Multi-specific binding agents targeting dopamine receptor D2 and methods of use thereof

Abstract

The present invention generally relates to binding agents capable of targeting tumor cells and immune cells. The binding agents of the present invention are multispecific and comprise antigen binding domains capable of binding to dopamine receptor D2 (DR2), binding to programmed cell death protein 1 (PD-1), and / or binding to cluster of differentiation 47 (CD47). The multispecific binding agents of the present invention are useful for treating an individual in need thereof.

Description

Technical Field

[0001] The present invention generally relates to binding agents capable of targeting tumor cells and immune cells. The binding agents of the present invention are multispecific and comprise antigen-binding domains capable of binding dopamine receptor D2 (DR2), binding programmed cell death protein 1 (PD-1) and / or binding cluster of differentiation 47 (CD47). The multispecific binding agents of the present invention can be used to treat an individual in need thereof. Background Art

[0002] Camels and cartilaginous fish naturally produce antibodies consisting of only functional homodimeric heavy-chain antibodies (HCAbs) (Hamers-Casterman et al., 1993; Muyldermans and Smider, 2016). The heavy chain of HCAbs lacks the first constant domain (CH1) and differs from classical antibodies only by some amino acid substitutions usually involved in light-chain pairing (Muyldermans et al., 1994; Vu et al., 1997). Such substitutions (Val37Phe / Tyr, Gly44Glu, Leu45Arg and Trp47Gly) are present in framework region 2 (FR2). The antigen-binding fragment of HCAbs is called VHH or The molecular weight of VHH is about 15 kDa, which makes them suitable for applications requiring enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic applications, it is generally necessary to increase the VHH half-life in order to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al., Antibodies 8(1), 1-22, 2019). Although some methods for increasing the VHH half-life have been employed, such as PEGylation, N-glycosylation, HSA or other carrier protein fusions, such constructs may cause immunogenicity or have limited success rates.

[0003] VHHs have been used as building blocks to prepare bispecific and multispecific antibodies. In some studies, bivalent constructs showed increased avidity or affinity compared to the monovalent form (Conrath et al., 2001; Coppieters et al., 2006; Hmila et al., 2008; Simmons et al., 2006 and Hultberg et al., 2011, et al. (2010), Fridy et al., 2014).

[0004] Multiple VHH-based therapeutic agents are currently in late-stage research or have been approved by the FDA. Such therapeutic agents include the bivalent monospecific antibody caplacizumab against the antigen vWF, which is approved for thrombotic thrombocytopenic purpura (Duggan, 2018). The trivalent nanobody complex ALX-0171 against RSV is in late-stage development for respiratory syncytial virus infection (Detallea et al., 2015). ALX-0061 is a monovalent drug against the antigen IL-6R but is attached to an HSA nanobody to extend its half-life and is in clinical development for RA and SLE indications (Van Roy et al., 2015). The investigational drug ALX-0761 contains three nanobodies against the antigens IL-17A, IL-17F, and HAS and is being developed for psoriasis (Svecova et al., 2019). Anti-RANKL, i.e., ALX-0141, is a bivalent drug against the antigen RANKL and is attached to HSA to extend its half-life (Schoen et al., 2013). Ozoralizumab is a bivalent nanobody against the antigen TNFα and is attached to HSA to extend its half-life (Fleischmann et al., 2012).

[0005] The dopamine receptor family contains five G protein-coupled receptors (GPCRs), namely dopamine receptor D1 (DRD1), dopamine receptor D2 (DRD2), dopamine receptor D3 (DRD3), dopamine receptor D4 (DRD4), and dopamine receptor D5 (DRD5). Based on the G protein that mediates intracellular signal transduction, it can be divided into the D1-like family of receptors, including DRD1 and DRD5; and the D2-like family of receptors, including DRD2, DRD3, and DRD5. Each dopamine receptor seems to show a specific expression pattern in different types of tumors. For example, DRD2 shows increased expression in glioblastoma tumor samples (Dolma S. et al., 2016; Prabhu V.V. et al. 2019). DRD2 is also upregulated in breast cancer, lung cancer, gastric cancer, acute myeloid leukemia (AML), and pancreatic cancer (Gholipour N. et al., 2018, Wu X.-Y. et al.,, 2018, Carl M. et al., 2021, Jandaghi P. et al., 2016).

[0006] The applicant has developed novel binders that target tumor cells and immune cells expressing DR2 for use in methods of treating an individual in need thereof. SUMMARY OF THE INVENTION

[0007] The present invention generally relates to binders capable of targeting tumor cells, immune cells, and the like.

[0008] Advantageously, the binder of the present invention reduces tumor growth and / or induces tumor regression in animal models.

[0009] Accordingly, the binder of the present invention can be used to treat an individual in need.

[0010] In some embodiments, the binder of the present invention can be used to induce tumor regression in an individual in need.

[0011] In some embodiments, the binder of the present invention can be used to delay cancer progression in an individual in need.

[0012] In some embodiments, the method of the present invention involves administering a binder to an individual suffering from cancer so as to promote the regression of one or more tumors or tumor lesions. Accordingly, administering the binder of the present invention can cause at least partial regression of one or more tumors or tumor lesions.

[0013] In other embodiments, the method of the present invention involves administering a binder to an individual suffering from cancer so as to reduce the size of one or more tumors or tumor lesions. Accordingly, administering the binder of the present invention can cause a reduction in the size of one or more tumors or tumor lesions.

[0014] In other embodiments, the method of the present invention involves administering a binder to an individual suffering from cancer so as to reduce the growth rate of one or more tumors or tumor lesions. Accordingly, administering the binder of the present invention can result in a slowdown in the growth of one or more tumors or tumor lesions.

[0015] In some embodiments, the binder of the present invention is administered to an individual suffering from a tumor expressing dopamine receptor D2 (DR2). In other embodiments, the binder of the present invention is administered to an individual suffering from a tumor expressing cluster of differentiation 47 (CD47).

[0016] In some embodiments, the binder of the present invention is multispecific and comprises at least one antigen-binding domain capable of binding dopamine receptor D2 (DR2), at least one antigen-binding domain capable of binding programmed cell death protein 1 (PD-1), and / or at least one antigen-binding domain capable of binding cluster of differentiation 47 (CD47). The antigen-binding domains of the binder can bind cells expressing DR2, bind cells expressing PD-1, and / or bind cells expressing CD47.

[0017] Accordingly, in some embodiments, the binder of the present invention is capable of binding dopamine receptor D2 (DR2), binding programmed cell death protein 1 (PD-1), and / or binding cluster of differentiation 47 (CD47). In other embodiments of the present invention, the binder can bind cells expressing DR2, cells expressing PD-1, and / or cells expressing CD47.

[0018] In an exemplary embodiment, the binder or antigen-binding domain of the present invention is capable of binding to a DR2 antigen comprising a sequence that is at least 80% identical to the human DR2 protein, binding to a PD-1 antigen comprising a sequence that is at least 80% identical to human PD-1, and / or binding to a CD47 antigen comprising a sequence that is at least 80% identical to human CD47.

[0019] In an exemplary embodiment, the binder or antigen-binding domain of the present invention is capable of binding to human DR2, binding to human PD-1, and / or binding to human CD47. In other embodiments of the present invention, the binder or antigen-binding domain may bind to human cells expressing DR2, bind to human cells expressing PD-1, and / or bind to human cells expressing CD47.

[0020] According to the present invention, the binder may further comprise an additional antigen-binding domain.

[0021] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2), at least one antigen-binding domain 2 (ABD2) that binds to an immune modulator, and at least one antigen-binding domain 3 (ABD3) that binds to an immune cell.

[0022] In some embodiments, the antigen-binding domain 2 (ABD2) is an antigen-binding domain capable of binding to PD-1 or cells expressing PD-1.

[0023] In some embodiments, the antigen-binding domain 3 (ABD3) is an antigen-binding domain capable of binding to CD47 or cells expressing CD47.

[0024] In some embodiments, the binder may comprise an antigen-binding domain that targets an epitope other than those covered by ABD1, ABD2, and ABD3. Accordingly, the binder may comprise antigen-binding domains having the same or different specificities as ABD1, ABD2, and / or ABD3.

[0025] In some embodiments, the binder may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, ten or more antigen-binding domains.

[0026] In some embodiments, the binder may comprise between two and twelve antigen-binding domains.

[0027] In some embodiments, the binder of the present invention may be multispecific.

[0028] In some embodiments, the binder of the present invention can be multivalent.

[0029] In some embodiments, the binder of the present invention can be in monomeric form.

[0030] In some embodiments, the binder of the present invention can be in dimeric or higher order forms, such as trimeric, tetrameric, pentameric, etc. (e.g., polymeric).

[0031] In some cases, the antigen-binding domain of the binder is derived from a heavy chain antibody. In some cases, the heavy chain antibody can be obtained by immunizing camels or transgenic animals.

[0032] In some embodiments, the binder comprises at least one antigen-binding domain that binds to DR2 and comprises the complementarity-determining regions set forth herein.

[0033] In some embodiments, the binder comprises at least one antigen-binding domain that binds to PD-1 and comprises the complementarity-determining regions set forth herein.

[0034] In some embodiments, the binder comprises at least one antigen-binding domain that binds to CD47 and comprises the complementarity-determining regions set forth herein.

[0035] In some embodiments, the antigen-binding domain is located on one or more polypeptide chains.

[0036] In some embodiments, the antigen-binding domain antigens are located on the same polypeptide chain.

[0037] In some embodiments, the binder comprises a single polypeptide chain.

[0038] In some embodiments, the binder comprises two polypeptide chains. Thus, the binder can be in dimeric form.

[0039] In some embodiments, the binder comprises more than two polypeptide chains, such as three or more polypeptide chains, four or more polypeptide chains, five or more polypeptide chains, six or more polypeptide chains, seven or more polypeptide chains, eight or more polypeptide chains, nine or more polypeptide chains, ten or more polypeptide chains. Thus, the binder can be in polymeric form. For example, a binder comprising three polypeptide chains is referred to herein as a trimer, a binder comprising four polypeptide chains is referred to herein as a tetramer, and the like.

[0040] In some embodiments, the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises one or more antigen-binding domains.

[0041] In some embodiments, two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen-binding domain.

[0042] In some embodiments, two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen-binding domain.

[0043] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 1 (ABD1).

[0044] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 2 (ABD2).

[0045] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 3 (ABD3).

[0046] In some exemplary embodiments, the binder comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1) and the same antigen-binding domain 2 (ABD2).

[0047] In other exemplary embodiments, the binder comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1) and the same antigen-binding domain 3 (ABD3).

[0048] In other exemplary embodiments, the binder comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 2 (ABD2) and the same antigen-binding domain 3 (ABD3).

[0049] In still other exemplary embodiments, the binder comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1), the same antigen-binding domain 2 (ABD2), and the same antigen-binding domain 3 (ABD3).

[0050] In some cases, the antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and antigen-binding domain 3 (ABD3) can occupy the same position on each of the two polypeptide chains.

[0051] In some cases, the antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and antigen-binding domain 3 (ABD3) can occupy different positions on each of the two polypeptide chains.

[0052] In some embodiments, each polypeptide chain of the binder is the same.

[0053] In some embodiments, each polypeptide chain of the binder is different.

[0054] In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.

[0055] In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.

[0056] In some embodiments, the antigen-binding domains are separated by an amino acid sequence.

[0057] In some embodiments, the amino acid sequence is a linker. In some embodiments, the amino acid sequence is an antibody hinge or a fragment thereof.

[0058] The binding agents of the present invention encompass, for example, the antigen-binding domains disclosed herein, the single polypeptide chains disclosed herein, the dimers of the polypeptide chains disclosed herein, or the multimers of the polypeptide chains disclosed herein.

[0059] Thus, the binding agents of the present invention can have the following forms: antibodies or antigen-binding fragments thereof, antibody-like molecules (Fc-fusions, CH3-fusions, and the like), fusions with protein scaffolds, immunocyte modulators, and the like.

[0060] In some embodiments, the binding agents of the present invention can have the forms disclosed in PCT / CA2020 / 051753 (filed on December 18, 2020, published as WO / 2021 / 119832 on June 24, 2021, the entire content of which is incorporated herein by reference), such as Form I, Form Ia, Form Ib, Form Ic, Form II, Form III, Form IIIa, and Form IIIb, Form IV, Form V, Form VI, Form VII, or Form VIII, and the like, or the forms disclosed herein.

[0061] In some embodiments, the binding agent is composed of polypeptide chains that include one or more antigen-binding domains and a dimerization domain that enables at least two polypeptide chains to form a dimer.

[0062] In some embodiments, the binding agent of the present invention can include one or more polypeptide chains, each of which independently includes, in an N-terminal to C-terminal manner, the amino acid sequence of Form I:

[0063] X - [(Ab a ) - (L b )] m - (DD) - [(L c ) - (Ab d )] n - Y

[0064] where m is 0, 1, 2, or an integer greater than 2;

[0065] where n is an integer of 0, 1, 2, or greater than 2;

[0066] where m and n are not both 0;

[0067] where Ab a 、Ab d each represents an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3);

[0068] where X or Y independently exists or does not exist and contains an amino acid sequence;

[0069] where L b 、L c each independently contains one or more linkers; and

[0070] where DD represents a dimerization domain.

[0071] In some embodiments, the polypeptide chain comprises two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, etc.

[0072] In some embodiments, the polypeptide chain comprises an antigen-binding domain between one and twelve.

[0073] In some embodiments, the binder comprises a single polypeptide chain.

[0074] In some embodiments, the binder comprises two polypeptide chains, three polypeptide chains, four polypeptide chains, five polypeptide chains, six polypeptide chains, seven polypeptide chains, eight polypeptide chains, nine polypeptide chains, ten polypeptide chains, or more than ten polypeptide chains.

[0075] In some embodiments, the polypeptide chains may be covalently linked.

[0076] In some embodiments, the polypeptide chains may be non-covalently linked.

[0077] In some embodiments, the polypeptide chains may associate via one or more electrostatic interactions.

[0078] In some embodiments where m is an integer of 2 or greater than 2, the [(Ab a )-(L b )] units are the same or different.

[0079] In some embodiments where n is an integer of 2 or greater than 2, the [(L c )-(Ab d )] units are the same or different.

[0080] In some embodiments where m is an integer of 2 or greater than 2, each Ab a is the same or different.

[0081] In some embodiments where n is an integer of 2 or greater than 2, each Ab d is the same or different.

[0082] In some embodiments, Ab a represents ABD1, ABD2, or ABD3.

[0083] In some embodiments, Ab d represents ABD1, ABD2, or ABD3.

[0084] In some embodiments, Ab d represents ABD1, ABD2, or ABD3.

[0085] In some embodiments, the one or more polypeptide chains further comprise the hinge region of an antibody or an antigen-binding fragment thereof. For example, in some embodiments, L b is the hinge region of an antibody or an antigen-binding fragment thereof.

[0086] In some embodiments, the hinge region is the native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0087] In some embodiments, the hinge region is a mutant hinge region that is at least 70% identical to the native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0088] In some embodiments, each of the one or more linkers independently has at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues in length.

[0089] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0090] In some embodiments, the flexible linker is a GS linker. In some embodiments, the flexible linker comprises one or more GGGGS units as described herein.

[0091] In some embodiments, the rigid linker comprises multiple PA repeat sequences as described herein.

[0092] In some embodiments, the helical linker comprises one or more EAAAK units as described herein.

[0093] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0094] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0095] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0096] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0097] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0098] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0099] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VIII).

[0100] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 each independently comprises an antigen-binding domain.

[0101] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 each independently represents an antigen-binding domain.

[0102] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3At least one of them is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0103] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 At least one of them is not antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0104] In some embodiments, L b1 comprises one or more linkers and / or the hinge region of an antibody or an antigen-binding fragment thereof. In some embodiments, the hinge region is a native hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.

[0105] In some embodiments, L b2 , L b3 , L c1 , L c2 and L c3 each independently comprises one or more linkers as disclosed herein.

[0106] In some embodiments, the polypeptide chain comprises the antigen-binding domain ABD1 as disclosed herein and an antigen-binding domain that binds to immune cells.

[0107] In some embodiments, the polypeptide chain comprises the antigen-binding domain ABD2 as disclosed herein and an antigen-binding domain that binds to tumor cells.

[0108] In some embodiments, the polypeptide chain comprises the antigen-binding domain ABD3 as disclosed herein and an antigen-binding domain that binds to tumor cells.

[0109] In some embodiments, the polypeptide chain comprises the antigen-binding domain ABD3 as disclosed herein and an antigen-binding domain that binds to immune cells.

[0110] In some embodiments, the polypeptide chain comprises at least one antigen-binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0111] In some embodiments, the polypeptide chain comprises at least two antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0112] In some embodiments, the polypeptide chain comprises at least three antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0113] In some embodiments, the dimerization domain comprises an immunoglobulin dimerization domain. Other dimerization domains known to those skilled in the art are also encompassed herein, including leucine zippers and the like.

[0114] In some embodiments, the dimerization domain comprises an IgG, IgM, IgA, IgD or IgE dimerization domain (from human or animal IgG, IgM, IgA, IgD or IgE).

[0115] In some embodiments, the dimerization domain comprises the CH3 domain of an antibody. The dimerization domain may also comprise the CH2 domain of an antibody.

[0116] In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.

[0117] In some exemplary embodiments, the dimerization domain comprises a mutant CH3 domain.

[0118] In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.

[0119] In some embodiments, the dimerization domain comprises a native CH2 domain and a mutant CH3 domain.

[0120] In some embodiments, the dimerization domain comprises a mutant CH2 domain and a mutant CH3 domain.

[0121] In some embodiments, the dimerization domain comprises a mutant CH2 domain and a native CH3 domain.

[0122] In some embodiments, the dimerization domain does not comprise the CH1 domain.

[0123] In some embodiments, the dimerization domain does not comprise the CH4 domain.

[0124] In some embodiments, the dimerization domain comprises the Fc region of an antibody or a portion thereof. In some embodiments, the dimerization domain comprises the constant region of an antibody heavy chain or a portion thereof.

[0125] In some embodiments, the antigen-binding domain comprises or consists of the antigen-binding domain of a single-domain antibody (sdAb).

[0126] In some embodiments, the antigen-binding domain comprises a heavy-chain variable region (VH or VHH).

[0127] In some embodiments, the VHH is derived from humans, mice, rats, etc.

[0128] In some embodiments, the VHH is from a transgenic mouse or rat capable of expressing a camelized mouse or rat VHH, a VHH from another species (such as a human, etc.), or a camelized VHH from another species (such as a camelized human VHH, etc.).

[0129] In some embodiments, the binder does not comprise a variable light chain region (VL or VLL).

[0130] In some embodiments, the binder comprises a variable light chain region (VL or VLL).

[0131] In some embodiments, the antigen-binding domain is a single-chain variable fragment (ScFv).

[0132] In some embodiments, the antigen-binding domain is from a V NAR fragment.

[0133] In other embodiments, the antigen-binding domain of the polypeptide chain comprises any combination of antigen-binding domains of a single-domain antibody (sdAb), a variable heavy chain region (VH or VHH), a variable light chain region (VL or VLL), a single-chain variable fragment (ScFv), and / or a V NAR fragment.

[0134] In some embodiments, the sdAb or VHH is from a camelid antibody.

[0135] In some embodiments, the camelid antibody is from a dromedary, a camel, a llama, an alpaca, etc.

[0136] In other embodiments, the sdAb or VHH is from a cartilaginous fish antibody.

[0137] In some embodiments, the cartilaginous fish antibody is a shark antibody.

[0138] In some embodiments, each antigen-binding domain specifically binds to a different epitope.

[0139] In some other embodiments, each antigen-binding domain specifically binds to a different antigen.

[0140] In still some other embodiments, each antigen-binding domain specifically binds to a different protein.

[0141] In some embodiments, the binder comprises a polypeptide chain that comprises at least one antigen-binding domain that modulates CD47 function.

[0142] In some embodiments, the binder comprises a polypeptide chain comprising at least one antigen-binding domain that binds to CD47 and enhances macrophage function by blocking the SIRPα / CD47 interaction.

[0143] In some embodiments, the antigen-binding domain that specifically binds to a tumor antigen is located at the N-terminus of the dimerization domain, and the antigen-binding domain that specifically binds to an immunomodulator is located at the C-terminus of the dimerization domain.

[0144] In some embodiments, the immunomodulator is an immune checkpoint protein, a cytokine, a chemokine, or an immune receptor or co-receptor.

[0145] It should be understood herein that a given antigen-binding domain can bind to epitopes present in different proteins. Thus, in some embodiments, the antigen-binding domain or the binder comprising it can bind to more than one protein. In some embodiments, the antigen-binding domain or binder can have an affinity for more than one protein.

[0146] In some embodiments, the binder is bispecific, trispecific, or tetravalent.

[0147] In some embodiments, the binder comprises an antibody or an antigen-binding fragment thereof.

[0148] In some embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

[0149] In some embodiments, one or more of the antigen-binding domains are humanized.

[0150] In some embodiments, one or more of the antigen-binding domains are partially humanized.

[0151] In some embodiments, the antigen-binding domain comprises one or more human frameworks.

[0152] In some embodiments, X or Y is independently selected from a linker, a cytokine, a chemokine, a tag, a masking domain, a phage coat protein (pIII, pVI, pV, pVII, or pIX), an antigen-binding domain, or a combination thereof.

[0153] In some embodiments, the binder of the present invention can have two polypeptide chains, the polypeptide chains comprising an amino acid sequence identical to the amino acid sequence of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027, or KC028.

[0154] In some embodiments, the binder of the present invention may have a VHH portion of any one of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027 or KC028 and a dimerization domain and / or linker different from those listed in Table 6 or Table 7.

[0155] In some embodiments, the binder of the present invention may have a VHH portion of any one of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027 or KC028 and a dimerization domain, such as a CH2-CH3 domain that allows the formation of a homodimer as described herein.

[0156] In some embodiments, the binder of the present invention may have a VHH portion as shown in any one of SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 and / or 266 and a dimerization domain, such as a CH2-CH3 domain that allows the formation of a homodimer as described herein.

[0157] In some embodiments, the dimerization domain is as shown in SEQ ID NO:168 or at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO:168.

[0158] In some embodiments, the binder is conjugated to a therapeutic moiety.

