Antibodies and bispecific binding proteins that bind OX40 and / or PD-L1

Abstract

Provided are novel antibodies that recognize OX40, a member of the TNF receptor superfamily, novel antibodies that recognize Programmed Death-Ligand 1 (PD-L1), and bispecific OX40 / PD-L1 binding proteins, such as FIT-Ig binding proteins, made using these antibodies. Such antibodies and bispecific binding proteins are useful for treating diseases, such as cancer.

Description

[Technical field]

[0001] The present disclosure relates to antibodies capable of recognizing the tumor necrosis factor receptor OX40 (CD134), and related bispecific binding proteins comprising at least one OX40 binding domain and at least one PD-L1 binding domain, such as bispecific OX40 / PD-L1 binding proteins (e.g., FIT-Ig binding proteins (Fab-in-tandem immunoglobulin)). The present disclosure also relates to antibodies capable of recognizing PD-L1, and related bispecific binding proteins comprising at least one PD-L1 binding domain and at least one OX40 binding domain, such as bispecific OX40 / PD-L1 binding proteins (e.g., FIT-Ig binding proteins). The antibodies and bispecific binding proteins disclosed herein may be useful for treating diseases, for example, in cancer immunotherapy. The present disclosure further relates to nucleic acids encoding said antibodies or bispecific binding proteins, and methods of making said antibodies or bispecific binding proteins. [Background technology]

[0002] The tumor necrosis factor (TNF) receptor superfamily (TNFR) is a large class of functionally diverse receptors capable of mediating a range of immune cell functions (Mayes PA, 2018). Many members of the TNFR superfamily are costimulatory receptors that can be expressed on multiple immune cell types, including T cells, B cells, and natural killer (NK) cells, as well as antigen-presenting cells (APCs), and have been shown to induce immune cell function, proliferation, and survival (Watts TH, 2005).

[0003] OX40 (CD134), a member of the TNFR superfamily with a type I transmembrane glycoprotein characterized by four cysteine-rich domains (CRDs), is expressed primarily on activated CD4 T cells and CD8 T cells, and on Foxp3 + CD4 +OX40L is expressed on regulatory T cells (Tregs), whereas its ligand, OX40L (CD252), is expressed on activated APCs, e.g., dendritic cells (DCs), B cells, and macrophages (Weinberg AD, 2011). Upon activation by TCR-MHC / peptide interaction, OX40L homotrimers form and bind to three OX40 receptors, resulting in receptor cross-linking (Watts, 2005; Jane Willoughby, 2017). High-order clustering of OX40 has been suggested to be necessary to mediate downstream signaling. Clustered OX40 receptors recruit TNF receptor-associated factors (TRAFs) to the intracellular domain of OX40. TRAF2 and TRAF3 activate the PI3K / PKB, nuclear factor kappa B1 (NF-kappa B1), and NFAT pathways that are responsible for T cell division, survival, and cytokine production (Croft, 2010; Kawamata, 1998; Song, 2008). Thus, signaling downstream of OX40 has the potential to enhance proliferation, inhibit apoptosis, and induce increased cytokine responses from T cells, all functional outcomes that OX40 agonistic antibodies are capable of eliciting when used in immunotherapy.

[0004] The mechanism of agonistic anti-OX40 antibodies in mediating anti-tumor efficacy has been extensively explored in various mouse tumor models. Most agonistic anti-OX40 mAbs employ human IgG1 isotypes for strong FcγR binding to trigger costimulatory signaling pathways on effector T cells, thereby supporting the survival and expansion of activated T cell subsets and the establishment of T cell memory for CD8 T cell responses by OX40 (Brendan D Curti, 2013; Glisson, 2020). Further data suggest that costimulation by OX40 inhibits FoxP3 expression and Treg induction via downstream signaling (Zhang X, 2018). Since OX40 is highly expressed on infiltrating Tregs, induction of antitumor responses by OX40 antibodies depends on depletion of intratumoral Treg cells by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) via Fc-mediated effector functions (Aspeslagh, 2016; Smyth, 2014). However, depletion of intratumoral Tregs may not be associated with increased CD8 T cells in the tumor microenvironment (TME), as evidenced by enhanced antitumor immune responses and improved survival in several mouse models. +It may improve the ratio of infiltration of effector T cells to infiltration of Tregs (Jacquemin, 2015; Bulliard, 2014). Most agonistic OX40 antibodies under clinical development are IgG1 isotype antibodies due to their desired antitumor efficacy (Choi, 2020; Brendan D Curti, 2013; Glisson, 2020). Clinical trials using OX40 targeting drugs have illustrated their safety when used as monotherapy or in combination with immune checkpoint blockade (ICB). OX40 targeting therapy has been supported with impressive results in preclinical mouse models, but according to preliminary clinical data, its efficacy as monotherapy in humans is modest (Glisson, 2020; Carolina, 2020; Martin Gutierrez, 2020). However, according to a recently published phase 1 / 2a study, costimulation with OX40 combined with anti-PD-1, anti-PD-L1, or anti-CTLA4 did not result in a clear improvement in efficacy (Martin Gutierrez, 2020). The low response to agonistic anti-OX40 antibodies in patients can be attributed to two causes. First, in some tumors, the efficiency of FcγR-dependent clustering may be reduced when limited by available infiltrating FcγRs in the TME (Willoughby, 2017) or in the presence of high concentrations of endogenous IgG competing for binding to FcγRs (Christian Gieffers, 2013). Second, as found by recent clinical studies, after anti-OX40 IgG1 antibody treatment, the efficiency of FcγR-dependent clustering may be reduced. + CD4 + The percentage of memory T cells is probably OX40 + Due to the reduced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of cells (Glisson, 2020), there is therefore a need to generate new anti-OX40 agonists that can mediate effective high-clustering despite limited availability of FcγR while maintaining low effector functions.

[0005] PD-L1 (CD274) is a 40 kDa type I transmembrane protein, and the PD-1 / PD-L1 signaling pathway plays an important role in immune tolerance and tumor immune evasion. PD-L1 is expressed in many human tumor tissues (e.g., lung, gastric, breast, and intestinal cancers). Blocking the PD-1 / PD-L1 inhibitory signaling pathway activates suppressed T cells to attack cancer cells. Most anti-PD-L1 mAbs inhibit tumor growth both in vivo and in patients by promoting the proliferation of tumor antigen-specific T cells (Julie, 2012; Brahmer, 2012).

[0006] Bispecific antibodies are a class of engineered antibodies that have dual affinities for two different antigens / epitopes. Various forms of bispecific antibodies have been reported and explored, including FIT-Ig (Fab-in-tandem immunoglobulin) disclosed in WO2015 / 103072. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure provides a new antibody that binds to PD-L1 with high affinity and a new antibody that binds to OX40 with high affinity. The present disclosure also provides a PD-L1 / OX40 bispecific FIT-Ig (Fab-in-tandem immunoglobulin) that simultaneously binds to both PD-L1 and OX40. The antibodies and bispecific binding proteins of the present disclosure can block inhibitory signaling by PD-L1 on tumor-infiltrating lymphocytes (TILs) and reactivate tumor-infiltrating cytotoxic T cells against tumor cells. [Means for solving the problem]

[0008] The bispecific antibody of the present disclosure comprises two antigen-binding regions with a dual mechanism. First, the bispecific antibody agent binds to tumor cells or APCs expressing PD-L1 through its PD-L1-binding region, while it binds to OX40 through its OX40-binding region, mediating high clustering, which may activate T cells under PD-L1-dependent conditions. Second, the bispecific antibody of the present disclosure blocks the binding of human PD-L1 to human PD-1, preventing PD-L1-mediated immune escape via PD-1. Thus, the bispecific antibody of the present disclosure activates T cells through binding to OX40, while preventing T cell exhaustion through PD-1 / PD-L1 interaction, resulting in enhanced T cell activation, proliferation of effector T cells and memory T cells to boost anti-tumor efficacy. Furthermore, the bispecific antibody of the present disclosure introduces a LALA mutation into the Fc region to alleviate ADCC and ADCP against OX40-positive T cells.

[0009] The disclosed PD-L1 / OX40 bispecific antibodies overcome the limitations of anti-OX40 monotherapy by inducing high-order OX40 clustering and inducing sufficient OX40 signaling through PD-L1 cross-linking. Concurrent binding of PD-L1 on tumor cells and OX40 on T cells results in both PD-L1-dependent activation of OX40 on T cells and inhibition of inhibitory signaling by PD-1 / PD-L1, which may result in efficient induction of anti-tumor immunity. Thus, OX40 / PD-L1 bispecific antibodies have utility in the treatment of cancer. [Brief description of the drawings]

[0010] [Figure 1a]Figure 1a shows the epitope identification of anti-OX40 antibodies. Figure 1a shows the binding of HuEM1007-044-16 (top), OX40-Tab1 (middle), and OX40-Tab2 (bottom) to full-length extracellular OX40 (CRD1-4, circles) and truncated OX40 mutants, ΔCRD1 (lacking CRD1, squares), ΔCRD1-2 (lacking CRD1 and CRD2, triangles), and ΔCRD1-3 (lacking CRD1, CRD2, and CRD3, diamonds). [Figure 1b] Figure 1b shows binding of OX40-Tab2 to extracellular OX40 full-length (CRD1-4, circles), mCRD1 (CRD1-4 in which the CRD1 domain has been replaced by mouse CRD1, squares), mCRD2 (CRD1-4 in which the CRD2 domain has been replaced by mouse CRD2, triangles), mCRD3 (CRD1-4 in which the CRD3 domain has been replaced by mouse CRD3, inverted triangles), and mCRD4 (CRD1-4 in which the CRD4 domain has been replaced by mouse CRD4, diamonds). [Diagram 2] Anti-OX40 antibody HuEM1007-044-16 (black) induced selective proliferation of effector T cells over Treg cells. Irrelevant human IgG (grey) was used as a negative control. [Diagram 3] Figure illustrates the binding of serially diluted antibodies, FIT1014-20a (diamonds) and HuEM0005-86-64 (squares), to CHO-PD-L1 along with an irrelevant human IgG (triangle) as a negative control, as measured by FACS. [Figure 4] Figure 1 shows FACS affinity results for binding to human OX40 transfected CHO cells with serially diluted antibody FIT1014-20a (diamonds) and its parent OX40 antibody HuEM1007-44-16 (squares). An irrelevant human IgG (triangles) is used for negative control. [Diagram 5]Figure 2 illustrates blockade of PD-1 / PD-L1 binding by bispecific FIT1014-20a (squares) and parental PD-L1 antibody (triangles), as well as irrelevant human IgG as a negative control (inverted triangles) in a cell-based receptor blocking assay. [Figure 6] Figure 2 illustrates blockade of PD-L1-mediated inhibitory signaling by bispecific FIT1014-20a (squares) and parental PD-L1 antibody (triangles), as well as irrelevant human IgG (inverted triangles) as a negative control. [Figure 7] (Top) Activation of OX40 downstream signaling by bispecific FIT1014-20a (squares) and a combination of two parent antibodies containing identical PD-L1 and OX40 binding domains, respectively (inverted triangles), as well as an irrelevant human IgG (diamond) as a negative control. In control assays (bottom), CHO cells that do not express PD-L1 show a lack of activation by FIT1014-20a or the combination of the two parent antibodies. [Figure 8] Figure 2 shows production of IL2 (top, 72 hours after incubation) and IFN-γ (bottom, 48 hours after incubation) from co-cultures of CHO-PD-L1-OS8 cells and primary human T cells upon co-incubation with FIT1014-20a (squares), parental antibody combinations (inverted triangles), or irrelevant human IgG (diamonds). [Figure 9] FIG. 1 presents T cell activation as assessed by IL2 levels observed from a mixed lymphocyte reaction (MLR) assay after 3 days of incubation with FIT1014-20a (dark) and parental antibody combinations (grey). [Figure 10] FIG. 1 shows T cell activation as assessed by IL2 levels observed from a Staphylococcus aureus endotoxin B (SEB) assay after 96 hours of incubation with FIT1014-20a (squares), parental antibody combinations (inverted triangles), or irrelevant human IgG as a negative control (diamonds). [Figure 11]FIG. 1 shows a complement dependent cytotoxicity assay for FIT1014-20a (circles) with anti-HLA-1 (triangles) as a positive control, and irrelevant human IgG (squares) as a negative control. [Figure 12] FIG. 1 shows the phagocytic effect of FIT1014-20a (solid black), HuEM1007-044-16-hIgG1 (solid grey), HuEM1007-044-16 (diagonal stripes), OX40-Tab2 (horizontal stripes), and irrelevant hIgG (checkerboard) on CHO-OX40. [Figure 13] Figure 1 shows evaluation of antitumor efficacy in humanized OX40 / PD-L1B6 mice bearing MC38-hPD-L1 tumor cells treated with FIT1014-20a (triangles), parental PD-L1 mAb HuEM0005-86-64 (squares), atezolizumab (squares), and vehicle as a negative control (circles). [Figure 14] Figure 2 shows tumor volume profiles for CT26-hPD-L1 syngeneic tumors established in human PD-1 / PD-L1 / OX40 knock-in mice treated with vehicle control (circles), the reference PD-L1 antibody atezolizumab (squares), and FIT1014-20a (triangles). Arrows indicate the assigned drug administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure relates to anti-OX40 antibodies, anti-PD-L1 antibodies, antigen-binding portions thereof, and multivalent bispecific binding proteins, such as FIT-Ig, that bind both OX40 and PD-L1. Various aspects of the present disclosure relate to anti-OX40 antibodies and antigen-binding fragments thereof, anti-PD-L1 antibodies and antigen-binding fragments thereof, FIT-Ig binding proteins that bind human OX40 and human PD-L1, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies, antigen-binding fragments, and binding proteins. Methods of using the antibodies, antigen-binding fragments, and bispecific binding proteins of the present disclosure to detect human OX40, human PD-L1, or both, modulate human OX40 activity and / or human PD-L1 activity in vitro or in vivo, induce and / or enhance adaptive immune responses against foreign antigens, such as tumors, and treat diseases, especially cancer, are also encompassed by the present disclosure.

[0012] definition Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Similarly, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising multiple subunits, unless otherwise specified.

[0013] As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), herein referred to as "numbering according to Kabat". Specifically, the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by referring to the numbering according to the Kabat EU index in this case).

[0014] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by reason of the origin or source of its derivation, is not associated with naturally associated components which accompany it in its natural state, is substantially free of other proteins from the same species, is expressed by cells from a different species, or is non-naturally occurring. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0015] The terms "specific binding" or "specifically binds" with respect to the interaction of an antibody, binding protein or peptide with a second chemical species means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. Generally, if an antibody is specific for epitope "A", in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody. In accordance with the present disclosure, a specific binding protein has a K of 10 nM or less, e.g., 1 nM or less. D The term "K D " means the equilibrium dissociation constant (the reciprocal of the equilibrium binding constant) and is used herein according to the definition provided in the art. The K D Values ​​can be determined by well-known methods, including but not limited to, fluorescence titration, competitive ELISA, calorimetric methods such as isothermal titration calorimetry (ITC), flow cytometry titration analysis (FACS titration), biolayer interferometry (BLI), surface plasmon resonance (BIAcore), and the like.

[0016] The term "antibody" refers broadly to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any antigen-binding fragment, mutant, variant, or derivative thereof that retains the essential epitope binding properties of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, and non-limiting embodiments are discussed below.

