemtuzumab (CD33), tositumomab 9CD22) or obinutuzumab, ocrelizumab, ofatumumab, rituximab, and ibritumomab (CD20).

[0067] Additionally, the antigen binding domains of the present invention may include various other tumor-specific antibody domains known in the art. Antibodies and their respective targets for the treatment of cancer include nivolumab (anti-PD-1 Ab), TA99 (anti-gp75), 3F8 (anti-GD2), 8H9 (anti-B7-H3), abagovomab (anti-CA-125 (mimetic)), adecatumumab (anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2), altumomab pentetate (anti-CEA), amatuximab (anti-mesothelin), AME-133 (anti-CD2 0), anatumomab mafenatox (anti-TAG-72), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA), bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab (anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab (anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab (anti-CD19), BMS-663513 (anti-CD137), brentuximab vedotin (anti-CD30 (TNFRSF8)), cantuzumab mertansine (anti-mucin CanAg), cantuzumab ravtansine (anti-MUC1), capromab pendetide (anti-prostate cancer cells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM, CD3), cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49 (anti-TAG-72), cedelizumab (anti-CD4), Ch.14.18 (anti-GD2), ch-TNT (anti-DNA-related antigen), sitatuzumab bogatox (anti-EpCAM), cizutumumab (anti-IGF-1 receptor), clivatuzumab tetraxetan (anti-MUC1), conatumumab (anti-TRAIL-R2), CP-8708 93 (anti-CD40), dacetuzumab (anti-CD40), daclizumab (anti-CD25), darotuzumab (anti-insulin-like growth factor I receptor), daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase)), demicizumab (anti-DLL4), detumomab (anti-B lymphoma cells), drozitumab (anti-DR5), durigotumab (anti-HER3), dusigitumab (anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab (anti-EpCAM), elotuzumab (anti-SLAMF7), elisirimomab (anti-IL-6), enavatuzumab (anti-TWEAK receptor),Enotimab (anti-DLL4), encituximab (anti-5AC), epitumomab-situxetan (anti-episialin), epratuzumab (anti-CD22), ertumaxomab (anti-HER2 / neu, CD3), etaracizumab (anti-integrin αvβ3), faralimomab (anti-interferon receptor), farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20), ficlatuzumab (anti-HGF), figitumumab (anti-IGF-1 receptor), framvotumab (anti-TYRP1 (glycoprotein 75)), fresolimumab (anti-TGFβ), futuximab (anti-EGFR), galiximab (anti-CD80), ganitumab (anti-IGF-I), gemtuzumab ozogamicin (anti-CD33), dilentuximab (anti-carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (anti-GPNMB), gaselkumaab (anti-IL13), ibalizumab (anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1), i Gobomab (anti-CA-125), IMAB362 (anti-CLDN18.2), IMC-CS4 (anti-CSF1R), IMC-TR1 (TGFβRII), igatuzumab (anti-EGFR), inlacumab (anti-selectin P), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin (anti-CD22), intetumumab (anti-CD51), ipilimumab (anti-CD152), iratumumab (anti-CD30 (TNFRSF8)), KM3 065 (anti-CD20), KW-0761 (anti-CD194), LY2875358 (anti-MET), labetuzumab (anti-CEA), lambrolizumab (anti-PDCD1), lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirilumab (anti-KIR2D), lobotuzumab mertansine (anti-CD56), lucatumumab (anti-CD40), rumiliximab (anti-CD23 (IgE receptor)), macatumumab (anti-TRAIL-R1), Rugetuximab (anti-ch4D5), matuzumab (anti-EGFR), mavrilimumab (anti-GMCSF receptor α chain), milatuzumab (anti-CD74), minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside), mogamulizumab (anti-CCR4), moxetumomab passudotox (anti-CD22), nacolomab butafenatox (anti-C242 antigen), naptomomab estafenatox (anti-5T4), nalutamumab (anti-RON),Necitumumab (anti-EGFR), nesbacumab (anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4), nofetumomab merpentane, ocrelizumab (anti-CD20), ocaratuzumab (anti-CD20), olatumab (anti-PDGF-Rα), onartuzumab (anti-c-MET), ontuxizumab (anti-TEM1), oportuzumab monotox (anti-EpCAM), oregovomab (anti-CA-125), otratuzumab (anti-CD37), pancomab (anti-tumor specific glycosylation of MUC1), palsatuzumab (anti-EGFL7), pascolizumab Mab (anti-IL-4), patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab (anti-HER2 / neu), pidilizumab (anti-PD-1), pinatuzumab vedotin (anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumab vedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20), kirisumab (anti-IGHE), racotumomab (anti-N-glycolylneuraminic acid), radeletumab (anti-fibronectin ectodomain B), ramucirumab (anti-VEGFR2), rilotumumab (anti-HGF), lobatumumab (anti-IGF-1 receptor), lordomab (anti-RHD), rovelizumab (anti-CD11 and CD18), samaryzumab (anti-CD200), satumomab pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A (anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab (anti-IL-6), solitomab (anti-EpCAM), sonutuzumab (anti-epicillin), tabalumab (anti-BAFF), tacatuzumab tetraxetan (anti-alpha-fetoprotein), tapritumomab paptox (anti-CD1 9), terimomab aritox, tenatumomab (anti-tenascin C), teneliximab (anti-CD40), teprotumumab (anti-CD221), TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20), tobetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4), tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab semoroquine (anti-EpCAM), ublituximab (anti-CD20),Urelumab (anti-4-1BB), vantictomab (anti-Frizzled receptor), bapaliximab (anti-AOC3 (VAP-1)), batelizumab (anti-ITGA2), veltuzumab (anti-CD20), besencumab (anti-NRP1), visilizumab (anti-CD3), boroximab (anti-integrin α5β1), borsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigen CTAA16.88), These include, but are not limited to, lutumumab (anti-EGFR), zanolimumab (anti-CD4), zatuximab (anti-HER1), dillalimumab (anti-CD147 (basigin)), RG7636 (anti-ETBR), RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL3280A (anti-PD-L1), RG7450 (anti-STEAP1), and GDC-0199 (anti-Bcl-2).

[0068] Exemplary amino acid sequences of the antigen-binding domains of the bispecific antibodies of the disclosure are shown in Table 1.

[0069] Table 1. Amino acid sequences of antigen-binding domains TIFF2025503109000001.tif218163TIFF2025503109000002.tif246161TIFF202 5503109000003.tif237161TIFF2025503109000004.tif248162TIFF20255031090 00005.tif247162TIFF2025503109000006.tif248161TIFF2025503109000007.t if248161TIFF2025503109000008.tif240161TIFF2025503109000009.tif101161

[0070] Binding arms can be combined in any combination to generate anti-IL2R x TAA bsAbs of the present disclosure. Exemplary bispecific antibodies of the present disclosure are shown in Table 2. Each bispecific antibody in the matrix is ​​a unique bsAb with a specific combination of antigen binding domains. The specific format (KiH or κλ-body) is also shown. For example, bispecific antibody "AL1N2-28" has a κλ-body format and includes an "AL1" anti-IL2Rβ antigen binding domain and an "N2-28" anti-HER2 antigen binding domain. For example, bispecific antibody "AM5O30" has a κλ-body format and includes an "AM5" anti-IL2Rγ antigen binding domain and an "O30" anti-MSLN antigen binding domain.

[0071] Exemplary κλ-body format bispecific antibodies described herein comprise a common heavy chain (HC), one kappa or one lambda chain for the anti-TAA and anti-first / second subunits of the cytokine receptor antigen binding domain, one kappa and one lambda light chain (LC) for the anti-TAA and anti-first / second subunits of the cytokine receptor antigen binding domain, as shown in the amino acid sequences listed below. Each of the exemplary anti-TAA and anti-first / second subunits of the cytokine receptor bispecific antibodies described below comprises a common variable heavy chain domain (VH), one kappa or one lambda variable light chain domain for the anti-TAA and anti-first / second subunits of the cytokine receptor antibody, and one kappa and one lambda variable light chain domain (VL) for the anti-TAA and anti-first / second subunits of the cytokine receptor antigen binding domain, as shown in the amino acid sequences listed below.

[0072] The following antibody sequences are provided herein as examples, but it should be understood that these sequences can be used to generate bispecific antibodies using any of a variety of art-recognized techniques. Examples of bispecific formats include bispecific IgG based on Fab arm exchange (Gramer et al., 2013 MAbs.5(6)), CrossMab formats (Klein C et al., 2012 MAbs 4(6)), multiple formats based on forced heterodimerization approaches such as SEED technology (Davis JH et al., 2010 Protein Eng Des Sel.23(4):195-202), electrostatic steering (Gunasekaran K et al., J Biol Chem.2010 285(25):19637-46) or knobs-into-holes (Ridgway JB et al., Protein Eng.1996 9(7):617-21.) or other sets of mutations that block homodimer formation (Von Kreudenstein TS et al., 2013 MAbs.5(5):646-54), fragment-based bispecific formats such as tandem scFvs (such as BiTEs) (Wolf E et al.,2005 Drug Discov.Today 10(18):1237-44.), bispecific tetravalent antibodies (Portner LM et al.,2012 Cancer Immunol Immunother.61(10):1869-75.), dual affinity retargeting molecules (Moore PA et al.,2011 Blood.117(17):4542-51), diabodies (Kontermann RE et al., Nat Biotechnol.1997 15(7):629-31).

[0073] The bsAb antibodies of the composition can be generated using different formats and technologies, either relying on forced pairing of antibody chains to achieve bispecificity (e.g., knob-into-hole / Cross-Mab technology) or using bsAb formats that maintain the native structure of human IgG (kappa-lambda body technology).

[0074] Table 2. Nomenclature of exemplary bispecific antibodies of the present disclosure TIFF2025503109000010.tif162165

[0075] Exemplary Anti-IL2Rγ and Anti-HER2 Bispecific Antibodies In some embodiments, the bispecific antibody P1A3 x trastuzumab comprises a P1A3 antigen-binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, CDRH3 comprising the amino acid sequence of SEQ ID NO: 21, CDRL1 comprising the amino acid sequence of SEQ ID NO: 15, CDRL2 comprising the amino acid sequence of SEQ ID NO: 16, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 17, and a trastuzumab antigen-binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 27, CDRH2 comprising the amino acid sequence of SEQ ID NO: 28, CDRH3 comprising the amino acid sequence of SEQ ID NO: 29, CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 25.

[0076] In some embodiments, the bispecific antibody P1A3 x trastuzumab comprises a P1A3 antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 14, and a trastuzumab antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0077] In some embodiments, the bispecific antibody P1A3 x Pertuzumab comprises a P1A3 antigen binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 19, CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, CDRH3 comprising the amino acid sequence of SEQ ID NO: 21, CDRL1 comprising the amino acid sequence of SEQ ID NO: 15, CDRL2 comprising the amino acid sequence of SEQ ID NO: 16, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 17, and a Pertuzumab antigen binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 35, CDRH2 comprising the amino acid sequence of SEQ ID NO: 36, CDRH3 comprising the amino acid sequence of SEQ ID NO: 37, CDRL1 comprising the amino acid sequence of SEQ ID NO: 31, CDRL2 comprising the amino acid sequence of SEQ ID NO: 32, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 33.