[0159] In some embodiments, the binder is conjugated to a detectable moiety.

[0160] In some embodiments, the binder is conjugated to a protein that allows for an extended half-life.

[0161] In some embodiments, the binder is attached to a nanoparticle.

[0162] Some other aspects and embodiments of the present invention relate to a composition comprising at least one of the binders disclosed herein.

[0163] In some embodiments, the composition comprises monomers, dimers and mixtures thereof.

[0164] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition in dimer form.

[0165] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition in homodimer form.

[0166] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first polypeptide chain and the second polypeptide chain are present in the composition in heterodimer form.

[0167] Some other aspects and embodiments of the present invention relate to a pharmaceutical composition comprising a binder disclosed herein and a pharmaceutically acceptable carrier.

[0168] Some other aspects and embodiments of the present invention relate to a nucleic acid or nucleic acid set encoding a polypeptide chain and / or binder disclosed herein.

[0169] The nucleic acid may be in the form of a DNA segment as disclosed herein.

[0170] Some additional aspects and embodiments of the present invention relate to a vector comprising a nucleic acid disclosed herein or a set of vectors each comprising a nucleic acid disclosed herein.

[0171] Some other aspects and embodiments of the present invention relate to a cell expressing a polypeptide chain or binder disclosed herein.

[0172] Some additional aspects and embodiments of the present invention relate to a cell comprising a nucleic acid or vector disclosed herein.

[0173] Some other aspects and embodiments of the present invention relate to a kit comprising a binder disclosed herein.

[0174] Some other aspects and embodiments of the present invention relate to a kit comprising a nucleic acid disclosed herein.

[0175] Some other aspects and embodiments of the present invention relate to a kit comprising a vector disclosed herein.

[0176] Some other aspects and embodiments of the present invention relate to a kit comprising a cell disclosed herein.

[0177] In another aspect and embodiment, the present invention relates to a method of treating a disorder or disease, the method comprising administering a binder disclosed herein.

[0178] In further aspects and embodiments, the present invention relates to a method of treating a disorder or disease, the method comprising administering a composition disclosed herein.

[0179] In further aspects and embodiments, the present invention relates to a method of treating a disorder or disease, the method comprising administering a pharmaceutical composition disclosed herein.

[0180] In some embodiments, the disorder or disease is cancer.

[0181] In some embodiments, the disorder or disease is a solid tumor. In some embodiments, the solid tumor is a neuroendocrine solid tumor.

[0182] In some embodiments, the disorder or disease is advanced metastatic solid cancer.

[0183] In some embodiments, the disorder or disease is neuroblastoma.

[0184] In some embodiments, the disorder or disease is gastric cancer.

[0185] In some embodiments, the disorder or disease is hematogenous cancer.

[0186] In some embodiments, the disorder or disease is lung cancer.

[0187] In some embodiments, the lung cancer is metastatic.

[0188] In some embodiments, the disease or disorder is small cell lung cancer.

[0189] In some embodiments, the disease or disorder is non-small cell lung cancer.

[0190] In some embodiments, the disorder or disease is myeloma.

[0191] In some embodiments, the disorder or disease is prostate cancer.

[0192] In some embodiments, the disorder or disease is ovarian cancer.

[0193] In some embodiments, the disorder or disease is breast cancer.

[0194] In some embodiments, the breast cancer is triple-negative breast cancer.

[0195] In some embodiments, the disorder or disease is rectal cancer.

[0196] In some embodiments, the disorder or disease is colorectal cancer.

[0197] In some embodiments, the disorder or disease is pancreatic cancer.

[0198] In some embodiments, the disorder or disease is glioblastoma.

[0199] In some embodiments, the disorder or disease is an infection.

[0200] In some embodiments, the disorder or disease is an immune disorder.

[0201] In other aspects and embodiments, the present invention relates to a method of preparing a binder disclosed herein, the method comprising transforming a cell with one or more vectors comprising a nucleic acid disclosed herein.

[0202] In some embodiments, the method may further comprise separating and / or purifying the binder from impurities.

[0203] In other embodiments, the method may further comprise separating and / or purifying the heterodimer from monomers and / or homodimers.

[0204] In other embodiments, the method may further comprise separating and / or purifying the homodimer from monomers and / or heterodimers.

[0205] In some embodiments, the method further comprises conjugating the binder to a therapeutic moiety, a detectable moiety, or a protein that allows for an extended half-life, or to a nanoparticle.

[0206] Other scopes, applications, and advantages of the present invention will be apparent from the non-limiting detailed description given below. However, it should be understood that this detailed description indicates some exemplary embodiments of the present invention, which are given by way of example only, with reference to the accompanying drawings. Brief Description of the Drawings

[0208] Figure 1 : A schematic diagram showing the binding curve of the D2-specific binder (VHH-hinge-CH2-CH3) on D2 protein liposomes. Mock liposomes were included as a negative control.

[0209] Figure 2 : A table listing the EC50 of the D2-specific binder (VHH-hinge-CH2-CH3) in GPCR BRET assays transfected with various G protein sensors. Dopamine was used as a positive control to show the GPCR response with G proteins including Gαz, Gαi2, GαoA, and Barr2. The negative control was the 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain shown in SEQ ID NO: 114.

[0210] Figure 3 : Heatmap of gene expression changes in NCI-H69 cells after treatment with a binder (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO:73) containing a D2-specific antigen-binding domain. The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain shown in SEQ ID NO:114.

[0211] Figure 4 : Representation according to Figure 3 The RNAseq results of the PANTHER analysis of downregulated signaling pathways and protein classes.

[0212] Figure 5 A to Figure 5 C: Representation of the downregulated genes verified by qRT-PCR in NCI-H69 ( Figure 5 A), NCI-H82 ( Figure 5 B), and HEK-D2 ( Figure 5 C) after treatment with a binder (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO:73) containing a D2-specific antigen-binding domain.

[0213] Figure 6 A to Figure 6 B: Representation of the pharmacokinetics of a binder (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO:73) containing a D2-specific antigen-binding domain and trastuzumab in SCID mice using a single-dose injection ( Figure 6 A: IV injection) ( Figure 6 B: IP injection).

[0214] Figure 7 A to Figure 7 C: Showing the NCI-H727 tumor prevention model ( Figure 7 A), NCI-H69 tumor prevention model ( Figure 7 B), or NCI-H510A tumor prevention model ( Figure 7Schema of tumor kinetics and in vivo efficacy of a binder (VHH-hinge-CH2-CH3, solid line) (KC013; SEQ ID NO:73) containing a D2-specific antigen-binding domain in NCG mice with a small cell lung cancer (SCLC) xenograft model compared to the isotype control (dashed line) in (C). The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain shown in SEQ ID NO:114.

[0215] Figure 8 : Table listing the percentage of tumor growth inhibition in various tumor prevention SCLC tumor models in SCID mice after treatment with a D2-specific binder (VHH-hinge-CH2-CH3) containing the amino acid sequence shown in SEQ ID NO:71 (KC011), SEQ ID NO:72 (KC012), SEQ ID NO:73 (KC013), or SEQ ID NO:74 (KC014).

[0216] Figure 9A : Schema showing the binding curves of a PD-1-specific binder (KC036; SEQ ID NO:94) on human, cynomolgus macaque, and rat recombinant PD1 proteins or a control protein (BSA). The negative control is a binder (KC035: SEQ ID NO:93) containing a HEWL-specific antigen-binding domain.

[0217] Figure 9B : Top panel: Schema showing the binding curve of a PD-1-specific binder (KC036; SEQ ID NO:94) on human recombinant PD1 protein compared to the anti-PD-1 antibody pembrolizumab (positive control). The negative control is a binder (KC035: SEQ ID NO:93) containing a HEWL-specific antigen-binding domain; Bottom panel: Schema showing the binding curves of PD-1-specific binders (KC036; SEQ ID NO:94; KC058; and KC067) on human recombinant PD-1 protein compared to the anti-PD-1 antibody pembrolizumab (positive control). The negative control is a binder (KC035: SEQ ID NO:93) containing a HEWL-specific antigen-binding domain.

[0218] Figure 9C: A diagram showing the binding curve of the CD47-specific binder (KC015; SEQ ID NO:75) to human recombinant CD47 protein. The negative control for KC015 was a binder containing the HEWL-specific antigen-binding domain (KC016: SEQ ID NO:76). The positive control was monoclonal antibody clone B6H12. The isotype control of clone B6H12 was used as the mouse IgG isotype control antibody.

[0219] Figure 9D : A diagram showing the binding curve of the CD47-specific binder KC015 to cynomolgus macaque CD47 protein. The negative control KC016 was a binder containing the HEWL-specific antigen-binding domain. The positive control was monoclonal antibody clone B6H12. The isotype control of clone B6H12 was used as the mouse IgG isotype control antibody.

[0220] Figure 9E : A diagram showing the binding curves of the CD47-specific binder KC015 to various blood cell subtypes including T cells, monocytes, granulocytes, red blood cells (RBC), and platelets.

[0221] Figure 10 : A diagram showing the cell-binding activity of the PD-1-specific binder (KC036; SEQ ID NO:94) obtained by flow cytometry on CHO-PD1 cells ( Figure 10 B) and CHO parental cells ( Figure 10 A). A commercially available PD-1 antibody was used as the positive control. The negative control was a binder containing the HEWL-specific antigen-binding domain (KC035: SEQ ID NO:93).

[0222] Figure 11 : A diagram showing the cytotoxicity data of PBMC-mediated THP-1 death induced by the PD-1-specific binder (KC036; SEQ ID NO:94) in the presence of CD33 BiTE (for activating T cells via the CD3 subunit) or in the presence of a control BiTE after 48 hours of incubation. The negative control was a binder containing the HEWL-specific antigen-binding domain (KC031: SEQ ID NO:89). The positive controls included pembrolizumab and nivolumab.

[0223] Figure 12A: Schematic showing the checkpoint inhibitory activity of a PD-1 specific binder (KC036; SEQ ID NO:94) using a PD-1 / PD-L1 blocking bioassay system. This system is an engineered Jurkat cell line expressing a luciferase reporter under the NFAT promoter. The negative control is a binder containing an HEWL specific antigen binding domain (KC035: SEQ ID NO:93). The anti-PD-1 antibody pembrolizumab is used as a positive control.

[0224] Figure 12B : Schematic showing the checkpoint inhibitory activity of PD-1 specific binders (KC036; SEQ ID NO:94, KC059, KC060 and KC067) using a PD-1 / PD-L1 blocking bioassay system. This system is an engineered Jurkat cell line expressing a luciferase reporter under the NFAT promoter. The negative control is a binder containing an HEWL specific antigen binding domain (KC035: SEQ ID NO:93). The anti-PD-1 antibody pembrolizumab is used as a positive control.

[0225] Figure 13 : Schematic showing the binding data of a trispecific binder containing anti-D2, anti-PD-1 and anti-CD47 antigen binding domains to HEK293T cells and HEK293T-CD47 KO cells (CD47 knockout cells) (KC020; SEQ ID NO:77) and a monospecific binder containing the same anti-CD47 antigen binding domain (KC015: SEQ ID NO:75 VHH-hinge-CH2-CH3). Negative controls include corresponding forms containing an anti-HEWL antigen binding domain (KC016 (SEQ ID NO:76) and KC039 (SEQ ID NO:97)). A commercially available anti-human CD47 antibody (clone CC2C6) is used as a positive control.

[0226] Figure 14 : Schematic showing the binding of a binder containing anti-D2 and anti-CD47 antigen binding domains to human T cells (KC040: SEQ ID NO:84) compared to control binders containing an anti-HEWL antigen binding domain (KC035: SEQ ID NO:93) or anti-CD3 and anti-HEWL antigen binding domains (KC034; SEQ ID NO:92).

[0227] Figure 15 A to Figure 15 B: Schematic showing T cell activation induced by a binder containing anti-D2 and anti-CD47 antigen binding domains at an E to T ratio of 5:1 on human T cells (KC040: SEQ ID NO:84) Figure 15A) and T cell-dependent cytotoxicity ( Figure 15 B). Controls included medium only (negative control), a binder comprising an anti-HEWL antigen-binding domain and the same anti-D2 antigen-binding domain (negative control KC032; SEQ ID NO:90), and OKT3 (positive control).

[0228] Figure 16 : A graph showing T cell activation after treatment with a binder (KC040: SEQ ID NO:84) comprising a D2-specific antigen-binding domain and a CD47-specific antigen-binding domain in the presence or absence of Concanavalin A. Controls included a binder comprising a HEWL-specific antigen-binding domain (KC035: SEQ ID NO:93) or a binder comprising anti-CD3 and anti-HEWL antigen-binding domains (positive control KC034; SEQ ID NO:92) or OKT3 (positive control).

[0229] Figure 17 : A graph showing checkpoint inhibitory activity of a CD47-specific binder (KC015; SEQ ID NO:75) using the Promega CD47 / SIRPα blockade bioassay system. This system is an engineered THP-1 cell line expressing a luciferase reporter induced by an FcγR protein conjugated to FcγR. The negative control was a binder comprising a HEWL-specific antigen-binding domain (KC016: SEQ ID NO:76). The positive control was the monoclonal antibody clone B6H12. The isotype control for clone B6H12 was a mouse IgG isotype control antibody.

[0230] Figure 18 : A schema showing tumor volume after treatment with a trispecific binder (KC021: SEQ ID NO:78) comprising anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains in an established NCI-H82 xenograft model in NCG mice. Negative controls included a binder comprising a HEWL-specific antigen-binding domain (KC031: SEQ ID NO:89).

[0231] Figure 19 A to Figure 19 B: A schema showing T cell infiltration into NCI-H69 cell clusters after treatment with a binder comprising anti-D2 and anti-PD-1 antigen-binding domains (KC037; SEQ ID NO:95), anti-PD-1 and anti-CD47 antigen-binding domains (KC038; SEQ ID NO:96), or a trispecific binder comprising the same anti-D2, anti-PD1, and anti-CD47 antigen-binding domains (KC020; SEQ ID NO:77 and KC021: SEQ ID NO:78)Figure 19 A), or activating T cells with the same binder Figure 19 B). Controls included a binder containing an antigen-binding domain specific for HEWL (KC035: SEQ ID NO:93).

[0232] Figure 20 A to Figure 20 B: Schematics showing tumor volume after treatment with trispecific binders (KC020; SEQ ID NO:77, KC022; SEQ ID NO:79, KC023; SEQ ID NO:80, KC024; SEQ ID NO:81, KC025; SEQ ID NO:82, KC026; SEQ ID NO:83) containing different anti-D2 and the same anti-PD-1 and anti-CD47 antigen-binding domains Figure 20 A) and a table showing tumor growth inhibition Figure 20 B).

[0233] Figure 21 A: Chart showing tumor volume after treatment with a CD47-specific binder (KC015; SEQ ID NO:75) in an NCI-H82 xenograft model pre-implanted with PBMCs in NCG mice. 10 million PBMCs were implanted into NCG mice 3 days before tumor cell implantation. Treatment started 10 days after PBMC implantation, twice a week for a total of 8 doses. The negative control used in the study was PBS.

[0234] Figure 21 B: Table showing the in vivo efficacy of KC020 (SEQ ID NO:77) using various tumor cells including NCI-H69, NCI-H82, SCLC-21H, and RKO in different models.

[0235] Figure 21 C: Schematic showing tumor volume after treatment with an anti-CD47-specific binder KC015 in an established MDA-MB-231 xenograft model in SCID mice. The negative control used in the study was PBS. 5 million MDA-MB-231 cells were injected subcutaneously (s.c.) into the right lower abdomen of SCID mice. Mice were treated intraperitoneally (i.p.) with the binder (8 mg / kg) once a week for a total of eight doses.

[0236] Figure 22: Graph showing tumor volume after treatment with an IgG1 or IgG4 isotype of a binder comprising antigen-binding domains against D2, anti-PD-1, and anti-CD47 (KC028; SEQ ID NO:86; KC029; SEQ ID NO:87, KC027: SEQ ID NO:85). Controls included a binder comprising an antigen-binding domain against HEWL (KC031: SEQ ID NO:89).

[0237] Figure 23 A to Figure 23 B: Illustration of biodistribution experiment ( Figure 23 A) and graph showing accumulation of a trispecific binder (KC021; SEQ ID NO:78) comprising antigen-binding domains against D2, anti-PD-1, and anti-CD47 in NCI-H82 (upper panel) and NCI-H69 (lower panel) xenograft models in NCG mice ( Figure 23 B).

[0238] Figure 24 A to Figure 24 B: Graph showing liver metastases in hCD34+-humanized NCG mice bearing established NCI-H69 tumors treated with PBS or KC020 (SEQ ID NO:77) ( Figure 24 A), and H&E staining of tumors, as indicated by the black box ( Figure 24 B), indicating disseminated tumor cells from the PBS group but not the KC020 (SEQ ID NO:77) group.

[0239] Figure 24 C: Graph showing tumor kinetics and in vivo efficacy of KC020 in an NCI-H82 / PBMC co-implantation model in NCG mice with an E:T ratio of 1:5.

[0240] Figure 25 A to Figure 25 C: Graph showing binding of KC020 (SEQ ID NO:77) subjected to stress conditions measured by ELISA analysis to D2 protein liposomes ( Figure 25 A), human recombinant PD-1 protein ( Figure 25 B), and human recombinant CD47 protein ( Figure 25 C). Stress conditions included stirring for 3 days, freeze-thawing five times, and storage at 40 °C for two weeks.

[0241] Figure 26 A to Figure 26 C: Graph showing binding of KC020 (SEQ ID NO:77) subjected to low pH stress measured by ELISA analysis to D2 protein liposomes ( Figure 26A), human recombinant PD-1 protein ( Figure 26 B) and human recombinant CD47 protein ( Figure 26 C) binding charts. Stress conditions include treatment at pH 3.5 for 4 hours and 48 hours.

[0242] Figure 27 : A schema representing the survival curve of animals treated with KC020 implanted in the NCI-H82 SCLC tail vein metastasis model. NCG mice were inoculated with 50,000 NCI-H82 cells and then treated with KC020 the next day. KC020 was administered every two weeks. The negative control used in the study was PBS.

[0243] Figure 28 A: Chart of the average tumor volume (mm 3 ) in MDA-MB-468 xenograft SCID mouse models treated once a week with 26 mg / kg KC020 compared to the PBS negative control, several days after implantation.

[0244] Figure 28 B: Chart of the average tumor volume (mm 3 ) in MDA-MB-231 xenograft SCID mouse models treated twice a week with 1 mg / ml, 3 mg / ml or 10 mg / kg KC020 compared to the PBS negative control, several days after implantation.

[0245] Figure 29 A: Chart of the average tumor volume (mm 3 ) in MDA-MB-453 xenograft models in SCID mice treated once a week with 1 mg / ml, 10 mg / ml or 28 mg / kg KC020 compared to the PBS negative control, several days after implantation.

[0246] Figure 29 B: Chart of the average tumor volume (mm 3 ) in T.Tn xenograft models in SCID mice treated once a week with 1 mg / ml, 10 mg / ml or 26 mg / kg KC020 compared to the PBS negative control, several days after implantation.

[0247] Figure 30A : Alignment of selected anti-D2 VHHs of Table 3.

[0248] Figure 30B : Alignment of selected anti-PD-1 VHHs of Table 4. Detailed Description of the Invention

[0250] Definitions

[0251] Unless otherwise indicated, the amino acid numbering indicated for the dimerization domain is according to the EU numbering system.

[0252] Unless otherwise indicated herein or clearly contradicted by the context, the terms "a / an" and "the", "said" and similar reference words used in the context of describing embodiments (especially in the context of the claims) shall be understood to cover the singular and plural.

[0253] Unless expressly stated or obvious from the context, the term "or" as used herein shall be understood to be inclusive, covering "or" and "and".

[0254] "And / or" as used herein shall be regarded as specifically disclosing each designated feature or component, with another feature or component present or absent.

[0255] Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" shall be understood as open terms (i.e., meaning "including but not limited to"). The term "consisting of" is regarded as closed.

[0256] For the purposes of the present invention, the term "treatment" refers to therapeutic treatment and prophylactic or tumor preventive measures. Individuals in need of treatment include individuals who already have a disorder and individuals who are predisposed to the disorder, or individuals to be protected from the disorder. Thus, individuals in need of treatment include individuals who already have cancer and individuals who are predisposed to cancer or individuals to be managed for cancer.

[0257] The term "about" or "approximately" with respect to a given value means covering variations of the value. In some embodiments, the term "about" or "approximately" generally means a range within + / - 10%, + / - 5%, + / - 4%, + / - 3%, + / - 2% or + / - 1% of the given value or range.

[0258] As used herein, the term "DR2" or "D2" refers to the dopamine receptor D2 protein or a polypeptide comprising the DR2 amino acid sequence. The term "DR2" covers the human DR2 protein and the DR2 antigen.

[0259] As used herein, the term "PD-1" refers to the programmed cell death protein 1 protein or a polypeptide comprising the PD-1 amino acid sequence. The term "PD-1" covers the human PD-1 protein and the PD-1 antigen.

[0260] As used herein, the term "CD47" refers to the cluster of differentiation 47 protein or a polypeptide comprising the CD47 amino acid sequence. The term "CD47" covers the human CD47 protein and the CD47 antigen.

[0261] The term "binding agent" refers to a molecule that contains a polypeptide moiety (such as a polypeptide chain) and is capable of specifically binding to an antigen or contains a domain (such as an antigen-binding domain) capable of specifically binding to an antigen.

[0262] The term "antibody" is used in the broadest sense and encompasses various antibody forms and structures, including any immunoglobulin, monoclonal antibody, polyclonal antibody, bivalent antibody, monovalent antibody, bispecific antibody, multispecific (polyspecific) antibody, conventional antibody, single-domain antibody, single-chain antibody, heavy-chain-only antibody, nanobody, full-length antibody, humanized antibody, chimeric antibody that binds a specific antigen, and any antigen-binding fragment that exhibits the desired antigen-binding activity. An antibody can be naturally occurring (native) or generated by recombinant techniques.

[0263] As used herein, the expressions "single-domain antibody" and "heavy-chain-only antibody" can be used interchangeably. The term "single-domain antibody" includes naturally occurring single-domain antibodies or heavy-chain-only antibodies, as well as single-domain antibodies in which the naturally occurring constant region has been replaced by another dimerization domain (such as the constant region of a human immunoglobulin (e.g., IgG)); and variants, such as humanized or chimeric single-domain antibodies.