[0017] In a full-length antibody, each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second and third CDRs of the VH domain are commonly enumerated as CDR-H1, CDR-H2 and CDR-H3. Similarly, the first, second and third CDRs of a VL domain are commonly enumerated as CDR-L1, CDR-L2 and CDR-L3. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0018] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally, in the case of the Fc region of an IgM and IgE antibody, for example, a CH4 domain. The Fc regions of IgG, IgA and IgD antibodies comprise a hinge region, a CH2 domain and a CH3 domain. In contrast, the Fc regions of IgM and IgE antibodies lack a hinge region, but comprise a CH2 domain, a CH3 domain and a CH4 domain. Variant Fc regions having substitutions of amino acid residues in the Fc portion to alter antibody effector functions are known in the art (see, for example, Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates one or more effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and / or half-life / clearance rate of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, while in other cases, they may be unnecessary or even harmful depending on the therapeutic purpose. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC through binding to FcγR and complement C1q, respectively. In yet another embodiment, at least one amino acid residue is replaced in the constant region of the antibody, e.g., the Fc region of the antibody, such that the effector function of the antibody is altered. Dimerization of two identical heavy chains of an immunoglobulin is mediated by dimerization of the CH3 domains and stabilized by disulfide bonds in the hinge region that connects the CH1 constant domain to the Fc constant domains (e.g., CH2 and CH3). The anti-inflammatory activity of IgG depends on sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity were determined such that a suitable IgG1 Fc fragment could be engineered, thereby generating a fully recombinant sialylated IgG1 Fc with greatly enhanced potency (see Anthony et al., Science, 320:373-376 (2008)).

[0019] The terms "antigen-binding portion" and "antigen-binding fragment" or "functional fragment" of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen (e.g., OX40, PD-L1) as the full-length antibody from which the portion or fragment is derived. It has been found that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Such antibody embodiments may be bispecific, dual specific, or multispecific formats that specifically bind to two or more different antigens (e.g., OX40 and a different antigen, e.g., PD-L1). Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) an F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment comprising a single variable domain (Ward et al., Nature, 341:544-546 (1989); PCT Publication No. WO 90 / 05144); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, which allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody and equivalent terms given above. Other forms of single-chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and form two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)). Such antibody binding moieties are known in the art (Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition, single-chain antibodies also include "linear antibodies" that comprise a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen-binding regions. (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and U.S. Patent No. 5,641,870).

[0020] Immunoglobulin constant (C) domain refers to either the heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0021] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method.

[0022] The term "human sequence" refers to a sequence that is or is derived from a human immunoglobulin sequence, with respect to the light chain constant domain CL, the heavy chain constant domain CH, and the Fc region of an antibody or binding protein according to the present application. The human sequence of the present disclosure may be a native human sequence or a variant thereof that contains one or more (e.g., up to 20, 15, 10) amino acid residue changes.

[0023] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0024] The term "CDR-grafted antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, e.g., an antibody having human heavy and light chain variable regions in which one or more of the human CDRs have been replaced with murine CDR sequences.

[0025] The term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which CDR sequences from a non-human species (e.g., mouse) have been introduced into human VH and VL framework sequences. A humanized antibody is an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and comprises framework and constant regions that have substantially the amino acid sequence of a human antibody, but comprises complementarity determining regions (CDRs) that have substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of CDRs refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In a specific embodiment, the humanized antibody comprises only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0026] The humanized antibody can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0027] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences (e.g., donor antibody CDRs), or the acceptor framework can be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in exemplary embodiments, such mutations will not be extensive. Usually, at least 80%, at least 85%, at least 90%, or at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. Back mutations at specific framework positions that restore the same amino acid that appears at that position in the donor antibody are often used to preserve a particular loop structure or to orient the CDR sequence correctly for contact with the target antigen.

[0028] The term "CDR" refers to the complementarity determining region in an antibody variable domain sequence. There are three CDRs in each of the heavy and light chain variable regions, which are referred to as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR set" as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Maryland (1991)) not only provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs.

[0029] The growth and analysis of extensive public databases of amino acid sequences of variable heavy and light chain regions over the past 20 years has allowed the typical boundaries between framework regions (FR) and CDR sequences within variable region sequences to be understood and allows one of skill in the art to precisely determine CDRs according to Kabat numbering, Chothia numbering, or other systems. See, e.g., Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages 432-433.

[0030] The term "multivalent binding protein" refers to a binding protein that contains two or more antigen binding sites. Multivalent binding proteins, in certain cases, are engineered to have three or more antigen binding sites and are generally not naturally occurring antibodies.

[0031] The term "bispecific binding protein" (unless otherwise specified, can be used interchangeably with the term "bispecific antibody") refers to a binding protein that can bind to two targets of different specificity. The FIT-Ig binding protein of the present disclosure contains four antigen binding sites and is typically a tetravalent binding protein. The FIT-Ig according to the present disclosure binds to both OX40 and PD-L1 and is bispecific.

[0032] FIT-Ig binding proteins containing two long (heavy) VCVC-Fc chain polypeptides and four short (light) VC chain polypeptides form a hexamer exhibiting four Fab antigen binding sites (VH-CH1 and VL-CL pairs, sometimes denoted as VH-CH1::VL-CL). Each half of FIT-Ig contains a heavy chain polypeptide and two light chain polypeptides, and the complementary immunoglobulin pairing of the VH-CH1 and VL-CL elements of the three chains results in two Fab structural antigen binding sites arranged in tandem. In the present disclosure, the immunoglobulin domains containing the Fab elements are preferably fused directly in the heavy chain polypeptide without the use of an interdomain linker. That is, the N-terminal VC element of the long (heavy chain) polypeptide is fused at its C-terminus directly to the N-terminus of another VC element, which in turn is linked to the C-terminal Fc region. In bispecific FIT-Ig binding proteins, the tandem Fab elements may react with different antigens. Each Fab antigen-binding site contains a heavy chain variable domain and a light chain variable domain, with a total of six CDRs per antigen-binding site.

[0033] A description of the design, expression and characterization of FIT-Ig molecules is provided in PCT Publication WO 2015 / 103072, which is incorporated herein in its entirety. One example of such a FIT-Ig molecule comprises a heavy chain and two distinct light chains. The heavy chain comprises a VH B Structural formula VL A -CL-VH B -CH1-Fc (i.e., "format LH") or CH1 is VL A The structural formula VH is directly fused to B-CH1-VL A -CL-Fc (i.e., "format HL"), and the two light chain polypeptides of FIT-Ig are correspondingly format VH A -CH1 and VL B Alternatively, the heavy chain has a CL in the VH A Structural formula VL B -CL-VH A -CH1-Fc (for "format LH"), or CH1 is VL B The structural formula VH is directly fused to A -CH1-VL B -CL-Fc (for "format HL"), and the two light chain polypeptides of FIT-Ig each have the corresponding formula VL A -CL and VH B -CH1; where VL A is the variable light domain from the parent antibody that binds antigen A, and VL B is the variable light domain from the parent antibody that binds antigen B, and VH A is the variable heavy domain from the parent antibody that binds antigen A, and VH B is a variable heavy domain from a parent antibody that binds antigen B, CL is a light chain constant domain, CH1 is a heavy chain constant domain, and Fc is an immunoglobulin Fc region (e.g., the C-terminal hinge-CH2-CH3 portion of the heavy chain of an IgG1 antibody). In a bispecific FIT-Ig embodiment, antigen A and antigen B are different antigens or different epitopes of the same antigen. In the present disclosure, one of A and B is OX40 and the other is PD-L1, e.g., A is OX40 and B is PD-L1.

[0034] As used herein, the term "k on (also "K on ", "k on "), as known in the art, refers to the on rate constant for association of a binding protein (e.g., an antibody) to an antigen to form an association complex, e.g., an antibody / antigen complex. As used interchangeably herein, "k on" is also known by the term "association rate constant" or "ka". This value indicates the rate of binding of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, as shown by the formula below: Antibody (“Ab”) + antigen (“Ag”) → Ab-Ag.

[0035] As used herein, the term "k off " (also "Koff", "koff"), as known in the art, refers to the off rate constant or "dissociation rate constant" for dissociation of a binding protein (e.g., an antibody) from an association complex (e.g., an antibody / antigen complex). This value indicates the rate of dissociation of an antibody from its target antigen or the separation of the Ab-Ag complex into free antibody and antigen over time, as shown by the formula below: Ab+Ag←Ab-Ag.

[0036] As used herein, the term "K D " (also "Kd") refers to the "equilibrium dissociation constant", measured by titration at equilibrium or by the association rate constant (k on ) to determine the dissociation rate constant (k off The association rate constant (k on ), dissociation rate constant (k off ) and the equilibrium dissociation constant (K D) is used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples in physiological buffers at equilibrium. Other experimental techniques and instruments, such as BIAcore® (Biomolecular Interaction Analysis) assays, can be used (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Biolayer Interferometry (BLI), using, for example, the Octet® RED96 system (Pall ForteBio LLC), is another affinity assay technique. Additionally, KinExA® (Kinetic Exclusion Assay) assays (available from Sapidyne Instruments, Boise, Idaho) can also be used.

[0037] The term "isolated nucleic acid" means a polynucleotide that is not associated, by human intervention, with all or a portion of polynucleotides with which it is found in nature, operably linked to polynucleotides with which it is not naturally linked, or that does not occur in nature as part of a larger sequence (e.g., of genomic, cDNA, or synthetic origin, or a combination of portions thereof).

[0038] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors that are useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0039] The term "operably linked" refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. As used herein, the term "expression control sequences" refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, where appropriate, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences. In eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0040] "Transformation", as referred to herein, refers to any process by which exogenous DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and includes, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Cells thus "transformed" include stably transformed cells in which the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of the host chromosome. Such cells also include cells that transiently express the inserted DNA or RNA for a limited period of time.

[0041] The term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous DNA has been introduced. In one embodiment, a host cell (such as a host cell described in U.S. Pat. No. 7,262,028) contains two or more (e.g., a plurality) of nucleic acids encoding an antibody. Such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; the mammalian cell lines CHO, HEK293, Jurkat, COS, NS0, SP2, and PER.C6; the insect cell line Sf9; and the fungal cell yeast (Saccharomyces cerevisiae).

[0042] As used herein, the term "effective amount" refers to an amount of a therapy that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof; prevent the progression of a disorder; cause regression of a disorder; prevent the recurrence, occurrence or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or improve the prophylactic or therapeutic effects of another therapy (e.g., a prophylactic or therapeutic agent).

[0043] As used herein, "T cell activation" or "T cell activation" refers to the core process of cell-mediated immunity in which a particular foreign antigen induces a cognate naive T cell to respond to it. T cell activation is reflected in the proliferation and / or differentiation of T cells and the production of large numbers of effector T cells (e.g., cytotoxic T lymphocytes, etc.), resulting in, for example, the reduction or elimination of foreign antigens. This process is complex and controlled by many factors, such as, for example, the immunosuppressive tumor microenvironment. Signs of T cell activation that can be measured include, but are not limited to, a significant increase in the secretion of IL-2 or IFN-γ from T cells, and / or an increase in antigen response (e.g., tumor clearance). Measurement methods are known to those skilled in the art.

[0044] The antibodies, antigen-binding fragments thereof, and binding proteins of the present disclosure may be purified (for the intended use) by using one or more of the various methods and materials available in the art for purifying antibodies and binding proteins. Such methods and materials include, but are not limited to, affinity chromatography (e.g., using Protein A, Protein G, Protein L, or resins, particles, or membranes bound to specific ligands of the antibody, antigen-binding fragment, or binding protein), ion exchange chromatography (e.g., using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g., using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (to inactivate contaminating viruses), and combinations thereof to obtain a purity acceptable for the intended use. A non-limiting example of a low pH treatment to inactivate contaminating viruses includes lowering the pH of a solution or suspension containing the antibody, antigen-binding fragment, or binding protein of the present disclosure to pH 3.5 using 0.5 M phosphoric acid at 18°C ​​to 25°C for 60 to 70 minutes.

[0045] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above can generally be performed by conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0046] Anti-OX40 and anti-PD-L1 monospecific antibodies The anti-OX40 and anti-PD-L1 antibodies of the present disclosure can be produced by any of a number of techniques known in the art. See, for example, WO2021 / 1034434, the contents of which are incorporated herein by reference. For example, expression from a host cell, where expression vectors encoding the heavy and light chains have been transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, such as mammalian host cells, is particularly contemplated, since such eukaryotic cells (e.g., mammalian cells) are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies.

[0047] In some embodiments, mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr- CHO cells, used with a DHFR selection marker (described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or even secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0048] The host cells can also be used to produce antigen-binding fragments, such as Fab fragments or scFv molecules. It will be understood that variations of the above procedures fall within the scope of the present disclosure. For example, it may be desirable to transfect host cells with DNA encoding an antigen-binding fragment of either the light and / or heavy chain of the antibody of the present disclosure. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure. In addition, bifunctional antibodies can be produced by crosslinking the antibodies of the present disclosure to a second antibody or another functional moiety by standard chemical crosslinking methods.

[0049] In an exemplary system for recombinant expression of the antibody, or antigen-binding portion thereof, of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high level transcription of the genes. The recombinant expression vector also carries a DHFR gene, allowing for the selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transfected host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transfectants, culture the host cells, and recover the antibody from the culture medium. The present disclosure also provides methods of producing a recombinant anti-OX40 or anti-PD-L1 antibody of the present disclosure by culturing a transfected host cell of the present disclosure in a suitable culture medium until a recombinant antibody of the present disclosure is produced. Optionally, the method can further include a step of isolating the recombinant antibody from the culture medium.

[0050] Anti-OX40 antibody In some embodiments, the present disclosure provides antibodies that bind to OX40 at the membrane proximal CRD of the OX40 Ig-like domain. In some embodiments, the antibodies disclosed herein are characterized by high cell binding avidity and / or low internalization rates, e.g., as measured by cell-based assays.

[0051] In some embodiments, the present disclosure discloses an isolated anti-OX40 antibody or antigen-binding fragment thereof that specifically binds to OX40. In further embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises the sequence SSWMN (SEQ ID NO: 1), CDR-H2 comprises the sequence RIYPGDEITNYNGKFKD (SEQ ID NO: 2) or RIYPGDEITNYNAKFKD (SEQ ID NO: 4), CDR-H3 comprises the sequence DLLMPY (SEQ ID NO: 3), CDR-L1 comprises the sequence RSSKSLLYSNGITYLY (SEQ ID NO: 5) or RSSKSLLYSNAITYLY (SEQ ID NO: 8), CDR-L2 comprises the sequence QMSNLAP (SEQ ID NO: 6), CDR-L3 comprises the sequence AQNLELPFT (SEQ ID NO: 7), The CDRs are defined according to the Kabat numbering system.

[0052] In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises, at positions H31 to H35, H50 to H66, and H99 to H104 according to the Kabat numbering, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of: (i) SEQ ID NO:1, 2, 3; or (ii) SEQ ID NO:1, 4, 3.

[0053] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof comprises the amino acid sequences of SEQ ID NOs: 5, 6, and 7, or SEQ ID NOs: 8, 6, and 7 for CDR-L1, CDR-L2, and CDR-L3, respectively, at positions L24-39, L55-61, and L94-102 according to the Kabat numbering.

[0054] In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a G62A mutation in the VH domain according to Kabat numbering. In certain embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises a G34A mutation in the VL domain according to Kabat numbering. In some embodiments, the mutation reduces the tendency of asparagine to be deamidated in the anti-OX40 antibody or antigen-binding fragment thereof. In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof with the mutation has increased stability compared to the parent antibody without the mutation.

[0055] In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in the CDR sequences of SEQ ID NOs: 1-3 and 5-7. In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in the CDR sequences of SEQ ID NOs: 1, 4, 3, and 5-7. In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in the CDR sequences of SEQ ID NOs: 1-3, and 8, 6, 7. In some embodiments, the anti-OX40 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in the CDR sequences of SEQ ID NOs: 1, 4, 3, and 8, 6, 7. The amino acid modifications can be amino acid substitutions, deletions, and / or additions, e.g., conservative substitutions.