[0078] In some embodiments, the bispecific antibody P1A3 x Pertuzumab comprises a P1A3 antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 14, and a Pertuzumab antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and a light chain comprising the amino acid sequence of SEQ ID NO: 30.

[0079] In some embodiments, the bispecific antibody AM1N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:64, a CDRL2 comprising the amino acid sequence of SEQ ID NO:65, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:66, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0080] In some embodiments, the bispecific antibody AM1N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:67, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0081] In some embodiments, the bispecific antibody AM1N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:63, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0082] In some embodiments, the bispecific antibody AM2N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:69, a CDRL2 comprising the amino acid sequence of SEQ ID NO:70, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:71, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0083] In some embodiments, the bispecific antibody AM2N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:72, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0084] In some embodiments, the bispecific antibody AM2N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:68, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0085] In some embodiments, the bispecific antibody AM3N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:74, a CDRL2 comprising the amino acid sequence of SEQ ID NO:75, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:76, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0086] In some embodiments, the bispecific antibody AM3N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:77, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0087] In some embodiments, the bispecific antibody AM3N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:73, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0088] In some embodiments, the bispecific antibody AM4N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:79, a CDRL2 comprising the amino acid sequence of SEQ ID NO:80, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0089] In some embodiments, the bispecific antibody AM4N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:82, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0090] In some embodiments, the bispecific antibody AM4N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:78, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0091] In some embodiments, the bispecific antibody AM5N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:84, a CDRL2 comprising the amino acid sequence of SEQ ID NO:85, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0092] In some embodiments, the bispecific antibody AM5N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0093] In some embodiments, the bispecific antibody AM5N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0094] In some embodiments, the bispecific antibody AM6N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:89, a CDRL2 comprising the amino acid sequence of SEQ ID NO:90, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:91, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0095] In some embodiments, the bispecific antibody AM6N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:92, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0096] In some embodiments, the bispecific antibody AM6N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0097] In some embodiments, the bispecific antibody AM7N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:94, a CDRL2 comprising the amino acid sequence of SEQ ID NO:95, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:96, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0098] In some embodiments, the bispecific antibody AM7N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:97, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0099] In some embodiments, the bispecific antibody AM7N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:93, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0100] In some embodiments, the bispecific antibody AM8N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:99, a CDRL2 comprising the amino acid sequence of SEQ ID NO:100, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:101, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0101] In some embodiments, the bispecific antibody AM8N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:102, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0102] In some embodiments, the bispecific antibody AM8N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:98, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0103] In some embodiments, the bispecific antibody AM9N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:104, a CDRL2 comprising the amino acid sequence of SEQ ID NO:105, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:106, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0104] In some embodiments, the bispecific antibody AM9N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0105] In some embodiments, the bispecific antibody AM9N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:103, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0106] In some embodiments, the bispecific antibody AM10N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:109, a CDRL2 comprising the amino acid sequence of SEQ ID NO:110, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:111, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0107] In some embodiments, the bispecific antibody AM10N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:112, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0108] In some embodiments, the bispecific antibody AM10N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:108, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0109] In some embodiments, the bispecific antibody AM11N2-19 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:114, a CDRL2 comprising the amino acid sequence of SEQ ID NO:115, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:116, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:146, a CDRL2 comprising the amino acid sequence of SEQ ID NO:147, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:148.

[0110] In some embodiments, the bispecific antibody AM11N2-19 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:117, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:149.

[0111] In some embodiments, the bispecific antibody AM11N2-19 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:113, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:145.

[0112] Exemplary Anti-IL2Rβ and Anti-HER2 Bispecific Antibodies In some embodiments, the bispecific antibody P2C4 x trastuzumab comprises a P2C4 antigen-binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 11, CDRH2 comprising the amino acid sequence of SEQ ID NO: 12, CDRH3 comprising the amino acid sequence of SEQ ID NO: 13, CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 9, and a trastuzumab antigen-binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 27, CDRH2 comprising the amino acid sequence of SEQ ID NO: 28, CDRH3 comprising the amino acid sequence of SEQ ID NO: 29, CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 25.

[0113] In some embodiments, the bispecific antibody P2C4 x trastuzumab comprises a P2C4 antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain comprising the amino acid sequence of SEQ ID NO: 6, and a trastuzumab antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0114] In some embodiments, the bispecific antibody P2C4 x Pertuzumab comprises a P2C4 antigen binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 11, CDRH2 comprising the amino acid sequence of SEQ ID NO: 12, CDRH3 comprising the amino acid sequence of SEQ ID NO: 13, CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, CDRL2 comprising the amino acid sequence of SEQ ID NO: 8, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 9, and a Pertuzumab antigen binding domain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO: 35, CDRH2 comprising the amino acid sequence of SEQ ID NO: 36, CDRH3 comprising the amino acid sequence of SEQ ID NO: 37, CDRL1 comprising the amino acid sequence of SEQ ID NO: 31, CDRL2 comprising the amino acid sequence of SEQ ID NO: 32, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 33.

[0115] In some embodiments, the bispecific antibody P2C4 x Pertuzumab comprises a P2C4 antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain comprising the amino acid sequence of SEQ ID NO: 6, and a Pertuzumab antigen-binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and a light chain comprising the amino acid sequence of SEQ ID NO: 30.

[0116] In some embodiments, the bispecific antibody AL1N2-28 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:39, a CDRL2 comprising the amino acid sequence of SEQ ID NO:40, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:41, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:151, a CDRL2 comprising the amino acid sequence of SEQ ID NO:152, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:153.

[0117] In some embodiments, the bispecific antibody AL1N2-28 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:42, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:154.

[0118] In some embodiments, the bispecific antibody AL1N2-28 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:38, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0119] In some embodiments, the bispecific antibody AL2N2-28 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:44, a CDRL2 comprising the amino acid sequence of SEQ ID NO:45, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:46, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:151, a CDRL2 comprising the amino acid sequence of SEQ ID NO:152, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:153.

[0120] In some embodiments, the bispecific antibody AL2N2-28 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:47, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:154.

[0121] In some embodiments, the bispecific antibody AL2N2-28 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:43, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0122] In some embodiments, the bispecific antibody AL3N2-28 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:49, a CDRL2 comprising the amino acid sequence of SEQ ID NO:50, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:51, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:151, a CDRL2 comprising the amino acid sequence of SEQ ID NO:152, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:153.

[0123] In some embodiments, the bispecific antibody AL3N2-28 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:52, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:154.

[0124] In some embodiments, the bispecific antibody AL3N2-28 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:48, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0125] In some embodiments, the bispecific antibody AL4N2-28 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:54, a CDRL2 comprising the amino acid sequence of SEQ ID NO:55, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:56, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:151, a CDRL2 comprising the amino acid sequence of SEQ ID NO:152, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:153.

[0126] In some embodiments, the bispecific antibody AL4N2-28 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:57, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:154.

[0127] In some embodiments, the bispecific antibody AL4N2-28 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:53, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0128] In some embodiments, the bispecific antibody AL5N2-28 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:59, a CDRL2 comprising the amino acid sequence of SEQ ID NO:60, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:61, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:151, a CDRL2 comprising the amino acid sequence of SEQ ID NO:152, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:153.

[0129] In some embodiments, the bispecific antibody AL5N2-28 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:62, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:154.

[0130] In some embodiments, the bispecific antibody AL5N2-28 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:58, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0131] Exemplary Anti-IL2Rγ and Anti-HER2 Bispecific Antibodies In some embodiments, the bispecific antibody AM5O30 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:84, a CDRL2 comprising the amino acid sequence of SEQ ID NO:85, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:126, a CDRL2 comprising the amino acid sequence of SEQ ID NO:127, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:128.

[0132] In some embodiments, the bispecific antibody AM5O30 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:129.

[0133] In some embodiments, the bispecific antibody AM5O30 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:125.

[0134] In some embodiments, the bispecific antibody AM5O35 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:84, a CDRL2 comprising the amino acid sequence of SEQ ID NO:85, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0135] In some embodiments, the bispecific antibody AM5O35 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:134.

[0136] In some embodiments, the bispecific antibody AM5O35 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:130.

[0137] In some embodiments, the bispecific antibody AM5O38 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:84, CDRL2 comprising the amino acid sequence of SEQ ID NO:85, and CDRL3 comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:136, CDRL2 comprising the amino acid sequence of SEQ ID NO:137, and CDRL3 comprising the amino acid sequence of SEQ ID NO:138.

[0138] In some embodiments, the bispecific antibody AM5O38 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:139.

[0139] In some embodiments, the bispecific antibody AM5O38 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:135.

[0140] In some embodiments, the bispecific antibody AM5O41 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:84, a CDRL2 comprising the amino acid sequence of SEQ ID NO:85, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:141, a CDRL2 comprising the amino acid sequence of SEQ ID NO:142, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:143.

[0141] In some embodiments, the bispecific antibody AM5O41 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0142] In some embodiments, the bispecific antibody AM5O41 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:140.

[0143] In some embodiments, the bispecific antibody AM9O30 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:104, a CDRL2 comprising the amino acid sequence of SEQ ID NO:105, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:106, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:126, a CDRL2 comprising the amino acid sequence of SEQ ID NO:127, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:128.

[0144] In some embodiments, the bispecific antibody AM9O30 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:129.

[0145] In some embodiments, the bispecific antibody AM9O30 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:103, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:125.

[0146] Exemplary Anti-IL2Rβ and Anti-HER2 Bispecific Antibodies In some embodiments, the bispecific antibody AL4O30 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:54, a CDRL2 comprising the amino acid sequence of SEQ ID NO:55, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:56, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:126, a CDRL2 comprising the amino acid sequence of SEQ ID NO:127, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:128.

[0147] In some embodiments, the bispecific antibody AL4O30 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:57, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:129.

[0148] In some embodiments, the bispecific antibody AL4O30 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:53, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:125.

[0149] In some embodiments, the bispecific antibody AL4O35 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:54, a CDRL2 comprising the amino acid sequence of SEQ ID NO:55, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:56, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0150] In some embodiments, the bispecific antibody AL4O35 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:57, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:134.

[0151] In some embodiments, the bispecific antibody AL4O35 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:53, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:130.

[0152] In some embodiments, the bispecific antibody AL4O38 comprises a common heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:54, a CDRL2 comprising the amino acid sequence of SEQ ID NO:55, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:56, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:136, a CDRL2 comprising the amino acid sequence of SEQ ID NO:137, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:138.

[0153] In some embodiments, the bispecific antibody AL4O38 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:57, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:139.

[0154] In some embodiments, the bispecific antibody AL4O38 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:53, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:135.