[0264] "Naturally occurring single-domain antibodies" include antibodies produced by camels (camelid antibodies) or by sharks. "Naturally occurring single-domain antibodies" also encompass antibodies produced by transgenic animals that have been modified to express heavy-chain-only antibodies. Exemplary embodiments of transgenic animals are provided in PCT / CA2021 / 050951, filed July 21, 2021, which was published as WO2022 / 011457 on January 20, 2022, the entire content of which is incorporated herein by reference.

[0265] As used herein, the term "subject in need" refers to an individual suffering from a disorder or disease associated with DR2, DR2 expression (such as expression in tissue, cells, or serum), or DR2 overexpression or DR2 upregulation, or suspected of suffering from such a disorder or disease. The term "subject in need" also refers to an individual suffering from a disorder or disease that can benefit from treatment with a binding agent that targets DR2, or suspected of suffering from such a disorder or disease. "Subject in need" includes individuals suffering from cancer or suspected of suffering from cancer.

[0266] As used herein, the term "humanized" means that a binding agent (such as an antibody or antigen-binding fragment) contains CDRs or CDR amino acid residues derived from a non-human animal antibody, FR regions derived from a human antibody, and, where applicable, a constant region derived from a human antibody.

[0267] In some cases, a humanized binder may comprise CDRs or CDR amino acid residues of a human antibody variable region (such as a human germline antibody variable region sequence).

[0268] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between a binder or its antigen-binding domain and an antigen. "Affinity" is represented, for example, by the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on ) when the binding between the antigen and the binder (such as an antibody, such as a single-domain antibody) reaches equilibrium. Affinity can be determined by using any conventional method known in the art, including (but not limited to) surface plasmon resonance methods, microscale thermophoresis methods, HPLC-MS methods, and flow cytometry (such as FACS) methods. The K D value of an antibody is typically ≤ 10 -6 M (such as ≤ 5 × 10 -7 M, ≤ 2 × 10 -7 M, ≤ 10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤ 10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M or ≤ 10 -9 M and any value ≤ 10 -6 ). Preferably, the K D value of the antibody is in the nanomolar concentration range or lower (such as, ≤ 9 × 10 -9 M, ≤ 8 × 10 -9 M, ≤ 7 × 10 -9 M, ≤ 6 × 10 -9 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, ≤ 1 × 10 -9 M or lower, such as ≤ 1 × 10 -10 M, ≤ 1 × 10 -11 M, ≤ 1 × 10 -12 M). Even more preferably, the K D value of the antibody is in the picomolar concentration range or lower (such as, ≤ 9 × 10 -12 M, ≤ 8 × 10 -12 M, ≤ 7 × 10 -12 M, ≤ 6 × 10 -12 M, ≤ 5 × 10 -12M, ≤ 4×10 -12 M, ≤ 3×10 -12 M, ≤ 2×10 -12 M, ≤ 1×10 -12 M or lower).

[0269] As used herein, the terms "specifically binds to" or "specifically binds" refer to a non-random binding reaction between two molecules (such as between an antibody and an antigen). Specific binding can be characterized by binding affinity, ≤ 10 -6 M of K D value (e.g., ≤ 5×10 -7 M, ≤ 2×10 -7 M, ≤ 10 -7 M, ≤ 5×10 -8 M, ≤ 2×10 -8 M, ≤ 10 -8 M, ≤ 5×10 -9 M, ≤ 4×10 -9 M, ≤ 3×10 -9 M, ≤ 2×10 -9 M or ≤ 10 -9 M and any ≤ 10 -6 value) can indicate specific binding between a binder (e.g., an antibody (such as a single-domain antibody)) and DR2 (e.g., human DR2).

[0270] As used herein, the terms "competing for binding / compete for binding", "compete(s) with / competing with" refer to the ability of a first antibody or its antigen-binding fragment (e.g., a single-domain antibody or its antigen-binding fragment) to inhibit the binding interaction between DR2 and a second anti-DR2 antibody, PD-1 and a second anti-PD-1 antibody, CD47 and a second anti-CD47 antibody (e.g., a single-domain antibody or its antigen-binding fragment) to any detectable extent. Means for reducing tumor size, for inhibiting tumor growth, and / or for inducing tumor regression can be achieved using competitive binders.

[0271] As used herein, the term "epitope" refers to a specific group of atoms or amino acid residues on an antigen to which an antibody (e.g., a single domain antibody) binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes within the antigen. Epitopes can be linear or conformational (i.e., including amino acid residues that are spaced apart). For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody (e.g., a single domain antibody) to an antigen by at least 85%, or at least 90%, or at least 95%, the antibody or antigen-binding fragment can be considered to bind the same / closely related epitope as the reference antibody. For example, monoclonal, chimeric, human or humanized antibodies or their antigen-binding fragments can compete with the single domain antibodies of the invention for binding to human DR2.

[0272] As used herein, the term "sequence identity" of the present invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and when mutations such as substitutions, insertions or deletions are appropriate. The sequence identity can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% and 100%.

[0273] The term "antigen-binding fragment" as used in the expression "antibody or its antigen-binding fragment" or "single domain antibody or its antigen-binding fragment" refers to a fragment of an antibody or a single antibody that encompasses the antigen-binding domain, and may or may not incorporate other parts of the antibody or single domain antibody, such as amino acid residues of the hinge region, amino acid residues of the constant region, parts of the Fc region. Regardless of structure, the antigen-binding fragment binds the same antigen recognized by the intact antibody (e.g., a single domain antibody).

[0274] Antigen-binding fragments of single domain antibodies include, for example, CDRs and sequences encompassing CDRs, such as the entire variable region or parts thereof or the entire heavy chain or parts thereof, and may or may not include other parts of the antibody.

[0275] The term "antigen-binding domain" refers to the part of an antibody involved in antigen binding, including, for example, one or more CDRs, one or more framework regions (FRs), or the entire variable region. In the case of its single-domain antibody, the term "antigen-binding domain" refers to the part of the single-domain antibody involved in antigen binding, including, for example, one or more of CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more of the framework regions FR1, FR2, FR3, FR4, or the entire variable region (VH or VHH). In the case of its native antibody, the term "antigen-binding domain" refers to the native antibody part involved in antigen binding and includes, for example, one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light chain or heavy chain framework regions FR1, FR2, FR3, FR4, or one or both variable regions (heavy chain variable region (VH) and / or light chain variable region (VL)). In some embodiments, the framework regions are generally not considered part of the antigen-binding domain. However, some amino acid residues of one or more framework regions may contribute to antigen binding.

[0276] The term "functional activity" with respect to an antigen-binding domain means that the antigen-binding domain is capable of binding to its target, and optionally, the antigen-binding domain has one or more biological activities.

[0277] As used herein, the term "flexible linker" refers to a peptide that includes at least a portion composed of flexible amino acid residues that enable adjacent modules to move relative to each other.

[0278] As used herein, the term "rigid linker" refers to a peptide that includes at least a portion composed of amino acids that exhibit a rigid structure such that a distance is maintained between two modules.

[0279] As used herein, the term "helical linker" means a linker composed of amino acid residues that adopt an α-helical conformation.

[0280] As used herein, the term "cleavable linker" refers to a peptide that includes an enzymatic cleavage site sensitive to a protease selected from the group consisting of ADAMS, ADAMTS, aspartic proteases, caspases, cathepsin cysteines, cysteine proteases, metalloproteases, serine proteases, coagulation factor proteases, type II transmembrane serine proteases (TTSP), and combinations thereof.

[0281] As used herein, the term "monospecific" with respect to a polypeptide chain or binder means a polypeptide chain or binder that binds a single antigen or epitope. Thus, a monospecific polypeptide chain or binder can have one antigen-binding domain or more than one binding domain (identical or different) with the same specificity for a given antigen or epitope.

[0282] As used herein, the term "multispecific" with respect to a polypeptide chain or a binder refers to a polypeptide chain or a binder that binds to more than one antigen or epitope.

[0283] The term "multispecific" encompasses "bispecific", "trispecific", "tetraspecific", "pentaspecific", "hexaspecific" and the like.

[0284] As used herein in the context of a binder, the term "bispecific" denotes a binder that binds to two different antigens or proteins or two epitopes of the same antigen or protein.

[0285] As used herein, the expression "at least two polypeptide chains" and similar expressions, such as in the expression "a binder comprises at least two polypeptide chains" and similar expressions, refer to a plurality of polypeptide chain species and not an absolute numerical value.

[0286] It should be understood herein that an expression referring to a numerical range in the form of "A to B" includes each individual value and composition, and includes any sub-range of such range. For example, the expression "1 to 10" includes sub-ranges such as and not limited to "2 to 10", "2 to 9", "3 to 6", "5 to 7" and any individual value included therein, including 1 and 10, namely 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0287] It should be understood herein that the term "at least" with respect to a given value is intended to include that value and more values. For example, the term "at least 80%" includes "at least 81%", "at least 82%", "at least 83%", "at least 84%", "at least 85%", "at least 86%", "at least 87%", "at least 88%", "at least 89%", "at least 90%", "at least 91%", "at least 92%", "at least 93%", "at least 94%", "at least 95%", "at least 96%", "at least 97%", "at least 98%", "at least 99%", "at least 99.1%", "at least 99.2%", "at least 99.3%", "at least 99.4%", "at least 99.5%", "at least 99.6%", "at least 99.7%", "at least 99.8%", "at least 99.9%" and 100%.

[0288] The term "codon optimization" refers to a sequence in which a codon has been changed to another codon that encodes the same amino acid and that codon is more preferred or performs better (increases expression, minimizes secondary structure in RNA, etc.) in a given organism. A "codon-optimized" sequence can be obtained using publicly available software or via a service provider, including GenScript (OptimumGene TM , U.S. Patent No. 8,326,547).

[0289] The term "single-chain form" with respect to an antigen-binding domain means that the domains required for antigen binding are located on a single polypeptide chain.

[0290] As used herein, the term "regression" refers to the partial or complete disappearance of a tumor or tumor lesion, which disappearance may be temporary or permanent.

[0291] As used herein, the term "tumor" encompasses "primary tumor" and "secondary tumor".

[0292] As used herein, the term "tumor lesion" refers to a lesion originating from or caused by a tumor.

[0293] It should be understood herein that any one of the terms "KC011", "KC012", "KC013", "KC014", "KC015", "KC016", "KC017", "KC018", "KC019", "KC020", "KC021", "KC022", "KC023", "KC024", "KC025", "KC026", "KC027", "KC028", "KC029", "KC030", "KC031", "KC032", "KC033", "KC034", "KC035", "KC036", "KC037", "KC038", "KC039", "KC040", "KC058", "KC059", "KC060", or "KC067" refers to a binder comprising two chains, each having the corresponding amino acid sequence shown in Table 6 or Table 7 or as described herein.

[0294] Dopamine receptor D2 antigen

[0295] The anti-DR2 antibody or antigen-binding fragment thereof of the present invention can be obtained, for example, by immunizing an animal with a DR2 antigen. More specifically, an anti-DR2 single-domain antibody or antigen-binding fragment thereof can be obtained by immunizing an animal with D2 proteoliposomes (i.e., proteoliposomes containing the D2 antigen).

[0296] Alternatively, the anti-DR2 antibody or antigen-binding fragment thereof of the present invention can be obtained from an antibody library.

[0297] In one embodiment, the DR2 antigen is a human DR2 protein (e.g., Uniprot accession number P14416-1, the entire content of which is incorporated herein by reference) or a fragment thereof.

[0298] Binders that bind to DR2 can be identified by computer simulation, in vitro, and / or in vivo methods. Some in vitro methods rely on the binding of a candidate antibody or its antigen-binding fragment to an antigen. Thus, anti-DR2 antibodies or their antigen-binding fragments can be identified by methods involving binding to the DR2 antigen or D2 protein liposomes disclosed herein and / or using the assay methods disclosed herein.

[0299] In one embodiment, the DR2 antigen is a human DR2 protein or a fragment thereof. In some embodiments, the DR2 antigen is a DR2 homolog having a sequence that is at least 80% identical to that of human DR2 or a fragment thereof. In some embodiments, the DR2 antigen is a DR2 variant having a sequence that is at least 80% identical to that of human DR2 or a fragment thereof.

[0300] In one illustrative embodiment, the DR2 homolog is from a primate.

[0301] In one illustrative embodiment, the DR2 homolog is cynomolgus monkey DR2.

[0302] In another illustrative embodiment, the DR2 homolog is rhesus monkey DR2.

[0303] According to the present invention, the DR2 antigen may comprise at least 10 amino acid residues of human DR2.

[0304] According to the present invention, the DR2 antigen comprises between 10 and 400 amino acid residues, between 10 and 390 amino acid residues, between 10 and 380 amino acid residues, between 10 and 370 amino acid residues, between 10 and 360 amino acid residues, between 10 and 350 amino acid residues, between 10 and 340 amino acid residues, between 10 and 330 amino acid residues, between 10 and 320 amino acid residues, between 10 and 310 amino acid residues, between 10 and 300 amino acid residues, between 10 and 290 amino acid residues, between 10 and 280 amino acid residues, between 10 and 270 amino acid residues, between 10 and 260 amino acid residues, between 10 and 250 amino acid residues, between 10 and 240 amino acid residues, between 10 and 230 amino acid residues, between 10 and 220 amino acid residues, between 10 and 210 amino acid residues, between 10 and 200 amino acid residues, between 10 and 190 amino acid residues, between 10 and 180 amino acid residues, between 10 and 170 amino acid residues, between 10 and 160 amino acid residues, between 10 and 150 amino acid residues, between 10 and 140 amino acid residues, between 10 and 130 amino acid residues, between 10 and 120 amino acid residues, between 10 and 110 amino acid residues of the human DR2 amino acid sequence, including for example between 10 and 100 amino acid residues, between 10 and 90 amino acid residues, between 10 and 80 amino acid residues, between 10 and 70 amino acid residues, between 10 and 60 amino acid residues, between 10 and 50 amino acid residues, between 10 and 40 amino acid residues, between 10 and 30 amino acid residues, between 10 and 29 amino acid residues, between 10 and 28 amino acid residues, between 10 and 27 amino acid residues, between 10 and 26 amino acid residues, between 10 and 25 amino acid residues, between 10 and 24 amino acid residues, between 10 and 23 amino acid residues, between 10 and 22 amino acid residues, between 10 and 21 amino acid residues, between 10 and 20 amino acid residues, between 10 and 19 amino acid residues, between 10 and 18 amino acid residues, between 10 and 17 amino acid residues, between 10 and 16 amino acid residues, between 10 and 15 amino acid residues, between 10 and 14 amino acid residues, between 10 and 13 amino acid residues, between 10 and 12 amino acid residues, between 10 and 11 amino acid residues.

[0305] An illustrative and non-limiting embodiment of the DR2 antigen is provided in SEQ ID NO:165.

[0306] Programmed cell death protein 1 antigen

[0307] The anti-PD-1 antibody or antigen-binding fragment of the present invention can be obtained, for example, by immunizing an animal with a PD-1 antigen.

[0308] Alternatively, the antibody or antigen-binding fragment of the present invention can be obtained from an anti-PD-1 antibody library, such as an anti-PD-1 single-domain antibody library.

[0309] In one embodiment, the PD-1 antigen is a human PD-1 protein (e.g., Uniprot accession number Q15116, the entire content of which is incorporated herein by reference) or a fragment thereof. In some embodiments, the PD-1 antigen is a PD-1 homolog having a sequence that is at least 80% identical to the human PD-1 protein or a fragment thereof. In some embodiments, the PD-1 antigen is a PD-1 variant having a sequence that is at least 80% identical to the human PD-1 protein or a fragment thereof. An exemplary embodiment of the PD-1 antigen is provided in SEQ ID NO: 166.

[0310] Exemplary embodiments of the PD-1 antigen include the extracellular domain of PD-1 or a portion thereof.

[0311] According to the present invention, the PD-1 antigen comprises between 10 and 250 amino acid residues, between 10 and 200 amino acid residues, between 10 and 165 amino acid residues of the human PD-1 amino acid sequence, including, for example, between 10 and 160 amino acid residues, between 10 and 150 amino acid residues, between 10 and 140 amino acid residues, between 10 and 130 amino acid residues, between 10 and 120 amino acid residues, between 10 and 110 amino acid residues, between 10 and 100 amino acid residues, between 10 and 90 amino acid residues, between 10 and 80 amino acid residues, between 10 and 70 amino acid residues, between 10 and 60 amino acid residues, between 10 and 50 amino acid residues, between 10 and 40 amino acid residues, between 10 and 30 amino acid residues, between 10 and 29 amino acid residues, between 10 and 28 amino acid residues, between 10 and 27 amino acid residues, between 10 and 26 amino acid residues, between 10 and 25 amino acid residues, between 10 and 24 amino acid residues, between 10 and 23 amino acid residues, between 10 and 22 amino acid residues, between 10 and 21 amino acid residues, between 10 and 20 amino acid residues, between 10 and 19 amino acid residues, between 10 and 18 amino acid residues, between 10 and 17 amino acid residues, between 10 and 16 amino acid residues, between 10 and 15 amino acid residues, between 10 and 14 amino acid residues, between 10 and 13 amino acid residues, between 10 and 12 amino acid residues, between 10 and 11 amino acid residues.

[0312] Cluster of differentiation 47 antigen

[0313] The anti-CD47 antibody or antigen-binding fragment of the present invention can be obtained, for example, by immunizing an animal with the CD47 antigen.

[0314] Alternatively, the anti-CD47 antibody or antigen-binding fragment thereof of the present invention can be obtained from an anti-CD47 antibody library, such as an anti-CD47 single-domain antibody library.

[0315] In one embodiment, the CD47 antigen is the human CD47 protein (e.g., Uniprot accession number Q08722-1, which represents the classical sequence and the entire content is incorporated herein by reference) or a fragment thereof. In some embodiments, the CD47 antigen is a CD47 homolog having a sequence that is at least 80% identical to human CD47 or a fragment thereof. In some embodiments, the CD47 antigen is a CD47 variant having a sequence that is at least 80% identical to human CD47 or a fragment thereof.

[0316] According to the present invention, the CD47 antigen comprises between 10 and 140 amino acid residues, between 10 and 130 amino acid residues, between 10 and 120 amino acid residues, between 10 and 110 amino acid residues, between 10 and 100 amino acid residues, between 10 and 90 amino acid residues, between 10 and 80 amino acid residues, between 10 and 70 amino acid residues, between 10 and 60 amino acid residues, between 10 and 50 amino acid residues, between 10 and 40 amino acid residues, between 10 and 30 amino acid residues, between 10 and 29 amino acid residues, between 10 and 28 amino acid residues, between 10 and 27 amino acid residues, between 10 and 26 amino acid residues, between 10 and 25 amino acid residues, between 10 and 24 amino acid residues, between 10 and 23 amino acid residues, between 10 and 22 amino acid residues, between 10 and 21 amino acid residues, between 10 and 20 amino acid residues, between 10 and 19 amino acid residues, between 10 and 18 amino acid residues, between 10 and 17 amino acid residues, between 10 and 16 amino acid residues, between 10 and 15 amino acid residues, between 10 and 14 amino acid residues, between 10 and 13 amino acid residues, between 10 and 12 amino acid residues, between 10 and 11 amino acid residues of the human CD47 amino acid sequence.

[0317] Exemplary embodiments of the CD47 antigen include the extracellular domain of CD47 or a portion thereof. Exemplary embodiments of the CD47 antigen include the soluble form of the extracellular domain of CD47. An exemplary embodiment of the CD47 antigen is provided in SEQ ID NO:167.

[0318] Antigen-binding domain (Ab)

[0319] As disclosed herein, the binding agent may comprise one or more antigen-binding domains.

[0320] The antigen-binding domains of the present invention can be selected for their ability to bind to a specific target. The antigen-binding domains can also be selected for their in vivo and / or in vitro functional properties or biological effects, including, for example, their ability to regulate cellular processes such as gene expression, signal transduction, cell growth, cell viability, and the like.

[0321] For example, the binding agent of the present invention comprises one or more antigen-binding domains, each independently comprising one or more complementarity-determining regions (CDRs) of an antibody.

[0322] Thus, the specificity of the binding agent of the present invention can be conferred by its antigen-binding domains.

[0323] The binder of the present invention may comprise an antigen-binding domain capable of binding DR2 or cells expressing DR2, an antigen-binding domain capable of binding PD-1 or cells expressing PD-1, and / or an antigen-binding domain capable of binding CD47 or cells expressing CD47.

[0324] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding DR2 or cells expressing DR2, including, for example, antigen-binding domain 1 (ABD1).

[0325] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding PD-1 or cells expressing PD-1, including, for example, antigen-binding domain 2 (ABD2).

[0326] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding CD47 or cells expressing CD47, including, for example, antigen-binding domain 3 (ABD3).

[0327] In some embodiments, the antigen-binding domain targets the same epitope or antigen as ABD1, ABD2, and / or ABD3. In some embodiments, the antigen-binding domain competes with ABD1, ABD2, and / or ABD3 for binding to their respective epitopes or antigens.

[0328] In some embodiments, the antigen-binding domain binds to at least one antigen other than those selected from DR2, PD-1, or CD47.

[0329] In some embodiments, the binder may comprise more than one antigen-binding domain.

[0330] For example, in some embodiments, the binder may comprise two or more antigen-binding domains.

[0331] In some embodiments, the binder may comprise three or more antigen-binding domains.

[0332] In some embodiments, the binder may comprise four or more antigen-binding domains.

[0333] In some embodiments, the binder may comprise five or more antigen-binding domains.

[0334] In some embodiments, the binder may comprise six or more antigen-binding domains.

[0335] In some embodiments, the binder may comprise 1 to 12 antigen-binding domains, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 4 to 11, 4 to 12, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 9 to 10, 9 to 11, 9 to 12, 10 to 11, 10 to 12, or 11 to 12.

[0336] In some embodiments, the binder of the present invention may comprise one or more antigen-binding domains, and at least one of the antigen-binding domains is capable of binding to tumor cells.

[0337] In some embodiments, the binder of the present invention may comprise one or more antigen-binding domains, and at least one of the antigen-binding domains is capable of binding to an immunomodulator. In some embodiments, at least one of the antigen-binding domains is capable of binding to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is PD-1.