[0056] In one embodiment, an anti-OX40 antibody or antigen-binding fragment thereof according to the present disclosure comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the heavy chain variable domain, VH, and the light chain variable domain, VL, selected from the group consisting of the following VH / VL sequence pairs: SEQ ID NOs: 11 / 19, 12 / 19, 13 / 19, 14 / 19, 11 / 20, 12 / 20, 13 / 20, 14 / 20, 10 / 17, 9 / 18, 10 / 18, 9 / 19, 11 / 17, 15 / 21, 15 / 18, 16 / 21, and 16 / 18. The CDRs can be determined by one of skill in the art using the most widely used CDR definition schemes, such as the Kabat, Chothia, or IMGT definitions.

[0057] In one embodiment, an anti-OX40 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain, VH, and a light chain variable domain, VL, where: the VH domain comprises a sequence of SEQ ID NO: 9 or 10 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and / or The VL domain comprises a sequence of SEQ ID NO: 17 or 18 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0058] In another embodiment, an anti-OX40 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain, VH, and a light chain variable domain, VL, where the VH domain comprises a sequence selected from SEQ ID NOs: 11 to 16 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and / or The VL domain comprises a sequence selected from SEQ ID NOs: 19-21 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0059] In some embodiments, an anti-OX40 antibody comprising a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence while retaining the ability to bind to OX40 with the same or improved binding characteristics, e.g., off-rates and / or on-rates. In some embodiments, a total of 1-11 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NO:9, 10, or SEQ ID NO:11-16. In certain embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-OX40 antibody comprises a VH sequence of any one of SEQ ID NO:9, 10, or SEQ ID NO:11-16, including post-translational modifications of this sequence. In certain embodiments, the VH comprises one, two, or three CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or 4, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VH sequence is a humanized VH sequence.

[0060] In some embodiments, an anti-OX40 antibody comprising a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence while retaining the ability to bind to OX40 with the same or improved binding characteristics, e.g., off-rate and / or on-rate. In some embodiments, a total of 1 to 5 amino acids are substituted, inserted, and / or deleted in any one of SEQ ID NO: 17, 18, or SEQ ID NO: 19-21. In certain embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-OX40 antibody comprises a VL sequence of any one of SEQ ID NO: 17, 18, or SEQ ID NO: 19-21, including post-translational modifications of this sequence. In certain embodiments, the VL sequence comprises one, two, or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 8, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VL sequence is a humanized VL sequence.

[0061] In one embodiment, an anti-OX40 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain, VH, comprising or consisting of SEQ ID NO: 16, and a light chain variable domain, VL, comprising or consisting of SEQ ID NO: 21.

[0062] In one embodiment, the isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure is a chimeric or humanized antibody. In some embodiments, the anti-OX40 antibody or antigen-binding fragment is a humanized antibody.

[0063] In some embodiments, the humanized isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure comprises one or more back mutations at positions in the framework region to improve binding characteristics. In some embodiments, the VH domain of the humanized anti-OX40 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human residues to one or more of the following residues according to Kabat numbering: Glu (1E) at position 1, and optionally Gln (5Q) at position 5, His (27H) at position 27, Ala (28A) at position 28, Lys (38K) at position 38, Arg (40R) at position 40, Lys (43K) at position 43, Ile (48I) at position 48, Lys (67K) at position 67, Ala (68A) at position 68, and Leu (70L) at position 70. In one embodiment, the VL domain of a humanized anti-OX40 antibody or antigen-binding fragment according to the present disclosure optionally includes a back mutation at residue position 69 from the human residue to Ser (69S) according to Kabat numbering.

[0064] In one embodiment, an isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody comprising a backmutated amino acid residue in the VH domain selected from the group consisting of: (i) 1E, (ii) 1E and 27H, (iii) 1E, 27H, 48I, and 70L, (iv) 1E, 27H, 38K, 43K, 48I, 67K, and 70L, (v) 1E, 40R, and 43K, (vi) 1E, 5Q, 27H, 28A, 38K, 40R, 43K, 48I, 67K, 68A, and 70L, all according to Kabat numbering; and / or a backmutated amino acid residue of 69S in the VL domain according to Kabat numbering.

[0065] In one embodiment, an isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody that includes amino acid residues 1E, 5Q, 27H, 28A, 38K, 40R, 43K, 48I, 67K, 68A, and 70L in the VH domain and amino acid residue 69S in the VL domain according to Kabat numbering. In a further embodiment, an isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure further includes a G62A mutation in the VH domain according to Kabat numbering and a G34A mutation in the VL domain according to Kabat numbering.

[0066] In some embodiments, an isolated anti-OX40 antibody or antigen-binding fragment according to the present disclosure comprises a combination of VH and VL sequences selected from the group consisting of:

[0067] TIFF2024523838000001.tif153162

[0068] In some embodiments, the antibody comprises a VH domain comprising or consisting of the sequence of SEQ ID NO:16, and a VL domain comprising or consisting of the sequence of SEQ ID NO:21.

[0069] In some embodiments of the anti-OX40 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native Fc region or a variant Fc region. In certain embodiments, the Fc region is a human Fc region derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to alter (e.g., reduce or eliminate) at least one effector function, such as ADCC and / or CDC. In some embodiments, the present disclosure provides an anti-OX40 antibody or antigen-binding fragment comprising an Fc region with one or more mutations, e.g., L234A and L235A, that alter at least one effector function.

[0070] In some embodiments, an antigen-binding fragment of an anti-OX40 antibody according to the present disclosure can be, for example, an Fv, Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; or a single-chain antibody molecule (e.g., scFv).

[0071] In one embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein bind to the OX40 extracellular domain or a portion thereof. In some embodiments, the OX40 extracellular domain is selected from the group consisting of the amino acid sequence L29 to A214 of the human OX40 protein under UniProt identification number P43489, or SEQ ID NO: 44: It includes the amino acid sequence of TIFF2024523838000002.tif40170, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0072] In one embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein bind to OX40 at the CRD3 region of the OX40 extracellular domain.

[0073] In one embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein have an antibody binding activity against human OX40 of at least 1×10 as measured by biolayer interferometry or surface plasmon resonance. 4 M -1 seconds -1 , at least 3 × 10 4 M -1 seconds -1 , at least 5 × 10 4 M -1 seconds -1 , at least 7 × 10 4 M -1 seconds -1 , at least 9×10 4 M -1 seconds -1 , at least 1 × 10 5 M -1 seconds -1 The on-rate constant (k on ).

[0074] In another embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein have an activity of 5×10 to human OX40 as measured by surface plasmon resonance or biolayer interferometry. -3 seconds -1 Less than 3×10 -3 seconds -1 Less than 2×10 -3 seconds -1 Less than 1×10 -3 seconds -1 Less than 9×10 -4 seconds -1 Less than 6×10 -4 seconds -1 Less than 3×10 -4 seconds -1 Less than 2.5×10 -4 seconds -1 Less than 2×10 -4 seconds -1 Less than 1×10 -4 seconds -1 Less than 8×10 -5 seconds -1 Less than 5×10 -5 seconds -1 The off-rate constant (k off In a further embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein are humanized antibodies, and have the kAb of an antibody with the VH / VL sequence pairs of SEQ ID NOs: 9 / 10 and 17 / 18 against human OX40, within the same antibody format. off k for human OX40 that is about 50-500%, for example, about 80-150%, of the value off Generally, a small off-rate correlates with a slow dissociation of the complex formed, whereas a large off-rate correlates with a rapid dissociation. In one embodiment, the anti-OX40 antibodies, or antigen-binding fragments thereof, described herein have an affinity for the target OX40 that is greater than the affinity of 1A7.gr.1 described in WO2015153513, as indicated by a small off-rate.

[0075] In one embodiment, the anti-OX40 antibodies or antigen-binding fragments thereof described herein have nanomolar to picomolar (10-8 ~10 -10 ) range, e.g., 8×10 -8 Less than M, 5×10 -8 Less than M, 3 x 10 -8 Less than M, 1×10 -8 Less than M, 8 x 10 -9 Less than M, 5×10 -9 Less than M, 3 x 10 -9 Less than M, 2 x 10 -9 Less than M, 1×10 -9 Less than M, 8 x 10 -10 Less than M, 6×10 -10 Less than M, 4×10 -10 Less than M, 2 x 10 -10 Less than M or 1×10 -10 Dissociation constant (K D ).

[0076] In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein is + Specifically binds to OX40 displayed on target cells, for example, CHO cell lines or T cell lines expressing OX40 (e.g., primary T cells and Jurkat cells). In cell-based assays, anti-OX40 antibodies show strong binding to OX40+ cells, as measured by flow cytometry, where the binding to the cells is reflected by an EC50 of about 5nM or less, 4nM or less, 3nM or less, 2nM or less, or 1nM or less. In further embodiments, the EC50 is 0.5nM or less. In some embodiments, the anti-OX40 antibodies or antigen-binding fragments described herein show equal or greater binding to OX40 displayed on target cells compared to antibodies with VH / VL sequence pairs of SEQ ID NOs: 9 / 10 and 17 / 18. In one embodiment, the binding of the antibodies to OX40-expressing cells is measured in a cell-based assay as described in Example 1.2. In some embodiments, binding of an anti-OX40 antibody or antigen-binding fragment thereof described herein to OX40 with the avidity described above is sufficient to induce an intracellular effect in vivo or in vitro. In further embodiments, the effect is T cell activation and / or proliferation.

[0077] In one embodiment, the antibody can bind to OX40 in the same manner that its ligand, OX40L, binds to OX40 on the cell surface of OX40-expressing cells. In another embodiment, the antibody can be used to enhance OX40 / OX40L signaling. In a further embodiment, the antibody can be used to induce and / or enhance T cell activation and proliferation associated with the OX40 / OX40L pathway.

[0078] Anti-PD-L1 antibody The disclosure also provides antibodies capable of binding to human PD-L1.

[0079] In some embodiments, the anti-PD-L1 antibodies according to the disclosure comprise a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises the sequence TYGIN (SEQ ID NO: 22), CDR-H2 comprises the sequence YIYIGNAYTEYNEKFKG (SEQ ID NO: 23) or YIYIGNGYTEYNEKFKG (SEQ ID NO: 25), CDR-H3 comprises the sequence DLMVIAPKTMDY (SEQ ID NO: 24), CDR-L1 comprises the sequence KASQDVGTAVA (SEQ ID NO: 26), CDR-L2 comprises the sequence WASTRHT (SEQ ID NO: 27), CDR-L3 comprises the sequence QQYSSYPYT (SEQ ID NO: 28), The CDRs are defined according to the Kabat numbering system.

[0080] In some embodiments, the anti-PD-L1 antibodies, or antigen-binding fragments thereof, in accordance with the application: a VH domain comprising a sequence according to SEQ ID NO: 29, 30 or 31, or a sequence having at least 80% to 90%, or 95% to 99% identity thereto, and / or - A VL domain comprising the sequence of SEQ ID NO: 32, 33, or 34, or a sequence having at least 80% to 90%, or 95% to 99% identity thereto.

[0081] In some embodiments, the anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises a VH domain comprising the sequence of SEQ ID NO:31, and a VL domain comprising the sequence of SEQ ID NO:34.

[0082] In some embodiments, an anti-OX40 antibody according to the present disclosure or an anti-PD-L1 antibody according to the present disclosure can be used to generate a binding protein derivative that recognizes the same target antigen, by techniques well established in the art. Such derivatives can be, for example, single chain antibodies (scFv), Fab fragments (Fab), Fab' fragments, F(ab')2, Fv, and disulfide-linked Fv. Such derivatives can be, for example, fusion proteins or conjugates comprising an anti-OX40 antibody according to the present disclosure or an anti-PD-L1 antibody according to the present disclosure. The fusion protein can be a multispecific antibody or a CAR molecule. The conjugate can be an antibody-drug conjugate (ADC), or an antibody conjugated to a detection agent, for example, a radioisotope.

[0083] In one embodiment, the anti-PD-L1 antibodies, or antigen-binding fragments thereof, described herein bind to PD-L1, e.g., human PD-L1, at sub-nanomolar levels, e.g., 1×10 -9 Less than M, 8×10 -10 Less than M, 6×10 -10 Less than M, 4×10 -10 Less than M, 3 x 10 -10 Dissociation constant (K D In one embodiment, the anti-PD-L1 antibodies, or antigen-binding fragments thereof, described herein specifically bind to PD-L1 displayed on PD-L1+ target cells. The anti-PD-L1 antibodies specifically bind to PD-L1 displayed on PD-L1+ target cells as measured by flow cytometry, biolayer interferometry, and / or surface plasmon resonance. +It exhibits strong binding ability to cells. EC50 by FACS binding method and / or K by BLI or BIAcore. D Accordingly, the binding affinity to human PD-L1 is comparable to that to cynomolgus PD-L1, e.g., <5-fold difference or <3-fold difference.

[0084] OX40×PD-L1 bispecific binding protein In another aspect, the present disclosure provides OX40 / PD-L1 bispecific binding proteins, particularly FIT-Ig (Fab-in-tandem immunoglobulin), capable of binding to both OX40 and PD-L1. Each variable domain (VH or VL) in FIT-Ig can be derived from one or more "parent" monoclonal antibodies that bind to one of the target antigens, i.e., OX40 or PD-L1. FIT-Ig binding proteins can be made using the variable domain sequences of anti-OX40 and anti-PD-L1 monoclonal antibodies disclosed herein, such as humanized anti-OX40 parent antibodies and humanized anti-PD-L1 parent antibodies.

[0085] One aspect of the present disclosure relates to the selection of parent antibodies that have at least one or more desired properties within the FIT-Ig molecule. In one embodiment, the antibody properties are selected from the group consisting of antigen specificity, affinity for antigen, dissociation rate, cell binding strength, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, binding to orthologous antigens, and the like.

[0086] In some embodiments, the bispecific FIT-Ig proteins according to the present disclosure are constructed without an inter-domain peptide linker. It is generally understood in the art that in multivalent engineered immunoglobulin formats with tandem binding sites, adjacent binding sites will interfere with each other unless a flexible linker is used to spatially separate the binding sites. However, for the OX40 / PD-L1 FIT-Ig of the present disclosure, it has been discovered that the arrangement of immunoglobulin domains according to the chain formulas disclosed herein results in polypeptide chains that are sufficient for expression in transfected mammalian cells and are properly assembled and secreted as intact, bispecific, multivalent immunoglobulin-like binding proteins that bind to the target antigens OX40 and PD-L1. See the Examples below. Furthermore, the omission of the synthetic linker sequence from the binding protein can avoid the creation of antigenic sites recognizable by the mammalian immune system, and thus the elimination of the linker reduces the immunogenic potential of FIT-Ig and results in a circulating half-life similar to that of natural antibodies.

[0087] In some embodiments, the OX40×PD-L1 bispecific binding protein in accordance with the present application comprises: a) a first antigen-binding site that specifically binds to OX40; and b) comprises a second antigen-binding site that specifically binds to PD-L1.

[0088] In one embodiment, the bispecific binding protein described herein comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-OX40 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the OX40 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding protein described herein comprises a VH / VL pair derived from any anti-OX40 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the OX40 binding site of the bispecific binding protein.

[0089] In one embodiment, the bispecific binding proteins described herein further comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-PD-L1 antibody, or antigen-binding fragment thereof, described herein in accordance with the application, to form the PD-L1 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins described herein comprise a VH / VL pair derived from any anti-PD-L1 antibody, or antigen-binding fragment thereof, described herein in accordance with the application, to form the PD-L1 binding site of the bispecific binding protein.

[0090] In one embodiment, the OX40 binding site and the PD-L1 binding site in a bispecific OX40 / PD-L1 binding protein according to the present application are humanized and comprise humanized VH / VL sequences, respectively.