[0155] In some embodiments, the bispecific antibody AL4O41 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:54, CDRL2 comprising the amino acid sequence of SEQ ID NO:55, and CDRL3 comprising the amino acid sequence of SEQ ID NO:56, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:141, CDRL2 comprising the amino acid sequence of SEQ ID NO:142, and CDRL3 comprising the amino acid sequence of SEQ ID NO:143.

[0156] In some embodiments, the bispecific antibody AL4O41 comprises a common heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:57, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0157] In some embodiments, the bispecific antibody AL4O41 comprises a common heavy chain comprising the amino acid sequence of SEQ ID NO:5, a kappa light chain comprising the amino acid sequence of SEQ ID NO:53, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:140.

[0158] How to use The present disclosure relates to a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. In some embodiments, the compositions are used to inhibit tumor growth. In some embodiments, the compositions are used to treat cancer. In some embodiments, the compositions are used to enhance T cell mediated cell killing. In some embodiments, the compositions are used for T cell activation.

[0159] The present disclosure provides a method for treating, preventing or alleviating at least one symptom of a cell proliferative disorder in a subject in need thereof. In some aspects, the method alleviates at least one symptom of a cell proliferative disorder in a subject in need thereof. In one aspect, the cell proliferative disorder is cancer.

[0160] In some aspects, a method for treating, preventing or alleviating at least one symptom of a cell proliferative disorder in a subject in need thereof includes: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. The method comprises administering a composition comprising:

[0161] In some aspects, the first bispecific antibody and the second bispecific antibody are administered separately to a subject. In some aspects, the first bispecific antibody and the second antibody are formulated separately and combined prior to administration to a subject. In some embodiments, the first bispecific antibody and the second bispecific antibody are co-formulated and administered to a subject.

[0162] As used herein, a "subject" can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a pig, a sheep, a goat, a camel. In a preferred embodiment, the subject is a human.

[0163] In some aspects, a "subject in need thereof" is a subject having a cell proliferative disorder or a subject at increased risk of developing a cell proliferative disorder compared to the overall population as a whole. In some aspects, a subject in need thereof has a precancerous condition. In one aspect, a subject in need thereof has cancer.

[0164] As used herein, "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, including reducing or alleviating symptoms or complications, or eliminating a disease, condition, or disorder. As used herein, "prevention" describes the arrest of the onset of symptoms or complications of a disease, condition, or disorder. As used herein, "amelioration" describes the reduction of symptoms or complications of a disease, condition, or disorder.

[0165] As used herein, the term "cell proliferative disorder" refers to a condition in which uncontrolled or abnormal growth of cells, or both, can lead to the development of an undesirable condition or disease, which may be cancerous or non-cancerous. Exemplary cell proliferative disorders of the present disclosure encompass a variety of conditions in which cell division is deregulated. Exemplary cell proliferative disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, precancerous conditions, primary tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immune tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. Cell proliferative disorders include precancers or precancerous conditions. Cell proliferative disorders include cancers. Preferably, the methods provided herein are used to treat or alleviate the symptoms of cancer. The term "cancer" includes solid tumors, as well as hematological tumors and / or malignant tumors. A "precancerous cell" or "precancerous cell" is a cell that exhibits a cell proliferative disorder that is a precancer or precancerous condition. A "cancer cell" or "cancerous cell" is a cell that exhibits a cell proliferation disorder that is cancer. Any reproducible means of measurement may be used to identify cancer cells or precancerous cells. Cancer cells or precancerous cells can be identified by histological classification or grading of a tissue sample (e.g., a biopsy sample). Cancer cells or precancerous cells can be identified through the use of appropriate molecular markers.

[0166] Exemplary cancers include adrenal cortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), cholangiocarcinoma, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, gastrointestinal system, nervous system cancer, neurological ... Central nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Sezary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, ovarian germ cell tumors, gestational trophoblastic neoplasms, head and neck cancer, hepatocellular (liver) carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma , eye cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, kidney cancer, pharyngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's hypergammaglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell neck cancer, oral cavity cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, Myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cavity cancer, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma of the Ewing system, Kaposi's sarcoma,Cancers include, but are not limited to, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, and Wilm's tumor. Exemplary cancers include, but are not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer and renal pelvis cancer, oral cavity and pharyngeal cancer, uterine cancer, and / or melanoma.

[0167] Treatment regimens are made by identifying a subject, e.g., a human patient suffering from (or at risk of developing) cancer, using standard methods. Effective treatment is determined in association with any known method for diagnosing or treating a particular immune-related disorder. Amelioration of one or more symptoms of the immune-related disorder indicates that the antibody provides clinical benefit.

[0168] In some embodiments, the treatment of cancer results in a reduction in the size of the tumor. The reduction in the size of the tumor may also be referred to as "tumor regression". Preferably, after treatment, the tumor size is reduced by 5% or more compared to its size before treatment, more preferably, the tumor size is reduced by 10% or more, more preferably, by 20% or more, more preferably, by 30% or more, more preferably, by 40% or more, even more preferably, by 50% or more, and most preferably, by 75% or more. The size of the tumor may be measured by any reproducible means of measurement.

[0169] In some embodiments, treating cancer results in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or more compared to its size before treatment, more preferably, tumor volume is reduced by 10% or more, more preferably, by 20% or more, more preferably, by 30% or more, more preferably, by 40% or more, even more preferably, by 50% or more, and most preferably, by 75% or more. Tumor volume may be measured by any reproducible means of measurement.

[0170] In some embodiments, treating cancer results in a reduction in tumor number. Preferably, after treatment, tumor number is reduced by 5% or more compared to its number before treatment, more preferably, tumor number is reduced by 10% or more, more preferably, by 20% or more, more preferably, by 30% or more, more preferably, by 40% or more, even more preferably, by 50% or more, and most preferably, by more than 75%. Tumor number may be measured by any reproducible means of measurement.

[0171] In some embodiments, the treatment of cancer leads to a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reduced by 5% or more compared to the number before treatment, more preferably, the number of metastatic lesions is reduced by 10% or more, more preferably, the number of metastatic lesions is reduced by 20% or more, more preferably, the number of metastatic lesions is reduced by 30% or more, more preferably, the number of metastatic lesions is reduced by 40% or more, even more preferably, the number of metastatic lesions is reduced by 50% or more, and most preferably, the number of metastatic lesions is reduced by more than 75%.The number of metastatic lesions may be measured by any reproducible measuring means.

[0172] In some embodiments, treating cancer results in an increase in the average survival time of the population of treated subjects compared to the population receiving only the carrier.Preferably, the average survival time increases by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days.The increase in the average survival time of the population can be measured by any reproducible means.In some embodiments, the increase in the average survival time of the population can be measured, for example, by calculating the average length of survival for the population after the start of treatment with the active compound.In some embodiments, the increase in the average survival time of the population can also be measured, for example, by calculating the average length of survival for the population after the start of the first round of treatment with the active compound.

[0173] In some embodiments, treating cancer results in an increase in the average survival time of a population of treated subjects compared to a population of untreated subjects.Preferably, the average survival time increases by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days.The increase in the average survival time of a population can be measured by any reproducible means.In a preferred embodiment, the increase in the average survival time of a population can be measured, for example, by calculating the average survival time of a population after the start of treatment with an active compound.In another preferred embodiment, the increase in the average survival time of a population can also be measured, for example, by calculating the average length of survival of a population after the start of the first round of treatment with an active compound.

[0174] In some embodiments, treating cancer results in an increase in the average survival time of a population of treated subjects compared to a population receiving a therapy that is not a recombinant polypeptide of the present disclosure. Preferably, the average survival time is increased by more than 30 days, more preferably by more than 60 days, more preferably by more than 90 days, and most preferably by more than 120 days. The increase in the average survival time of a population can be measured by any reproducible means. In a preferred embodiment, the increase in the average survival time of a population can be measured, for example, by calculating the average survival time of a population after the start of treatment with an active compound. In another preferred embodiment, the increase in the average survival time of a population can also be measured, for example, by calculating the average length of survival of a population after the start of a first round of treatment with an active compound.

[0175] In some aspects, treating cancer results in a reduction in mortality of a population of treated subjects compared to a population receiving carrier alone. In some aspects, treating cancer results in a reduction in mortality of a population of treated subjects compared to a population receiving no treatment. In further aspects, treating cancer results in a reduction in mortality of a population of treated subjects compared to a population receiving monotherapy with a drug that is not a recombinant polypeptide of the present disclosure. Preferably, the mortality rate is reduced by more than 2%, more preferably, more than 5%, more preferably, more than 10%, and most preferably, more than 25%. In some aspects, the reduction in mortality of a population of treated subjects can be measured by any reproducible means. In some aspects, the reduction in mortality of a population can be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the start of treatment with an active compound. In some aspects, the reduction in mortality of a population can also be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the completion of a first round of treatment with an active compound.

[0176] In some embodiments, the treatment of cancer results in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5% compared to the number before treatment, more preferably, tumor growth rate is reduced by at least 10%, more preferably, by at least 20%, more preferably, by at least 30%, more preferably, by at least 40%, more preferably, by at least 50%, even more preferably, by at least 50%, and most preferably, by at least 75%.Tumor growth rate may be measured by any reproducible measurement means.In some embodiments, tumor growth rate is measured by the change in tumor diameter per unit time.

[0177] In some embodiments, the treatment of cancer results in a reduction in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%, more preferably, tumor regrowth is less than 10%, more preferably, less than 20%, more preferably, less than 30%, more preferably, less than 40%, more preferably, less than 50%, even more preferably, less than 50%, and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. In some embodiments, tumor regrowth is measured, for example, by measuring the increase in tumor diameter after a previous tumor shrinkage after treatment. In some embodiments, the reduction in tumor regrowth is indicated by the absence of tumor recurrence after treatment is stopped.

[0178] In some embodiments, the treatment, prevention or alleviation of cancer results in the reduction of cell proliferation rate.Preferably, after treatment, cell proliferation rate is reduced by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 50%, and most preferably at least 75%.Cell proliferation rate may be measured by any reproducible measuring means.In some embodiments, cell proliferation rate is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0179] In some embodiments, treating, preventing or alleviating cancer results in a reduction in the population of proliferating cells. Preferably, after treatment, the population of proliferating cells is reduced by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 50%, and most preferably at least 75%. The population of proliferating cells may be measured by any reproducible measuring means. In some embodiments, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells compared to the number of non-dividing cells in a tissue sample. In some embodiments, the proportion of proliferating cells is equivalent to the mitotic index.

[0180] In some embodiments, the treatment, prevention, or alleviation of cancer results in a reduction in the size of the area or zone of cell proliferation.Preferably, after treatment, the size of the area or zone of cell proliferation is reduced by at least 5% compared to its size before treatment, more preferably, by at least 10%, more preferably, by at least 20%, more preferably, by at least 30%, more preferably, by at least 40%, more preferably, by at least 50%, even more preferably, by at least 50%, and most preferably, by at least 75%.The size of the area or zone of cell proliferation may be measured by any reproducible measuring means.In some embodiments, the size of the area or zone of cell proliferation may be measured as the diameter or width of the area or zone of cell proliferation.