[0338] In some embodiments, the binder of the present invention may comprise one or more antigen-binding domains, and at least one of the antigen-binding domains is capable of binding to immune cells. In some embodiments, at least one of the antigen-binding domains is capable of binding to a protein expressed on the surface of immune cells (such as T cells, NK cells, monocytes, macrophages, etc.).

[0339] In some embodiments, the binder of the present invention may be multivalent and may comprise at least one antigen-binding domain that binds to tumor cells and at least one antigen-binding domain that binds to immune cells.

[0340] In some embodiments, the protein expressed on the surface of immune cells is selected from CD47 or CD3.

[0341] In some embodiments, the protein expressed on the surface of immune cells is CD47.

[0342] In some embodiments, the protein expressed at the surface of the immune cell is CD3.

[0343] The antigen-binding domain can be derived from natural antibodies (human or animal origin) or synthetic antibodies.

[0344] In some embodiments, the antigen-binding domain of a natural antibody is engineered to form a single chain.

[0345] In some embodiments, the antigen-binding domain can be obtained from IgG, such as IgG1, IgG2, IgG3 or IgG4. In some specific embodiments, the antigen-binding domain is derived from the human IgG heavy chain.

[0346] In some embodiments, the antigen-binding domain can be obtained from a heavy chain only antibody (HCAb).

[0347] Exemplary embodiments of the antigen-binding domain include, for example and without limitation, single domain antibodies (sdAb), heavy chain variable regions (VH or VHH), light chain variable regions (VL or VLL), single chain variable fragments (scFv), V NAR fragments and combinations thereof.

[0348] In some embodiments, the antigen-binding domain of the binder disclosed herein is VHH.

[0349] In some embodiments, the antigen-binding domain of the binder disclosed herein is VHH, which contains the complementarity determining regions of the VHH disclosed herein.

[0350] According to the present invention, CDRs can be identified using the Kabat numbering scheme (e.g., Kabat, J Immunol., 147:1709-19 (1991); Chothia C, Lesk AM, J Mol Biol. Aug 20; 196(4):901-17 (1987)).

[0351] In some embodiments, the complementarity determining regions (CDRs) of the VHH disclosed herein correspond to Kabat CDRs.

[0352] In some cases, CDR1, CDR2, CDR3 and / or FR2 correspond to Kabat CDR1, CDR2, CDR3 and / or FR2.

[0353] Alternatively, CDRs can be identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

[0354] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to the IMGT CDRs.

[0355] In other instances, CDR1, CDR2, CDR3, and / or FR2 correspond to IMGT CDR1, CDR2, CDR3, and / or FR2.

[0356] In some embodiments, the binder comprises two polypeptide chains, each comprising, from N-terminus to C-terminus: a) an antigen-binding domain comprising an antigen-binding fragment of a single-domain antibody, b) a linker, and c) a dimerization domain.

[0357] In some embodiments, the binder comprises two polypeptide chains, each comprising, from N-terminus to C-terminus: a) an antigen-binding domain of a single-domain antibody, b) a linker, and c) a dimerization domain.

[0358] In some embodiments, the binder comprises two polypeptide chains, each comprising, from N-terminus to C-terminus: a) a dimerization domain, b) a linker, and c) an antigen-binding domain comprising an antigen-binding fragment of a single-domain antibody.

[0359] In some embodiments, the binder comprises two polypeptide chains, each comprising, from N-terminus to C-terminus: a) a dimerization domain, b) a linker, and c) an antigen-binding domain of a single-domain antibody.

[0360] In other embodiments, the binder comprises two polypeptide chains, each comprising, from N-terminus to C-terminus: a) an antigen-binding domain of a single-domain antibody, b) a linker, c) a dimerization domain, d) a linker, and e) an antigen-binding domain of a single-domain antibody. In some embodiments, the antigen-binding domain of a) is different from the antigen-binding domain of e). In some embodiments, the antigen-binding domain of a) is the same as the antigen-binding domain of e). In some embodiments, the linker of b) is the same as the linker of d). In some embodiments, the linker of b) is different from the linker of d).

[0361] According to the invention, the binder may comprise additional amino acid sequences or moieties (small molecules, labels, etc.) at the N-terminus and / or C-terminus. For example, according to the invention, the binder may comprise an additional antigen-binding domain of a single-domain antibody at the N-terminus or C-terminus. The additional antigen-binding domain may be separated from the core by one or more linkers.

[0362] In a particular embodiment, the binder of the invention may comprise an antigen-binding domain VHH derived from human or mouse or rat or from a transgenic mouse or rat (wherein the mouse or rat VHH has been camelized); a human VHH; a human VHH that has been camelized; a VHH of IgG1, IgG2a, IgG2b, IgG2c or IgG3; or a combination thereof. The antibody may be obtained by immunizing a mouse or rat or a transgenic mouse or rat with the antigen of interest, the mouse or rat or transgenic mouse or rat lacking a functional CH1 domain in any of its heavy chains, IgG1, IgG2a, IgG2b, IgG2c or IgG3 or a combination thereof, or a combination of the VHHs described above.

[0363] In a particular embodiment, the polypeptide chain of the invention may comprise an antigen-binding domain of a camelid antibody, such as a VHH of IgG2 or IgG3. The camelid antibody may be obtained by immunizing a dromedary, camel, llama or alpaca with the antigen of interest.

[0364] In some embodiments, the camelid antibody may be derived from so-called Old World camelids, such as the Bactrian camel (Camelus bactrianus), the dromedary camel (Camelus dromaderus), or from New World camelids, such as the alpaca (Lama pacos), the llama (Lama glama) and the vicuña (Lama vicugna).

[0365] In another particular embodiment, the polypeptide chain of the invention may comprise an antigen-binding domain of a chondrichthyan, such as a V NAR fragment. The V NAR fragment may be derived from a shark antibody.

[0366] If desired, the antigen-binding domain of a non-human antibody may be humanized. For example, the framework regions of a non-human VH, VHH or HCAb may be modified to make them more human-like. Humanization of camelid antibodies is described, for example, in Vincke C. et al. (J. Biol. Chem. 2009, 284(5):3273-3284), the entire content of which is incorporated herein by reference. Humanized camelid antibodies may be obtained, for example, by CDR grafting onto a general humanized nanobody scaffold (e.g., h-NbBcII10 FGLA ) disclosed in Vincke C. et al. V NARThe antibody can be humanized by converting non-CDR residues to residues of the human germline Vκ1 sequence DPK9, as discussed by Kovalenko OV et al. (J Biol Chem. 2013, 288: 17408-17419), the entire content of which is incorporated herein by reference. Accordingly, the polypeptide chains of the present invention encompass humanized antigen-binding domains.

[0367] In yet another specific embodiment, the antigen-binding domain can comprise a human VH (modified or unmodified). The human VH can be obtained, for example, from a synthetic human VH library. Modified human VHs include those in which some amino acid residues have been modified to make them more camel-like (i.e., camelized).

[0368] Those skilled in the art will understand that the antigen-binding domain can be incorporated into an antibody, antigen-binding fragment, or antibody-like molecule, including but not limited to single-domain antibodies (sdAbs), VHHs, conventional antibodies or their antigen-binding fragments, bispecific antibodies, single-chain Fv-CH3 (scFv-CH3) fusions, tandem scFv-CH3 (TaFv-CH3) fusions, bifunctional antibody-CH3 (Db-CH3) fusions, tandem Db-CH3 (TaDb-CH3) fusions, single-chain Db-CH3 fusions (scDb-CH3), Fab-CH3 fusions, single-chain Fab-CH3 fusions, Fab-scFv-CH3 fusions, dual-affinity retargeting (DART)-CH3 fusions, Fab-DART-CH3 fusions, single-chain Fv-Fc (scFv-Fc) fusions, tandem scFv-Fc (TaFv-Fc) fusions, bifunctional antibody-Fc (Db-Fc) fusions, tandem Db-Fc (TaDb-Fc) fusions, single-chain Db-Fc fusions (scDb-Fc), Fab-Fc fusions, single-chain Fab-Fc fusions, Fab-scFv-Fc fusions, dual-affinity retargeting (DART)-Fc fusions, Fab-DART-Fc fusions, etc. Such antibody formats can have the native CH3, mutant CH3 domains, native CH2-CH3 domains, or mutant CH2-CH3 domains disclosed herein.

[0369] Exemplary embodiments of ABD1

[0370] Antigen-binding domain 1 (ABD1) can be selected, for example, from antigen-binding domains that bind DR2, some non-limiting illustrative embodiments of which are provided herein.

[0371] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a heavy-chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:1, a heavy-chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:3.

[0372] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO:4, a CDRH2 having the amino acid sequence shown in SEQ ID NO:5, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:6.

[0373] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO:8, a CDRH2 having the amino acid sequence shown in SEQ ID NO:9, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:10.

[0374] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO:11, a CDRH2 having the amino acid sequence shown in SEQ ID NO:12, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:13.

[0375] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO:15, a CDRH2 having the amino acid sequence shown in SEQ ID NO:16, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:17.

[0376] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 18, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 19, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 20.

[0377] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 22, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 23, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 24.

[0378] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 25, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 26, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 27.

[0379] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 29, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 30, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 31.

[0380] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 32, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 33, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 34.

[0381] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 36, CDRH2 having the amino acid sequence shown in SEQ ID NO: 37, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 38.

[0382] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 39, CDRH2 having the amino acid sequence shown in SEQ ID NO: 40, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 41.

[0383] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45.

[0384] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 46, CDRH2 having the amino acid sequence shown in SEQ ID NO: 47, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 48.

[0385] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 50, CDRH2 having the amino acid sequence shown in SEQ ID NO: 51, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 52.

[0386] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO:53, a CDRH2 having the amino acid sequence shown in SEQ ID NO:54, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:55.

[0387] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises the amino acid sequence shown in SEQ ID NO:280, wherein X 1a to X 1k is any (e.g., natural) amino acid residue, or

[0388] wherein X 1a is H or Q, wherein X 1b is L or R, wherein X 1c is F or L, wherein X 1d is A or T, wherein X 1e is V or I, wherein X 1f is R or K, wherein X 1g is D or E, wherein X 1h is R or K, wherein X 1j is V or L, and / or wherein X 1k is A or V.

[0389] QVQLQESGGGLVX 1a PGGSLX 1b LSCAASGX 1c X 1d FSQRAMSW VRQAPGKGLEWX 1e SDIX 1f STGX 1g TSYADSVKGRFTISRDNAX 1h N TX 1j YLQMNSLKPEDTAVYYCALGLWKILPSX 1k RGRGTQVTVSS(SEQ ID NO:280)

[0390] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:7.

[0391] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:7 and CDRs that are identical to the Kabat CDRs of SEQ ID NO:7.

[0392] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:7 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:7.

[0393] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises the amino acid sequence shown in SEQ ID NO:7.

[0394] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:14.

[0395] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:14 and CDRs that are identical to the Kabat CDRs of SEQ ID NO:14.

[0396] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:14 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:14.

[0397] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO:14.

[0398] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:21. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:21.

[0399] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:21 and CDRs that are identical to the Kabat CDRs of SEQ ID NO:21.

[0400] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:21 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:21.

[0401] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO:21.

[0402] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:28. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:28.

[0403] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:28 and CDRs that are identical to the Kabat CDRs of SEQ ID NO:28.

[0404] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 28 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 28.

[0405] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO: 28.

[0406] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 35.

[0407] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 35 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 35.

[0408] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 35 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 35.

[0409] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO: 35.

[0410] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 42.

[0411] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 42 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 42.

[0412] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 42 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 42.

[0413] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO: 42.

[0414] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 49.

[0415] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 49 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 49.

[0416] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 49 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 49.

[0417] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence shown in SEQ ID NO: 49.

[0418] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 56.

[0419] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 56 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 56.

[0420] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 56 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 56.

[0421] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises the amino acid sequence shown in SEQ ID NO: 56.

[0422] Exemplary embodiments of ABD2

[0423] Antigen-binding domain 2 (ABD2) may be selected, for example, from antigen-binding domains that bind to PD-1, and some non-limiting exemplary embodiments thereof are provided herein.

[0424] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 57, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 58, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 59.

[0425] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 60, CDRH2 having the amino acid sequence shown in SEQ ID NO: 61, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 62.

[0426] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 176, CDRH2 having the amino acid sequence shown in SEQ ID NO: 177, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 178.

[0427] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 179, CDRH2 having the amino acid sequence shown in SEQ ID NO: 180, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 181.

[0428] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 183, CDRH2 having the amino acid sequence shown in SEQ ID NO: 184, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 185.

[0429] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 186, CDRH2 having the amino acid sequence shown in SEQ ID NO: 187, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 188.

[0430] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 190, CDRH2 having the amino acid sequence shown in SEQ ID NO: 191, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 192.

[0431] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 193, CDRH2 having the amino acid sequence shown in SEQ ID NO: 194, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 195.

[0432] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 197, CDRH2 having the amino acid sequence shown in SEQ ID NO: 198, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 199.

[0433] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 200, CDRH2 having the amino acid sequence shown in SEQ ID NO: 201, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 202.

[0434] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 204, CDRH2 having the amino acid sequence shown in SEQ ID NO: 205, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 206.

[0435] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 207, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 208, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 209.

[0436] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 211, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 212, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 213.

[0437] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 214, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 215, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 216.

[0438] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 218, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 219, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 220.

[0439] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 221, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 222, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 223.

[0440] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 225, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 226, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 227.

[0441] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 228, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 229, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 230.

[0442] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 232, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 233, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 234.

[0443] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 235, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 236, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 237.

[0444] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 239, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 240, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 241.

[0445] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 242, CDRH2 having the amino acid sequence shown in SEQ ID NO: 243, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 244.

[0446] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 246, CDRH2 having the amino acid sequence shown in SEQ ID NO: 247, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 248.

[0447] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 249, CDRH2 having the amino acid sequence shown in SEQ ID NO: 250, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 251.

[0448] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 253, CDRH2 having the amino acid sequence shown in SEQ ID NO: 254, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 255.

[0449] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence shown in SEQ ID NO: 256, CDRH2 having the amino acid sequence shown in SEQ ID NO: 257, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 258.

[0450] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 260, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 261, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 262.

[0451] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 263, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 264, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 265.

[0452] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence shown in SEQ ID NO: 281, wherein X 1a to X 1k is any (e.g., natural) amino acid residue, or wherein X 1a is L or E, wherein X 1b is A or P, wherein X 1c is D or G, wherein X 1d is N or S, wherein X 1e is S or T, wherein X 1f is K or R, wherein X 1g is D or G, wherein X 1h is K or Q, wherein X 1j is T or A and / or wherein X 1k is L or V.

[0453] QLQESGGGX 1a VQX 1b GX 1c SLRLSCAASGFTFSX 1d YGMSWVR QAPGEGLEWVSSIDSX 1e GGTTX 1f YAX 1g SVX 1h GRFTISRDNAKNX 1j X 1k YLQMNSLKPEDTAVYYCAKDFLSWMPRGQGTQVTVSS (SEQ ID NO: 281)

[0454] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 63.

[0455] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 63 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 63.

[0456] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 63 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 63.

[0457] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence shown in SEQ ID NO: 63.

[0458] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 182.

[0459] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 182.

[0460] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 182.

[0461] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 182.

[0462] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 189.

[0463] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 189.

[0464] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 189.

[0465] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 189.

[0466] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 196. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 196.

[0467] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 196 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 196.

[0468] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 196 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 196.

[0469] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 196.

[0470] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 203. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 203.

[0471] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 203 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 203.

[0472] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 203 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 203.

[0473] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 203.

[0474] In some illustrative embodiments, the binding agent of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 210. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 210.

[0475] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 210 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 210.

[0476] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 210 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 210.

[0477] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 210.

[0478] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 217. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 217.

[0479] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 217 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 217.

[0480] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 217 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 217.

[0481] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 217.

[0482] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 224. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 224.

[0483] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 224 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 224.

[0484] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 224 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 224.

[0485] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence shown in SEQ ID NO: 224.

[0486] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 231. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 231.

[0487] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 231 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 231.

[0488] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 231 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 231.

[0489] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence shown in SEQ ID NO:231.

[0490] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:238. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:238.

[0491] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:238 and CDRs identical to the Kabat CDRs of SEQ ID NO:238.

[0492] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:238 and CDRs identical to the IMGT CDRs of SEQ ID NO:238.

[0493] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence shown in SEQ ID NO:238.

[0494] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:245. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:245.

[0495] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:245 and CDRs identical to the Kabat CDRs of SEQ ID NO:245.

[0496] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 245 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 245.

[0497] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 245.

[0498] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 252. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 252.

[0499] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 252 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 252.

[0500] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 252 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 252.

[0501] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO: 252.

[0502] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 259. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 259.

[0503] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:259 and CDRs identical to the Kabat CDRs of SEQ ID NO:259.

[0504] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:259 and CDRs identical to the IMGT CDRs of SEQ ID NO:259.

[0505] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO:259.

[0506] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:266. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:266.

[0507] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:266 and CDRs identical to the Kabat CDRs of SEQ ID NO:266.

[0508] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:266 and CDRs identical to the IMGT CDRs of SEQ ID NO:266.

[0509] In some exemplary embodiments, the binder of the invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence shown in SEQ ID NO:266.

[0510] Exemplary embodiments of ABD3

[0511] Antigen-binding domain 3 (ABD3) may be selected, for example, from antigen-binding domains that bind CD47, and some non-limiting exemplary embodiments thereof are provided herein.

[0512] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises a heavy-chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 64, a heavy-chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 65, and a heavy-chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 66.

[0513] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises a CDRH1 having the amino acid sequence shown in SEQ ID NO: 67, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 68, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 69.

[0514] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 70.

[0515] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 70 and CDRs that are identical to the Kabat CDRs of SEQ ID NO: 70.

[0516] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 70 and CDRs that are identical to the IMGT CDRs of SEQ ID NO: 70.

[0517] In some exemplary embodiments, the binder of the present invention may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises the amino acid sequence shown in SEQ ID NO: 70.

[0518] Exemplary embodiments of other antigen-binding domains

[0519] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3), and wherein at least one antigen-binding domain binds to a different antigen.

[0520] In some embodiments, the binder may thus comprise an antigen-binding domain that binds CD3. Such antigen-binding domains include those known to those of skill in the art. An illustrative embodiment of an antigen-binding domain that binds CD3 is provided in SEQ ID NO:115.

[0521] Thus, in some embodiments, the binder may comprise an antigen-binding domain that binds DR2, PD-1, and / or CD3. In other embodiments, the binder may comprise an antigen-binding domain that binds DR2 and / or CD3. In still other embodiments, the binder may comprise an antigen-binding domain that binds PD-1 and / or CD3.

[0522] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3), and wherein at least one of the antigen-binding domains is selected from, for example and without limitation, antigen-binding domains that specifically bind to: CD3, CD36, DRD1, DRD2, DRD3, DRD4, DRD5, PD-L1, TROP2, CD147, MCT1, IL1RAP, AMIGO2, PTK7, MCT2, MCT4, NHE1, H+ / K+-ATPase, LAP, HLA-I A2, CD73, CD98, CEACAM5 / 6, ICAM-1, MCSP, fibronectin, β1 integrin, tetraspanin 8, CD164, CD59, CD63, CD44, CD166, cWF, TNF, IL-17A, IL17-F, IL-6R, BCMA, TNF, RANKL, ADAMTS5, VEGF, Ang2, CX3CR1, CXCR4, TfR1 (CD71), CXCR2, CD3, PD1, PDL-1, CTLA-4, CD8, LAG-3, OX40, CD27, CD122 / IL2RB, TLR8 / CD288, TIM-3, ICOS / CD278, NKG2A, A2AR, B7-H3, B7-H4, GITR / TNFRSF18, 4-IBB / CD137, KIR2DL1, KIR3DL2, SIRPα, CD47, VISTA, CD40, CD112, CD96, TOGOT, BTLA, TIGIT, CD4, VEGFR2, CD19, IGFR1, EpCAM, EGFR, DLL3, CGRP, CD79b, CD28, CCR5, ErbB3, ErbB2, TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, IDO1, IDO2, TLR-4, TLR-7, TLR-8, TLR-9, NOX2, or SIGLEC-7.

[0523] polypeptide chain

[0524] The binder of the present invention comprises one or more polypeptide chains.

[0525] The DNA segments encoding the desired polypeptide chain sequences can be synthesized in vitro. Different DNA modules are assembled into a single piece in an organized and directed manner and then cloned into an expression vector. Thus, the resulting polypeptide chain is composed of different modules that form a single chain.

[0526] The polypeptide chains of the present invention include, for example and without limitation, antigen-binding domains, linkers, and dimerization domains that facilitate the assembly of at least two polypeptide chains.

[0527] In some embodiments, the polypeptide chain of the present invention can be monospecific.

[0528] In some embodiments, the polypeptide chain of the present invention can be multispecific.

[0529] In some embodiments, the polypeptide chain of the present invention can be monovalent.

[0530] In some embodiments, the polypeptide chain of the present invention can be multivalent.

[0531] In some cases, the polypeptide chain does not contain a dimerization domain.

[0532] In some cases, the polypeptide chain contains a dimerization domain.

[0533] In some aspects of the present invention, the polypeptide chain can be monospecific.

[0534] An exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising one antigen-binding domain. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but the antigen-binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but the antigen-binding domains have the same CDRs and different framework regions. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising an antigen-binding domain, wherein the amino acid sequences of one or more CDRs of the antigen-binding domain are different (e.g., conservative substitutions in one or more CDRs), without affecting its ability to bind the same antigen or epitope.

[0535] In some aspects of the present invention, the polypeptide chain can be multispecific. The polypeptide chain can encompass, for example, bispecific polypeptide chains, trispecific polypeptide chains, tetraspecific polypeptide chains, pentaspecific polypeptide chains, hexaspecific polypeptide chains, bispecificity polypeptide chains, multispecificity polypeptide chains, etc.

[0536] In an exemplary configuration, one or more antigen-binding domains can be located at the N-terminus, C-terminus, or each side of the dimerization domain.

[0537] In another exemplary configuration, the polypeptide chain can include at least one antigen-binding domain at the N-terminus of the dimerization domain and at least one antigen-binding domain at the C-terminus of the dimerization domain.