[0091] Bispecific FIT-Ig binding protein In one embodiment, the OX40×PD-L1 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein capable of binding to OX40 and PD-L1. FIT-Ig binding protein (Fab-in-tandem immunoglobulin) is a bispecific tetravalent binding protein that comprises six polypeptide chains and has four functional binding regions, Fab, with two external binding regions, Fab, and two internal binding regions, Fab. The binding protein adopts a (external Fab-internal Fab-Fc)×2 format and binds to both antigen A and antigen B. In one aspect, the OX40×PD-L1 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein, where the two Fab domains of the FIT-Ig protein provide a first antigen binding site that specifically binds to OX40, and the other two Fab domains of the FIT-Ig protein provide a second antigen binding site that specifically binds to PD-L1. In some embodiments, the FIT-Ig binding proteins in accordance with the present disclosure do not utilize a linker between the immunoglobulin domains.

[0092] In one embodiment, the binding protein comprises, from amino terminus to carboxyl terminus, the V A -CL-VH B -CH1-Fc or VH B -CH1-VL A a first polypeptide comprising, from the amino terminus to the carboxyl terminus, a VH A a second polypeptide comprising, from the amino terminus to the carboxyl terminus, a VL B or alternatively, the binding protein comprises a third polypeptide comprising, from amino terminus to carboxyl terminus, VL B -CL-VH A -CH1-Fc or VH A -CH1-VL B a first polypeptide comprising, from the amino terminus to the carboxyl terminus, a VH B a second polypeptide comprising, from the amino terminus to the carboxyl terminus, a VL Awherein VL represents a light chain variable domain, CL represents a light chain constant domain, VH represents a heavy chain variable domain, CH1 represents the first constant domain of the heavy chain, A represents OX40, and B represents PD-L1. Each bispecific binding protein comprises two of said first polypeptides, two of said second polypeptides, and two of said third polypeptides, two of which are Fabs for binding to OX40 (VL A - VH paired with CL A -CH1, VH A -CH1::VL A -CL), and two are Fabs for binding to PD-L1 (VL B - VH paired with CL B -CH1, VH B -CH1::VL B -CL) is a hexamer exhibiting four Fab antigen-binding sites.

[0093] In some embodiments, a Fab that binds OX40 (e.g., when A is OX40, VL A -CL and VH A - Fab formed by CH1; or when B is OX40, VL B -CL and VH B The Fab (formed by -CH1) comprises a set of six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, from any anti-OX40 antibody or antigen-binding fragment thereof described herein in accordance with the present application, so as to form the OX40 binding site of the bispecific binding protein. In some further embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 1, 2, 3, and 5, 6, 7; the sequences of SEQ ID NOs: 1, 4, 3, and 5, 6, 7; the sequences of SEQ ID NOs: 1, 2, 3, and 8, 6, 7; or the sequences of SEQ ID NOs: 1, 4, 3, and 8, 6, 7, respectively.

[0094] In some embodiments, the OX40-binding Fab in the FIT-Ig binding protein comprises a VH / VL pair derived from any anti-OX40 antibody or antigen-binding fragment thereof described herein in accordance with the present application. In some further embodiments, the VH / VL pair comprises a sequence selected from the group consisting of the following VH / VL sequence pairs: SEQ ID NOs: 11 / 19, 12 / 19, 13 / 19, 14 / 19, 11 / 20, 12 / 20, 13 / 20, 14 / 20, 10 / 17, 9 / 18, 10 / 18, 9 / 19, 11 / 17, 15 / 21, 15 / 18, 16 / 21, and 16 / 18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the OX40-binding Fab in the FIT-Ig binding protein comprises a VH sequence of SEQ ID NO: 16, and a VL sequence of SEQ ID NO: 21.

[0095] In some embodiments, a Fab that binds to PD-L1 (e.g., when A is PD-L1, VL A -CL and VH A - Fab formed by CH1; or when B is PD-L1, VL B -CL and VH BThe PD-L1 binding Fab formed by pairing of the VL-CL with the VH-CH1 in the FIT-Ig binding protein comprises a set of six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, from any anti-PD-L1 antibody or antigen-binding fragment thereof described herein in accordance with the present application, so as to form the PD-L1 binding site of the bispecific binding protein. In some embodiments, the PD-L1 binding Fab formed by pairing of the VL-CL with the VH-CH1 in the FIT-Ig binding protein comprises a set of six CDRs, where CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 22, 23, 24, and 26, 27, 28; or the sequences of SEQ ID NOs: 22, 25, 24, and 26, 27, 28, respectively. In some further embodiments, the Fab that binds to PD-L1 comprises a VH / VL pair comprising a sequence of SEQ ID NO: 31 and 34, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0096] In the present disclosure, an OX40 / PD-L1FIT-Ig binding protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain is arranged, from the amino terminus to the carboxyl terminus, with a CL of VH PD-L1 VL directly fused to OX40 -CL-VH PD-L1 -CH1-Fc or CH1 is VL OX40 VH directly fused to PD-L1 -CH1-VL OX40 the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH OX40 the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL PD-L1 In an alternative embodiment, the OX40 / PD-L1FIT-Ig binding protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises, from amino terminus to carboxyl terminus, CH1 comprises VL PD-L1 VH directly fused to OX40 -CH1-VLPD-L1 -CL-Fc or CL is VH OX40 VL directly fused to PD-L1 -CL-VH OX40 the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH PD-L1 the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL OX40 In some embodiments, the VL OX40 is the light chain variable domain of the anti-OX40 antibody, CL is the light chain constant domain, and VH OX40 is the heavy chain variable domain of the anti-OX40 antibody, CH1 is the heavy chain constant domain, and VL PD-L1 is the light chain variable domain of the anti-PD-L1 antibody; VH PD-L1 is a heavy chain variable domain of an anti-PD-L1 antibody; optionally, the domain V PD-L1 -CL is the same as the light chain of the parent anti-PD-L1 antibody and has the domain VH PD-L1 -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the parent anti-PD-L1 antibody, and the domain VL OX40 -CL is the same as the light chain of the anti-OX40 parent antibody and has the domain VH OX40 -CH1 is identical to the heavy chain variable and heavy chain constant domains of the anti-OX40 parent antibody.

[0097] In one embodiment, the VH OX40 -CH1 comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:38.

[0098] SEQ ID NO:38 TIFF2024523838000003.tif26170TIFF2024523838000004.tif4170

[0099] In one embodiment, the V OX40-CL comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:37.

[0100] SEQ ID NO:37 TIFF2024523838000005.tif32170

[0101] In one embodiment, the VH PD-L1 -CH1 comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:36.

[0102] SEQ ID NO:36 TIFF2024523838000006.tif32170

[0103] In one embodiment, the V PD-L1 -CL comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:39.

[0104] SEQ ID NO:39 TIFF2024523838000007.tif31170

[0105] In the above formula for the FIT-Ig binding protein, the Fc region is a human Fc region derived from IgG1 in which at least one Fc effector function (e.g., Fc binding to FcγR, ADCC, and / or CDC) has been reduced or eliminated, for example by introduction of LALA mutations (Leu234 to Ala234, Leu235 to Ala235, according to the EU numbering system). In a further embodiment, the amino acid sequence of the Fc region is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 40. In one embodiment, the amino acid sequence of the Fc region further comprises the triple mutation M252Y / S254T / T256E (YTE, numbering according to the EU numbering system). In further embodiments, the amino acid sequence of the Fc region is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:41.

[0106] SEQ ID NO:40 TIFF2024523838000008.tif31170

[0107] SEQ ID NO:41 TIFF2024523838000009.tif31170

[0108] In one embodiment, the FIT-Ig binding proteins of the present disclosure retain one or more properties of the parent antibody. In some embodiments, FIT-Ig retains binding affinity for target antigens (i.e., PD-L1 and OX40) that is comparable to the binding affinity of the parent antibody means that the binding affinity of the FIT-Ig binding protein for antigen targets OX40 and PD-L1 does not vary by more than 10-fold compared to the binding affinity of the parent antibody for its respective target antigens, as measured by surface plasmon resonance or biolayer interferometry.

[0109] In one embodiment, a FIT-Ig binding protein of the disclosure binds to OX40 and PD-L1 and is comprised of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 35 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 36 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 37 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0110] In one embodiment, a FIT-Ig binding protein of the disclosure binds to OX40 and PD-L1 and is composed of a first polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:35; a second polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:36; and a third polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:37.

[0111] Characteristics of bispecific binding proteins In one embodiment, a bispecific OX40 / PD-L1 FIT-Ig binding protein capable of binding both PD-L1 and OX40 described herein comprises a humanized OX40 binding site, or a chimeric OX40 binding site, e.g., a humanized OX40 binding site. In one embodiment, the humanized OX40 binding site in a FIT-Ig protein format has a slower off-rate for binding to OX40 compared to a chimeric OX40 binding site in the same FIT-Ig format consisting of the VH / VL pair of SEQ ID NOs: 10 and 18. In a further embodiment, the off-rate ratio of the humanized OX40 binding site compared to the chimeric OX40 binding site is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% as measured by surface plasmon resonance or biolayer interferometry. In one embodiment, the off-rate of a FIT-Ig binding protein described herein on OX40 is greater than or equal to 5×10 as measured by surface plasmon resonance or biolayer interferometry. -3 seconds -1 Less than 3×10- 3 seconds -1 Less than 2×10- 3 seconds -1 Less than 1×10 -3 seconds -1 Less than 9×10 -4 seconds -1 Less than 6×10 -4 seconds -1 Less than 3×10 -4 seconds -1 Less than 2.5×10- 4 seconds- 1 Less than 2×10 -4 seconds -1 Less than 1×10 -4 seconds -1 Less than 8×10 -5 seconds -1 Less than 5×10 -5 seconds -1 In one embodiment, the FIT-Ig binding protein antibody or antigen-binding fragment thereof described herein has an antibody activity against OX40 of less than 10 -8 ~10 -10 range, e.g., 8×10 -8 Less than M, 5×10-8 Less than M, 3 x 10 -8 Less than M, 2 x 10 -8 Less than M, 1×10 -8 Less than M, 8 x 10 -9 Less than M, 5×10 -9 Less than M, 3 x 10 -9 Less than M, 2 x 10 -9 Less than M or 1×10 -9 Less than M, 8 x 10 -10 Less than M, 6×10 -10 Less than M, 4×10 -10 Less than M, 2 x 10 -10 Less than M or 1×10 -10 Dissociation constant (K D In one embodiment, the FIT-Ig binding protein antibody or antigen-binding fragment thereof described herein has an antibody binding activity of 1×10 -3 seconds- 1 ~1×10 -4 seconds- 1 , e.g., 5×10 -4 seconds- 1 Off-rates in the range of less than 1 x 10 -8 seconds- 1 ~1×10 -9 seconds- 1 range, e.g., 7×10 -9 seconds- 1 Less than K D has.

[0112] In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding protein capable of binding to PD-L1 and OX40 described herein has a purity of 90% or greater as detected by SEC-HPLC following one-step purification from cell culture medium using Protein A affinity chromatography. In one embodiment, the one-step purified binding protein has a purity of 91%, 92%, 93%, 95%, 97%, 99% or greater as detected by SEC-HPLC.

[0113] In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding protein described herein is capable of binding to both PD-L1 expressing cells and OX40 expressing cells. In one embodiment, the PD-L1 expressing cells are human PD-L1 transfected CHO cell lines or tumor cells. In one embodiment, the OX40 expressing cells are OX40 expressing T cells / cell lines, such as CD8+ T cells, CD4+ T cells, Treg cells, or Jurkat cells.

[0114] In one embodiment, the binding avidity of the bispecific FIT-Ig binding protein to OX40-expressing cells is equal or comparable to a corresponding parent anti-OX40 IgG monoclonal antibody that comprises the same VH / VL sequence pair as the bispecific FIT-Ig protein for binding to OX40 as measured by flow cytometry in a cell-based assay. In one embodiment, the binding avidity of the bispecific FIT-Ig binding protein to PD-L1-expressing cells is equal or comparable to a corresponding parent anti-PD-L1 IgG monoclonal antibody that comprises the same VH / VL sequence pair as the bispecific binding protein for binding to PD-L1 as measured by flow cytometry, for example, in the assays described in Examples 3 and 4.

[0115] In one embodiment, the bispecific binding proteins described herein are capable of modulating the biological function of OX40, PD-L1, or both. In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding proteins described herein are capable of activating OX40 signaling in a PD-L1-dependent manner. In one embodiment, the bispecific binding proteins of the disclosure exhibit activation of T cells by the OX40 signaling pathway. In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding proteins described herein exhibit cytotoxicity against tumor cells by OX40-activated T cells in a PD-L1-dependent manner. In one embodiment, the bispecific binding proteins of the disclosure are used to enhance the cytokine secretion activity of T cells against tumor cells in a PD-L1-dependent manner.

[0116] In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding protein described herein exhibits PD-L1-dependent OX40 activation. In one embodiment, the ratio of PD-L1 expressing cells to OX40 expressing T cells is about 1:1. In further embodiments, the bispecific OX40 / PD-L1 binding proteins exhibit activation of OX40 in T cells in the presence of PD-L1-expressing cells compared to much less activation of OX40 in T cells in the absence of PD-L1-expressing cells, and exhibit activation of OX40 in T cells in the presence of PD-L1-expressing cells compared to much less activation of OX40 in T cells in the presence of PD-L1-expressing cells induced by the combination of a corresponding parent anti-PD-L1 IgG monoclonal antibody that comprises the same VH / VL sequence pair for binding to PD-L1 as the bispecific binding protein and a corresponding parent anti-OX40 IgG monoclonal antibody that comprises the same VH / VL sequence pair for binding to OX40 as the bispecific FIT-Ig protein.

[0117] In one embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding proteins described herein result in cytotoxicity or cytokine secretion activity against tumor cells by T cells. In a further embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding proteins described herein enhance anti-tumor immunity and / or prevent tumor immune escape. In another embodiment, the bispecific OX40 / PD-L1 FIT-Ig binding proteins described herein exhibit anti-tumor activity, e.g., reducing tumor burden, inhibiting tumor growth, or suppressing neoplastic cell spread. In some embodiments, the bispecific OX40 / PD-L1 FIT-Ig binding proteins are capable of mediating high levels of clustering. In some embodiments, the bispecific OX40 / PD-L1 FIT-Ig binding proteins are capable of inducing high order OX40 clustering. In some embodiments, the bispecific OX40 / PD-L1 FIT-Ig binding proteins are capable of activating T cells under PD-L1-dependent conditions. In some embodiments, the bispecific OX40 / PD-L1 FIT-Ig binding protein induces sufficient OX40 signaling through cross-linking of PD-L1, e.g., which can overcome the limitations of anti-OX40 monotherapy. In some embodiments, the bispecific OX40 / PD-L1 FIT-Ig binding protein synergistically stimulates T cell activity, e.g., IL-2 production, as measured, e.g., by methods known in the art, compared to an appropriate control, e.g., the additive effect of a combination of both parent antibodies.

[0118] Nucleic Acids, Vectors, and Host Cells In a further aspect, the present disclosure provides isolated nucleic acids encoding one or more amino acid sequences of the anti-OX40 antibodies or antigen-binding fragments thereof of the present disclosure; isolated nucleic acids encoding one or more amino acid sequences of the anti-PD-L1 antibodies or antigen-binding fragments thereof of the present disclosure; and isolated nucleic acids encoding one or more amino acid sequences of bispecific binding proteins, including FIT-Ig (Fab-in-tandem immunoglobulin) binding proteins capable of binding to both OX40 and PD-L1. Such nucleic acids may be inserted into vectors to perform various genetic analyses or to express, characterize, or improve one or more properties of the antibodies or binding proteins described herein. The vector may include one or more nucleic acid molecules encoding one or more amino acid sequences of the antibodies or binding proteins described herein, operably linked to appropriate transcription and / or translation sequences that allow expression of the antibodies or binding proteins in a particular host cell harboring the vector. Examples of vectors for cloning or expressing nucleic acids encoding the amino acid sequences of the binding proteins described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, and derivatives thereof.