[0181] In some embodiments, the treatment, prevention, or alleviation of cancer results in a decrease in the number or percentage of cells with abnormal appearance or morphology. Preferably, after treatment, the number of cells with abnormal morphology is reduced by at least 5% compared to its size before treatment, more preferably by at least 10%, more preferably by at least 20%, more preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 50%, and most preferably by at least 75%. Abnormal cell appearance or morphology can be measured by any reproducible measuring means. In some embodiments, abnormal cell morphology is measured by microscopy, for example, using an inverted tissue culture microscope. In some embodiments, abnormal cell morphology takes the form of nuclear polymorphism.

[0182] In some embodiments, treating cancer or cell proliferation disorder results in cell death, preferably cell death results in at least a 10% reduction in the number of cells in the population. More preferably, cell death means at least a 20% reduction, more preferably at least a 30% reduction, more preferably at least a 40% reduction, more preferably at least a 50% reduction, and most preferably at least a 75% reduction. The number of cells in the population may be measured by any reproducible means. In some embodiments, the number of cells in the population is measured by fluorescence activated cell sorting (FACS). In some embodiments, the number of cells in the population is measured by immunofluorescence microscopy. In some embodiments, the number of cells in the population is measured by light microscopy. In some embodiments, methods for measuring cell death are shown in Li et al., (2003) Proc Natl Acad Sci USA. 100(5):2674-8.

[0183] The present disclosure provides a method for treating a population of cells, comprising: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. The present disclosure provides a method of enhancing immune cell-mediated cell killing, comprising providing a composition comprising: a. In some aspects, the present disclosure provides a method of enhancing T cell-mediated cell killing. In some aspects, the cancer T cell-mediated cell killing results in cell death, and preferably, cell death results in at least a 10% reduction in cell number in the population. More preferably, cell death means at least a 20% reduction, more preferably at least a 30% reduction, more preferably at least a 40% reduction, more preferably at least a 50% reduction, and most preferably at least a 75% reduction. The cell number in the population may be measured by any reproducible means.

[0184] The present disclosure provides a method for the treatment of a population of immune cells, comprising: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor. A method of enhancing immune cell activation is provided, comprising providing a composition comprising: a composition comprising: a) a cellular component comprising: ...

[0185] Pharmaceutical Compositions The antibody of the present invention (also referred to herein as "active compound"), as well as its derivatives, fragments, analogs and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include an antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0186] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. The solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and agents for adjusting osmolality such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0187] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent in the composition, such as sugar, mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition.

[0188] Sterile injection solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the preparation of sterile powder for sterile injection solution, the preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0189] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, active compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swallowed, expectorated, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0190] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0191] Systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0192] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0193] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0194] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.As used herein, dosage unit form refers to a physically separate unit suitable as a unit dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the unique characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for individual treatment.

[0195] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0196] Combination therapy The composition of the present disclosure, comprising a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor, and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of a cytokine receptor, is administered in combination with any other standard therapy, such methods being known to those skilled in the art and described in Remington's Pharmaceutical Sciences by EW Martin. If desired, the composition of the present disclosure is administered in combination with any conventional anti-tumor therapy, including but not limited to immunotherapy, therapeutic antibodies, targeted therapy, surgery, radiation therapy, or chemotherapy.

[0197] In some embodiments, the composition of the present disclosure is administered in combination with c) a third bispecific antibody comprising an antigen binding domain that binds a third tumor-associated antigen and an antigen binding domain that binds an antigen expressed on T cells. In some aspects, the antigen expressed on T cells is a CD3 antigen. Without being bound by theory, the addition of a third bispecific antibody can improve the clinical outcome of the composition by further activating a T cell subpopulation using a bispecific engager that targets the TAA and TCR complex. Without being bound by theory, the inventors believe that combining the bispecific antibodies of the present disclosure can stimulate both a primary signaling pathway that promotes T cell-mediated lysis of tumor cells (e.g., by clustering TCRs) and a second co-stimulatory pathway that induces T cell proliferation to overcome anergy.

[0198] definition As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0199] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be commonly represented by the dissociation constant (K.sub.D). Affinity can be measured by common methods known in the art, including KinExA and Biacore.

[0200] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0201] As used herein, unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab').sub.2, Fv fragments and individual antibody heavy or light chains, as well as individual heavy or light chain variable regions.

[0202] A "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0203] The "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0204] A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that also contains the VH and CH1 domains, and the region between the CH1 and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form an F(ab').sub.2 molecule.

[0205] An "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab').sub.2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody. The "Fv region" contains the variable regions of both the heavy and light chains, but lacks the constant region.

[0206] "Isolated antibody" refers to a purified state, and in such context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0207] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences within the variable domains, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0208] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0209] Generally, the basic "antibody" structural unit comprises a tetramer. In monospecific antibodies, each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a "variable region" or "variable domain" of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.

[0210] Typically, human constant light chains are classified as kappa and lambda light chains. Furthermore, human constant heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. These IgG subtypes include, for example, IgG1 and IgG4.

[0211] As used herein, "variable region", "variable domain", "V region", or "V chain" refers to a segment of an IgG chain whose sequence is variable among different antibodies. An antibody "variable region" refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable region of a heavy chain may be referred to as "V.sub.H.". The variable region of a light chain may be referred to as "V.sub.L.". Typically, both heavy and light chain variable regions contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, which allow binding to a specific epitope. Generally, from N-terminus to C-terminus, both light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally follows the definition for the sequence of proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th ed., NIH Publ. No. 91-3242 (1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia, et al., (1987) J Mol. Biol. 196:901-917, or Chothia, et al., (1989) Nature 342:878-883.

[0212] "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of an antibody V.sub.H. beta-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of an antibody V.sub.L. beta-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable regions within an antibody variable domain. CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved beta-sheet framework and therefore can accommodate different structures. Both terms are well known in the art. CDR region sequences are also defined by AbM, Contact, and IMGT. The positions of CDRs within standard antibody variable regions have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues within the hypervariable region varies in different antibodies, additional residues relative to the standard positions are conventionally numbered with a, b, c, etc. next to the residue number in the standard variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those of skill in the art. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT specific numbering systems, is well known to those of skill in the art. In some embodiments, the CDRs are as defined by the Kabat numbering system. In other embodiments, the CDRs are as defined by the IMGT numbering system. In still other embodiments, the CDRs are as defined by the AbM numbering system. In still other embodiments, the CDRs are as defined by the Chothia numbering system. In yet other embodiments, the CDRs are as defined by the Contact numbering system.

[0213] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same in equivalent positions when the two sequences are optimally aligned.

[0214] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.

[0215] "Conservatively modified variant" or "conservative substitution" refers to the substitution of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein.Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th Ed.)).In addition, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity.

[0216] As used herein, the term "epitope" refers to an area or region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen-binding fragment thereof disclosed herein to an epitope means that the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within the epitope.

[0217] An "isolated" nucleic acid molecule or polynucleotide refers to DNA or RNA, e.g., DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, in which the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature, or associated with a polynucleotide with which it is not associated in nature. For purposes of this disclosure, it should be understood that a polynucleotide "comprising (or similar to)" a particular nucleotide sequence does not encompass an intact chromosome. An isolated polynucleotide "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10, or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0218] The term "control sequence" refers to a polynucleotide sequence necessary or useful for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers. In one embodiment of the present invention, the polynucleotide is operably linked to a promoter, such as a viral promoter, a CMV promoter, an SV40 promoter, or a non-viral promoter or an elongation factor (EF)-1 promoter, and / or an intron.

[0219] A nucleic acid is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not necessarily, "operably linked" means that the polynucleotide sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0220] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transformations. It is also understood that not all progeny have exactly the same DNA content due to deliberate or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where separate designations are intended, it will be clear from the context.

[0221] Host cells include eukaryotic and prokaryotic host cells, including mammalian cells. Host cells include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells, among others. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or nettle looper), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophia, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnen), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia piperi, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucnowens lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.Pichia spp., any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucnowens, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa. The invention includes any host cell (e.g., a CHO cell or a Pichia cell, e.g., Pichia pastoris) containing an anti-ILT4 antibody or antigen-binding fragment thereof, or containing a polynucleotide containing such an antibody or fragment, or containing a vector containing a polynucleotide.

[0222] "Treat" or "treating" means administering an antibody or antigen-binding fragment thereof of the present invention to a subject having one or more symptoms of a disease for which the antibody and antigen-binding fragment are effective, for example, in treating a subject having or suspected of having cancer or an infectious disease for which the agent has therapeutic activity. Typically, the antibody or fragment is administered in an "effective amount" or "effective dose" that will alleviate one or more symptoms (e.g., symptoms of cancer or infectious disease) in the subject or population being treated by inducing regression or elimination of such symptoms, or to any clinically measurable extent, by inhibiting the progression of such symptoms, such as cancer symptoms, such as tumor growth or metastasis. The effective amount of the antibody or fragment may vary depending on factors such as the disease stage, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. EXAMPLES

[0223] Example 1: Construction and production of KiH bispecific antibodies 1.1.BsAb construction The complementarity determining regions (CDRs) of anti-IL-2Rγ (P1A3), anti-IL-2Rβ (P2C4) and anti-HER2 (Pertuzumab and Trastuzumab) were obtained from published sequences (WO2017 / 021540Al and US2006 / 0275305A1, respectively) and used for proof of concept. CDR sequences were synthesized by Eurofins and engineered into IL-2R×HER2 or IL-2Rβ×IL-2Rγ bsAbs using knob-in-the-hole (KiH) and CrossMAb technologies (US2017 / 0129962A1) with an inactive Fc (LALA PA mutation: leucine 234, leucine 235 and proline 329 were replaced with alanine residues). In all IL-2RxHER2 bsAb constructs, the CDR sequences of the anti-HER2 arm (both pertuzumab and trastuzumab) were inserted into the knob arm. In the P1A3xP2C4 construct, the CDR sequences of the anti-IL-2Rβ were inserted into the knob arm. The gene fragments were cloned into an expression vector under the control of the hCMV promoter by restriction enzymes (BsrGI and PshAI, XbaI and EcoRI, AscI and BamHI, AfeI and Cla I). ​​After each cloning step, the newly engineered plasmids were transfected into XL1 bacteria for amplification and selection using ampicillin resistance. After plasmid purification, the desired constructs were verified by sequencing.

[0224] 1.2. Expression and purification Plasmids were transiently transfected into PEAK cells using Lipofectamine 2000 transfection reagent (Invitrogen, #11668-019). BsAbs were purified from supernatants 5-6 days post-transfection using CaptureSelect™ FcXL affinity matrix (Thermo Fisher, #194328050) and CaptureSelect™ IgG-CH1 affinity matrix (Thermo Fisher, #194320005) successively, eluted using glycine pH 3.0 buffer, and desalted against 25 mM histidine / 125 mM NaCI pH 6.0 buffer in 50 kDa Amicon Ultra Centrifugal filter units (#ACS505024). The final bsAb product was quantified using Nanodrop and its quality was verified by SEC-HPLC, electrophoresis under denaturing conditions, isoelectric focusing gel and Agilent 2100 Bionalayzer using Protein 80 kit.