[0538] In another exemplary configuration, the polypeptide chain can include one antigen-binding domain at the N-terminus of the dimerization domain and at least two antigen-binding domains at the C-terminus of the dimerization domain.

[0539] In yet another illustrative configuration, the polypeptide chain may include two antigen-binding domains at the N-terminus of the dimerization domain and two antigen-binding domains at the C-terminus of the dimerization domain.

[0540] In some embodiments, the polypeptide chain may be included in Formula I, Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII, etc.

[0541] The polypeptide chain of the present invention includes an antigen-binding domain that is functionally active as a single chain or when part of a binder disclosed herein.

[0542] For example, the antigen-binding domain of the polypeptide chain may bind its target and may have biological activity.

[0543] In some embodiments, the biological activity of the antigen-binding domain includes, for example and without limitation, blocking the binding of the target to its natural receptor or ligand. Alternatively, the biological activity of the antigen-binding domain includes its ability to chelate the target. In addition, the biological activity of the antigen-binding domain includes its ability to induce signal transduction.

[0544] A polypeptide chain comprising more than one antigen-binding domain is characterized as multivalent.

[0545] The polypeptide chain of the present invention may include additional amino acid sequences (defined by X and Y, respectively, in the formulas disclosed herein) at its N-terminus or C-terminus or both termini.

[0546] In some embodiments, the amino acid sequence at the N-terminus (defined by X) may include a signal peptide, an illustrative embodiment of which is provided in SEQ ID NO: 133.

[0547] In some embodiments, the amino acid sequence at the N-terminus (defined by X) or C-terminus (defined by Y) may independently include a linker, a cytokine, a chemokine, a tag (such as a His tag (such as SEQ ID NO: 134)), a masking domain, a phage coat protein, an antigen-binding domain, or a combination thereof.

[0548] An illustrative embodiment of a multispecific polypeptide chain includes a polypeptide chain comprising at least two antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs, thereby resulting in different binding specificities.

[0549] When a polypeptide chain binds to two different epitopes or antigens, it can be more specifically characterized as bispecific. When a polypeptide chain binds to three different epitopes or antigens, it can be characterized as trispecific. When a polypeptide chain binds to four different epitopes or antigens, it can be characterized as tetra-specific. When a polypeptide chain binds to five different epitopes or antigens, it can be characterized as penta-specific. When a polypeptide chain binds to six different epitopes or antigens, it can be characterized as hexa-specific.

[0550] A polypeptide chain containing two antigen-binding domains that bind to two non-overlapping epitopes on the same target is characterized as bispecific. A polypeptide chain containing an antigen-binding domain that binds to three, four, or more epitopes on the same target is characterized as multispecific.

[0551] The antigen-binding domains of a given polypeptide chain will be selected based on the intended use, such as for detection, diagnostic, and / or therapeutic uses. Each of the antigen-binding domains of a specific polypeptide chain can be selected to produce an additive or synergistic effect.

[0552] In some embodiments, the antigen-binding domains can be selected for their ability to specifically bind to proteins involved in a disease or condition.

[0553] For example, the polypeptide chain of the present invention can include at least one antigen-binding domain that specifically binds to an antigen expressed by a tumor cell or the tumor cell environment (i.e., a tumor-specific antigen-binding domain).

[0554] In some other aspects and embodiments of the present invention, the polypeptide chain can include at least one antigen-binding domain that specifically binds to an immunomodulator.

[0555] For example, the polypeptide chain can include one or more antigen-binding domains that bind to an immune checkpoint protein, cytokine, chemokine, or immune receptor or co-receptor, etc. (e.g., an immune-specific antigen-binding domain).

[0556] In some exemplary embodiments, the antigen-binding domain can bind to dopamine receptor D2 (DR2).

[0557] In some exemplary embodiments, the antigen-binding domain can bind to PD-1.

[0558] In some exemplary embodiments, the antigen-binding domain can bind to CD47.

[0559] In one exemplary embodiment, the polypeptide chain of the present invention can include at least one tumor-specific antigen-binding domain and at least one immune-specific antigen-binding domain.

[0560] In some embodiments, the tumor-specific antigen-binding domain can be located at the N-terminus of the dimerization domain.

[0561] In some embodiments, the tumor-specific antigen-binding domain can be located at the C-terminus of the dimerization domain.

[0562] In some embodiments, the tumor-specific antigen-binding domain can be located at both the N-terminus and the C-terminus of the dimerization domain.

[0563] In some embodiments, the multi-immunospecific antigen-binding domain can be located at the N-terminus of the dimerization domain.

[0564] In some embodiments, the immunospecific antigen-binding domain can be located at the C-terminus of the dimerization domain.

[0565] In some embodiments, the immunospecific antigen-binding domain can be located at both the N-terminus and the C-terminus of the dimerization domain.

[0566] In some illustrative and non-limiting embodiments, the polypeptide chain or binder can comprise two immunospecific antigen-binding domains at the C-terminus of the dimerization domain. In some embodiments, the immunospecific antigen-binding domain adjacent to the C-terminal portion of the dimerization domain can be linked via a non-cleavable linker.

[0567] Dimerization domain (DD)

[0568] In some embodiments, the polypeptide chain of the present invention comprises a dimerization domain. Thus, two polypeptide chains can assemble to form a binder. Exemplary embodiments of the binder include homodimers and heterodimers.

[0569] The dimerization domain can comprise, for example and without limitation, the constant region of an immunoglobulin, including, for example, the Fc, CH2, and / or CH3 domains of a heavy-chain immunoglobulin.

[0570] In certain embodiments and aspects of the present invention, the dimerization domain can have a sequence identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or identical to its corresponding CH2 and / or CH3 domain.

[0571] The present invention particularly encompasses a dimerization domain having the same sequence as a native human antibody sequence. Exemplary embodiments of the dimerization domain include, for example, the CH2-CH3 domain of a native human heavy chain antibody.

[0572] Thus, in some embodiments, the dimerization domain comprises the native constant region of an antibody, such as a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, or a native human IgG4 constant region.

[0573] Thus, in some embodiments, the dimerization domain comprises a native CH3 domain.

[0574] In some illustrative embodiments, the dimerization domain comprises a native human CH3 domain.

[0575] In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.

[0576] In some embodiments, the native CH3 domain is a native IgG1 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG1 CH3 (e.g., SEQ ID NO: 116).

[0577] In some embodiments, the native CH3 domain is a native IgG2 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG2 CH3 domain.

[0578] In some embodiments, the native CH3 domain is a native IgG3 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG3 CH3 domain.

[0579] In some embodiments, the native CH3 domain is a native IgG4 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG4 CH3 domain.

[0580] When the two polypeptide chains of the binder are composed of the same amino acid sequence, the binder will form a homodimer. However, co-expression of polypeptide chains having the CH2-CH3 domains of a native antibody but different amino acid sequences may produce a mixture of homodimers and heterodimers. The different binders present in the mixture can be separated by methods known in the art, including, for example, size exclusion chromatography.

[0581] Thus, illustrative heterodimers of the invention include those heterodimers having the CH3 domain or CH2-CH3 domain of a native antibody and formed from two polypeptide chains having different sequences or conformations.

[0582] In some embodiments, the polypeptide chain may have a mutated dimerization domain that contains, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 amino acid substitutions compared to the native or wild-type sequence.

[0583] In some illustrative embodiments, the mutated dimerization domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. The amino acid substitutions may be conservative or non-conservative as outlined in Table 2.

[0584] In some exemplary embodiments, the polypeptide chain may have a mutated dimerization domain having a sequence that is 80% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or to the CH2 and / or CH3 domains. Polypeptide chains encompassed by the present invention include those polypeptide chains comprising a mutated dimerization domain that is 85% to 99% identical, 90% to 99% identical, 95% to 99% identical to a mutated dimerization domain of a native IgG1, IgG2, IgG3, or IgG4 constant region or to the CH2 and / or CH3 domains.

[0585] In some embodiments, the polypeptide chain of the present invention may comprise a mutated dimerization domain that comprises an amino acid substitution that favors heterodimer formation. Thus, heterodimers of the present invention can be formed from polypeptide chains comprising such mutations.

[0586] Thus, in some embodiments, the dimerization domain comprises a mutated constant region of an antibody, such as a mutated human IgG1 constant region, a mutated human IgG2 constant region, a mutated human IgG3 constant region, or a mutated human IgG4 constant region. Compared to the native constant region, the mutated constant region may have one or more amino acid substitutions, amino acid insertions, or amino acid deletions.

[0587] Thus, in some embodiments, the dimerization domain comprises a mutated CH3 domain. Compared to the native CH3 domain, the mutated CH3 domain may have one or more amino acid substitutions, amino acid insertions, or amino acid deletions.

[0588] In some embodiments, the dimerization domain comprises a native CH2 domain and a mutated CH3 domain.

[0589] In some embodiments, the dimerization domain comprises a mutated CH2 domain and a mutated CH3 domain. Compared to the native CH2 domain, the mutated CH2 domain may have one or more amino acid substitutions, amino acid insertions, or amino acid deletions.

[0590] In some embodiments, the mutated CH3 domain is a mutated IgG1 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG1 CH3 domain.

[0591] In some embodiments, the mutated CH3 domain is a mutated IgG2 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG2 CH3 domain.

[0592] In some embodiments, the mutant CH3 domain is a mutant IgG3 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG3 CH3 domain.

[0593] In some embodiments, the mutant CH3 domain is a mutant IgG4 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG4 CH3 domain.

[0594] In some embodiments, the mutant CH2 domain is a mutant IgG1 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG1 CH2 domain.

[0595] In some embodiments, the mutant CH2 domain is a mutant IgG2 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG2 CH2 domain.

[0596] In some embodiments, the mutant CH2 domain is a mutant IgG3 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG3 CH2 domain.

[0597] In some embodiments, the mutant CH2 domain is a mutant IgG4 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG4 CH2 domain.

[0598] In some embodiments, the Fc region can be modified to prevent glycosylation, extend its half-life, modulate receptor binding, or effector function. Exemplary mutations are described in Saunders K.O. (Front. Immunol. 10:1296, 2019, the entire content of which is incorporated herein by reference) and include, for example, mutations at asparagine 297 (e.g., N297).

[0599] In some embodiments, the dimerization domain comprises the Fc region of an antibody or a portion thereof.

[0600] In some embodiments, the dimerization domain comprises the Fc region or a portion thereof, which may or may not include Fc modifications.

[0601] In other embodiments, the dimerization domain comprises an altered or mutant Fc region or a portion thereof. For example, the Fc region or a portion thereof can be altered or mutated to modify one or more characteristics of the binder. In an exemplary embodiment, the Fc region or a portion thereof is not glycosylated. In other exemplary embodiments, the Fc region or a portion thereof comprises one or more of the Fc modifications of Table 1.

[0602] In other illustrative embodiments, the Fc region or a portion thereof is altered or mutated to increase ADCC activity. For example, defucosylation (e.g., N297 according to the EU numbering system) can result in increased binding of FcRγIII on NK cells and can effectively increase ADCC. Thus, in some embodiments, the Fc region or a portion thereof may have a reduced number of fucose residues. In other embodiments, the binding agent comprises a defucosylated Fc region or a portion thereof. In still other embodiments, the Fc region or a portion of the binding agent lacks fucose residues. In additional embodiments, the Fc region or a portion of the binding agent lacks core fucose residues. In other embodiments, the Fc region or a portion of the binding agent completely lacks core fucose residues.

[0603] Illustrative and non-limiting embodiments of mutations in constant regions (e.g., Fc regions) that improve one or more effector functions are encompassed by the present invention, and illustrative embodiments thereof are provided in Table 1 (List of Mutations Antibodies (Basel). November 17, 2020; 9(4):64, the entire content of which is incorporated herein by reference).

[0604] Table 1

[0605]

[0606] According to the present invention, the mutated CH3 domain comprises one or more mutations compared to the native CH3 domain. Thus, in some embodiments, the dimerization domain comprises a mutated CH3 domain that contains one or more mutations compared to the native CH3 domain.

[0607] For example, the mutated CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG1. In another example, the mutated CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG2. In yet another example, the mutated CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG3. In another example, the mutated CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG4.

[0608] According to the present invention, the binding agent can be composed of two different polypeptide chains that associate to form a dimer, herein referred to as a heterodimer.

[0609] The heterodimer can be prepared by co-expressing two different polypeptide chains (e.g., chain A and chain B).

[0610] In some embodiments, the polypeptide chain may comprise a dimerization domain that includes wild-type human CH2 and mutant human CH3 that favors heterodimer formation. In some cases, the polypeptide chain may be capable of forming homodimers when expressed alone or heterodimers (or a mixture of homodimers and heterodimers) when expressed with a complementary chain.

[0611] Accordingly, monomers, heterodimers, and homodimers comprising the CH3 mutations disclosed herein, as well as mixtures of such monomers, heterodimers, and / or homodimers, are encompassed by the present invention.

[0612] In other embodiments, the polypeptide chains disclosed herein may comprise a mutant dimerization domain that includes mutations known in the art that favor heterodimer formation.

[0613] For example, the polypeptide chains of the present invention may comprise the configurations shown in Formulas I, II, III, IIIa, and IIIb, IV, V, VI, VII, or VIII disclosed herein, as well as mutations known in the art that favor heterodimer formation.

[0614] Exemplary embodiments of CH3 mutations are disclosed, for example, in Ha, J-H et al. (Front Immunol, 2016; 7:394) or Godar M et al. (Expert Opinion on Therapeutic patents, 2018; 28(3):251-276), the entire contents of which are incorporated by reference, including, for example, the mortise-tenon (the first CH3 domain mutation T366Y and the second CH3 domain mutation Y407T; the first CH3 domain mutation T366W and the second CH3 domain mutations T366S, L368A, Y407V; or the first CH3 domain mutations S354C, T366W and the second CH3 domain mutations Y349C, T366S, L368A, Y407V), DD / KK mutations (the first CH3 domain mutations K409D, K392D; the second CH3 domain mutations D399K, E356K), asymmetric re-engineering techniques (the first CH3 domain mutations E356K, E357K, D399K and the second CH3 domain mutations K439E, K370E, K409D), BiMAb mutations (the first CH3 domain mutations K249E, K288E, the second CH3 domain mutations E236K, D278K), XmAb mutations (the first CH3 domain mutations S364H, F405A, the second CH3 domain mutations Y349T, T394F), DuoBody mutations (the first CH3 domain mutation F405L, the second CH3 domain mutation K409R), asymmetric mutations (the first CH3 domain mutations T350V, L351Y, S400E, F405A, Y407V, the second CH3 domain mutations T350V, T366L, N390R, K392M, T394W), Biclonics mutations (the first CH3 domain mutation T366K(+L351K), Y349, L368 or the second CH3 domain mutation at Y349+R355 L351D or E or D), ZW1 mutations (the first CH3 domain mutations T350V, L351Y, F405A, Y407V, the second CH3 domain mutations T350V, T366L, K392L, T394W), 7.8.60 mutations (the first CH3 domain mutations K360D, D399M, Y407A, the second CH3 domain mutations E345R, Q347R, T366V, K409V), EW-RVT mutations (the first CH3 domain mutations K360E, K409W and the second CH3 domain mutations Q347R, D399V, F405T), EW-RVTs-s mutations (the first CH3 domain mutations K360E, K409W, Y349C and the second CH3 domain mutations Q347R, D399V, F405T, S354C), SEED mutations (the first CH3 domain mutations, 45 residues derived from IgA on IgG1 CH3 and the second CH3 domain mutations, 57 residues derived from IgG1 on IgA CH3), A107 mutations (the first CH3 domain mutations K370E, K409W, the second CH3 domain mutations E357N, D399V, F405T), etc. Other exemplary embodiments of CH3 mutations are disclosed in International Application No. PCT / CA2020 / 051753 filed on December 18, 2020, which was published as WO2021 / 119832 on June 24, 2021.

[0615] Linker (L)

[0616] The different modules of the polypeptide chains disclosed herein can be associated with each other via a linker.

[0617] In some embodiments, the linker for connecting one or more modules of the polypeptide chain is not a cleavable linker.

[0618] In an exemplary embodiment, the linker (Lc) located adjacent to the C-terminus of the dimerization domain does not contain a cleavable linker.

[0619] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains does not contain a cleavable linker.

[0620] In other embodiments, the linker for connecting one or more modules of the polypeptide chain may include a non-cleavable linker.

[0621] In an exemplary embodiment, the linker located adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.

[0622] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains is a non-cleavable linker.

[0623] In another illustrative embodiment, the linker positioned immediately C-terminal to the dimerization domain and the linker connecting the first two antigen-binding domains located at the C-terminus of the dimerization domain are non-cleavable linkers.

[0624] In some embodiments, the linker immediately N-terminal to the dimerization domain may preferably comprise the hinge region of an antibody.

[0625] In some embodiments, the hinge region is a native hinge region.

[0626] In some embodiments, the native hinge region is a native IgG1 hinge region. In some embodiments, the native hinge region is a native human IgG1 hinge region.

[0627] In some embodiments, the native hinge region is a native IgG2 hinge region. In other embodiments, the native hinge region is a native human IgG2 hinge region.

[0628] In some embodiments, the native hinge region is a native IgG3 hinge region. In other embodiments, the native hinge region is a native human IgG3 hinge region.

[0629] In some embodiments, the native hinge region is a native IgG4 hinge region. In some embodiments, the native hinge region is a native human IgG4 hinge region.

[0630] In some embodiments, the hinge region is a mutated hinge region.

[0631] In some embodiments, the mutated hinge region is a mutated IgG1 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG1 hinge region.

[0632] In some embodiments, the mutated hinge region is a mutated IgG2 hinge region. In other embodiments, the mutated hinge region is a mutated human IgG2 hinge region.

[0633] In some embodiments, the mutated hinge region is a mutated IgG3 hinge region. In other embodiments, the mutated hinge region is a mutated human IgG3 hinge region.

[0634] In some embodiments, the mutated hinge region is a mutated IgG4 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG4 hinge region.

[0635] In some embodiments, all modules of the polypeptide chain are connected via non-cleavable linkers.

[0636] Exemplary embodiments of non-cleavable linkers include linkers that remain substantially intact during protein expression or during preparation. As used herein, "substantially intact" means that linker cleavage occurs in 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the total polypeptide content in a given solution or composition.

[0637] Other exemplary embodiments of non-cleavable linkers also include linkers that do not contain specific cleavage sites for one or more proteases present in human or animal blood or serum.

[0638] Additional exemplary embodiments of non-cleavable linkers further include linkers that remain intact for at least one, two, three, four, five, six, twelve, twenty-four, forty-eight hours or longer after administration of the binder to an individual.

[0639] In other exemplary embodiments, the linker comprises both a non-cleavable linker and a cleavable linker.

[0640] In some embodiments, the linker is non-cleavable.

[0641] In some cases, cleavable linkers can be used to release in vivo a drug (e.g., a cell growth inhibitory molecule, a cytotoxic molecule, a chemotherapeutic agent, etc.) or a label attached to a polypeptide chain of the present invention.

[0642] Exemplary embodiments of cleavable linkers are provided, for example, in US2019 / 0010242, and include linkers that are sensitive to cleavage by proteases, typically extracellular proteases such as those produced by tumors or activated immune effector cells, including linkers having a site specifically cleaved by a protease selected from: ADAMs; ADAMTS (e.g., ADAMS; ADAMS; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5); aspartic proteases, such as BACE or Renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine cathepsins, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine proteases, such as Cruzipain; Legumain; Otubain-2; KLKs, such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteases, such as Meprin; Neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27; serine proteases, such as activated protein C, cathepsin A, cathepsin G, Chymase, coagulation factor proteases (e.g., FVIIa, FIXa, Fxa, FXIa, FXIIa), elastase, granzyme B, Guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA;Type II transmembrane serine proteases (TTSPs), such as DESC1, DPP-4, FAP, Hepsin, Matrilysin-2, Matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof. In some embodiments, the polypeptide chain of the invention does not include such a linker at the position corresponding to Lc.;

[0643] Exemplary embodiments of linkers include flexible linkers, rigid linkers, helical linkers, and combinations thereof. For example, linkers are discussed in Chen X et al. (Adv Drug Deliv Rev. 2013; 65(10):1357-1369), the entire content of which is incorporated herein by reference.

[0644] In some embodiments, the hinge region or a portion thereof can be used to connect a module to a dimerization domain and is considered a linker herein. The hinge region can be derived from a natural antibody (human or animal origin) or a synthetic antibody. The hinge region can be obtained, for example, from IgG, such as IgG1, IgG2, IgG3, or IgG4. Exemplary embodiments of the hinge region are provided in SEQ ID NO:98, SEQ ID NO:121, SEQ ID NO:125, and SEQ ID NO:129.

[0645] In some cases, the hinge region can have one or more amino acid substitutions, amino acid insertions, and / or amino acid deletions compared to the native hinge region. Mutant hinge regions include, for example, sequences that are 80% to 99% identical to the sequence of the native IgG1, IgG2, IgG3, or IgG4 hinge region (mutant hinge regions). Exemplary and non-limiting embodiments of mutant hinge regions include the hinge region of IgG4, wherein S228 is replaced by P (EU numbering) (Angal, S. et al., Mol Immunol 30, 105-108, 1993). Other exemplary embodiments of mutant hinges are provided in SEQ ID NO:118-120, 122-124, 126-128, and 130-132.

[0646] Flexible linkers are typically composed of small polar amino acids, such as threonine or serine and glycine. Exemplary and non-limiting embodiments of flexible linkers include GS linkers (glycine / serine repeats), such as (GGGS) n (GGGGS) m (SEQ ID NO:172), (GS) n , (GGS) n , (GGGS) n (SEQ ID NO:173), (GGGGS) n(SEQ ID NO:104), (GGSG) n (SEQ ID NO:174), (GGGSS) n (SEQ ID NO:175), where n and m can independently be integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, such as 15, 20 or 25.

[0647] Specific exemplary and non-limiting embodiments of the flexible linker include linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103 or SEQ ID NO:104.

[0648] Other exemplary and non-limiting embodiments of the linker include linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO:172, 173, 174 or 175.

[0649] It should be understood that SEQ ID NO:104 can be represented by (GGGGS) n where n is an integer selected from 1 to 10, or alternatively by the formula GGGGSX1, where X1 is absent, or if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO:104.