[0119] The present disclosure also provides host cells that express or are capable of expressing vectors that include a nucleic acid encoding one or more amino acid sequences of the antibodies or binding proteins described herein. Host cells useful in the present disclosure can be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells in the present disclosure include protist cells, animal cells, plant cells, and fungal cells. An exemplary fungal cell is a yeast cell, including Saccharomyces cerevisiae. Exemplary animal cells useful as host cells according to the present disclosure include, but are not limited to, mammalian cells, avian cells, and insect cells. Exemplary mammalian cells include, but are not limited to, CHO cells, HEK cells, Jurkat cells, and COS cells.

[0120] Method for preparation In another aspect, the disclosure provides a method of making an anti-OX40 antibody, or antigen-binding fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding to OX40.

[0121] In another aspect, the disclosure provides a method of making an anti-PD-L1 antibody, or antigen-binding fragment thereof, comprising culturing a host cell containing an expression vector encoding the antibody or antigen-binding fragment, in a culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding to PD-L1.

[0122] In another aspect, the disclosure provides a method of making a bispecific multivalent binding protein capable of binding to OX40 and PD-L1, particularly a FIT-Ig binding protein that binds to OX40 and PD-L1, comprising culturing a host cell comprising an expression vector encoding the FIT-Ig binding protein in a culture medium under conditions sufficient to cause the host cell to express the binding protein capable of binding to OX40 and PD-L1. The proteins made by the methods disclosed herein may be isolated by and used in a variety of compositions and methods described herein.

[0123] Uses of antibodies and binding proteins Given their ability to bind human OX40 and / or PD-L1, the antibodies, antigen-binding fragments thereof, and bispecific multivalent binding proteins described herein can be used to detect OX40 or PD-L1, or both, in biological samples containing cells expressing, for example, one or both of these target antigens. The antibodies, antigen-binding fragments, and binding proteins of the present disclosure can be used in conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or immunohistochemistry. The present disclosure provides a method of detecting OX40 or PD-L1 in a biological sample, comprising contacting the biological sample with an antibody, antigen-binding portion thereof, or binding protein of the present disclosure, and detecting whether binding to the target antigen occurs, thereby detecting the presence or absence of the target in the biological sample. The antibody, antigen-binding fragment, or binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody / fragment / binding protein. Suitable detection substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; an example of a suitable radioactive material includes 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Contains Sm.

[0124] In some embodiments, the antibodies, or antigen-binding fragments thereof, of the present disclosure are capable of neutralizing human PD-L1 activity both in vitro and in vivo, and thus the antibodies, or antigen-binding fragments thereof, of the present disclosure may be used to inhibit human PD-L1 activity, for example, to inhibit PD-L1-associated cell signaling in a cell culture containing PD-L1-expressing cells in a human subject or other mammalian subject having a PD-L1 with which the antibodies, or antigen-binding fragments thereof, or binding proteins of the present disclosure cross-react.

[0125] In another embodiment, the disclosure provides an antibody or bispecific binding protein of the disclosure for use in treating a subject suffering from a disease or disorder in which PD-L1 activity is detrimental, where the antibody or binding protein is administered to the subject such that PD-L1-mediated activity in the subject is reduced. As used herein, the term "disorder in which PD-L1 activity is detrimental" is intended to include diseases and other disorders in which interaction of PD-L1 with its receptor (e.g., PD-1) in a subject suffering from the disorder contributes to the pathophysiology of the disorder or is a factor contributing to the exacerbation of the disorder. Examples of such diseases or disorders are tumors associated with immune evasion or tumors that exhibit tumor immune evasion. Thus, a disorder in which PD-L1 activity is detrimental is a disorder in which inhibition of PD-L1 activity is predicted to alleviate symptoms and / or progression of the disorder. In one embodiment, an anti-PD-L1 antibody, antigen-binding fragment thereof, or bispecific binding protein of the disclosure is used in a method of inhibiting the proliferation or survival of malignant cells or reducing tumor burden.

[0126] In some embodiments, the bispecific binding proteins (FIT-Ig) of the present disclosure are capable of enhancing T cell cytotoxicity or cytokine secretion activity against PD-L1 expressing tumor cells both in vitro and in vivo. Thus, the bispecific binding proteins of the present disclosure may be used to inhibit the growth or spread of PD-L1 expressing malignant cells in a human subject or other mammalian subject having a PD-L1 with which an antibody, antigen-binding fragment thereof, or bispecific binding protein of the present disclosure cross-reacts.

[0127] In another embodiment, the disclosure provides a method for treating a disease or disorder in which OX40-mediated signaling activity is beneficial (e.g., OX40 + The present disclosure provides an antibody or bispecific binding protein for use in treating a subject suffering from a tumor (T cell infiltrating tumor). As used herein, the term "disorder in which OX40-mediated signaling activity is beneficial" is intended to include diseases and other disorders in which high clustering and / or activation of OX40 in a subject suffering from the disorder activates T cells, thereby reversing the effects / alleviating symptoms / slowing the progression of the disease or disorder, e.g., tumor. In one embodiment, the present disclosure's anti-OX40 antibody, antigen-binding fragment thereof, or bispecific binding protein is used in a method of inhibiting the proliferation or survival of malignant cells or reducing tumor burden.

[0128] In another embodiment, the present disclosure provides a PD-L1 / OX40 bispecific (FIT-Ig) binding protein for use in treating a PD-L1-expressing malignancy in a subject via activation of OX40 by T cells, where the binding protein is administered to the subject. In some embodiments, the malignancy is a tumor, e.g., a solid tumor, e.g., colon cancer.

[0129] In some further embodiments, the antibodies (including antigen-binding fragments thereof) and binding proteins of the present disclosure are incorporated into or used in the manufacture of pharmaceutical compositions (described supra) suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibodies or binding proteins of the present disclosure and a pharma- ceutically acceptable carrier. As used herein, a "pharma- ceutically acceptable carrier" includes any / all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Examples of pharma- ceutical acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent, such as a sugar, a polyalcohol (e.g., mannitol or sorbitol), or sodium chloride in the composition. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody or binding protein present in the composition. A pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration.

[0130] The disclosed method may include administering a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with added preservatives. The compositions may take such forms, for example, as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the main active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0131] The use of the present disclosure may include administration of the composition formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. For example, the composition may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), may be formulated with ion exchange resins, or may be formulated as a poorly soluble derivative (e.g., as a poorly soluble salt).

[0132] The antibody, its antigen-binding fragment, or binding protein of the present disclosure may also be administered with one or more additional therapeutic agents that are useful in treating various diseases. The antibody, its antigen-binding fragment, and binding protein described herein may be used alone or in combination with an additional agent, e.g., an additional therapeutic agent, an additional agent selected by a person skilled in the art for its intended purpose. For example, the additional agent may be a therapeutic agent recognized in the art as being useful for treating the disease or condition treated by the antibody or binding protein of the present disclosure. The additional agent may also be an agent that imparts a beneficial attribute to the therapeutic composition, e.g., an agent that affects the viscosity of the composition.

[0133] Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, or a bispecific multivalent binding protein (i.e., the main active ingredient) of the present disclosure, and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical composition of the present disclosure may comprise two or more antibodies of the present disclosure, e.g., an anti-OX40 antibody and an anti-PD-L1 antibody. In a further embodiment, the pharmaceutical composition of the present disclosure may comprise at least one antibody according to the present disclosure and at least one bispecific binding protein. In a specific embodiment, the composition comprises one or more antibodies or binding proteins of the present disclosure. The present disclosure also provides a pharmaceutical composition comprising a combination of antibodies (such as, e.g., an anti-OX40 antibody and an anti-PD-L1 antibody), or antigen-binding fragment(s) thereof, described herein, and a pharma- ceutically acceptable carrier. In particular, the present disclosure provides a pharmaceutical composition comprising at least one FIT-Ig binding protein capable of binding to OX40 and PD-L1, and a pharma- ceutically acceptable carrier. The pharmaceutical composition of the present disclosure may further comprise at least one additional active ingredient. In some embodiments, such additional components include, but are not limited to, prophylactic and / or therapeutic agents, detection agents, such as anti-tumor drugs, cytotoxic agents, antibodies of different specificities or antigen-binding fragments thereof, detectable labels or detectable reporters. In one embodiment, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents, i.e., agents other than the antibodies or binding proteins of the present disclosure, for treating disorders in which PD-L1 activity is detrimental and / or disorders in which OX40 activity is beneficial. In one embodiment, the additional prophylactic or therapeutic agents are known to be useful, used, or currently being used in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof.

[0134] The pharmaceutical composition comprising the protein of the present disclosure is a pharmaceutical composition for, but not limited to, diagnosis, detection, or monitoring of a disorder; treatment, management, or amelioration of a disorder or one or more symptoms thereof; and / or use in research studies. In some embodiments, the composition may further comprise a carrier, diluent, or excipient. An excipient is generally any compound or combination of compounds that also provides a desired characteristic to a composition other than the composition of the main active ingredient (i.e., a composition other than the antibody, its antigen-binding portion, or binding protein of the present disclosure).

[0135] Methods for treatment and medical uses In one embodiment, the present disclosure provides a method of modulating an immune response in a subject comprising administering to the subject at least one antibody and / or at least one bispecific binding protein according to the present disclosure.

[0136] In some embodiments, the present disclosure provides methods for activating T cells. In some further embodiments, activation of T cells can result in induction and / or enhancement of T cell-mediated anti-tumor activity. In some further embodiments, the anti-tumor activity is cytotoxicity against tumor cells and / or cytokine production, where the cytokine is, for example, IL-2 or IFN-γ. In some further embodiments, the T cells are CD8+ T cells. In some other embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are effector T cells.

[0137] In some embodiments, the disclosure provides a method of treating cancer in a subject, comprising administering to the subject at least one antibody and / or at least one bispecific binding protein according to the disclosure. In some embodiments, the cancer is a tumor immune evasion, or a tumor exhibiting tumor immune evasion. In some further embodiments, the cancer is a cancer that responds to T cell activation, e.g., a cancer associated with T cell dysfunction. In some further embodiments, the cancer is a cancer that has elevated levels of PD-L1 protein expression, or elevated levels of a nucleic acid encoding PD-L1, e.g., compared to levels in a normal subject or normal cell. In one embodiment, the disclosure provides a method of treating cancer in a subject in need thereof, for example, a tumor that exhibits tumor immune evasion, or ... + The present disclosure provides a method of treating a tumor (T cell infiltrating tumor) comprising administering to a subject an anti-OX40 antibody, or an OX40-binding fragment thereof, as described herein, wherein the antibody or binding fragment is capable of binding to OX40 and activating OX40-mediated signaling in cells expressing OX40. In another embodiment, the present disclosure provides the use of an effective amount of an anti-OX40 antibody, or an antigen-binding fragment thereof, as described herein, in the treatment of such a disorder. In another embodiment, the present disclosure provides the use of an anti-OX40 antibody, or an antigen-binding fragment thereof, as described herein, in the manufacture of a composition for the treatment of such a disorder. In another embodiment, the present disclosure provides the anti-OX40 antibody, or an antigen-binding fragment thereof, as described herein, for use in the treatment of such a disorder.

[0138] In further embodiments of the methods or uses described herein, the anti-OX40 antibody, or antigen-binding fragment, of the disclosure binds to OX40 and comprises a VH domain comprising, essentially consisting of, or consisting of the sequence of SEQ ID NO: 16, and a VL domain comprising, essentially consisting of, or consisting of the sequence of SEQ ID NO: 21.

[0139] In some embodiments, the disclosure provides a method of treating a disorder in which PD-L1 activity is detrimental in a subject in need thereof, comprising the step of administering to the subject an anti-PD-L1 antibody, or PD-L1-binding fragment thereof, as described herein, wherein the antibody or binding fragment is capable of binding to PD-L1 and blocking the interaction of PD-L1, e.g., with a PD-L1 receptor, e.g., PD-1, thereby inhibiting PD-L1-associated signaling in cells that express the PD-L1 receptor.

[0140] In a further embodiment of the methods or uses described herein, the anti-PD-L1 antibody, or antigen-binding fragment, of the disclosure binds to PD-L1 and comprises a VH domain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:31, and a VL domain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:34.

[0141] In another embodiment, the disclosure provides a method for treating a disorder in a subject in need thereof in which OX40-mediated signaling activity is beneficial (e.g., OX40 + The present disclosure provides a method of treating a tumor (T cell infiltrating tumor) and / or a disorder in which PD-L1 activity is detrimental, comprising administering to a subject a bispecific FIT-Ig binding protein capable of binding to PD-L1 and OX40 as described herein. In another embodiment, the present disclosure provides the use of an effective amount of a bispecific FIT-Ig binding protein as described herein in the treatment of such a disorder. In another embodiment, the present disclosure provides the use of a bispecific FIT-Ig binding protein as described herein in the manufacture of a composition for the treatment of such a disorder. In another embodiment, the present disclosure provides the bispecific FIT-Ig binding protein as described herein for use in the treatment of such a disorder.

[0142] In further embodiments of the methods or uses described herein, the FIT-Ig binding protein of the disclosure binds to OX40 and PD-L1 and is composed of a first polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:35; a second polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:36; and a third polypeptide chain comprising, consisting essentially of, or consisting of a sequence of SEQ ID NO:37.

[0143] In some embodiments, disorders that may be treated with an antibody or binding protein in accordance with the disclosure include a variety of malignancies that express PD-L1 on the cell surface of malignant cells. In some further embodiments, disorders that may be treated with an antibody or binding protein in accordance with the disclosure include tumors that exhibit tumor immune evasion, e.g., via PD-L1 / PD-1 interactions. In another embodiment, the antibody or binding protein inhibits the proliferation or survival of malignant cells. In another embodiment, the antibody or binding protein reduces tumor burden. In another embodiment, the cancer is colon cancer.

[0144] The treatment methods described herein may further comprise a step of administering to a subject in need thereof an additional active ingredient, which is suitably present in combination with the antibody or binding protein of the present disclosure for the intended treatment purpose, e.g., another drug having anti-tumor activity. In the treatment methods of the present disclosure, the additional active ingredient may be incorporated into a composition comprising the antibody or binding protein of the present disclosure and administered to the subject in need of treatment. In another embodiment, the treatment methods of the present disclosure may comprise a step of administering to a subject in need of treatment an antibody or binding protein described herein, and a separate step of administering to the subject an additional active ingredient before, contemporaneously with, or after the step of administering to the subject an antibody or binding protein of the present disclosure.

[0145] Having now described the present disclosure in detail, the same will be more clearly understood with reference to the following examples, which are incorporated for purposes of illustration only and are not intended to be limitations of the disclosure.

[0146] [Example] [Example 1] Generation of anti-OX40 antibodies [Example 1.1] Screening, cloning, and sequence analysis of anti-OX40 monoclonal antibody 8G9D5C5 Anti-OX40 monoclonal antibodies were generated by standard hybridoma screening protocols. For immunization, cell immunization and gene gun (DNA immunization) were used, where the immunogens were HEK293 cells overexpressing human OX40 and an expression plasmid containing human OX40 gene, respectively. Hybridoma clones were screened for binding activity and biological activity using CHO-K1 cells expressing human OX40. Clone 8G9D5C5 was selected for further characterization.