[0225] Example 2: Selection and screening of phage display of IL2-Rb or IL2-Rg Fv using a human scFv library containing fixed variable heavy chain domains The general procedure for constructing and handling a human scFv library displayed on M13 bacteriophage is described in Vaughan et al.,

[20] , which is incorporated herein by reference in its entirety. The libraries for selection and screening all encode scFvs that share the same VH domain and are diversified only in the VL domain. Methods for generating fixed VH libraries and their use for identifying and assembling bispecific antibodies are described in US2012 / 0184716 and WO2012 / 023053, each of which is incorporated herein by reference in its entirety. The procedure for identifying scFvs that bind to human IL-2Rb or IL-2Rg is described below. Selection was performed using biotinylated human IL-2Rb or IL-2Rg proteins pre-coated on magnetic beads. The selection strategy included up to four rounds of selection.

[0226] 2.1. Protein Selection: Aliquots of scFv phage libraries were blocked with PBS containing 2% (w / v) skim milk. Blocked phages were first deselected on streptavidin / neutravidin magnetic beads (Dynabeads™ M-280 streptavidin magnetic beads or Sera-Mag SpeedBeads Neutravidin™ coated magnetic particles) and then incubated with 100 nM, 10 nM or 1 nM biotinylated recombinant human and cynomolgus IL2Rb or IL2Rg (commercially available or produced in-house) previously captured on the same type of beads used for deselection. The mixture was washed five times with PBS / 1% BSA / 0.1% Tween® 20 and twice with PBS only. Phages were eluted with 1 mg / mL trypsin and, after adding AEBSF to block trypsin activity, the eluate was added directly to exponentially growing TG1 bacterial cells. The results were rescued and used for the next selection round.

[0227] 2.2. Screening for scFv binding / non-binding to human IL-2R: Screening of scFvs for binding to IL-2Rb or IL-2Rg was tested by ELISA using biotinylated recombinant proteins (and biotinylated huMSLN protein as an irrelevant control).

[0228] 2.3.ELISA: For the binding ELISA, neutravidin-coated plates were blocked with a 1% casein solution in PBS. Biotinylated IL-2Rb, IL-2Rg and huMSLN (mesothelin) were captured at 5 nM. Dilutions of freshly prepared periplasmic extracts containing selected scFvs were applied to the plate and detected using a combination of mouse anti-c-myc and donkey anti-mouse IgG-HRP antibodies. After addition of TMB, the OD at 450 nm was measured using a spectrophotometer plate reader. The OD on IL-2Rb or IL-2Rg was measured in the absence of binding to the irrelevant huMSLN protein and in the absence of binding to the unrelated huMSLN protein. 450 Background OD 450 Hits were classified as specific binders if their binding was at least 3-fold higher than 1. Hits were sequenced after DNA extraction from single clones.

[0229] Example 3: Fixed VH candidates reformatted into IgG and transiently expressed in mammalian cells After screening and sequencing, scFv candidates with the desired binding properties were reformatted into IgG and expressed by transient transfection into PEAK cells. The VH and VL sequences of the selected scFvs were amplified with specific oligonucleotides and cloned into an expression vector containing the heavy and light chain constant regions. The expression vector was verified by sequencing and transfected into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 4 × 10 6PEAK cells were cultured in 25 ml of culture medium containing fetal bovine serum in a T75 flask. The transfected cells were cultured at 37°C for 5-6 days and IgG production was quantified using an Octet RED96 instrument. The supernatant was harvested for IgG purification on FcXL affinity resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the supernatant from the transfected cells was incubated overnight at 4°C with an appropriate amount of FcXL resin. After washing the resin with PBS, the sample was loaded onto an Amicon Pro column and the resulting IgG was eluted with 50 mM glycine pH 3.5. The eluted IgG fraction was then dialyzed by Amicon 50 kDa against histidine NaCl pH 6.0 buffer and the IgG content was quantified by absorbance at 280 nm. Purity and IgG integrity were verified by electrophoresis using an Agilent Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies).

[0230] Example 4: Expression and purification of a bispecific antibody carrying lambda and kappa light chains Co-expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Co-expression can be achieved in different ways, such as by transfection of multiple vectors expressing one of the co-expressed chains, or by using vectors driving multiple gene expression.

[0231] Here, the two light chains were cloned into the vector pNovi κHλ, which was previously generated to allow for the co-expression of one heavy chain, one kappa light chain and one lambda light chain, as described in US20120184716 and WO2012023053, each of which is incorporated herein by reference in its entirety. Expression of the three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains the glutamine synthetase gene (GS), which allows for the selection and establishment of stable cell lines. The common VH and VL genes of IL-2Rb or IL-2Rg IgG (both described in this patent) and anti-MSLN IgG (O30, O35, O38, O41, according to this patent WO2018215835A1) or anti-HER2 IgG (N2-19, N2-28, as described in this patent) were cloned into the vector pNovi κHλ for transient expression in mammalian cells. All constructs were generated based on an inactive Fc, referred to as / N in the documentation (LALA PA mutation: leucine 234, leucine 235 and proline 329 were replaced by alanine residues). Expi293 cells were cultured in suspension in appropriate Erlenmeyer flasks with the appropriate number of cells and culture medium volume. Plasmid DNA was transfected into Expi293 cells using PEI. Antibody concentrations in the supernatants of transfected cells were measured during production using an Octet RED96. According to the antibody concentration, the supernatants were harvested 5-7 days after transfection and clarified by filtration after the addition of diatomaceous earth (Sartorius). Purification was based on a three-step purification process. First, CaptureSelect™ FcXL affinity substrate (Thermo Fisher Scientific) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4°C and 20 rpm, the supernatant was centrifuged at 2000g for 10 min, the flow-through was saved and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column and a solution containing 50 mM glycine at pH 3.5 was used for elution. Several elution fractions were generated, neutralized with Tris-HCl pH 7.4 and pooled.The pool containing total human IgG (bispecific and two monospecific antibodies) was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies). A small aliquot was saved for further analysis and the remaining sample was incubated with an appropriate volume of CaptureSelect™ Kappa XL affinity substrate (Thermo Fisher Scientific) for 30 min at room temperature at 20 rpm. Resin recovery and washing, elution and neutralization steps were performed as described above. The final affinity purification step was performed using CaptureSelect™ Lambda Fab affinity substrate (Thermo Fisher Scientific) applying the same process as the Kappa purification step. Alternatively, purification was based on a two-step purification process using only CaptureSelect™ Kappa XL affinity substrate and CaptureSelect™ Lambda Fab affinity substrate. All elution fractions were pooled and desalted against His-NaCl pH 6.0 formulation buffer using 50 kDa Amicon Ultra centrifugal filter units (Merck Millipore). The final product was quantified using a Nanodrop.

[0232] The purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer with the Protein 80 kit as described by the manufacturer (Agilent Technologies). Aggregate levels were determined by SEC-UPLC. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories).

[0233] Example 5: Assessment of binding of bsAbs to their targets by ELISA The binding specificity of all IL-2RxTAA bsAbs was assessed by direct ELISA (Figure 2). 50μl / well of biotinylated human recombinant proteins IL-2Rβ, IL-2Rγ, HER2, MSLN, and an unrelated protein (recombinant human carcinoembryonic antigen) were added at a concentration of 1μg / ml (concentration was adjusted to compensate for incomplete biotinylation) onto streptavidin-coated 96-well black plates (Greiner, #655997). Binding of bsAbs to the coated proteins was assessed using HRP-conjugated goat anti-human IgG-Fcγ (#109-036-098) and Amplex™ UltraRed Reagent (#A36006). Data were acquired using a Synergy HT (Biotek) at the following wavelengths: Excitation: 530 / 25, Emission: 590 / 35. Binding to IL-2Rβ was confirmed for KiH bsAbs carrying the P2C4 arm, i.e., P2C4×Pertuzumab, P2C4×Trastuzumab and P1A3×P2C4 (Figure 2A), binding to IL-2Rγ was confirmed for KiH bsAbs carrying the P1A3 arm, i.e., P1A3×Pertuzumab, P1A3×Trastuzumab and P1A3×P2C4 (Figure 2B), while binding to HER2 was confirmed for all bsAbs carrying the anti-HER2 arm, i.e., all bsAbs except P1A3×P2C4 (Figure 2C). None of the bsAbs bind to unrelated proteins (Figure 2D).

[0234] IL-2R κλ-bodies were also generated targeting two different TAAs: HER2 and MSLN. A first set of IL-2R×HER2 κλ-bodies was generated by pairing the same HER2 arm (N2-28) with different IL-2Rb arms (AL1-AL5) and another HER2 arm (N2-19) with different IL-2Rg arms (AM1-AM11). Another set of IL-2R×MSLN κλ-bodies was generated by pairing one IL-2Rb arm (AL4) and one IL-2Rg arm (AM5) with different MSLN arms (O30, O35, O38, O41 according to patent WO2018215835A1). Similar ELISA assays were performed to verify binding to each target. Binding to IL2Rb was confirmed for all κλ-bodies carrying IL-2Rb arms (AL1-AL5) (Figure 2E). Binding to IL2Rg was confirmed for all κλ-bodies carrying IL2Rg arms (AM1-AM11) (Figure 2F). Binding to HER2 was shown for all IL-2R×HER2 κλ-bodies (Figure 2G) and binding to MSLN was shown for all IL-2R×MSLN κλ-bodies (Figure 2H).

[0235] Example 6: Evaluation of co-engagement of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs with their targets by biolayer interferometry Binding of KiH bsAbs to their targets was assessed using Biolayer Interferometry (BLI) technology (OctetRED96). BsAbs were diluted to a concentration of 10 μg / ml in kinetic buffer (Sartorius, #18-1105) loaded onto a Protein A biosensor (Sartorius, #18-5010). Biosensors were incubated simultaneously in wells containing either individual targets or the two targets of the corresponding bsAb. Results showed that P1A3×Pertuzumab bound both IL-2Rγ and HER2 separately and simultaneously (Fig. 3A), whereas P2C4×Pertuzumab and P2C4×Trastuzumab bound IL-2Rβ and HER2 separately and simultaneously (Fig. 3B, C). In agreement with the ELISA results, BLI experiments confirmed the binding specificity of the bsAbs to their targets. Furthermore, the higher signal (wavelength shift) obtained for P1A3 x Pertuzumab, P2C4 x Pertuzumab and P2C4 x Trastuzumab bsAbs indicates the ability to bind both targets simultaneously when both targets were present compared to conditions with a single target (Figure 3).