[0650] It should be understood that SEQ ID NO:172 can be represented by the formula (GGGS) n (GGGGS) m where n and / or m are independently integers selected from 1 to 10, or alternatively by the formula GGGSX1GGGGSX2, where X1 is absent, or if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO:172; and where X2 is absent, or if present, is 1 to 9 repeats of amino acid residues 6 to 10 of SEQ ID NO:172.

[0651] It should be understood that SEQ ID NO:173 can be represented by the formula (GGGS) n where n is an integer selected from 1 to 10, or alternatively by the formula GGGSX1, where X1 is absent, or if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO:173.

[0652] It should be understood that SEQ ID NO:174 can be represented by the formula (GGSG) nis represented, where n is an integer selected from 1 to 10 or alternatively is represented by the formula GGSGX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 174.

[0653] It should be understood that SEQ ID NO: 175 can be represented by the formula (GGGSS) n is represented, where n is an integer selected from 1 to 10 or alternatively is represented by the formula GGGSSX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 175.

[0654] According to the present invention, n can be an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 15, 20 or 25.

[0655] According to the present invention, m can be an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 15, 20 or 25.

[0656] The rigid linker of the present invention is generally composed of a proline-rich sequence (XP) n composed, where X represents any amino acid, preferably Ala, Lys or Glu, and n is an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. (Chen X et al., 2013).

[0657] Specific exemplary and non-limiting embodiments of the rigid linker include linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 or SEQ ID NO: 108.

[0658] It should be understood that SEQ ID NO: 108 can be represented by the formula (X(PAPAP)) n KA is represented, where n is an integer selected from 1 to 10, where X is present or absent and, if present, is A, or alternatively, SEQ ID NO: 108 can be represented by (XPAPAP)X2KA, where X can be present or absent and, if present, is A; and where X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 6 of SEQ ID NO: 108.

[0659] Helical linkers can sometimes be characterized as rigid, but are herein classified into different linker families. Exemplary embodiments of helical linkers are discussed in Chen X et al., 2013 and include, for example, repeat sequences of alanine residues flanked by positively and negatively charged amino acid residues.

[0660] Specific exemplary and non-limiting embodiments of the helical linker include linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112.

[0661] It is understood that SEQ ID NO: 112 can be represented by the formula X(EAAAK) n X2, where n is an integer selected from 1 to 10, more preferably 2 - 5, where X and X2 are independently present or absent, and if present, are preferably A. Alternatively, SEQ ID NO: 112 can be represented by the formula X(EAAAK)X3X2, where X and X2 are independently present or absent, and if present, are preferably A; and X3 is absent, or if present, is 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO: 112.

[0662] In one exemplary embodiment, the linker adjacent to the C-terminus of the dimerization domain (labeled L c1 ) in Formulas II to VIII) can comprise a flexible linker, a rigid linker, or a helical linker. Linkers that can be specifically selected to occupy this position include, for example and without limitation, linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO: 100 - 109, SEQ ID NO: 111, or SEQ ID NO: 112, where n is 1.

[0663] In one exemplary embodiment, the linker connecting the first two antigen-binding domains located at the C-terminus of the dimerization domain (labeled L c2 ) in Formulas II to VIII) can comprise a flexible linker, a rigid linker, or a helical linker. Linkers that can be specifically selected to occupy this position include, for example and without limitation, linkers comprising or consisting of the amino acid sequences shown in SEQ ID NO: 100 - 110 or SEQ ID NO: 112, where n is 1.

[0664] The present invention also provides linkers with the addition of 1 to 10 amino acids (and any range or value within 1 - 10, such as 1 to 5) at one or both of the N-terminus or C-terminus of any one of SEQ ID NO: 100 to 112. Such additional amino acid residues can each independently be selected from any amino acid residue. Such additional amino acid residues preferably form a non-cleavable sequence.

[0665] The present invention also provides linkers having a deletion of 1, 2, 3, 4, or 5 amino acids (and any value within 1 - 5) at one or both of the N-terminus or C-terminus of any one of SEQ ID NO: 100 to 112.

[0666] Suitable linkers can include, for example, amino acid sequences comprising from about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 25, about 3 to about 20, about 3 to about 15, or about 3 to about 10 amino acid residues.

[0667] In exemplary embodiments, the length of each linker can independently range from about 5 to about 50 amino acid residues, including, for example, from about 5 to about 40 amino acid residues, from about 10 to about 40 amino acid residues, from about 20 to about 40 amino acid residues, from about 20 to about 35 amino acid residues, from about 25 to about 30 amino acid residues, and any sub-ranges subsuming and including such ranges.

[0668] In some embodiments, the "n" value in the linker comprising the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:108, or SEQ ID NO:112 is preferably from 1 to 10, more preferably from 2 to 5, including 2, 3, 4, or 5.

[0669] Binding agent

[0670] The binding agents of the present invention encompass, for example, the antigen-binding domains disclosed herein.

[0671] The binding agents of the present invention encompass, for example, the polypeptide chains disclosed herein.

[0672] The binding agents of the present invention encompass, for example, dimers of the polypeptide chains disclosed herein.

[0673] The binding agents of the present invention encompass, for example, multimers of the polypeptide chains disclosed herein.

[0674] The binding agents of the present invention can have the following forms: antibodies or antigen-binding fragments thereof, antibody-like molecules (Fc-fusions, CH3-fusions, etc.), fusions with protein scaffolds, immunocyte modulators, and the like.

[0675] In some embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof.

[0676] In some embodiments, the binding agent comprises an antibody-like molecule.

[0677] In some embodiments, the binding agent can be fused to a protein scaffold.

[0678] In some embodiments, the binding agent comprises an immunocyte modulator.

[0679] Thus, in some embodiments, the binding agent includes antibodies and antigen-binding fragments thereof, such as and without limitation single-domain antibodies from camels or sharks; human antibodies, including IgG (including human IgG1, human IgG2, human IgG3, human IgG4), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD; animal antibodies, including for example IgG (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE and IgD.

[0680] In other embodiments, the binding agent includes antigen-binding fragments, such as and without limitation Fab, Fab', F(ab')2, complementarity-determining regions, variable regions including VH, VHH, VL and the like.

[0681] The binding agent further includes immunocyte modulators, such as bispecific retargeting molecules (DARTs), chimeric antigen receptor (CAR) constructs, bispecific T cell engager constructs (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs) containing scFv or VHH.

[0682] The binding agent further includes fusions with protein scaffolds, including ankyrin repeat proteins, the Z domain of staphylococcal protein-A, type III fibronectin, knottins, etc. Exemplary embodiments of the binding agent include monospecific, bispecific (symmetric or asymmetric), trispecific or multispecific antibodies, as well as monovalent, divalent, trivalent or multivalent antibodies, single-chain FVs (scFVs) and derivatives, such as bispecific antibodies, trispecific antibodies, tetravalent antibodies, tandem di-scFvs, tandem tri-scFvs, scFV-Fc, minibodies (scFV-CH3), tandem bispecific antibodies, di-bispecific antibodies, diabodies, etc., VH or VHH and derivatives, such as tandem bispecific or multispecific VHHs, divalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, divalent CH3 fusions, VHH pentamers, decamers, etc.

[0683] In some embodiments, the binding agent of the present invention may have the forms disclosed in PCT / CA2020 / 051753 (filed on December 18, 2020), such as formula Ia, formula Ib, formula Ic, formula II, formula III, formula IV, formula V, formula VI, formula VII or formula VIII and similar formulas, or formula I, formula II, formula III, formula IIIa and formula IIIb, formula IV, formula V, formula VI, formula VII or formula VIII disclosed herein.

[0684] The binder of the present invention can be formed by the assembly of two polypeptide chains having the same structure (having the same or different amino acid sequences) or having different structures, wherein the same or different structures include the structures shown in Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII disclosed herein.

[0685] The binder of the present invention may comprise, for example, one or more polypeptide chains, and the one or more polypeptide chains independently comprise the amino acid sequence of Formula I in an N-terminal to C-terminal manner:

[0686] X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y

[0687] wherein m is an integer of 0, 1, 2 or greater than 2;

[0688] wherein n is an integer of 0, 1, 2 or greater than 2;

[0689] wherein m and n are not both 0;

[0690] wherein Ab a and Ab d each represent an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3);

[0691] wherein X or Y independently exists or does not exist and comprises an amino acid sequence;

[0692] wherein L b and L c each independently comprise one or more linkers; and

[0693] wherein DD represents a dimerization domain.

[0694] In some embodiments, L b may be absent. In some embodiments, L b2 and / or L b3 may be absent.

[0695] In some embodiments, L c may be absent. In some embodiments, L c1 and L c2 and / or L c3 may be absent.

[0696] In some embodiments, L band L c Both may not exist.

[0697] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0698] In some embodiments, the polypeptide chain comprises two or more antigen-binding domains.

[0699] In some embodiments, the polypeptide chain comprises three or more antigen-binding domains.

[0700] In some embodiments, the polypeptide chain comprises four or more antigen-binding domains.

[0701] In some embodiments, the polypeptide chain comprises five or more antigen-binding domains.

[0702] In some embodiments, the polypeptide chain comprises six or more antigen-binding domains.

[0703] In some embodiments, the polypeptide chain comprises from one to twelve antigen-binding domains.

[0704] In some embodiments, the binder may comprise from one to twelve antigen-binding domains, such as from one to two, from one to three, from one to four, from one to five, from one to six, from one to seven, from one to eight, from one to nine, from one to ten, from one to eleven, from one to twelve, from two to three, from two to four, from two to five, from two to six, from two to seven, from two to eight, from two to nine, from two to ten, from two to eleven, from two to twelve, from three to four, from three to five, from three to six, from three to seven, from three to eight, from three to nine, from three to ten, from three to eleven, from three to twelve, from four to five, from four to six, from four to seven, from four to eight, from four to nine, from four to ten, from four to eleven, from four to twelve, from five to six, from five to seven, from five to eight, from five to nine, from five to ten, from five to eleven, from five to twelve, from six to seven, from six to eight, from six to nine, from six to ten, from six to eleven, from six to twelve, from seven to eight, from seven to nine, from seven to ten, from seven to eleven, from seven to twelve, from eight to nine, from eight to ten, from eight to eleven, from eight to twelve, from nine to ten, from nine to eleven, from nine to twelve, from ten to eleven, from ten to twelve, or from eleven to twelve.

[0705] In some embodiments, the binder comprises a polypeptide chain.

[0706] In some embodiments, the binder comprises two polypeptide chains.

[0707] In some embodiments, the binder comprises three polypeptide chains.

[0708] In some embodiments, the binder comprises four polypeptide chains.

[0709] In some embodiments, the binder comprises five polypeptide chains.

[0710] In some embodiments, the binder comprises six polypeptide chains.

[0711] In some embodiments, the binder comprises seven polypeptide chains.

[0712] In some embodiments, the binder comprises eight polypeptide chains.

[0713] In some embodiments, the binder comprises nine polypeptide chains.

[0714] In some embodiments, the binder comprises ten polypeptide chains.

[0715] In some embodiments, the binder comprises more than ten polypeptide chains.

[0716] In some embodiments, the binder comprises at least one polypeptide chain having Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0717] In some embodiments, the binder comprises at least one polypeptide chain having Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0718] In some embodiments, the binder comprises at least one polypeptide chain having Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0719] In some embodiments, the binder comprises at least one polypeptide chain having Formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0720] In some embodiments, the binder comprises at least one polypeptide chain having Formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0721] In some embodiments, the binder comprises at least one polypeptide chain having Formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0722] In some embodiments, the binder comprises at least one polypeptide chain having Formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VIII).

[0723] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises an amino acid sequence of Formula III in an N-terminal to C-terminal manner

[0724] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0725] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 1 (ABD1).

[0726] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises an amino acid sequence of Formula III in an N-terminal to C-terminal manner

[0727] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0728] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 2 (ABD2).

[0729] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises an amino acid sequence of Formula III in an N-terminal to C-terminal manner

[0730] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0731] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 3 (ABD3).

[0732] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises, in an N-terminal to C-terminal manner, an amino acid sequence of Formula III

[0733] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0734] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 1 (ABD1), one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 2 (ABD2), and one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 3 (ABD3).

[0735] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises, in an N-terminal to C-terminal manner, an amino acid sequence of Formula III

[0736] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0737] wherein Ab a1 represents antigen-binding domain 1 (ABD1), Ab d1 represents antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3).

[0738] In some embodiments, the binder comprises one or more polypeptide chains, each of which independently comprises, in an N-terminal to C-terminal manner, an amino acid sequence of Formula III, wherein Ab a1 is an antigen-binding domain that binds to DR2, Ab d1 is an antigen-binding domain that binds to PD-1, and Ab d2 is an antigen-binding domain that binds to CD47.

[0739] In some embodiments, the binder comprises one or more polypeptide chains, each independently comprising, in an N-terminal to C-terminal manner, an amino acid sequence of Formula III, wherein Ab a1 is an antigen-binding domain that binds to DR2, Ab d1 is an antigen-binding domain that binds to CD47, and Ab d2 is an antigen-binding domain that binds to PD-1.

[0740] In some embodiments, the binder comprises one or more polypeptide chains, each independently comprising, in an N-terminal to C-terminal manner, an amino acid sequence of Formula IIIa:

[0741] X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa).

[0742] In some embodiments, the binder comprises one or more polypeptide chains, each independently comprising, in an N-terminal to C-terminal manner, an amino acid sequence of Formula IIIb:

[0743] X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb).

[0744] In some embodiments, the binder comprises two identical polypeptide chains.

[0745] In some embodiments, the two polypeptide chains of the binder may have the structure shown in Formula II (with the same or different amino acid sequences).

[0746] In some embodiments, the two polypeptide chains of the binder may have the structure shown in Formula III (with the same or different amino acid sequences).

[0747] In some embodiments, the two polypeptide chains of the binder may have the structure shown in Formula IIIa (with the same or different amino acid sequences).

[0748] In some embodiments, the two polypeptide chains of the binder may have the structure shown in Formula IIIb (with the same or different amino acid sequences).

[0749] In some embodiments, one polypeptide chain may have the structure shown in Formula IIIa and one polypeptide chain may have the structure shown in Formula IIIb (antigen-binding domains with the same or different amino acid sequences).

[0750] In some embodiments, one of the polypeptide chains may have the structure described in Formula II, while the other may have the structure shown in Formula III.

[0751] In some embodiments, one of the polypeptide chains may have the structure shown in Formula II, while the other has the structure shown in Formula IV.

[0752] In some embodiments, one of the polypeptide chains may have the structure shown in Formula III, while the other has the structure shown in Formula IV.

[0753] In some embodiments, one of the polypeptide chains may have the structure shown in Formula IV, while the other has the structure shown in Formula IV.

[0754] Multispecific binding agents encompass binding agents that are specific for more than one epitope (epitopes of the same antigen or different antigens) or more than one antigen. For example, a multispecific polypeptide chain or binding agent may thus have more than one antigen-binding domain, where at least two bind different antigens or epitopes.

[0755] The binding agents of the present invention can thus be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc. In some embodiments, each antigen-binding domain may be specific for a given antigen. In some embodiments, two or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, three or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, four or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, five or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, six or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. Specificity can depend on the number of antigen-binding domains present in a given binding agent.

[0756] Exemplary non-limiting embodiments of multispecific binding agents include binding agents composed of multispecific polypeptide chains. Other exemplary non-limiting embodiments of multispecific binding agents include binding agents having two antigen-binding domains of different specificities. Still other exemplary non-limiting embodiments of multispecific binding agents include binding agents having more than two antigen-binding domains that bind two different antigens, proteins, or two different epitopes on the same antigen or protein.

[0757] In some embodiments, the binding agents of the present invention are monovalent or multivalent.

[0758] Exemplary non-limiting embodiments of the multivalent binder include binders composed of multivalent polypeptide chains. Other non-limiting exemplary embodiments of the multivalent binder include binders composed of more than one monovalent polypeptide chain.

[0759] According to the present invention, the bispecific binder can be bivalent or multivalent, depending on the number of antigen-binding domains it contains. Exemplary non-limiting embodiments of the bispecific binder include binders comprising two identical bispecific polypeptide chains that form a dimer.

[0760] Exemplary embodiments of multispecific binding agents

[0761] As indicated herein, the binders of the present invention can be multispecific.

[0762] Exemplary and non-limiting exemplary embodiments of multispecific binders are provided herein.

[0763] For example, in some embodiments, the binder can comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein.

[0764] In other embodiments, the binder can comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding a protein expressed on the surface of an immune cell.

[0765] In still some other embodiments, the binder can comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding a protein expressed on the surface of a tumor cell.

[0766] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 2 (ABD2).

[0767] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 3 (ABD3).

[0768] In some embodiments, the binder comprises at least one antigen-binding domain 2 (ABD2) and at least one antigen-binding domain 3 (ABD3).

[0769] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

[0770] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:56; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:63, SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:70.

[0771] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:70.

[0772] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 14; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0773] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 21; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0774] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 28; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0775] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 35; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0776] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 42; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0777] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 49; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0778] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 56; an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63; and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70.

[0779] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is an antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 7, at least one of the antigen-binding domains is an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and at least one of the antigen-binding domains is an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70.

[0780] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 14, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70.

[0781] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 21, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70.

[0782] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 28, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70.

[0783] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 35, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70.

[0784] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO:42, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO:63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO:70.

[0785] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO:49, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO:63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO:70.

[0786] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO:56, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO:63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO:70.

[0787] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:77. In other embodiments, the binder comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:77. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:77.

[0788] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:78. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:78. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:78.

[0789] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:79. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:79. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:79.

[0790] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:80. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:80. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:80.

[0791] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:81. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:81. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:81.

[0792] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:82. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:82. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:82. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:82.

[0793] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:83. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:83. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:83. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:83.

[0794] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:85. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:85. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:85. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:85.

[0795] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:86. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:86. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:86. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:86.

[0796] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:87. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:87. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:87. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:87.

[0797] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:88. In some embodiments, the binder comprises one polypeptide chain having the amino acid sequence shown in SEQ ID NO:88. In other embodiments, the binder comprises two polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:88. In still other embodiments, the binder comprises two polypeptide chains having the amino acid sequence shown in SEQ ID NO:88.

[0798] Other illustrative embodiments of the multispecific binders are provided herein, including those shown in Table 7.

[0799] In still other embodiments, the anti-PD-1 VHH moiety (SEQ ID NO:63) of KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027, KC028, KC029 or KC030 can be replaced by another anti-PD-1 VHH moiety, such as SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259 or SEQ ID NO:266.

[0800] In some embodiments, the binder of the present invention comprises an antigen-binding domain that binds to DR2, an antigen-binding domain that binds to PD1, and an antigen-binding domain that binds to CD47. In some embodiments, the binder of the present invention may be able to bring T cells close to tumor cells expressing DR2, may be able to restore T cell function and / or may be able to enhance macrophage function by blocking the SIRPα / CD47 interaction. In some embodiments of the present invention, the binder can exert its anti-tumor activity via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0801] Variant

[0802] Variants of the sequences disclosed herein are also encompassed by the present invention.

[0803] Variants covered by the present invention include variants that may include insertions of one or more amino acid residues at one or more positions, deletions of one or more amino acid residues at one or more positions, or substitutions (conservative or non-conservative substitutions) of one or more amino acid residues at one or more positions.

[0804] For example, naturally occurring residues are grouped according to shared side-chain properties. Conservative substitutions can be made by replacing an amino acid from one of the following groups (Group 1 to Group 6) with another amino acid in the same group. Non-conservative substitutions would require replacing a member within one of such groups with a member from another group.

[0805] (Group 1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile)

[0806] (Group 2) Neutral hydrophilic: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln),

[0807] (Group 3) Acidic: aspartic acid (Asp), glutamic acid (Glu)

[0808] (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg)

[0809] (Group 5) Residues affecting chain orientation: glycine (Gly), proline (Pro); and

[0810] (Group 6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe)

[0811] Other exemplary embodiments of conservative substitutions are shown in the "Preferred Substitutions" column in Table 2. If such substitutions result in undesirable properties, more substantial changes can be introduced, named "Exemplary Substitutions" in Table 2, or further described below with reference to amino acid classes, and the products screened.

[0812] Those skilled in the art will recognize that some amino acids are less positively charged, neutral, negatively charged, or have a reduced charge compared to other amino acids. Amino acids can be classified according to the net charge indicated by the isoelectric point of the amino acid. The isoelectric point is the pH value at which the average net charge of an amino acid molecule is zero. When pH > pI, the amino acid has a net negative charge, and when pH < pI, the amino acid has a net positive charge. In some embodiments, the measured pI value of the antibody is between about 3 and 9 (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, and 9) and any value therebetween. In some embodiments, the measured pI value of the antibody is between about 4 and 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) and any value therebetween. Exemplary isoelectric points of amino acids are shown in Table 2 below. Amino acids that typically have a positively charged side chain include, for example, arginine (R), histidine (H), and lysine (K). Amino acids that have a negatively charged side chain include, for example, aspartic acid (D) and glutamic acid (E). Amino acids with polar properties include, for example, serine (S), threonine (T), asparagine (N), glutamine (Q), and cysteine (C), tyrosine (Y), and tryptophan (W). Non-polar amino acids include, for example, alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), glycine (G), and proline (P).

[0813] In some embodiments, the isoelectric point of the antibody is altered via amino acid substitution. See, for example, US20110076275. In some embodiments, adjusting the isoelectric point of the polypeptide that makes up the antibody involves causing a change in the half-life of the antibody.

[0814] Table 2. Exemplary Amino Acid Substitutions

[0815]

[0816]

[0817] Generally, the degree of similarity and identity between variable chains is determined herein using the Blast2 Sequences program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) with default settings, namely the blastp program, the BLOSUM62 matrix (gap opening 11 and gap extension penalty 1; opening × decay 50, expectation 10.0, word size 3) and the activated filter.

[0818] Thus, the percentage of identity will indicate the amino acids that are identical and likely occupy the same or similar positions compared to the original peptide.

[0819] The percentage of similarity will indicate the identical amino acids and the amino acids replaced by conservative amino acid substitutions at the same or similar positions compared to the original peptide.

[0820] Thus, variants of the invention can comprise a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the original or reference sequence or a portion of the original sequence.