[0147] To amplify the heavy and light chain variable regions of antibodies, cells >5 × 10 were cultured using TRIzol reagent (product number: 15596, Invitrogen). 6From each of the 8G9D5C5 clones, total RNA was isolated and reverse transcribed using SuperScript™ III First-Strand Synthesis SuperMix (18080, Invitrogen) to generate cDNA that was applied as a template in PCR using Mouse Ig-Primer Set (product no. 69831-3, Novagen). PCR products were analyzed by electrophoresis on a 1.2% agarose gel with SYBR Safe DNA gel stain. DNA fragments with the correct size were purified by NucleoSpin™ Gel and PCR Clean-up (product no. 740609, MACHEREY-NAGEL) and individually subcloned into pMD18-T vector, which were then transformed into competent E. coli cells. From each transformation, 15 colonies were selected and the sequences of the inserted fragments were analyzed by DNA sequencing. A sequence was confirmed if the majority of the sequenced colonies (at least 8 of 15 colonies) yielded the same sequence. The amino acid sequence of the variable region of clone 8G9D5C5 is listed in Table 1. Complementarity determining regions (CDRs) are underlined according to the Kabat numbering system.

[0148] [Table 1]

[0149] [Example 1.2] Generation and characterization of chimeric antibodies The VH and VK genes of 8G9D5C5 presented in Table 1 were synthesized and cloned into vectors containing human IgG1 and human kappa constant domains, respectively. 293E cells co-transfected with both the heavy and light chain vectors were cultured for 7 days, then the supernatants were harvested and purified by Protein A chromatography.

[0150] The purified chimeric antibody was named EM1007-44c. The binding activity to human OX40 or cynomolgus monkey OX40 on the cell surface was evaluated by FACS. Briefly, 5 × 10 cells were 5 1 was seeded into each well of a 96-well plate (Corning, model number: 3799). The cells were centrifuged at 400g for 5 minutes and the supernatant was discarded. Then, 100 μl of a 3-fold serial dilution of the antibody starting from 100 nM was added to each well and mixed with the cells. After incubation at 4°C for 60 minutes, the plate was washed to remove excess antibody. Secondary Alexa Fluor® 647-conjugated goat anti-human IgG antibody (fresh dilution of 1:500, Jackson ImmunoResearch, model number: 109-606-098) was then added and incubated with the cells for 20 minutes at room temperature. After another centrifugation / washing round, the cells were resuspended in FACS buffer for reading on a CytoFLEX Flow Cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readouts were plotted versus antibody concentration and analyzed with GraphPad Prism 8.0.

[0151] The ability of OX40 to activate downstream signaling was detected in a Jurkat-OX40-NF-κB luciferase assay. Briefly, high-binding plates (Corning, model no. 3361) were coated overnight at 4° C. with 3-fold serial dilutions of EM1007-44c starting at 100 nM, washed, and then 1×10 per well of OX40-NF-κB reporter was added. 5 Cells were seeded and incubated for 6 hours at 37° C. At the end of the incubation, ONE-Glo™ Luminescence Assay Kit (Promega, Cat. No. E6130) reagents were prepared and added according to the manufacturer's instructions. Plates were read for luminescence signal on a Varioskan™ LUX microplate reader (ThermoFisher Scientific).

[0152] EM1007-44c was further evaluated for its ability to activate primary T cells and promote T cell proliferation. Briefly, primary T cell stimulation was measured in high binding plates (Corning, model no.: 3361) co-coated with 3-fold serial dilutions of EM1007-44c starting at 100 nM and 1 μg / ml OKT3 (Biolegend, model no.: 317326) by overnight incubation at 4°C. PD-L1+ T cells were purified from human PBMCs by a commercially available human T cell isolation kit (Stemcell Technologies, model no.: 17951) and 1 x 10 per well. 5 100 ul of cells were added to the freshly coated and PBS washed plates. The plates were incubated at 37° C. and 5% CO2 for 96 hours. For each well, 100 ul of supernatant was collected for IFN-γ quantification, and then 50 ul of CellTiter-Glo® Luminescent Cell Viability Assay (Promega, model number: G7570) mix was added per well and incubated at room temperature for 10 minutes for cell viability detection according to the manufacturer's instructions.

[0153] The data summarized in Table 2 confirm that EM1007-44c has similar binding activity to human and cynomolgus monkey OX40 on the cell surface and is capable of activating OX40 signaling and primary T cells.

[0154] [Table 2]

[0155] [Example 1.3] Humanization of EM1007-44c The variable region genes of EM1007-mAb044c presented in Table 1 were used to create a humanized antibody. First, the amino acid sequences of the VH and VK (VL kappa) domains of EM1007-mAb044c were compared against the human Ig V gene sequences available from the V BASE database (https: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php) to find the human germline Ig V gene sequence with the best overall match. The VH and VK framework segments were also compared against the FR sequences in the J region sequences available in V BASE to find the human frameworks with the highest degree of homology to the mouse VH and VK regions, respectively. For the light chain, the closest human V gene match was the O1 gene, and for the heavy chain, the closest human V gene match was the VH1-69 gene. Humanized variable domain sequences were then designed such that CDR-L1, CDR-L2, and CDR-L3 of the EM1007-044c light chain were grafted into the framework sequences of the O1 gene and the JK2 framework 4 sequence, while CDR-H1, CDR-H2, and CDR-H3 of the EM1007-044c heavy chain were grafted into the framework sequences of VH1-69 and the JH1 framework 4 sequence.

[0156] Meanwhile, a three-dimensional Fv model of EM1007-mAb044c was generated to identify any framework positions where mouse amino acids are crucial to support loop structure or the VH / VK interface. The corresponding residues in the human framework sequences should be backmutated to mouse residues at such identified positions to retain affinity / activity. As shown in Table 3 below, several backmutations desired in the VH and VK of EM1007-mAb044c were shown, and alternative designs of VH and VK were constructed. Because the "NG" (Asn-Gly) pattern found in CDR-H2 and CDR-L1 of EM1007-mAb044c is susceptible to deamination reactions and may result in heterogeneity during production, VH and VL domains containing NG (Asn-Gly) to NA (Asn-Ala) mutations (highlighted in bold italics), designated, for example, "EM1007-mAb044VH(GA)" and "EM1007-mAb044VK(GA)", were also designed and evaluated.

[0157] [Table 3] TIFF2024523838000013.tif42168

[0158] The humanized VH and VK (VL kappa) genes were synthesized and then cloned into vectors (sequences shown below) containing the human IgG1 heavy chain constant domain with the LALA mutation, and the human kappa light chain constant domain, respectively.

[0159] Amino acid sequence of human IgG1 heavy chain constant domain with LALA mutation (SEQ ID NO:42): TIFF2024523838000014.tif30170

[0160] Amino acid sequence of the human kappa light chain constant domain (SEQ ID NO:43): TIFF2024523838000015.tif11170

[0161] Pairing of the humanized VH chain with the humanized VK chain created 10 humanized antibodies named "HuEM1007-044-1" to "HuEM1007-044-8," "HuEM1007-044-14," and "HuEM1007-044-16," as shown in Table 4 below, along with chimeric antibodies named "EM1007-mAb044c-9" to "EM1007-mAb044c-13," "EM1007-mAb044c-15," and "EM1007-mAb044c-17," due to the potential impact on evaluation due to GA mutations in CDR-H2 and CDR-L1.

[0162] [Table 4]

[0163] All 17 antibodies in Table 4 were expressed by transient transfection of HEK293 cells, purified by Protein A chromatography, and the dissociation rate constants (k off) and ranked accordingly. Briefly, the binding affinity and kinetics of the antibodies were characterized by Octet® RED96 biolayer interferometry (Pall ForteBio LLC). The antibodies were captured by Anti-hIgG Fc Capture (AHC) Biosensors (Pall) at a concentration of 100 nM for 120 seconds. After this, the sensors were immersed in running buffer (1× pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to check the baseline, then immersed in recombinant human OX40 / His fusion protein (Novoprotein, CB17) at the assigned concentration for 200 seconds to measure binding, followed by immersion in running buffer for 600 seconds for dissociation. The assay was carried out in four test groups (listed in Table 5), all containing EM1007-044c chimeric antibody as a basis for normalization. Using ForteBio Data Analysis software (Pall), the association and dissociation curves were fitted to a 1:1 Langmuir binding model to obtain the off-rate constants shown in Table 5 below. The off-rate of each antibody was compared with the off-rate of EM1007-mAb044c chimeric antibody in the same test group obtained in parallel to obtain the corresponding off-rate ratio, which was used as the normalization index. The normalization index of the antibody indicates high affinity for human OX40.

[0164] [Table 5]

[0165] The off-rate of EM1007-mAb044c-11 is similar to that of EM1007-mAb044c, suggesting that the concomitant mutations of NG (Asn-Gly) to NA (Asn-Ala) in the former's VH and VL do not impair binding affinity. Therefore, the humanized design, HuEM1007-044-16, is used for the bispecific molecule construction, as it best retains affinity while also containing both the NG to NA mutations.

[0166] [Example 1.4] Characterization of anti-OX40 antibodies [Example 1.4.1] Epitope Identification Four cysteine-rich domains (CRDs) derived from the OX40 extracellular domain were identified from UniProt (identification number: P43489), and the full-length form of extracellular OX40 (CRD1-4) and the truncated OX40 mutants, ΔCRD1 (lacking CRD1), ΔCRD1-2 (lacking CRD1 and CRD2), ΔCRD1-3 (lacking CRD1, CRD2, and CRD3), mCRD1 (CRD1-4 in which the CRD1 domain is replaced by mouse CRD1), mCRD2 (CRD1-4 in which the CRD2 domain is replaced by mouse CRD2), mCRD3 (CRD1-4 in which the CRD3 domain is replaced by mouse CRD3), and mCRD4 (CRD1-4 in which the CRD4 domain is replaced by mouse CRD4) were synthesized by Biointron. The binding of HuEM1007-044-16, OX40-Tab1 (WO2015153513), OX40-Tab2 (WO2020151761) to OX40 or OX40 truncated proteins was analyzed using ELISA to identify the relevant binding epitopes. Briefly, 96-well plates (Corning, model number: 3361) were coated with each of the OX40 variants at 1ug / ml, incubated overnight at 4°C, washed with PBS containing 0.05% Tween20, and blocked with blocking buffer (PBS containing 0.05% Tween20 and 2% BSA) for 2 hours at 37°C. Once the plates were coated and blocked, serially diluted antibodies were added, incubated at 37°C for 1 hour, washed three times, and then HRP-labeled secondary antibodies were added. For color development, a chromophore solution of tetramethylbenzidine (TMB) was added for 5 min, and then the reaction was quenched with 1 M HCl. The optical density at 450 nm (OD450) was measured on a microplate reader.Figure 1a shows the results of the ELISA assay described above, suggesting that OX40-mAb targets the CRD3 domain, while OX40-Tab1 and OX40-Tab2 target a conformational epitope and the CRD1 domain or a conformational epitope containing the CRD1 domain, respectively. Another ELISA assay was performed similarly, except using OX40 mutants in which the designated CRD domains were replaced with their corresponding mouse counterparts, and the results shown in Figure 1b further suggest that the human CRD1 domain, human CRD2 domain, and human CRD4 domain are crucial for binding to Tab2.

[0167] [Example 1.4.2] Selective expansion of effector T cells The agonistic effect of anti-OX40 antibody was measured by differentiation of Treg from naive CD4 cells. Briefly, naive CD4 T cells isolated using a commercially available kit (Stemcells, model number: 17555) were cultured at 5 × 10 per well. 6 Cells were seeded into 6-well plates pre-coated with 2μg / ml OKT3 and 30nM HuEM1007-044-16. After 5 days of incubation supplemented with 2μg / ml CD28 (Biolegend, Cat. No.: 302934) and 5ng / ml TGF-beta, FACS analysis was used to evaluate Treg cell populations defined by CD25+ Foxp3+ T cell populations and non-Treg T cell populations. Figure 2 shows that treatment with HuEM1007-044-16 induced selective proliferation of effector T cells over Treg cells and reduced Treg polarization even at a concentration of 30nM.

[0168] [Example 1.4.3] OX40 agonist mAb internalization assay Briefly, 5 × 10 CHO cells overexpressing human OX40 (CHO-hOX40) were cultured in vitro. 5Cells were seeded into each well of a 96-well plate (Corning, model number: 3799) and subjected to various concentrations of HuEM1007-044-16 treatment with or without internalization inhibitor. After incubation at 37°C for 60 minutes, the plate was washed multiple times to remove excess antibody, and then added with secondary Alexa Fluor® 647-conjugated goat anti-human IgG antibody (freshly diluted 1:500, Jackson ImmunoResearch, model number: 109-606-098) and incubated at room temperature for 20 minutes. After another centrifugation / washing round, cells were resuspended in FACS buffer for reading on a CytoFLEX Flow Cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readouts can be plotted against antibody concentration and analyzed by GraphPad Prism 8.0.

[0169] [Example 2] Generation and characterization of anti-PD-L1 antibodies The anti-PD-L1 antibody EM0005-mAb86 was obtained as described in WO2021 / 104434. After expression in HEK293 cells and purification by Protein A chromatography, EM0005-mAb86 showed an aggregation percentage of over 10%, indicating challenges in CMC development of bispecific molecules utilizing antibodies as themselves or as binding domains. For antibodies with good developability, the VH and VL sequences of EM0005-mAb86 (listed in Table 6) were utilized and framework sequence changes were introduced to alter the physicochemical properties of the full-length antibody, for example to alter the overall charge, disrupt hydrophobic patches, and / or increase hydrophilicity, with minimal or no impact on its biological activity.

[0170] [Table 6]

[0171] Briefly, the humanized variable domain sequences of EM0005-mAb86 were designed such that its CDR-L1, CDR-L2, and CDR-L3 (for the VL of EM0005-mAb86, presented in Table 6) were derived from a V BASE and grafted onto diverse germline gene framework sequences with a JK4 framework 4 sequence after CDR-L3, and its CDR-H1, CDR-H2, and CDR-H3 (for the VH of EM0005-mAb86, presented in Table 6) were derived from a V BASE and grafted onto diverse VH framework sequences with a JH6 framework 4 sequence after CDR-H3.

[0172] The designed VH and VK (VL kappa) genes were synthesized and then cloned into a vector containing a human IgG1 heavy chain constant domain and a human kappa light chain constant domain, respectively (sequences presented in Example 1.3). Pairing of the humanized VH and VK chains created 50 humanized antibodies, 49 of which were named "HuEM0005-86-15" through "HuEM0005-86-63", along with HuEM0005-86-64, which was designed to have the same sequence as HuEM0005-86-21, except for the mutations Q (Gln) to E (Glu) at position 1 and C (Cys) to S (Ser) at position 82a (Kabat numbering). An additional chimeric mutant, EM0005-86c-1, was designed to evaluate the impact on the binding properties of the antibody by mutating "NG" (Asn-Gly) to "NA" (Asn-Ala), which has a G55A mutation in CDR-H2 of "EM0005-86c" and is hypothesized to be desirable to avoid "NG" (Asn-Gly) in CDR-H2 of EM0005-mAb86, a pattern that is susceptible to deamination reactions and may result in heterogeneity during manufacturing.

[0173] All antibodies were transiently expressed in HEK293, purified by one-step Protein A purification, and assessed for expression titer and purity by SEC-HPLC. Since the impurities of purified antibodies are mainly aggregated fractions, high purity indicates low aggregation tendency of the corresponding antibody.

[0174] As shown in Table 7, 10 of the 50 humanized antibodies were selected based on potency and purity, and the dissociation rate constants (k off ) were further assayed. EM0005-86c, a chimeric antibody with identical VH / VL sequences to EM0005-mAb86 (presented in Table 6), was used as a positive control in each group and was used as a basis for normalization. Briefly, antibodies were characterized for affinity and binding kinetics by Octet® RED96 biolayer interferometry (Pall ForteBio LLC). Anti-hIgG Fc Capture (AHC) Biosensors (Pall) captured antibodies at a concentration of 100 nM for 120 seconds, immersed in running buffer (1x pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to establish a baseline, then immersed in a single concentration of recombinant human PD-L1 / His fusion protein (Novoprotein, product code: C315) to measure binding for 200 seconds, followed by immersion in running buffer to measure dissociation for 600 seconds. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis software (Pall). The off-rate ratios shown in Table 7 were calculated by the off-rates of the humanized antibodies relative to the off-rates of EM0005-86c in the same test group. The off-rate ratio is used as a normalization index so that humanized antibodies can be compared to each other across study groups. A low off-rate ratio indicates high affinity of the antibody for human PD-L1. HuEM0005-86-21 was selected for further exploration based on purity and off-rate data.