[0236] Example 7: Evaluation of IL-2 and IL-15 signaling neutralization by IL-2R x TAA bsAb Once binding of each IL-2RxTAA bsAb of the invention was confirmed, experiments were performed to determine whether engaged IL-2Rb or IL-2Rg interfered with native IL-2 or IL-15 signaling. To this end, an IL-2-reporter cell line (expressing a dimeric IL-2R and responding to both IL-2 and IL-15) was stimulated with a fixed dose of IL-2 (0.2 or 1 nM, respectively, Figure 4A,C) or IL15 (0.003 nM, Figure 4B,D) in the presence of a dose range of IL-2RbxHER2 bsAb (Figure 4A,B), or IL-2RgxHER2 bsAb (Figure 4C,D).

[0237] 7.1. Cell culture: HEK Blue IL-2 cells (Invivogen, #hkb-il2bg), which express IL-2Rb and IL-2Rg, were used as a reporter cell line for IL-2 and IL-15 signaling assays. The cell line is engineered to express secreted embryonic alkaline phosphatase (SEAP) downstream of IL-2 signaling (SEAP expression is controlled by a pSTAT5 inducible promoter). Reporter cells were cultured in DMEM 4.5 g / l glucose, 2 mM L-glutamine (#11965-084), fetal bovine serum (10% v / v, #F7524), gentamicin (25 μg / ml, #G1397-10ML), normocin (100 μg / ml, #ant-nr-2), 1× HEK-Blue CLR-Selection (#hb-csm), and puromycin (1 μg / ml, #ant-pr-1) at 37°C with 5% CO2.

[0238] 7.2. IL-2 Reporter Cell Assay: The assay was performed by incubating cells (HEK blue IL-2Rb / g) in flat-bottom 96-well plates (100000 cells / well) in DMEM 4.5g / l glucose 2mM L-glutamine (#11965-084), fetal bovine serum (10% v / v, #F7524), gentamicin (25μg / ml, #G1397-10ML) for 20-24 hours at 37°C and 5% CO2 under different test conditions. SEAP levels were measured in clear-bottom 96-well plates by incubating 20μl of supernatant from each well with 180μl of Quanti Blue™ solution (Invivogen, #rep-qbs) for 60 minutes at 37°C. Absorbance at 630nm was acquired using a Synergy HT (Biotek).

[0239] The results showed that in the dose range tested, IL-2RbxHER2 bsAb (Fig. 4A,B) or IL-2RgxHER2 bsAb (Fig. 4C,D) did not interfere with IL-2 (Fig. 4A,C) or IL-15 signaling (Fig. 4B,D).

[0240] Example 8: Activation of IL-2 signaling by combinations of IL-2Rβ×HER2 and IL-2Rγ×HER2 KiH bsAbs in the presence of soluble HER2 To demonstrate the IL-2-agonist activity of the IL-2R×HER2 bsAb combination in the presence of a soluble TAA (e.g., HER2), an experiment was performed in which soluble HER2 was mixed with a dose range of the IL-2Rb×HER2+IL-2Rg×HER2 KiH bsAb combination (Figure 5A). As expected, incubation of P1A3×P2C4 KiH bsAb with the reporter cell line induced IL-2 signaling (Figure 5B-D). However, the combination of the two IL-2R×HER2 bsAbs did not induce IL-2 signaling in the absence of HER2 (data not shown). In the presence of soluble HER2, the combination of two IL-2R×HER2 bsAbs targeting the same epitope on HER2 (P1A3×Pertuzumab+P2C4×Pertuzumab or P1A3×Trastuzumab+P2C4×Trastuzumab) did not induce IL-2 signaling (Figure 5B, C). In contrast, the combination of two IL-2R×HER2 bsAbs targeting two different epitopes on HER2 (P1A3×Trastuzumab+P2C4×Pertuzumab) induced IL-2 signaling when incubated with soluble HER2 (Figure 5D). Moreover, pathway activation was dependent on soluble HER2 concentration.

[0241] Activation of IL-2 signaling requires that IL-2Rβ (CD122) and IL-2Rγ (CD132) be brought into close proximity to each other. Since soluble HER2 monomer can only bring together two KiH bsAbs that target two distinct epitopes on HER2 (Figure 5A), the results obtained with the combination of KiH bsAbs in the presence of soluble HER2 are consistent with expectations.

[0242] Example 9: Activation of IL-2 signaling by combinations of IL-2Rβ×TAA and IL-2Rγ×TAA bsAbs in the presence of TAA-coated microparticles 9.1. Cell culture: HEK Blue IL-2 cells (Invivogen, #hkb-il2), which express IL-2Ra, IL-2Rb and IL-2Rg, were used as a reporter cell line for IL-2 signaling assays. The cell line is engineered to express secreted embryonic alkaline phosphatase (SEAP) downstream of IL-2 signaling (SEAP expression is controlled by a pSTAT5 inducible promoter). Reporter cells were cultured in DMEM 4.5 g / l glucose, 2 mM L-glutamine (#11965-084), fetal bovine serum (10% v / v, #F7524), gentamicin (25 μg / ml, #G1397-10ML), normocin (100 μg / ml, #ant-nr-2), 1× HEK-Blue CLR-Selection (#hb-csm), and puromycin (1 μg / ml, #ant-pr-1) at 37°C with 5% CO2.

[0243] 9.2. IL-2 Reporter Cell Assay: Assays were performed in flat-bottom 96-well plates (50000 cells / well) by incubating streptavidin-biot-TAA coated microparticles (300000 beads / well) and cells in DMEM 4.5g / l glucose 2mM L-glutamine (#11965-084), fetal bovine serum (10% v / v, #F7524), gentamicin (25μg / ml, #G1397-10ML) for 20-24 hours at 37°C and 5% CO2 under different test conditions. SEAP levels were measured in clear-bottom 96-well plates by incubating 20μl of supernatant from each well with 180μl of Quanti Blue™ solution (Invivogen, #rep-qbs) for 120 minutes at 37°C. Absorbance at 630nm was acquired using a SpectraMax-i3x (Molecular Devices).

[0244] As shown in the previous examples, in the presence of soluble HER2 monomer, only the combination of two IL-2R×HER2 bsAbs targeting two different epitopes on HER2 can induce IL-2 signaling. Because the clustering of HER2 on the cell surface has the potential to bring two IL-2R×HER2 bsAbs targeting the same HER2 epitope into close proximity with each other, it is expected that in the presence of surface-clustered HER2, the combination of two IL-2R×HER2 bsAbs targeting the same HER2 epitope can also induce IL-2 signaling. To test this hypothesis, HER2-coated microparticles (#24158-5) were used to mimic HER2 expressed on the cell surface in an IL-2 reporter assay (Figure 6A). In the presence of HER2-coated microparticles, both the combination of two IL-2R×HER2 bsAbs targeting two different HER2 epitopes (Fig. 6B,C) and the combination targeting the same HER2 epitope (Fig. 6D,E) induced IL-2 signaling activation. With both types of combinations, we observed a positive correlation between the level of IL-2 signaling measured and the concentration of HER2-coated microparticles. Importantly, the agonistic effect of the combination of two IL-2R×HER2 bsAbs was much more potent compared to that observed with P1A3×P2C4 bsAb (non-TAA targeting), reaching levels close to those induced by recombinant human IL-2 (Fig. 6B-E). Incubation of the bsAb combination with high concentrations of microparticles coated with an unrelated protein did not activate IL-2 signaling.

[0245] Similar findings were obtained with various combinations of IL-2R x TAA κλ-bodies (Figure 7). A first set of IL-2R x HER2 bsAbs was tested by combining κλ-bodies that share the same HER2 arm (N2-28) but paired with different IL-2Rb arms (AL1-AL5) with κλ-bodies that share the same HER2 arm (N2-19, targeting a different HER2 epitope than N2-28) but paired with different IL-2Rg arms (AM1-AM5). All κλ-bodies were cross-combined and tested as shown in Figure 6A in the presence of HER2-coated beads (fixed 6:1, bead:IL-2 reporter cell line ratio). The data showed strong IL-2 agonist activity regardless of the combination tested (Figure 7). Interestingly, the IL-2 agonist activity can be fine-tuned across different combinations. When AL1N2-28 / N was combined with AM52-29 / N, IL-2 signaling could be induced, but was consistently weaker when combined with AM1N2-19 / N (Figure 7A). Similar data were generated by combining AM5N2-19 / N with AL2N2-28 / N (Figure 7B), AL3N2-28 / N (Figure 7C), AL4N2-28 / N (Figure 7D) or AL5N2-28 / N (Figure 7E). The resulting IL-2 signaling could be modulated either by decreasing the concentration of the bsAb combination or by combining different pairs of IL-2R×HER2 κλ-bodies.

[0246] To show that IL-2R-bsAb combinations can also mediate IL-2 signaling by engaging another TAA, an additional set of κλ-bodies was generated by pairing one IL-2Rb arm (AL4) and one IL-2Rg arm (AM5) with various MSLN targeting arms (O30, O35, O38, O41), each of which binds to a non-overlapping epitope (patent WO2018215835A). Their activity in combination was tested using the same method as above, but replacing the HER2-coated beads with MSLN-coated beads (Figure 6A). All κλ-bodies cross-combined and showed potent IL-2 agonist activity regardless of the MSLN arm used (Figure 7F-J). As observed for IL-2R×HER2 bsAb, IL-2 agonist activity can be fine-tuned across different combinations. When AM5O30 / N was combined with AL4O38 / N, higher IL-2 signaling could be induced (Figure 7F). Similar data was generated by combining AM5O30 / N with AL4O35 (Figure 7G), AL4O30 / N (Figure 7H) or AL4O41 / N (Figure 7I). Importantly, as observed for the IL-2R×HER2 bsAb, combination of two IL-2R×MSLN bsAbs targeting the same MSLN epitope with O35 (Figure 8A), O41 (Figure 8B) or O30 (Figure 8C) induced IL-2 signaling to levels close to those induced by recombinant human IL-2 (Figures 8A-C).

[0247] Example 10: TAA + Activation of IL-2 signaling by combinations of IL-2Rβ × TAA and IL-2Rγ × TAA bsAbs in the presence of tumor cells The results described above show that in the presence of TAA-coated microparticles and regardless of the bsAb format used (KiH construct or κλ-body), the IL-2Rb×TAA and IL-2Rg×TAA bsAb combinations can induce IL-2 signaling activation, suggesting that IL-2 agonist activity can also be mediated in the presence of TAA-expressing tumor cells. Both HER2- and MSLN-expressing cell lines were used as sources of TAAs.