[0821] In some embodiments, polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% identical or less than 100% identical to a given amino acid sequence can have amino acid substitutions, additions or deletions, which are generally outside the complementarity determining regions.

[0822] In some embodiments, the variant can have at least 80% sequence identity with the sequences disclosed herein. In other embodiments, the variant can have at least 85% sequence identity with the sequences disclosed herein. In still other embodiments, the variant can have at least 90% sequence identity with the sequences disclosed herein. In other embodiments, the variant can have at least 95% sequence identity with the sequences disclosed herein. In other embodiments, the variant can have at least 99% sequence identity with the sequences disclosed herein.

[0823] Exemplary embodiments of the variants include polypeptide chains or binders that comprise a hinge, Fc, CH3, CH2 / CH3 region derived from a natural antibody but containing one, two, three, four, five, six, seven, eight, nine, ten or more amino acid differences (including amino acid substitutions, insertions or deletions).

[0824] In some embodiments, the polypeptide chain of the invention may thus comprise a mutant hinge region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to the hinge region of a natural antibody.

[0825] In some embodiments, the polypeptide chain of the invention may thus comprise a mutant Fc portion that is at least 80% identical to the Fc of a natural antibody.

[0826] In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH2 domain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to the CH2 domain of a natural antibody.

[0827] In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH3 domain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to the CH3 domain of a natural antibody.

[0828] In some embodiments, the polypeptide chain of the invention may thus comprise a mutant CH2 / CH3 domain that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to the CH2 / CH3 domain of a natural antibody.

[0829] Competitive binding agent

[0830] The binders of the invention also encompass those binders that compete with the binders of Table 6 or Table 7 or with binders having CDRs or variable domains (VHHs) shown in any of Tables 3, 4 or 5.

[0831] The binders of the invention may comprise one or more antigen-binding domains of a single-domain antibody that competes with the binders of Table 6 or Table 7 or with binders having CDRs or variable domains (VHHs) shown in any of Tables 3, 4 or 5.

[0832] In one exemplary embodiment, the competition between the binders (e.g., between single-domain antibodies) can be determined using the DR2 antigen or cells expressing the DR2 antigen or D2 proteoliposomes (expressing the D2 antigen) based on any type of binding assay (FACS, ELISA, SPR, etc.).

[0833] In an exemplary embodiment, competition between the binding agents (e.g., between single-domain antibodies) can be determined using the PD-1 antigen or cells expressing the PD-1 antigen based on any type of binding assay (FACS, ELISA, SPR, etc.).

[0834] In an exemplary embodiment, competition between the binding agents (e.g., between single-domain antibodies) can be determined using the CD47 antigen or cells expressing the CD47 antigen based on any type of binding assay (FACS, ELISA, SPR, etc.).

[0835] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single-domain antibody that is capable of competing with a single-domain antibody comprising an antigen-binding domain 1 (ABD1) as described herein (competitive single-domain antibody).

[0836] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single-domain antibody that is capable of competing with a single-domain antibody comprising an antigen-binding domain 2 (ABD2) as described herein (competitive single-domain antibody).

[0837] In some embodiments, the competitive binding agent comprises the CDRs of a single-domain antibody.

[0838] In some embodiments, the competitive binding agent comprises the variable region of a single-domain antibody.

[0839] In some embodiments, the competitive binding agent is a single-domain antibody or an antigen-binding fragment thereof.

[0840] In some embodiments, the competitive binding agent is an antibody or an antigen-binding fragment thereof.

[0841] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single-domain antibody that is capable of competing with a single-domain antibody comprising an antigen-binding domain 3 (ABD3) as described herein (competitive single-domain antibody).

[0842] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single-domain antibody comprises the CDRH1, CDRH2, and / or CDRH3 amino acid sequences of the anti-DR2 single-domain antibody of Table 3.

[0843] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single-domain antibody comprises the CDRH1, CDRH2, and / or CDRH3 amino acid sequences of the anti-PD-1 single-domain antibody of Table 4.

[0844] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single-domain antibody comprises the CDRH1, CDRH2, and / or CDRH3 amino acid sequences of the anti-CD47 single-domain antibody of Table 5.

[0845] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single-domain antibody comprises a variable region that is at least 80% identical to the variable region of Table 3.

[0846] In some embodiments, the competitive binder may have a K -6 value of ≤ 10 D M for the DR2 antigen.

[0847] In some embodiments, the competitive binder may have a K -6 value of ≤ 10 D M for the PD-1 antigen.

[0848] In some embodiments, the competitive binder may have a K -6 value of ≤ 10 D M for the CD47 antigen.

[0849] In some embodiments, the method comprises administering a competitive binder.

[0850] In some embodiments, the competitive binder is capable of competing with KC001, KC013, KC020, KC021, and / or KC027 for binding to DR2, PD-1, and / or CD47.

[0851] In some embodiments, the method comprises administering a competitive binder that is capable of competing with KC001, KC013, KC020, KC021, and / or KC027 for binding to DR2, PD-1, and / or CD47.

[0852] In one embodiment, the method comprises administering a competitive binder that is capable of competing with KC020 for binding to DR2, PD-1, and / or CD47.

[0853] In some embodiments, the method comprises administering a competitive binder that is capable of competing with KC020 for binding to DR2, PD-1, and / or CD47.

[0854] Nucleic acids, vectors, kits, cells, and methods for preparing polypeptide chains

[0855] The nucleic acid molecules of the invention can be single-stranded or double-stranded. The nucleic acid molecules disclosed herein can comprise deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, or modified ribonucleotides. The nucleic acid molecules of the invention can comprise, for example, DNA.

[0856] The present invention particularly provides DNA segments and vectors encoding one or more modules or an entire polypeptide chain.

[0857] The DNA segments and / or vectors may be provided in separate vials and sold in the form of a kit.

[0858] More specifically, the present invention relates to nucleic acid molecules encoding antigen-binding domains comprising the amino acid sequences shown in any one of SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0859] In some embodiments, the nucleic acid molecules of the present invention may encode binders comprising the amino acid sequences shown in any one of SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0860] In other embodiments, the nucleic acid molecules of the present invention may encode binders comprising the amino acid sequences shown in any one of SEQ ID NO:71 - 75.

[0861] In still other embodiments, the nucleic acid molecules of the present invention may encode binders comprising the amino acid sequences shown in any one of SEQ ID NO:77 - 83 or 85 - 86.

[0862] In exemplary embodiments, the nucleic acid molecule may comprise a sequence that is at least 50% identical to any one of SEQ ID NO:151 - 164 or any one of SEQ ID NO:267 - 279.

[0863] The present invention particularly relates to nucleic acid molecules having codon-optimized sequences. In some embodiments, the codon-optimized sequences may improve the expression of the polypeptide chain in mammalian cells (e.g., including but not limited to human cells).

[0864] In exemplary embodiments, the nucleic acid molecule may comprise a sequence that is at least 50% identical to any one of SEQ ID NO:151 - 164 or any one of SEQ ID NO:267 - 279, and wherein the sequence is codon-optimized.

[0865] The present invention also relates to a vector comprising a nucleic acid molecule disclosed herein. In some embodiments, the vector comprises a codon-optimized sequence and expresses a polypeptide chain comprising an amino acid sequence as shown in any of SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70-75, 77-83, 85-86, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0866] The present invention also relates to a method for preparing a multispecific binder. The method comprises transforming a cell with an expression vector comprising a nucleic acid molecule disclosed herein. In some embodiments, the method is used to prepare a binder by transforming a cell with an expression vector comprising a codon-optimized sequence that allows the expression of a polypeptide chain comprising an amino acid sequence as shown in SEQ ID NO:7, 14, 21, 28, 35, 42, 49, 56, 63, 70-75, 77-83, 85-86, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0867] Due to the inherent degeneracy of the genetic code, DNA sequences encoding the same, substantially the same, or functionally equivalent amino acid sequences can be produced and used. The nucleotide sequences of the present invention can be engineered using methods well known in the art to alter the nucleotide sequences for various purposes, including but not limited to alterations for cloning, processing, and / or expressing gene products. DNA shuffling by random fragmentation of gene fragments and synthetic oligonucleotides and PCR reassembly can be used to engineer nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preferences, generate splice variants, etc. The present invention encompasses codon-optimized nucleic acids encoding the polypeptide chains described herein.

[0868] The polypeptide chains and binders disclosed herein can be prepared by a variety of methods familiar to those skilled in the art, including by recombinant DNA methods or by in vitro transcription / translation.

[0869] Generally, the polypeptide chains described herein are expressed from a nucleic acid sequence inserted into an expression vector, i.e., a vector containing elements for transcriptional and translational control of the inserted coding sequence in a particular host. Such elements can include regulatory sequences such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions.

[0870] A variety of expression vector / host cell systems known to those skilled in the art can be used to express the polypeptide chains described herein. In the case where the binder is composed of different polypeptide chains, each such polypeptide chain can be provided by a separate expression vector or by a unique expression vector. According to the present invention, the two chains of the binder can be encoded by a single vector or by separate vectors (vector sets).

[0871] Polypeptides are generally expressed in mammalian cells. For long-term production of recombinant proteins, stable expression systems can be used, in which the DNA segment is integrated into the host cell genome or maintained episomally by using selectable markers. The host cell type can be selected according to the ability to regulate the expression of the inserted sequence or to process the expressed polypeptide in the desired manner. Different host cells with specific cellular mechanisms and post-translational activity characteristic mechanisms (e.g., CHO, HeLa, MDCK, HEK293, and W138) are commercially available or obtainable from the American Type Culture Collection (ATCC), and can be selected to ensure the correct modification and processing of the expressed polypeptide.

[0872] Other types of expression systems can be used. Such types include, for example, microorganisms, such as bacteria transformed with recombinant phage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; or animal cell systems.

[0873] The present invention thus relates to isolated cells transformed or transfected with a vector, nucleic acid, vector set, or nucleic acid set encoding at least one polypeptide chain described herein. Thus, the present invention also relates to isolated cells capable of expressing the polypeptide chains or binders disclosed herein.

[0874] The present invention also relates to a method for preparing a binder. The method can comprise providing a cell (e.g., a mammalian cell) with a vector or vector set encoding one or more of the polypeptide chains disclosed herein and allowing expression.

[0875] The method can further comprise purifying the binder from the cells or cell debris.

[0876] In some embodiments, the preparation method allows for a purity level of at least 80%, at least 85%, at least 90%, at least 99% (dimer).

[0877] In some embodiments, the purity level of the binder is between 80.0% and 99.9%.

[0878] In some embodiments, the purity level of the binder is between 95.0% and 99.9%.

[0879] In some embodiments, the purity level of the binder is at least 90.0 + / - 5.0%.

[0880] In some embodiments, the purity level of the binder is at least 95.0 + / - 5.0%.

[0881] In some embodiments, the purity level of the binder is equal to or higher than 95%.

[0882] In some embodiments, the purity level of the binder is 97.0 + / - 1.0%.

[0883] In some embodiments, the purity level of the binder is equal to or higher than 97%.

[0884] In some embodiments, the purity level of the binder is 99.0 + / - 1.0%.

[0885] In some embodiments, the purity level of the binder is equal to or higher than 99%.

[0886] In some embodiments, the titer of the binder produced by cells can be 0.1 g / L or higher. In some cases, the titer of the binder produced by cells can be 0.5 g / L or higher. In some cases, the titer of the binder produced by cells can be 1 g / L or higher. In some cases, the titer of the binder produced by cells can be 2 g / L or higher. In some cases, the titer of the binder produced by cells can be 3 g / L or higher. In some cases, the titer of the binder produced by cells can be 4 g / L or higher. In some cases, the titer of the binder produced by cells can be 5 g / L or higher. In some cases, the titer of the binder produced by cells can be 6 g / L or higher. Generally, the homodimer is prepared by transfecting cells with a vector containing a nucleic acid sequence encoding one of the polypeptide chains disclosed herein. The collected supernatant can contain the homodimer or a mixture of monomers and / or homodimers.

[0887] Generally, the heterodimer is prepared by co-transfecting cells with at least two types of vectors (vector set), each of which contains a nucleic acid sequence encoding two different polypeptide chains. The appropriate ratio of chain A to chain B usually depends on the protein expression level obtained from each individual plasmid and can vary, for example, from about 1:10 to about 10:1. For some constructs disclosed herein, a DNA ratio of approximately 1:1 is particularly preferred.

[0888] The heterodimer can also be prepared by transfecting cells with a single vector encoding two polypeptide chains. The collected supernatant can contain the heterodimer or a mixture of monomers, heterodimers, and / or homodimers.

[0889] The method for preparing a polypeptide chain of the present invention may further include the step of separating or isolating monomers, homodimers, and heterodimers from a mixture containing monomers, homodimers, and heterodimers. The homodimers or heterodimers can be purified and separated, for example, by size exclusion chromatography or by means of a tag or by other methods known to those skilled in the art.

[0890] The method may also include the step of separating and / or purifying the binding agent from impurities.

[0891] Thus, the method of the present invention will produce a composition comprising a mixture of homodimers, heterodimers, or monomers, heterodimers, and / or homodimers.

[0892] In some exemplary embodiments, the composition may mainly comprise homodimers. In one exemplary embodiment, the composition may comprise homodimers in a proportion of at least about 80%, at least 85%, at least 90%, at least 99%, or 100%.

[0893] In other exemplary embodiments, the composition may mainly comprise heterodimers. In one exemplary embodiment, the composition may comprise heterodimers in a proportion of at least about 80%, at least 85%, at least 90%, at least 99%, or 100%.

[0894] Conjugate

[0895] The polypeptide chain or binding agent of the present invention can be conjugated, for example, with a therapeutic moiety (for therapeutic purposes) or a detectable moiety (i.e., for detection or diagnostic purposes), or conjugated with a protein that allows for an extended half-life, or attached to a nanoparticle. In some cases, the therapeutic or detectable moiety can be linked to at least one amino acid residue of the polypeptide chain.

[0896] In one exemplary embodiment, the polypeptide chain or binding agent of the present invention is conjugated with a therapeutic moiety such as, but not limited to, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an anticancer drug (e.g., a small molecule), etc.

[0897] The therapeutic moiety may include, for example, and not limited to, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and many other substances recognized by those skilled in the art (e.g., lutetium (e.g., Lu 177 )), bismuth (e.g., Bi 213 )), copper (e.g., Cu 67), 5-fluorouracil, adriamycin, irinotecan, taxanes, pseudomonas endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), maytansinoids (e.g., mertansine), and other toxins.

[0898] In another illustrative embodiment, the polypeptide chain or binder of the invention is conjugated to a detectable moiety, which includes, for example and without limitation, moieties detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, and / or other physical means. The detectable moiety can be coupled to the polypeptide chain or binder directly and / or indirectly (e.g., via linkages such as, without limitation, DOTA or NHS linkages) using methods well known in the art. A variety of detectable moieties can be used, and the selection depends on the desired sensitivity, ease of conjugation, stability requirements, and available instrumentation. Suitable detectable moieties include, but are not limited to, fluorescent labels, radioactive labels (e.g., without limitation 125 I, In 111 、Tc 99 、I 131 , and including positron-emitting isotopes for PET scanners, etc.), nuclear magnetic resonance-active labels, luminescent labels, chemiluminescent labels, chromophore labels, enzyme labels (e.g., without limitation, horseradish peroxidase, alkaline phosphatase, etc.), quantum dots, and / or nanoparticles. The detectable moiety can elicit and / or generate a detectable signal, thereby allowing detection of the signal from the detectable moiety.

[0899] Pharmaceutical composition

[0900] The invention also encompasses pharmaceutical compositions comprising the polypeptide chain or binder of the invention. The pharmaceutical compositions can also comprise a pharmaceutically acceptable carrier.

[0901] In some embodiments, the pharmaceutical composition comprises a conjugated binder as disclosed herein. In some embodiments, the pharmaceutical composition comprises a binder conjugated to a therapeutic moiety. In some embodiments, the pharmaceutical composition comprises a binder conjugated to a detectable label.

[0902] In addition to the active ingredient, the pharmaceutical composition can contain a pharmaceutically acceptable carrier, including water, PBS, saline solutions, gelatin, oils, alcohols, and other excipients, as well as auxiliaries that facilitate processing of the active compound into a pharmaceutically usable preparation. In other cases, such preparations can be sterilized.

[0903] As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of an agent and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. As used herein, "therapeutically effective amount" refers to the amount that provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquid or lyophilized or otherwise dried formulations, and include diluents having various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, additives such as albumin or gelatin to prevent absorption by surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts). Solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent conjugates of polymers such as polyethylene glycol with proteins, chelates with metal ions, or materials incorporated into particulate formulations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or on or incorporated into liposomes, microemulsions, micelles, monolayers or multilayers of vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will affect the physical state, solubility, stability, rate of release in vivo, and rate of clearance in vivo. Controlled-release or sustained-release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). The present invention also encompasses particulate compositions coated with polymers (e.g., poloxamer or poloxamine). Other embodiments of the compositions of the present invention combine particulate forms with protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment, the pharmaceutical composition is administered by parenteral, paracancerous, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, and intratumoral routes.

[0904] In addition, as used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" is known in the art and includes, but is not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol; polyethylene glycol; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, collating agents, inert gases, and the like.

[0905] For any compound, a therapeutically effective dose can initially be estimated in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. Animal models can also be used to determine dosage ranges and routes of administration. Such information can then be used to determine useful dosages and routes for human administration. Such techniques are well known to those of skill in the art, and a therapeutically effective dose refers to the amount of the active ingredient that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by calculating and comparing the ED 50 (therapeutically effective dose in 50% of the population) and LD 50 (lethal dose in 50% of the population) statistics. Any of the above-described pharmaceutical compositions can be applied to any individual in need of treatment, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and especially humans.

[0906] The pharmaceutical compositions described herein can be administered by a variety of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0907] In some embodiments, the pharmaceutical composition is made from a binder having a purity level between 80.0% and 99.9%. Thus, in some embodiments, the pharmaceutical composition contains a binder substantially free of impurities. For example, the pharmaceutical composition contains a binder having a purity of at least 90.0 + / - 5.0%. In other instances, the pharmaceutical composition contains a binder having a purity of at least 95.0 + / - 5.0%. In other embodiments, the pharmaceutical composition contains a binder having a purity of at least 99.0 + / - 1.0%.

[0908] In some embodiments, the pharmaceutical composition comprises a binder that is stable in solution under one or more pressure conditions as described herein.

[0909] In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In other embodiments, the pharmaceutical composition is formulated for intraperitoneal administration.

[0910] In some embodiments, the pharmaceutical composition is formulated at a concentration suitable for administering to a human a dose that is between 0.01 and 150 mg / kg, between 0.01 and 100 mg / kg, between 0.01 and 50 mg / kg, between 0.05 and 150 mg / kg, between 0.05 and 100 mg / kg, between 0.05 and 50 mg / kg, between 0.05 and 30 mg / kg, between 0.05 and 10 mg / kg, between 0.1 and 50 mg / kg, between 0.1 and 30 mg / kg, between 0.1 and 10 mg / kg, or between 1.0 and 10 mg / kg.

[0911] Method of use

[0912] The binder of the present invention can be used to treat a disorder or disease.

[0913] The method of the present invention can be particularly used to treat cancer or delay cancer progression in an individual in need thereof.

[0914] In one embodiment, the method of the present invention comprises administering a pharmaceutical composition comprising means for reducing tumor size and a pharmaceutically acceptable carrier, wherein the means for reducing tumor size comprises means for binding to dopamine receptor D2 (DR2) and / or means for blocking the interaction between PD-1 and PD-L1 and / or means for blocking the interaction between CD47 and the SIRPα carrier.

[0915] In one embodiment, the method of the present invention comprises administering a pharmaceutical composition comprising means for inhibiting tumor growth and a pharmaceutically acceptable carrier, wherein the means for inhibiting tumor growth comprises means for binding to dopamine receptor D2 (DR2) and / or means for blocking the interaction between PD-1 and PD-L1 and / or means for blocking the interaction between CD47 and the SIRPα carrier.

[0916] In one embodiment, the method of the present invention comprises administering a pharmaceutical composition comprising means for inducing tumor regression and a pharmaceutically acceptable carrier, wherein the means for inducing tumor regression comprises means for binding to dopamine receptor D2 (DR2) and / or means for blocking the interaction between PD-1 and PD-L1 and / or means for blocking the interaction between CD47 and the SIRPα carrier.

[0917] Agents comprising one or more antigen-binding domains that specifically bind dopamine receptor D2 (DR2) and / or one or more antigen-binding domains that specifically bind programmed cell death protein 1 (PD-1) and / or one or more antigen-binding domains that specifically bind cluster of differentiation 47 (CD47) can achieve means for reducing tumor size, for inhibiting tumor growth, and / or for inducing tumor regression.

[0918] The amino acid sequence of the antigen-binding domain can correspond to the amino acid sequence of an antibody or an antigen-binding fragment thereof (including a single-domain antibody or an antigen-binding fragment thereof) that binds the desired antigen and / or inhibits the interaction between the antigen and one of its ligands.

[0919] The amino acid sequence of the antigen-binding domain can be the same as the amino acid sequence of the original antibody isolated from a non-human animal (such as a mouse, rat, rabbit, camel), such as a transgenic animal capable of expressing a human, humanized, or camelized antibody. The amino acid sequence of the antigen-binding domain can also be modified by design to make it more human-like or more animal-like. Thus, the amino acid sequence of the antigen-binding domain can comprise a non-humanized antibody, a human antibody, a humanized antibody, or a camelid antibody amino acid sequence.

[0920] In some embodiments, the amino acid sequence of one or more antigen-binding domains of the agent is in a single-chain format.

[0921] In some embodiments, the antigen-binding domain can be from a single-domain antibody or an antigen-binding fragment thereof that is capable of binding D2 and inducing downstream signal transduction. In some embodiments, the antigen-binding domain can be from a single-domain antibody or an antigen-binding fragment thereof that is capable of binding D2 and blocking downstream signal transduction. In some embodiments, the antigen-binding domain can be from a single-domain antibody or an antigen-binding fragment thereof that is capable of downregulating the expression of one or more cancer-related pathways. Downstream D2 signal transduction can be evaluated using, for example and without limitation, the GPCR BRET assay and / or gene expression assays as described herein.