[0175] [Table 7]

[0176] Based on HuEM0005-86-21, HuEM0005-86-64 was further designed to include the C82aS mutation and used for FIT-Ig construction. The sequence of HuEM0005-86-64 is shown in Table 8.

[0177] [Table 8]

[0178] [Example 3] Generation and characterization of PDL1 / OX40 FIT-Ig [Example 3.1] Construction of PDL1 / OX40 FIT-Ig, FIT1014-20a The coding sequences for the immunoglobulin domains of the parent antibodies HuEM0005-86-64 (humanized anti-PD-L1, see Table 8) and HuEM1007-44-16 (humanized anti-OX40, see Tables 3 and 4) were utilized to construct a PD-L1 / OX40 FIT-Ig, designated FIT1014-20a. The FIT-Ig, FIT1014-20a, is a hexamer composed of three polypeptide chain components. Polypeptide chain 1 has the domain formula: VL-CL of HuEM0005-86-64 fused directly to VH-CH1 of HuEM1007-44-16 fused directly to the Fc of a mutant human IgG1 constant domain; they have a triple mutation M252Y / S254T / T256E ("YTE", EU numbering) in the CH2 domain that causes an approximately 10-fold increase in binding to the human neonatal Fc receptor (FcRn), which may extend the serum half-life of FIT1014-20a. Polypeptide chain 2 has a VH-CH1 of domain formula: HuEM0005-86-64; Polypeptide chain 3 has the light chain (VL-CL) of the domain formula: HuEM1007-44-16. The amino acid sequences of the three polypeptide chains of expressed FIT1014-20a are shown in Table 9 below.

[0179] [Table 9]

[0180] DNA molecules encoding the amino acid sequences of each of the three polypeptide chain components were synthesized and cloned into the mammalian expression vector pcDNA3.1. HEK293E cells were co-transfected with the three recombinant pcDNA3.1 expression vectors to express each of the three polypeptide chain components. Approximately six days after transfection, cell culture supernatants were harvested and subjected to Protein A affinity chromatography to obtain purified PD-L1 / OX40 FIT-Ig bispecific binding protein.

[0181] [Example 3.2] Binding activity of PDL1 / OX40 FIT-Ig FACS binding method The binding affinity of PD-L1 / OX40 antibodies to CHO cells overexpressing human PD-L1 (ATCC, #CCL-61) (CHO-PD-L1) and CHO cells overexpressing OX40 (CHO-OX40) was measured. 51 was seeded into each well of a 96-well plate (Corning, model number: 3799). The cells were centrifuged at 400g for 5 minutes and the supernatant was discarded. Then, 100 μl of a 3-fold serial dilution of the antibody starting from 100 nM was added to each well and mixed with the cells. After incubation at 4°C for 60 minutes, the plate was washed multiple times to remove excess antibody. Secondary Alexa Fluor® 647-conjugated goat anti-human IgG antibody (fresh dilution of 1:500, Jackson ImmunoResearch, model number: 109-606-098) was then added and incubated with the cells for 20 minutes at room temperature. After another centrifugation / washing round, the cells were resuspended in FACS buffer for reading on a CytoFLEX Flow Cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readouts were plotted versus antibody concentration and analyzed with GraphPad Prism 8.0.

[0182] As shown in Figure 3, the binding affinity of FIT-1014-20a to CHO-PD-L1 was similar to that of its parent anti-PD-L1 monoclonal antibody (HuEM0005-86-64), and the negative control unrelated human IgG showed no binding.

[0183] FACS affinity results for binding to human OX40 transfected CHO cells, shown in FIG. 4, indicate that FIT1014-20a has a relatively lower binding affinity (EC50 of 4.3 nM vs. 1.8 nM) than that of its parent OX40 antibody HuEM1007-44-16.

[0184] Affinity for PD-L1 and OX40 The binding activity of FIT1014-20a to PD-L1 was detected by biolayer interferometry using an Octet® Red sensing device (ForteBio, Red96). FIT1014-20a (YTE) was captured at a concentration of 100 nM for 30 seconds on Anti-hIgG Fc Capture (AHC) Biosensors (Pall) which were immersed in running buffer (1× pH 7.2 PBS, 0.05% Tween 20, 0.1% BSA) for 60 seconds to establish a baseline, and then immersed in serial dilutions of recombinant human PD-L1-his protein or recombinant cynomolgus PD-L1-his protein (100 nM, 33.3 nM, 11.1 nM, 3.7 nM) to measure association for 200 seconds, followed by immersion in running buffer to measure dissociation for 1200 seconds. The reaction rate constant, K, was calculated using ForteBio Data Analysis software (Pall). on and K off The association and dissociation curves were fitted to a 1:1 Langmuir binding model to determine the equilibrium dissociation constant, KD(M), for the reaction between the antibody and the relevant target protein, as follows: KD=k off / k on The affinity for OX40 was also detected similarly, except that human OX40 or cynomolgus OX40 (300 nM, 100 nM, 33.3 nM, 11.1 nM) was used instead of PD-L1-his protein. The results are shown in Table 10 below.

[0185] [Table 10]

[0186] [Example 4] PD-L1 blockade [Example 4.1] Blockade of PD-1 / PD-L1 binding Blockade of PD-1 / PD-L1 binding was assessed by cell-based receptor blocking assay (RBA) using 2 × 10 cells per well. 5 100μl of CHO-PD-L1 cells were added to a 96-well round-bottom plate (Corning, model no. 3799), 50μl of serially diluted antibodies from 0.016nM to 50nM, and 50μl of 50μg / ml PD-1-mFc (Novoprotein, model no. C754) were added to each well, mixed gently, and then incubated at 4℃ for 1 hour. Cells were washed and stained with Alexa Fluor® 647 anti-mouse IgG (1:500, Jackson ImmunoResearch, model no. 115-606-008). Signals were read by FACS and curves were fitted by GraphPad Prism 8.0. As shown in Figure 5, FIT1014-20a demonstrated similar potency in blocking PD-1 protein binding to PD-L1 overexpressing cells as its parent anti-PD-L1 antibody, HuEM0005-86-64.

[0187] [Example 4.2] Blockade of PD-L1-mediated inhibitory signaling Blockade of inhibitory signaling by PD-L1 was examined by co-culture of CHO-PD-L1-OS8 (disclosed in US8735553, stably transduced with PD-L1 using OS8 as a T cell activation molecule) with Jurkat-PD-1-NFAT-luciferase reporter cell line expressing both human PD-1 and a luciferase reporter driven by NFAT response element. Briefly, logarithmic growth phase CHO-PD-L1-OS8 were harvested, washed, resuspended in assay medium (RPMI1640 with 10% FBS) and 1x10 cells / well were cultured. 5 50 μl of cells were seeded into a 96-well plate (Corning, model number: 3799) and then 1 × 10 cells were plated per well. 550μl of Jurkat-PD-1-NFAT-luciferase reporter cells were added. 50ul of serially diluted sample antibodies were added and incubated with the cell mixture at 37℃ for 6 hours. After incubation, ONE-Glo™ Luminescence Assay Kit (Promega, model number: E6130) reagents were prepared and added according to the manufacturer's instructions. The plate was read for luminescence signal on a Varioskan™ LUX microplate reader (ThermoFisher Scientific). As shown in Figure 6, FIT1014-20a demonstrated similar efficacy as its parent anti-PD-L1 antibody, HuEM0005-86-64, in blocking PD-L1-mediated PD-1 downstream signaling.

[0188] [Example 5] PD-L1-dependent activation of OX40 downstream signaling The ability to induce PD-L1-dependent activation of OX40 downstream signaling was assessed by co-culturing CHO-PD-L1 with a Jurkat-OX40-NFκB-luciferase reporter cell line and examining activation of OX40 via cross-linking of PD-L1. 4 100 μl of PD-L1 expressing CHO cells and 1 × 10 cells per well 5100 μl of OX40-expressing NFκB-luciferase reporter cell lines were co-seeded into 96-well plates (Corning, model number: 3799) and incubated with 50 μl of serially diluted antibodies for 6 hours at 37°C. At the end of the incubation, ONE-Glo™ Luminescence Assay Kit (Promega, model number: E6130) reagents were prepared and added according to the manufacturer's instructions. Plates were read for luminescence signal on a Varioskan™ LUX microplate reader (ThermoFisher Scientific). As shown in the figure, FIT1014-20a induced activation of OX40 downstream NF-K signaling pathway in the presence of PD-L1 positive cells in a dose-dependent manner (Figure 7, top), whereas the parental mAb combination did not. Parallel assays using the same antibody and reporter cell line but with PD-L1 negative CHO cells were performed as a control, and as shown in Figure 7 (bottom), the lack of activation indicates that FIT1014-20a-induced activation is PD-L1 dependent.

[0189] [Example 6] T cell activation [Example 6.1] IFN-γ and IL2 production by primary T cells T cell activation was measured by IFN-γ and IL2 production in co-cultures of CHO-PD-L1-OS8 cells with primary human T cells. Briefly, CHO-PD-L1-OS8 cells were harvested, washed, and cultured at 4 x 10 per ml in assay medium (RPMI1640 with 10% FBS). 5 The cells were resuspended to 10 cells and 100 μl of cells were added to a 96-well plate (Corning, model number: 3799). T cells were purified from human PBMCs using a commercially available human T cell isolation kit (Stemcell Technologies, model number: 17951) and were diluted to 4 × 10 cells per ml. 5100 μl of cells were added to the cell plate. Test antibodies and negative control irrelevant human IgG were added and incubated with the cell mixture for 48 hours at 37° C., and the supernatants were collected for measurement of IFN-γ production using a PerkinElmer IFN-γ detection kit (PerkinElmer, model number: TRF1217M). After 72 hours of incubation, additional supernatants were collected for measurement of IL-2 production using a PerkinElmer IL-2 detection kit (PerkinElmer, model number: TRF1221M). According to the production of IFN-γ and IL2 shown in FIG. 8, FIT1014-20a was able to activate T cells to produce both IFN-γ and IL2 in a dose-dependent manner compared to the monospecific parent antibody combination.

[0190] [Example 6.2] Mixed lymphocyte reaction (MLR) assay To determine whether the bispecific antibodies (BsAbs) described herein can synergistically stimulate T cells by simultaneous activation of OX40 and PD-L1 / PD-1 blockade, a mixed lymphocyte reaction (MLR) assay was set up to evaluate the effect on T cell activation as reported (Tourkva et al., 2001). Briefly, monocytes isolated from PBMCs by Monocyte Enrichment Kit (Stemcell, 19058) were maintained in medium (RPMI1640+10% FBS) supplemented with 50ng / ml GM-CSF (R&D, model number: 215-GM-050 / CF), 35ng / ml IL-4 (R&D, model number: 204-IL-050 / CF) for 6 days. Mature DCs were induced by incubation in medium supplemented with 20ng / ml TNF-a (R&D, #210-TA-005 / CF), 50μg / ml Poly I:C (Sigma, #I3036) and incubated for an additional 2 days at 37°C and 5% CO2. Allogeneic human CD4+ T cells were isolated from PBMCs by EasySep™ Human CD4+ T Enrichment Kit (Stemcell, model no: 17952). 1x10 per well 5100 μl of CD4+ T cells were seeded into a 96-well round-bottom plate (Corning, model number: 3799) at 1 × 10 per well. 5 100 μl of mature DCs were added to the plate and incubated with serially diluted antibodies. After 3 days, supernatants were harvested to detect IL-2 production as a readout of the MLR response. The IL2 levels derived from the MLR assay, shown in FIG. 9, suggest that FIT1014-20a is more potent in promoting IL-2 production than the combination of both parental antibodies.

[0191] [Example 6.3] Staphylococcal endotoxin B (SEB) assay To further evaluate the effect on T cell activation, a bacterial toxin stimulation assay was performed using the superantigen Staphylococcus aureus endotoxin B (SEB). Briefly, 100 μl of PBMCs from healthy human donors were plated into a 96-well assay plate at 2×10 per well. 5 Cells were seeded onto the plates, then 50μl of serial dilutions of the test antibodies were added and incubated with the PBMCs at 37℃ for 30 minutes. 50μl of SEB solution at a final concentration of 10ng / ml was added and the plates were further incubated for 96 hours. 100μl of cell culture supernatant was harvested for measurement of IL-2 using PerkinElmer IL-2 detection kit (PerkinElmer, model no: TRF1221M). FIT1014-20a is more potent than its parental anti-OX40 / PD-L1 antibody combination in enhancing T cell activation as shown by the IL-2 levels shown in Figure 10.

[0192] [Example 7] Searching for Fc that mediates effector functions [Example 7.1] Complement-dependent cytotoxicity against activated CD4+ cells Human CD4+ T cells were isolated from PBMCs using the EasySep™ Human CD4+ T Enrichment Kit (Stemcell, model number: 17952) and activated with T cell activator (Stemcell, model number: 10971) for 3 days. The activated CD4+ T cells were harvested at a concentration of 1 × 10 per ml. 6 The cells were diluted to 1000 cells, and 50 μL of the activated CD4+ T cell solution was added to a 96-well cell plate (Corning, model number: 3799), 50 μL of normal human serum complement (Quidel, model number: A113) was added to the cell plate, and then 50 μL of serially diluted antibodies were added along with irrelevant human IgG (as a negative control) or anti-HLA (AntibodyGenie, AGEL1612 as a positive control). After incubation at 37° C. and 5% CO2 for 6 hours, a cell cytotoxicity assay was performed using alamarBlue™ Cell Viability Reagent (Thermofisher, model number: DAL1100). As shown in FIG. 11, FIT1014-20a did not produce any cytotoxic effect on activated human CD4+ T cells, whereas the positive control anti-HLA showed a dose-dependent cytotoxic effect on activated human CD4+ T cells.

[0193] [Example 7.2] Phagocytic effect on CHO-OX40 Briefly, CD14+ monocytes were isolated from fresh PBMCs and differentiated into macrophages by incubation with 100 ng / ml M-CSF (R&D, 216-MC-010 / CF) for 6 days, then labeled with CellTrace™ Far Red (Thermo Fisher Scientific, C34564). 2×10 per well were plated into 96-well plates (Corning, 7007). 5 100 μL of CellTrace™ CFSE (Thermo Fisher Scientific, C34554) labeled CHO-OX40 cells, density 4 × 10 550 μL of Far red-labeled human macrophages and 50 μL of the assigned concentration of antibody were added and then incubated for 3 hours at 37° C. Phagocytosis was monitored by flow cytometry using the Far red + CFSE in cells + As shown in Figure 12, FIT1014-20a and HuEM1007-044-16 (parental OX40 mAb) produced little phagocytic effect, whereas its parent HuEM1007-044-16 with hIgG1 and reference antibody OX40-Tab2 showed some phagocytic effect.

[0194] [Example 8] Antitumor efficacy in MC38 colon tumor-bearing humanized PD-L1 / OX40 mouse model The antitumor efficacy of FIT1014-20a was investigated in a hPD-L1 / hOX40 transgenic syngeneic mouse model (Biocytogen, Beijing, China) harboring MC38 tumor cells expressing human PD-L1 (Shanghai Model Organisms Center Inc., Shanghai, China). PD-L1-expressing MC38 cells (5 × 10 cells) suspended in 0.1 ml of PBS were 6 ) were injected subcutaneously into the dorsal right flank of hPD-L1 / hOX40tg female mice. Five days later (day 0), mice were cultured at 100°C for 12 h after tumor volume (average 70 mm 3 Mice were randomly assigned into groups (n=6) based on the age of the subjects (defined as age group 1-4 years or older). Intraperitoneal injections with the test antibodies were administered on days 0, 3, 6, and 9. Tumor dimensions and body weights were measured twice weekly. The results in Figure 13 confirm that mice in the FIT1014-20a treatment group showed superior tumor growth inhibition compared to atezolizumab or parental PD-L1 mAb (HuEM0005-86-64) monotherapy.