[0248] 10.1. Tumor cell line culture: Three HER2+ tumor cell lines were used: BT-474, SK-BR-3 and NCI-N87. BT-474 (ATCC, #HTB-20) is an invasive ductal carcinoma. SK-BR-3 (ATCC, #HBT-30) is a breast adenocarcinoma cell line. NCI-N87 (ATCC, #CRL-5822) is a gastric carcinoma cell line. HER2 is overexpressed in all three above-mentioned tumor cells, BT-474, SK-BR-3 and NCI-N87 expressing 624'000, 587'000 and 552'000 HER2 / cell, respectively. As a source of MSLN, three tumor cell lines were used: NCI-H226, OVCAR-3 and NCI-N87. NCI-H226 (ATCC, #CRL-5826) is a lung squamous cell carcinoma. OVCAR-3 (ATCC, #HBT-161) is an epithelial ovarian cancer cell line. In contrast to HER2, MSLN is expressed at lower levels in NCI-H226, OVCAR-3 and NCI-N87 cell lines, expressing 180'000, 67'000 and 24'000 MSLN / cell, respectively. These five adherent cell lines were cultured at 37°C with 5% CO2 and detached for subculture using trypsin-EDTA solution. BT-474 was cultured in Hybridare medium (ATCC, #46-X), fetal bovine serum (10% v / v, Thermo Fisher #F7524), L-glutamine (2 mM, Sigma, #G7513-100ML) and sodium bicarbonate (1.5 g / L, Sigma #S8761-100mL). SK-BR-3 was cultured in McCoy's 5A (Sigma, #M9309), 10% fetal bovine serum and L-glutamine. NCI-N87 was cultured in RPMI 1640 (Thermo Fisher, #11875-093), 10% fetal bovine serum and 2 mM L-glutamine.

[0249] As shown in the previous examples, in the presence of HER2-coated microparticles, the combination of two IL-2R x HER2 bsAbs can induce IL-2 signaling. The combination of two IL-2R x HER2 bsAbs also inhibits HER2 +In the presence of HER2-overexpressing tumor cells, it is predicted that IL-2 signaling will be activated. To test this hypothesis, we inject HER2-coated microparticles into HER2 + We replaced the tumor cells and used an IL-2 reporter assay in which 100'000 tumor cells were mixed with 100'000 HEK Blue IL-2 cells (Figure 9A). + In the presence of tumor cells overexpressing NCI-N87, both tested IL-2R x HER2 KiH bsAb combinations induced dose-dependent IL-2 signaling activation (Figure 9B,C), approaching that induced by recombinant human IL-2 in the presence of NCI-N87. This observation confirms that our IL-2R x HER2 bsAb combinations are capable of activating IL-2 signaling in the presence of HER2-expressing cells.

[0250] In the previous example, we showed that IL-2 signaling can be induced in reporter cells in the presence of HER2-overexpressing tumor cells through our IL-2R×HER2 bsAb combination. To extend this observation to non-transduced cells of immune origin, we chose NK-92 (DSMZ, #ACC 488), a natural killer lymphoma cell line. To quantify IL-2 signaling activation in NK-92 cells, we measured the levels of pSTAT5 by flow cytometry, a method widely used to evaluate IL-2 signaling activation. STAT5 is a transcription factor involved in the signaling pathway of IL-2. Upon engagement of IL-2 to IL-2Rβ and IL-2Rγ, JAK1 and JAK3 are recruited and can phosphorylate STAT5. pSTAT5 can dimerize and function as a transcription factor (Figure 10A).

[0251] 10.2.NK92 cell culture: Tumor cell culture was as described in 6.1. NK-92 were cultured in MEM alpha medium (Thermo Fisher, #22561-021), 12.5% ​​fetal bovine serum, horse serum (12.5% ​​v / v, Sigma, #H1270-500ML), 2 mM L-glutamine and human recombinant IL-2 (5 ng / ml, Peprotech, #200-02) at 37°C with 5% CO2.

[0252] 10.2. pSTAT5 Measurement in NK-92 Cells: 24 hours prior to the assay, NK-92 cells were washed four times in DPBS (Thermo Fisher, #14190-144) and cultured for 24 hours under the same conditions but without human recombinant IL-2.

[0253] 10.2.1. Figure 10: NK-92 cells were washed in DPBS and plated at 5 x 10 in DPBS, Fc Block (30 μl / ml BD Biosciences, #564220) and Fixable Viability Stain 620 (1 μl / ml BD Biosciences, #564996). 6 NK-92 cells are washed once and incubated at 2 × 10 cells / ml in anti-human CD45-V500 (1 μl / 100 μl BD Biosciences, #560777) in DPBS, bovine serum albumin (2% m / v, Sigma #A3912-100G) at 4 °C. 6 Stain for 20–30 min at 150,000 cells / ml. NK-92 cells and harvested tumor cells are washed twice and transferred to a 96-well round-bottom plate at 150,000 cells / well, respectively. Cells are cultured in RPMI 1640 (Thermo Fisher, #11875-093), 10% fetal bovine serum, 2 mM L-glutamine, Hepes (10 mM, Sigma, #H0887), non-essential amino acids (1×, Sigma, #M7145-100mL), β-mercaptoethanol (0.050 mM, Thermo Fisher, #31350-010), gentamicin (25 μg / ml, Sigma, #G1397-10ML) at 5% CO under different test conditions. 2、Incubate at 37°C for 1-2 hours. Fix cells with 0.4% formaldehyde for 12 minutes at 37°C, then wash and permeabilize in MetOH for 30 minutes on ice. Wash once in DPBS and stain with Alexa Fluor 488 anti-STAT5 (pY694) (3 μl / well BD Biosciences, #562075), 2% bovine serum albumin in DPBS for 1 hour at room temperature in the dark. Wash cells twice with DPBS, 2% bovine serum albumin and resuspend in 150 μl DPBS for flow cytometry analysis. pSTAT5 + pSTAT5 - The threshold for distinguishing cells is determined based on control unstimulated wells. Data are presented as the percentage of pSTAT5 positive cells.

[0254] In the presence of either HER2-overexpressing cells, both the combination of two IL-2R×HER2 bsAbs targeting two different HER2 epitopes (Fig. 10B, C) and the combination targeting the same HER2 epitope (Fig. 10D, E) induced IL-2 signaling activation. Furthermore, we also observed activation of the pathway when all four IL-2R×HER2 bsAbs were used simultaneously (Fig. 10F). Importantly, the pSTAT5 upregulation observed with some of the IL-2R×HER2 bsAb combinations was not observed. + The percentage of cells that were transduced with HER2 was close to that induced by recombinant human IL-2 (Figure 10B-F). The results confirm that in the presence of HER2-overexpressing tumor cells, all combinations of KiH bsAbs tested are able to induce IL-2 signaling in immunogenic non-transduced cells.

[0255] 10.2.2. We obtained similar findings with various combinations of proprietary IL-2RxTAA κλ-bodies using different protocols (Figure 11). On the day of the assay, NK-92 and tumor cells are washed twice in DPBS and transferred to 96-well flat-bottom plates at 150,000 cells / well, respectively. Cells are incubated in MEM alpha medium (Thermo Fischer #22561-021), 12.5% ​​fetal bovine serum, horse serum (12.5% ​​v / v, Sigma #H1270-500ML), 2 mM L-glutamine, under different test conditions, at 5% CO2, 37°C for 30 minutes. After incubation, cells are transferred to V-bottom 96-well plates and washed with Fixable Viability Stain 620 (1 μL / mL BD Biosciences, #564996) for 5 min at 37°C, Fc Block (30 μL / mL BD Biosciences, #564220) for 10 min at room temperature and anti-human CD45-V500 (1 μL / 100 μL BD Biosciences, #560777) for 30 min at 4°C, respectively, before incubation. Cells were washed with DPBS, 2% bovine serum albumin between each step of incubation. Finally, cells are fixed with 0.4% formaldehyde for 12 min at 37°C, then washed and permeabilized in MetOH for 30 min on ice. After one wash, cells are stained with Alexa Fluor 488 anti-STAT5 (pY694) (3 μl / well BD Biosciences, #562075) in DPBS, 2% bovine serum albumin for 1 hour at room temperature in the dark, washed twice with DPBS, 2% bovine serum albumin, and then resuspended in 150 μl DPBS for flow cytometry analysis. + pSTAT5 - The threshold for distinguishing cells is determined based on control unstimulated wells. Data are presented as median fluorescence intensity (MFI) levels of pSTAT5 in CD45+ cells.

[0256] A first set of IL-2RxHER2 bsAbs was tested by combining κλ-bodies sharing the same HER2 arm (N2-28) with two different IL-2Rb arms (AL2 and AL4) and κλ-bodies sharing the same HER2 arm (N2-19, targeting another HER2 epitope than N2-28) with two different IL-2Rg arms (AM5 and AM9). All κλ-bodies were cross-combined and tested as shown in Figure 6A in the presence of HER2-coated beads (fixed ratio 6:1, beads:IL-2 reporter cell line). The data showed pSTAT5 induction in NK92 cells regardless of the combination tested. IL-2 signaling is increased in the presence of both HER2-overexpressing tumor cell lines: BT-474 (Figure 11A) and NCI-N87 (Figure 11B). IL-2 signaling induction is similar across all κλ-body combinations tested.

[0257] The combination of IL-2R-bsAb was + To show that they can mediate IL2 signaling in the presence of tumor cells, another set of IL-2R×MSLN κλ-bodies were tested in combination in the presence of three MSLN-expressing cell lines (Figure 11C-E). All combinations of bsAbs tested induced a similar increase in pSTAT5 in NK92 cells in the presence of NCI-H226 (Figure 11C) or OVCAR-3 (Figure 11D). Interestingly, NCI-N87, while expressing weaker levels of MSLN (24,000 MSLN / cell), is also able to mediate IL-2 signaling, regardless of the pair of IL-2R×MSLN κλ-bodies tested (Figure 11E). Importantly, all κλ-bodies tested as single agents (i.e., not combined) did not induce IL-2 signaling, highlighting that IL-2Rb- and IL-2Rg-bsAbs are only active when combined (Figure 11C-E).

[0258] In the previous example, we showed that our IL-2RxTAA bsAb combination can induce IL-2 signaling in immunogenic target non-transduced cells. To extend this observation to primary human immune cells, we used human peripheral blood mononuclear cells (PBMCs). hIL-2 is known to preferentially activate regulatory T cells because they constitutively express a high affinity trimeric receptor. As our bsAb combination targets only the β and γ subunits of the IL-2R, we were able to induce IL-2 signaling in CD8+ T cells. + We expect to observe similar sensitivity of T cells and regulatory T cells to our bsAbs. To test this hypothesis, we measured the levels of pSTAT5 in different cell populations by flow cytometry (Figure 12A).