[0195] [Example 9] Antitumor efficacy in a CT26 colon tumor-bearing humanized PD-L1 / PD-1 / OX40 mouse model The in vivo antitumor activity of FIT1014-20a after intraperitoneal administration was further explored against CT26-hPD-L1 syngeneic tumors established in human PD-1 / PD-L1 / OX40 knock-in mice (GemPharmatech, China). PD-L1-expressing CT26 cells (2.5 × 10 cells) suspended in 0.1 ml PBS were 6 ) were injected subcutaneously into the dorsal right flank of hPD-L1 / hPD-1 / hOX40 transgenic female mice. Five days later (day 0), mice were cultured to measure tumor volumes (average 80 mm 3 Mice were randomized into groups (n=8) based on the number of mice treated with FIT1014-20a and intraperitoneally injected with the test antibodies on days 0, 3, and 6. Tumor dimensions and body weights were measured twice weekly. The results in Figure 14 confirm that mice treated with FIT1014-20a showed superior tumor growth inhibition compared to monotherapy with the mAb atezolizumab.

[0196] TIFF2024523838000023.tif241161TIFF2024523838000024.tif246161TIFF2024523838000025.tif194161

[0197] References: Aspeslagh S. et al., Eur. J. Cancer. (2016). Rationale for anti-OX40 cancer immunotherapy. 52:50-66. Brahmer JR. et al., N. Engl. J. Med (2012). Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. 366(26):2455-65. Bulliard Y. et al., Immunol Cell Biol. (2014). OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy. 92(6):475-80. Choi Y. et al., J Immunother Cancer. (2020). T-cell agonists in cancer immunotherapy. Carolina Alves Costa Silva. et al., ESMO Open (2020). New pathways in immune stimulation:targeting OX40. 5(1):e000573. Croft M., Annu. Rev. Immunol.(2010). Control of immunity by the TNFR-related molecule OX40 (CD134). 28: 57-78. Curti BD. et al., Cancer Res. (2013). OX40 is a potent immune-stimulating target in late-stage cancer patients. 73(24):7189-7198 Gieffers C. et al., Mol Cancer Ther (2013). APG350 induces superior clustering of TRAIL receptors and shows therapeutic antitumor efficacy independent of cross-linking via Fcγ receptors. 12(12):2735-47. Glisson BS. et al., Clin Cancer Res. 2020. Safety and Clinical Activity of MEDI0562, a Humanized OX40 Agonist Monoclonal Antibody, in Adult Patients with Advanced Solid Tumors. 26(20):5358-5367. Gutierrez M. et al., Clin Cancer Res. (2021). OX40 Agonist BMS-986178 Alone or in Combination with Nivolumab and / or Ipilimumab in Patients with Advanced Solid Tumors. 27(2):460-472. Jacquemin C. et al., Immunity. (2015). OX40 Ligand Contributes to Human Lupus Pathogenesis by Promoting T Follicular Helper Response. 42(6):1159-70. Kawamata S. et al., J. Biol. Chem (1998). Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- andTRAF5-mediated NF-kappaB activation. 273 (10), 5808-5814. Mayes PA, Hance KW, Hoos A. Nat Rev Drug Discov. (2018). The promise and challenges of immune agonist antibody development in cancer. 17(7):509-527. Schaer DA. Et al., J. Immunother. Cancer (2014) Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy. 2, 7. Smyth MJ. et al., Immunol Cell Biol. (2014). Targeting regulatory T cells in tumor immunotherapy. 92(6):473-4. Song J. et al., Immunol (2008). Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival. 180 (11), 7240-7248. Tourkova IL. et al., Immunol Lett. (2001). Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay. 78(2):75-82. Watts TH. Annu. Rev. Immunol. (2005). TNF / TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol. 23, 23-68. Weinberg AD. et al., Immunol Rev. (2011). Science gone translational: the OX40 agonist story. 2011, 244:218-231 Willoughby J. et al., Mol Immunol (2017). Mar; 83:13-22. Zhang X, et al., Cell Rep. (2018). OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms. 24(3): 607-618.

Claims

1. An isolated antibody or an antigen-binding fragment thereof that specifically binds to OX40 and comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises the sequence of SSWMN (SEQ ID NO: 1), CDR-H2 comprises the sequence of RIYPGDEITNYNAKFKD (SEQ ID NO: 4) or RIYPGDEITNYNGKFKD (SEQ ID NO: 2), CDR-H3 comprises the sequence of DLLMPY (SEQ ID NO: 3), CDR-L1 comprises the sequence of RSSKSLLYSNAITYLY (SEQ ID NO: 8) or RSSKSLLYSNGITYLY (SEQ ID NO: 5), CDR-L2 comprises the sequence of QMSNLAP (SEQ ID NO: 6), CDR-L3 comprises the sequence of AQNLELPFT (SEQ ID NO: 7), optionally, the CDRs are defined according to Kabat numbering, the isolated antibody or an antigen-binding fragment thereof.

2. The antibody comprises VH, which is a heavy chain variable domain, and VL, which is a light chain variable domain, the VH domain comprises a sequence selected from any one of SEQ ID NOs: 16 and 11 - 15, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NOs: 21, 19 and 20, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, or The VH domain comprises the sequence of SEQ ID NO: 9 or 10, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 17 or 18, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. The isolated antibody or antigen-binding fragment according to claim 1.

3. The antibody is a chimeric antibody or a humanized antibody, optionally a humanized antibody. Optionally, the VH domain of the antibody comprises amino acid residues 1E according to Kabat numbering and 1 to 10 residues, for example 10 residues, selected from 5Q, 27H, 28A, 38K, 40R, 43K, 48I, 67K, 68A, 70L, and the VL domain comprises amino acid residue 69G or 69S, for example 69S according to Kabat numbering. The isolated antibody or antigen-binding fragment according to claim 1.

4. The antibody comprises a combination of a VH sequence and a VL sequence selected from the group consisting of optionally, a VH domain comprising the sequence of SEQ ID NO: 16 and a VL domain comprising the sequence of SEQ ID NO:

21. The isolated antibody or antigen-binding fragment according to claim 1.

5. The antibody has the following characteristics: (i) When binding to the cell surface of OX40-expressing cells (e.g., OX40-expressing T cells), it shows strong binding affinity to OX40+ cells, and the binding affinity to said cells is reflected by an EC50 of about 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less as measured by flow cytometry in a cell-based assay; (ii) It binds to human OX40 in CRD3 of the extracellular domain of OX40. (iii) The binding of the antibody to OX40 induces anti-tumor immunity of T cells, for example, reduction of tumor mass / tumor growth / tumor cell expansion, and optionally, said anti-tumor immunity includes anti-tumor cytotoxic effects and secretion of anti-tumor cytokines The isolated antibody or antigen-binding fragment according to claim 1, having one or more of the following.

6. The isolated antibody or antigen-binding fragment according to claim 1, wherein the antibody comprises an Fc region having the amino acid sequence of SEQ ID NO:

40.

7. A fusion or conjugate comprising the isolated antibody or antigen-binding fragment according to claim 1.

8. An isolated antibody or its antigen-binding fragment that contains a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and specifically binds to PD-L1, wherein CDR-H1 contains the sequence of TYGIN (SEQ ID NO: 22), CDR-H2 contains the sequence of YIYIGNAYTEYNEKFKG (SEQ ID NO: 23) or YIYIGNGYTEYNEKFKG (SEQ ID NO: 25); CDR-H3 contains the sequence of DLMVIAPKTMDY (SEQ ID NO: 24), CDR-L1 contains the sequence of KASQDVGTAVA (SEQ ID NO: 26), CDR-L2 contains the sequence of WASTRHT (SEQ ID NO: 27), CDR-L3 contains the sequence of QQYSSYPYT (SEQ ID NO: 28), Optionally, the CDRs are defined according to Kabat numbering, The isolated antibody or its antigen-binding fragment.

9. The antibody comprises a VH that is a heavy chain variable domain and a VL that is a light chain variable domain, The VH domain comprises a sequence selected from any one of SEQ ID NO: 31 or 30, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NO: 34 or 33, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Or The VH domain comprises the sequence of SEQ ID NO: 29, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 32, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. The isolated antibody or antigen-binding fragment according to claim 8.

10. The isolated antibody or antigen-binding fragment according to claim 8, wherein the antibody is a chimeric antibody or a humanized antibody, optionally a humanized antibody.

11. The isolated antibody or antigen-binding fragment according to claim 8, wherein the antibody comprises an Fc region having the amino acid sequence of SEQ ID NO:

40.

12. A fusion or conjugate comprising the isolated antibody or antigen-binding fragment according to claim 8.

13. A nucleic acid molecule encoding the isolated antibody or antigen-binding fragment according to claim 1.

14. A nucleic acid molecule encoding the isolated antibody or antigen-binding fragment according to claim 8.

15. A vector comprising the nucleic acid molecule according to claim 13.

16. A vector comprising the nucleic acid molecule according to claim 14.

17. A host cell that expresses a nucleic acid molecule encoding the isolated antibody or antigen-binding fragment according to claim 1.

18. A host cell that expresses a nucleic acid molecule encoding the isolated antibody or antigen-binding fragment according to claim 8.

19. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment according to any one of claims 1 and 8, the fusion or conjugate according to claim 7 or 12, the nucleic acid molecule according to claim 13 or 14, the vector according to claim 15 or 16, or the host cell according to claim 17 or 18.

20. A method for detecting OX40 in a biological sample, the method comprising contacting the biological sample with the isolated antibody or antigen-binding fragment according to claim 1, or the fusion or conjugate according to claim 7.

21. A method for detecting PD-L1 in a biological sample, the method comprising contacting the biological sample with the isolated antibody or antigen-binding fragment according to claim 8, or the fusion or conjugate according to claim 12.

22. A bispecific binding protein that specifically binds to OX40 and PD-L1, comprising a first antigen-binding site that specifically binds to OX40 and a second antigen-binding site that specifically binds to PD-L1, wherein the first antigen-binding site comprises a set of 6 CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, CDR-H1 comprises the sequence of SSWMN (SEQ ID NO: 1), CDR-H2 comprises the sequence of RIYPGDEITNYNAKFKD (SEQ ID NO: 4) or RIYPGDEITNYNGKFKD (SEQ ID NO: 2), CDR-H3 comprises the sequence of DLLMPY (SEQ ID NO: 3), CDR-L1 comprises the sequence of RSSKSLLYSNAITYLY (SEQ ID NO: 8) or RSSKSLLYSNGITYLY (SEQ ID NO: 5), CDR-L2 contains the sequence of QMSNLAP (SEQ ID NO: 6), CDR-L3 contains the sequence of AQNLELPFT (SEQ ID NO: 7), Optionally, the first antigen-binding site comprises the VH domain and the VL domain according to any one of claims 2 to 4; And / or the second antigen-binding site comprises a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, which are six CDRs, CDR-H1 contains the sequence of TYGIN (SEQ ID NO: 22), CDR-H2 contains the sequence of YIYIGNAYTEYNEKFKG (SEQ ID NO: 23) or YIYIGNGYTEYNEKFKG (SEQ ID NO: 25), CDR-H3 contains the sequence of DLMVIAPKTMDY (SEQ ID NO: 24), CDR-L1 contains the sequence of KASQDVGTAVA (SEQ ID NO: 26), CDR-L2 contains the sequence of WASTRHT (SEQ ID NO: 27), CDR-L3 contains the sequence of QQYSSYPYT (SEQ ID NO: 28), Optionally, the second antigen-binding site comprises a VH domain containing the sequence of SEQ ID NO: 31, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or a VL domain containing the sequence of SEQ ID NO: 34, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, The CDR is a bispecific binding protein defined according to Kabat numbering.

23. It comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, (i) The first polypeptide chain, from the amino terminus to the carboxyl terminus, has VL directly fused to VH B to CL A -CL-VH B-CH1-Fc, or VH directly fused to VL via CH1 A to form -CH1-VL B -CH1-VL A -CL-Fc; the second polypeptide chain contains VH from the amino terminus to the carboxyl terminus A -CH1; the third polypeptide chain contains VL from the amino terminus to the carboxyl terminus B -CL; or (ii) The first polypeptide chain contains VH directly fused to VL via CH1 from the amino terminus to the carboxyl terminus B to form -CH1-VL A -CH1-VL B -CL-Fc, or VL directly fused to VH via CL A to form -CL-VH B -CL-VH A -CH1-Fc; the second polypeptide chain contains VH from the amino terminus to the carboxyl terminus B -CH1; the third polypeptide chain contains VL from the amino terminus to the carboxyl terminus A -CL; VL is the light chain variable domain, CL is the light chain constant domain, VH is the heavy chain variable domain, CH1 is the heavy chain constant domain, and Fc is the immunoglobulin Fc region, for example, the Fc of IgG1 (optionally including hinge-CH2-CH3 from the amino terminus to the carboxyl terminus), VL A -CL pairs with VH A -CH1 to form a first Fab that specifically binds to a first antigen A, and VL B -CL pairs with VH B -CH1 to form a second Fab that specifically binds to a second antigen B, The first antigen A is OX40 and the second antigen B is PD-L1, Two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains associate to form a FIT-Ig protein, The bispecific binding protein according to claim 22.

24. The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, The second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, The third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, The bispecific binding protein according to claim 22.

25. The bispecific binding protein has the following characteristics: (i) When binding to the cell surface of OX40-expressing cells (e.g., OX40-expressing T cells), it shows strong binding affinity to OX40+ cells, and the binding affinity to said cells is reflected by an EC50 of about 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less as measured by flow cytometry in a cell-based assay; (ii) It binds to human OX40 in CRD3 of the extracellular domain of OX40; (iii) The binding of the binding protein to OX40 induces the promotion of anti-tumor immunity of T cells, e.g., reduction of tumor mass / tumor growth / tumor cell expansion, and optionally, said anti-tumor immunity includes anti-tumor cell cytotoxicity and / or secretion of anti-tumor cytokines having one or more of: The bispecific binding protein according to claim 22.

26. A nucleic acid molecule encoding the bispecific binding protein according to claim 22.

27. A vector comprising the nucleic acid molecule according to claim 26.

28. A host cell comprising the nucleic acid molecule according to claim 26. **Claim 29** A method for preparing the isolated antibody or antigen-binding fragment according to any one of claims 1 and 8, or the bispecific binding protein according to claim 22, comprising: culturing the host cell according to claim 17 or 18 or claim 28 under conditions that allow the production of an antibody, an antigen-binding fragment, or a bispecific binding protein; and recovering the antibody, antigen-binding fragment, or bispecific binding protein from the culture. A method comprising the above steps. **Claim 30** A pharmaceutical composition comprising the bispecific binding protein according to claim 22, the nucleic acid according to claim 26, the vector according to claim 27, or the host cell according to claim 28. **Claim 31** The pharmaceutical composition according to claim 19 for use in the treatment of a disorder in which the activity associated with PD-L1 is harmful and / or a disorder in which the activity mediated by OX40 is beneficial. **Claim 32** The pharmaceutical composition according to claim 30 for use in the treatment of a disorder in which the activity associated with PD-L1 is harmful and / or a disorder in which the activity mediated by OX40 is beneficial. **Claim 33** The pharmaceutical composition according to claim 31, wherein the disorder is cancer. **Claim 34** The pharmaceutical composition according to claim 32, wherein the disorder is cancer.

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