[0259] 10.3. PBMC isolation, stimulation, and pSTAT5 staining: The protocol used was adapted from that described in Example 7. PBMC cells were washed in DPBS and incubated at 5×10 in DPBS, Fc Block (30 μl / ml BD Biosciences, # 564220) and Fixable Viability Dye eFluor 506 (1 μl / ml Thermo Fisher, # 65-0866-18). 6Resuspend at 10 cells / ml and incubate for 15 minutes at room temperature in the dark. PBMC cells were washed once and incubated in DPBS, 2% bovine serum albumin with Alexa Fluor 594 anti-human CD3 antibody (1 μl / 100 μl, Biolegend, #300446), APC-Cy7 mouse anti-human CD8 (1 μl / 100 μl, BD Biosciences, #557834), BV421 mouse anti-human CD4 (1 μl / 100 μl, BD Biosciences, #562424), eFluor 506 eBioscience CD19 monoclonal antibody (1 μl / 100 μl, Thermo Fisher, #69-0199-42), eFluor 506 eBioscience CD14 monoclonal antibody (1 μl / 100 μl, Thermo Fisher, #69-0149-42), eFluor 506 eBioscience CD56 (NCAM) monoclonal antibody (1 μl / 100 μl, Thermo Fisher, #69-0566-42), 2 × 10 6Cells are stained at 250,000 cells / ml for 20-30 min at 4°C. PBMC cells and harvested tumor cells are washed twice and transferred to 96-well round-bottom plates at 250,000 PBMCs / well and 25,000 tumor cells / well. Cells are incubated for 1 h at 37°C with 5% CO2 in RPMI 1640, 10% fetal bovine serum, 2 mM L-glutamine, 10 mM Hepes, 1x non-essential amino acids, 0.050 mM β-mercaptoethanol, 25 μg / ml gentamicin under different test conditions. Cells are resuspended in 100 μl / well Fix buffer I (BD Biosciences, #557870) and incubated at 37°C for 10 min. Cells are washed and permeabilized in 100 μl / well pre-chilled Perm buffer III (BD Biosciences, #558050) for 30 min on ice. Wash once in DPBS and stain with Alexa Fluor 488 anti-STAT5 (pY694) (3 μl / well BD Biosciences, #562075), Alexa Fluor 647 mouse anti-human FoxP3 (10 μl / 100 μl, BD Biosciences, #560045), 2% bovine serum albumin in DPBS for 1 hour at room temperature in the dark. Wash cells twice with DPBS, 2% bovine serum albumin and resuspend in 150 μl DPBS for flow cytometry analysis. pSTAT5 + pSTAT5 - The threshold for distinguishing cells is determined based on control unstimulated wells.

[0260] Both hIL-2 and the IL-2R × HER2 KiH bsAb pair activated IL-2 signaling in T cells in conjunction with HER2-overexpressing cells. As expected, regulatory T cells were CD8 + T cells were significantly more sensitive to hIL-2 than T cells, which remained activated at >100-fold lower doses (Figure 12B). Importantly, regulatory T cells and CD8 +The difference in sensitivity between T cells was abrogated by our bsAb pair in both HER2-overexpressing tumor cells tested (Figure 12B). These results indicate that our strategy, like other approaches that circumvent preferential engagement of the trimeric IL-2 receptor, has enhanced selectivity for immune stimulatory capacity.

[0261] Example 11: T-cell dependent cytotoxicity (TDCC) mediated by IL-2R×TAA bsAb in combination with CD3×TAA bsAb The costimulatory IL-2R×TAA bsAb, which can induce IL-2R signaling, is expected to synergize with CD3×TAA bsAb in enhancing T cell activation, which may result in superior tumor cell killing in T cell-dependent cytotoxicity (TDCC) assays.To avoid binding competition between CD3×TAA and IL-2R×TAA bsAb, the IL-2R×MSLN bsAb pair was combined with CD3×HER2 bsAb, while the IL-2R×MSLN bsAb pair was combined with CD3×HER2 bsAb in TDCC assays.The NCI-N87 tumor cell line was selected as the target cell based on its dual expression of HER2 and MSLN.

[0262] 11.1. TDCC Assay: In vitro killing of the HER2 / MSLN double positive cell line NCI-N87 induced by MSLNxCD3 or HER2xCD3 bsAb was evaluated in combination with IL-2RxHER2 bsAb or IL-2RxMSLN bsAb pairs, respectively. For TDCC assays, PBMCs were added to target cells at a final E:T ratio of 1:1. After washing twice with PBS, target cells were detached with trypsin or cell dissociation solution. After a centrifugation step, cells were resuspended in assay medium (RPMI 1640 (Thermo Fisher, #11875-093), 10% fetal bovine serum, 2 mM L-glutamine, Hepes (10 mM, Sigma #H0887), non-essential amino acids (1×, Sigma #M7145-100mL), sodium pyruvate (1 mM, Sigma #S8636-100mL), β-mercaptoethanol (0.050 mM, Thermo Fisher #31350-010), gentamicin (25 μg / ml, Sigma #G1397-10ML), adjusted to the required concentration, and seeded into 96-well plates. Effector cells were cultured in SepMate™ tubes (Stemcell Technologies) containing Lymphoprep™ buffer (Stemcell Technologies). Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats derived from healthy human donors using Promega's CellTiter-Glo® (G7570) from a range of doses of the IL-2R x TAA bsAb pairs of the present invention (0.3, 3, 10 and 30 nM) and a fixed dose of CD3 x TAA bsAb (0.1 or 0.5 nM) were added to pre-seeded target and effector cells. As a control, single agent CD3 x TAA bsAb was used. Target cell killing was assessed by quantifying the number of viable adherent cells in culture using Promega's CellTiter-Glo® (G7570) after 6 days of incubation at 37°C, 5% CO2.Two different TDCC assay formats were tested: all bsAbs were combined simultaneously for 6 days (Figure 13A, referred to as mixed TDCC assay) or T cells were first pre-stimulated with the IL-2RxTAA bsAb pair for 3 days, followed by sequential testing with CD3xTAA bsAb for an additional 3 days (Figure 13C, referred to as sequential TDCC assay). Instead of IL-2RxTAA bsAb, CD3xTAA in combination with IL-2 was used as a positive control. Single treatment of CD3xTAA bsAb was used as a reference control (dashed line). Single treatment with hIgG1 or IL-2R-bsAb pair (e.g., in the absence of CD3xTAA bsAb) was used as a negative control.

[0263] Mixed TDCC killing assays showed that two different paired IL-2R×MSLN bsAb combinations (AL4O35 / N+AM5O30 / N or AL4O38 / N+AM5O30 / N) enhanced HER2×CD3 bsAb-induced killing of MSLN / HER2 double-positive NCI-N87 tumor target cells in a dose-dependent manner (FIG. 13B). Similar synergy was observed using sequential killing assays with a dose range of IL-2R×HER2 bsAb pairs (P1A3×trastuzumab+P2C4×pertuzumab) followed by a fixed dose of CD3×MSLN bsAb. Importantly, the combination of CD3×MSLN with 30 nM IL-2R×HER2 bsAb was at least as potent as the combination of CD3×MSLN bsAb with IL-2 in inducing tumor cell killing (FIG. 13D). In the absence of CD3×MSLN bsAb, no killing was induced by the IL-2R×HER2 bsAb pair alone (FIG. 13D).

[0264] Overall, the TDCC data presented herein demonstrate that IL-2Rβ×TAA and IL-2Rγ×TAA bsAb pairs can increase TDCC mediated by CD3×TAA bsAb to levels approaching those induced by the combination of IL-2 and CD3×TAA bsAb.

[0265] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. 1. A composition for use in inhibiting tumor growth, treating cancer, enhancing T cell mediated cell killing, and / or activating T cells, the composition comprising: a) a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor; and b) a second bispecific antibody comprising an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of said cytokine receptor. A composition comprising:

2. a) a first composition comprising a first bispecific antibody comprising an antigen-binding domain that binds to a first tumor-associated antigen and an antigen-binding domain that binds to a first subunit of a cytokine receptor; and b) a second composition comprising a bispecific antibody having an antigen-binding domain that binds to a second tumor-associated antigen and an antigen-binding domain that binds to a second subunit of said cytokine receptor. A composition comprising:

3. The composition for use according to claim 1 or the composition according to claim 2, wherein the first tumor-associated antigen and the second tumor-associated antigen are different tumor-associated antigens.

4. The composition for use according to claim 1 or the composition according to claim 2, wherein the first tumor-associated antigen and the second tumor-associated antigen are the same tumor-associated antigen.

5. The composition for use according to claim 1 or the composition according to claim 2, wherein the antigen-binding domain that binds to the first tumor-associated antigen and the antigen-binding domain that binds to the second tumor-associated antigen bind to two different epitopes of the same tumor-associated antigen.

6. The composition for use according to claim 1 or the composition according to claim 2, wherein the antigen-binding domain that binds to the first tumor-associated antigen and the antigen-binding domain that binds to the second tumor-associated antigen are identical.

7. The composition for use according to claim 1 or the composition according to claim 2, wherein the first tumor-associated antigen and the second tumor-associated antigen are expressed on the surface of the same tumor cell.

8. The composition for use according to claim 1 or the composition according to claim 2, wherein the first subunit of a cytokine receptor and the second subunit of a cytokine receptor are expressed on the surface of the same immune cell.

9. The composition for use according to claim 8 or the composition according to claim 8, wherein the immune cells are T cells.

10. The composition for use according to claim 1 or the composition according to claim 2, wherein the first tumor-associated antigen and / or the second tumor-associated antigen is human epidermal growth factor receptor 2 (HER2).

11. The composition for use according to claim 1 or the composition according to claim 2, wherein the first tumor-associated antigen and / or the second tumor-associated antigen is mesothelin (MSLN).

12. 3. The composition for use of claim 1 or the composition of claim 2, wherein the cytokine receptor binds to IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-12, IL-23, IFNα, IFNβ, IFNε, IFNk, IFNo, IFNδ, IFNτ, IFNω, IFNζ, IFNγ, or IFNλ.

13. The composition for use according to claim 1 or the composition according to claim 2, wherein the first subunit of the cytokine receptor or the second subunit of the cytokine receptor is IL-2Rγ.

14. The composition for use according to claim 1 or the composition according to claim 2, wherein the first subunit of the cytokine receptor is IL-2Rβ and the second subunit of the cytokine receptor is IL-2Rγ.

15. c) a third bispecific antibody comprising an antigen-binding domain that binds to a third tumor-associated antigen and an antigen-binding domain that binds to an antigen expressed on T cells.

3. The composition for use according to claim 1 or the composition according to claim 2, further comprising:

16. 16. The composition for use according to claim 15 or the composition according to claim 15, wherein the antigen expressed on the T cells is CD3.

17. The composition for use according to claim 15 or the composition according to claim 15, wherein the third tumor-associated antigen is different from the first tumor-associated antigen and the second tumor-associated antigen.

18. 3. The composition for use according to claim 1 or the composition according to claim 2, wherein the first bispecific antibody and / or the second bispecific antibody has an IgG isotype.

19. 16. The composition for use according to claim 15 or the composition according to claim 15, wherein the first bispecific antibody, the second bispecific antibody, and / or the third bispecific antibody have an IgG isotype.

20. 3. The composition for use of claim 1 or the composition of claim 2, wherein the first bispecific antibody and / or the second bispecific antibody is a chimeric antibody, a humanized antibody, or a human antibody.

21. 16. The composition for use of claim 15 or the composition of claim 15, wherein the first bispecific antibody, the second bispecific antibody, and / or the third bispecific antibody is a chimeric antibody, a humanized antibody, or a human antibody.

22. The composition for use according to claim 1 or the composition according to claim 2, which enables antigen-dependent activation of IL-2 receptor signaling or IL-15 receptor signaling in immune cells expressing IL-2Rγ and IL-2Rβ.

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