[0922] In some embodiments, the antigen-binding domain can be from a single-domain antibody or an antigen-binding fragment thereof that is capable of blocking the interaction between PD-1 and PD-L1. Thus, the antigen-binding domain can be from a single-domain antibody or an antigen-binding fragment thereof that binds PD-1. Alternatively, the antigen-binding domain can thus be from a single-domain antibody or an antigen-binding fragment thereof that binds PD-L1. The PD-1 / PD-L1 blocking assay can be performed using a reporter system, such as and without limitation, the luminescence reporter system described herein.

[0923] In some embodiments, the antigen binding domain may be from a single domain antibody or antigen binding fragment thereof that is capable of blocking the interaction of CD47 and SIRPα. The antigen binding domain may therefore be from a single domain antibody or antigen binding fragment thereof that binds CD47. Alternatively, the antigen binding domain may therefore be from a single domain antibody or antigen binding fragment thereof that binds SIRPα. The CD47 / SIRPα blocking assay may be performed using a reporter system, such as and not limited to the luminescent reporter system described herein.

[0924] Conventional binding assays include, for example and without limitation, ELISA, flow cytometry, surface plasmon resonance, electrochemiluminescence methods, mesoscale discovery, radioimmunoassay, fluorescence immunoassay, LC-MS detection, thermal shift analysis, biolayer interferometry, etc. Binding assays can be performed between recombinant proteins (or portions), with cell lines expressing the proteins on their surfaces, or with liposomes or nanoparticles comprising the proteins. In general, binding of the binding agent to its target results in the emission or reduction of a detectable signal.

[0925] Binding can be assessed by competition analysis, where candidate binding agents that can inhibit the interaction of previously identified binding agents (with desired binding and / or activity) with their targets (DR2, PD-1 or CD47) can be identified. Alternatively, binding can be assessed in silico using the three-dimensional structure of the antibody / antigen complex (Zhao J. et al., Antibodies 2018, 7, 22; doi10.3390 / antib7030022).

[0926] The methods of the invention involve administering a binding agent disclosed herein to an individual in need thereof.

[0927] In some embodiments, the individual in need thereof has an established tumor.

[0928] In some embodiments, the subject in need is a subject who has previously failed a treatment comprising an anti-PD-1 or anti-PD-L1 antibody. For example, the binding agent can be administered alone or in combination with chemotherapy to a subject who has previously failed treatment with pembrolizumab.

[0929] In some embodiments, the binding agent is multispecific, comprising at least one antigen binding domain capable of binding to dopamine receptor D2 (DR2), at least one antigen binding domain capable of binding to programmed cell death protein 1 (PD-1), and at least one antigen binding domain capable of binding to cluster of differentiation 47 (CD47).

[0930] In some embodiments, the individual in need thereof has a solid tumor.

[0931] In other embodiments, the subject in need thereof has a neuroendocrine solid tumor.

[0932] In other embodiments, the subject in need thereof has cancer.

[0933] In still other embodiments, the subject in need thereof has metastatic cancer.

[0934] In other embodiments, the subject in need thereof has advanced cancer.

[0935] In some embodiments, the cancer comprises tumor cells that express dopamine receptor D2 (DR2).

[0936] In exemplary embodiments, the cancer is neuroendocrine carcinoma, neuroblastoma, gastric cancer, hematogenous cancer, lung cancer, myeloma, prostate cancer, ovarian cancer, breast cancer, rectal cancer, pancreatic cancer, or glioblastoma.

[0937] In some embodiments, the lung cancer is small cell lung cancer.

[0938] In other embodiments, the lung cancer is non-small cell lung cancer.

[0939] In some embodiments, the breast cancer is triple negative breast cancer.

[0940] In some embodiments, the rectal cancer is colorectal cancer.

[0941] In one exemplary embodiment, the subject in need thereof is treated with a binder disclosed herein. In some embodiments, the binder may be conjugated to a therapeutic moiety and used in a treatment method.

[0942] In one exemplary embodiment, the subject in need thereof is treated with a multispecific binder disclosed herein. In some embodiments, the multispecific binder may be conjugated to a therapeutic moiety and used in a treatment method.

[0943] According to the invention, the method comprises administering to a subject in need thereof a multispecific binder comprising at least one antigen-binding domain 1 (ABD1) that binds dopamine receptor D2 (DR2), at least one antigen-binding domain 2 (ABD2) that binds an immunomodulator, and at least one antigen-binding domain 3 (ABD3) that binds an immune cell.

[0944] More specifically, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein ABD1 is an antigen-binding domain that binds to dopamine receptor D2 (DR2), wherein ABD2 is an antigen-binding domain that binds to programmed cell death protein 1 (PD-1), and wherein ABD3 is an antigen-binding domain that binds to cluster of differentiation 47 (CD47).

[0945] In some embodiments, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3),

[0946] wherein ABD1 comprises:

[0947] a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:1, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:2, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:3;

[0948] b. a CDRH1 having the amino acid sequence shown in SEQ ID NO:4, a CDRH2 having the amino acid sequence shown in SEQ ID NO:5, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:6;

[0949] c. a CDRH1 having the amino acid sequence shown in SEQ ID NO:8, a CDRH2 having the amino acid sequence shown in SEQ ID NO:9, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:10;

[0950] d. a CDRH1 having the amino acid sequence shown in SEQ ID NO:11, a CDRH2 having the amino acid sequence shown in SEQ ID NO:12, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:13;

[0951] e. a CDRH1 having the amino acid sequence shown in SEQ ID NO:15, a CDRH2 having the amino acid sequence shown in SEQ ID NO:16, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:17;

[0952] f. CDRH1 having the amino acid sequence shown in SEQ ID NO:18, CDRH2 having the amino acid sequence shown in SEQ ID NO:19, and CDRH3 having the amino acid sequence shown in SEQ ID NO:20;

[0953] g. CDRH1 having the amino acid sequence shown in SEQ ID NO:22, CDRH2 having the amino acid sequence shown in SEQ ID NO:23, and CDRH3 having the amino acid sequence shown in SEQ ID NO:24;

[0954] h. CDRH1 having the amino acid sequence shown in SEQ ID NO:25, CDRH2 having the amino acid sequence shown in SEQ ID NO:26, and CDRH3 having the amino acid sequence shown in SEQ ID NO:27;

[0955] i. CDRH1 having the amino acid sequence shown in SEQ ID NO:29, CDRH2 having the amino acid sequence shown in SEQ ID NO:30, and CDRH3 having the amino acid sequence shown in SEQ ID NO:31;

[0956] j. CDRH1 having the amino acid sequence shown in SEQ ID NO:32, CDRH2 having the amino acid sequence shown in SEQ ID NO:33, and CDRH3 having the amino acid sequence shown in SEQ ID NO:34;

[0957] k. CDRH1 having the amino acid sequence shown in SEQ ID NO:36, CDRH2 having the amino acid sequence shown in SEQ ID NO:37, and CDRH3 having the amino acid sequence shown in SEQ ID NO:38;

[0958] l. CDRH1 having the amino acid sequence shown in SEQ ID NO:39, CDRH2 having the amino acid sequence shown in SEQ ID NO:40, and CDRH3 having the amino acid sequence shown in SEQ ID NO:41;

[0959] m. CDRH1 having the amino acid sequence shown in SEQ ID NO:43, CDRH2 having the amino acid sequence shown in SEQ ID NO:44, and CDRH3 having the amino acid sequence shown in SEQ ID NO:45;

[0960] n. CDRH1 having the amino acid sequence shown in SEQ ID NO:46, CDRH2 having the amino acid sequence shown in SEQ ID NO:47, and CDRH3 having the amino acid sequence shown in SEQ ID NO:48;

[0961] o. CDRH1 having the amino acid sequence shown in SEQ ID NO:50, CDRH2 having the amino acid sequence shown in SEQ ID NO:51, and CDRH3 having the amino acid sequence shown in SEQ ID NO:52;

[0962] p. CDRH1 having the amino acid sequence shown in SEQ ID NO:53, CDRH2 having the amino acid sequence shown in SEQ ID NO:54, and CDRH3 having the amino acid sequence shown in SEQ ID NO:55;

[0963] q. The amino acid sequence shown in SEQ ID NO:280;

[0964] r. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:7;

[0965] s. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:14;

[0966] t. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:21;

[0967] u. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:28;

[0968] v. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:35;

[0969] w. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:42;

[0970] x. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:49; or

[0971] y. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 56.

[0972] Wherein ABD2 comprises:

[0973] a. A heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 57, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 58, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 59;

[0974] b. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 60, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 61, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 62;

[0975] c. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 176, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 177, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 178;

[0976] d. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 179, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 180, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 181;

[0977] e. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 183, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 184, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 185;

[0978] f. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 186, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 187, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 188;

[0979] g. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 190, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 191, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 192;

[0980] h. CDRH1 having the amino acid sequence shown in SEQ ID NO: 193, CDRH2 having the amino acid sequence shown in SEQ ID NO: 194, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 195;

[0981] i. CDRH1 having the amino acid sequence shown in SEQ ID NO: 197, CDRH2 having the amino acid sequence shown in SEQ ID NO: 198, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 199;

[0982] j. CDRH1 having the amino acid sequence shown in SEQ ID NO: 200, CDRH2 having the amino acid sequence shown in SEQ ID NO: 201, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 202;

[0983] k. CDRH1 having the amino acid sequence shown in SEQ ID NO: 204, CDRH2 having the amino acid sequence shown in SEQ ID NO: 205, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 206;

[0984] l. CDRH1 having the amino acid sequence shown in SEQ ID NO: 207, CDRH2 having the amino acid sequence shown in SEQ ID NO: 208, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 209;

[0985] m. CDRH1 having the amino acid sequence shown in SEQ ID NO: 211, CDRH2 having the amino acid sequence shown in SEQ ID NO: 212, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 213;

[0986] n. CDRH1 having the amino acid sequence shown in SEQ ID NO: 214, CDRH2 having the amino acid sequence shown in SEQ ID NO: 215, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 216;

[0987] o. CDRH1 having the amino acid sequence shown in SEQ ID NO: 218, CDRH2 having the amino acid sequence shown in SEQ ID NO: 219, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 220;

[0988] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 221, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 222, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 223;

[0989] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 225, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 226, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 227;

[0990] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230;

[0991] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234;

[0992] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237;

[0993] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241;

[0994] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244;

[0995] CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248;

[0996] x. CDRH1 having the amino acid sequence shown in SEQ ID NO: 249, CDRH2 having the amino acid sequence shown in SEQ ID NO: 250, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 251;

[0997] y. CDRH1 having the amino acid sequence shown in SEQ ID NO: 253, CDRH2 having the amino acid sequence shown in SEQ ID NO: 254, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 255;

[0998] z. CDRH1 having the amino acid sequence shown in SEQ ID NO: 256, CDRH2 having the amino acid sequence shown in SEQ ID NO: 257, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 258;

[0999] aa. CDRH1 having the amino acid sequence shown in SEQ ID NO: 260, CDRH2 having the amino acid sequence shown in SEQ ID NO: 261, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 262;

[1000] bb. CDRH1 having the amino acid sequence shown in SEQ ID NO: 263, CDRH2 having the amino acid sequence shown in SEQ ID NO: 264, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 265;

[1001] cc. The amino acid sequence shown in SEQ ID NO: 281;

[1002] dd. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 63;

[1003] ee. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182;

[1004] ff. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189;

[1005] gg. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 196;

[1006] hh. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 203;

[1007] ii. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 210;

[1008] jj. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 217;

[1009] kk. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 224;

[1010] ll. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 231;

[1011] mm. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 238;

[1012] nn. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 245;

[1013] oo. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 252;

[1014] pp. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 259; or

[1015] qq. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 266.

[1016] Wherein ABD3 comprises:

[1017] a. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 64, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 65, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 66;

[1018] b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or

[1019] c. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1020] In one exemplary embodiment, the method comprises administering to an individual in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1021] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 14, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 70.

[1022] In yet another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 21, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 70.

[1023] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 28, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1024] In one illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 35, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1025] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 42, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1026] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 49, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1027] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 56, wherein the antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 70.

[1028] In some embodiments, the multispecific binder may comprise one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one to two amino acid substitutions or one amino acid substitution, addition in any one or more of the ABD1, ABD2, and / or ABD3 units.

[1029] In other embodiments, the multispecific binder may comprise one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one to two amino acid deletions or one amino acid deletion in any one or more of the ABD1, ABD2, and / or ABD3 units.

[1030] In still other embodiments, the multispecific binder may comprise one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, one to two amino acid additions or one amino acid addition in any one or more of the ABD1, ABD2, and / or ABD3 units.

[1031] In one illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 7.

[1032] In another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 14.

[1033] In another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 21.

[1034] In another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 28.

[1035] In yet another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 35.

[1036] In another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 42.

[1037] In another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 49.

[1038] In yet another illustrative embodiment, the antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 56.

[1039] In one illustrative embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 63.

[1040] In one illustrative embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 182.

[1041] In one illustrative embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 189.

[1042] In one illustrative embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 196.

[1043] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 203.

[1044] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 210.

[1045] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 217.

[1046] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 224.

[1047] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 231.

[1048] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 238.

[1049] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 245.

[1050] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 252.

[1051] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 259.

[1052] In an exemplary embodiment, the antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 266.

[1053] In an illustrative embodiment, the antigen-binding domain 3 (ABD3) may comprise an amino acid sequence having one to ten amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:70. The amino acid substitutions, deletions, and / or additions may be contiguous or non-contiguous.

[1054] In some embodiments, the amino acid substitutions, deletions, and / or additions may be outside of one or more of the CDRs.

[1055] In some embodiments, the amino acid substitutions, deletions, and / or additions may be outside of CDR3.

[1056] In yet another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:56, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1057] In yet another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:7, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1058] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:14, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1059] In one illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:21, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1060] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 28, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1061] In yet another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 35, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1062] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 42, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1063] In another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:49, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1064] In yet another illustrative embodiment, the method comprises administering to an individual in need thereof a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein the antigen-binding domain 1 (ABD1) comprises the amino acid sequence shown in SEQ ID NO:56, wherein the antigen-binding domain 2 (ABD2) comprises the amino acid sequence shown in SEQ ID NO:63, and wherein the antigen-binding domain 3 (ABD3) comprises the amino acid sequence shown in SEQ ID NO:70.

[1065] In some embodiments, in a binder comprising ABD3 (such as a binder comprising ABD1, ABD2, and ABD3) and its methods or uses, the amino acid sequence shown in SEQ ID NO:63 can be replaced with the amino acid sequence shown in SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266.

[1066] In some embodiments, the multispecific binders of the present invention can be used for in vivo targeting of tumors.

[1067] In some embodiments, the polypeptide chains and multispecific binders can be used to promote or induce tumor regression and / or reduce tumor size and / or volume in vivo.

[1068] Regression can be evaluated by measuring the size and / or volume of a tumor or tumor lesion. The size and / or volume of the tumor is generally determined prior to treatment and monitored at least once during or after the treatment phase.

[1069] A decrease in the size and / or volume of the tumor, compared to the initial tumor size and / or volume, can indicate tumor regression.

[1070] In some embodiments, a decrease in the size and / or volume of the tumor occurring at least once (e.g., at a single time point) during the treatment or monitoring phase is considered tumor regression. For example, for the purposes of the present invention, a decrease in the size and / or volume of the tumor, followed by an increase in the size or volume of the tumor, is still considered tumor regression.

[1071] In some embodiments, a decrease in the size and / or volume of the tumor is observed at several time points (continuously or discontinuously) during the treatment or monitoring phase.

[1072] A decrease in tumor size and / or volume of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% compared to the initial tumor size and / or volume is considered tumor regression.

[1073] In some embodiments, the size and / or volume of the tumor can be further decreased by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% compared to one or more prior measurements of the tumor size and / or volume.

[1074] The means for measuring tumor size and / or volume include imaging techniques. In some embodiments, the imaging techniques can involve computer methods.

[1075] In other embodiments, a decrease in the metabolic activity of the tumor (with or without a decrease in size and / or volume) can indicate tumor ...

Claims

1. A method of treating an individual suffering from cancer, the method comprising administering a multispecific binder, wherein the multispecific binder comprises at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2), at least one antigen-binding domain 2 (ABD2) that binds to programmed cell death protein 1 (PD-1), and / or at least one antigen-binding domain 3 (ABD3) that binds to cluster of differentiation 47 (CD47), wherein the method causes a reduction in the size and / or volume of the tumor.

2. The method according to claim 1, wherein the size and / or volume of the tumor is reduced by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to the initial size and / or volume of the tumor.

3. The method according to any one of the preceding claims, wherein the cancer is neuroendocrine cancer, neuroblastoma, gastric cancer, hematogenous cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, myeloma, prostate cancer, ovarian cancer, breast cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, pancreatic cancer, or glioblastoma.

4. A method of treating triple-negative breast cancer in an individual in need thereof, the method comprising administering a multispecific binder, wherein the multispecific binder comprises at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2) and at least one antigen-binding domain 2 (ABD2) that binds to programmed cell death protein 1 (PD-1) and / or at least one antigen-binding domain 3 (ABD3) that binds to cluster of differentiation 47 (CD47).

5. The method according to any one of the preceding claims, wherein the individual has metastatic cancer or advanced cancer.

6. The method according to any one of the preceding claims, wherein the multispecific binder is administered once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, or once every six months.

7. The method according to any one of claims 1 to 6, wherein the multispecific binder is administered at a dose between about 1 mg / kg and about 50 mg / kg.

8. The method according to any one of the preceding claims, wherein the method comprises administering more than 8 doses of the multispecific binder.

9. The method according to any one of the preceding claims, wherein the method comprises administering the multispecific binder until the size and / or volume of one or more tumors or tumor lesions is reduced.

10. The method according to any one of the preceding claims, wherein the method comprises administering the multispecific binder until one or more tumors or tumor lesions show signs of regression.

11. The method according to any one of the preceding claims, wherein the administration causes a decrease in the size and / or volume of one or more tumors or tumor lesions.

12. The method according to any one of the preceding claims, wherein the administration causes the regression of one or more tumors or tumor lesions.

13. The method according to any one of the preceding claims, wherein the multispecific binding agent comprises two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

14. The method according to any one of the preceding claims, wherein ABD1 comprises: a. a heavy-chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 1, a heavy-chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 2, and a heavy-chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 3; b. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 4, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 5, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 6; c. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 8, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 9, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 10; d. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 11, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 12, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 13; e. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 15, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 16, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 17; f. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 18, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 19, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 20; g. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 22, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 23, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 24; h. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 25, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 26, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:

27. i. CDRH1 having the amino acid sequence shown in SEQ ID NO: 29, CDRH2 having the amino acid sequence shown in SEQ ID NO: 30, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 31; j. CDRH1 having the amino acid sequence shown in SEQ ID NO: 32, CDRH2 having the amino acid sequence shown in SEQ ID NO: 33, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence shown in SEQ ID NO: 36, CDRH2 having the amino acid sequence shown in SEQ ID NO: 37, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence shown in SEQ ID NO: 39, CDRH2 having the amino acid sequence shown in SEQ ID NO: 40, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 41; m. CDRH1 having the amino acid sequence shown in SEQ ID NO: 43, CDRH2 having the amino acid sequence shown in SEQ ID NO: 44, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence shown in SEQ ID NO: 46, CDRH2 having the amino acid sequence shown in SEQ ID NO: 47, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence shown in SEQ ID NO: 50, CDRH2 having the amino acid sequence shown in SEQ ID NO: 51, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 52; p. CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55; q. The amino acid sequence shown in SEQ ID NO: 280; r. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 7; s. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 14; t. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 21; u. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 28; v. an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO:35; w. an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO:42; x. an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO:49; or y. an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO:

56.

15. The method according to any one of the preceding claims, wherein ABD2 comprises: a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:57, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:58, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:59; b. a CDRH1 having the amino acid sequence shown in SEQ ID NO:60, a CDRH2 having the amino acid sequence shown in SEQ ID NO:61, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:62; c. a CDRH1 having the amino acid sequence shown in SEQ ID NO:176, a CDRH2 having the amino acid sequence shown in SEQ ID NO:177, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:178; d. a CDRH1 having the amino acid sequence shown in SEQ ID NO:179, a CDRH2 having the amino acid sequence shown in SEQ ID NO:180, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:181; e. a CDRH1 having the amino acid sequence shown in SEQ ID NO:183, a CDRH2 having the amino acid sequence shown in SEQ ID NO:184, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:185; f. a CDRH1 having the amino acid sequence shown in SEQ ID NO:186, a CDRH2 having the amino acid sequence shown in SEQ ID NO:187, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:188; g. a CDRH1 having the amino acid sequence shown in SEQ ID NO:190, a CDRH2 having the amino acid sequence shown in SEQ ID NO:191, and a CDRH3 having the amino acid sequence shown in SEQ ID NO:

192. h. CDRH1 having the amino acid sequence shown in SEQ ID NO: 193, CDRH2 having the amino acid sequence shown in SEQ ID NO: 194, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 195; i. CDRH1 having the amino acid sequence shown in SEQ ID NO: 197, CDRH2 having the amino acid sequence shown in SEQ ID NO: 198, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 199; j. CDRH1 having the amino acid sequence shown in SEQ ID NO: 200, CDRH2 having the amino acid sequence shown in SEQ ID NO: 201, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 202; k. CDRH1 having the amino acid sequence shown in SEQ ID NO: 204, CDRH2 having the amino acid sequence shown in SEQ ID NO: 205, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 206; l. CDRH1 having the amino acid sequence shown in SEQ ID NO: 207, CDRH2 having the amino acid sequence shown in SEQ ID NO: 208, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 209; m. CDRH1 having the amino acid sequence shown in SEQ ID NO: 211, CDRH2 having the amino acid sequence shown in SEQ ID NO: 212, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 213; n. CDRH1 having the amino acid sequence shown in SEQ ID NO: 214, CDRH2 having the amino acid sequence shown in SEQ ID NO: 215, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 216; o. CDRH1 having the amino acid sequence shown in SEQ ID NO: 218, CDRH2 having the amino acid sequence shown in SEQ ID NO: 219, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 220; p. CDRH1 having the amino acid sequence shown in SEQ ID NO: 221, CDRH2 having the amino acid sequence shown in SEQ ID NO: 222, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 223; q. CDRH1 having the amino acid sequence shown in SEQ ID NO: 225, CDRH2 having the amino acid sequence shown in SEQ ID NO: 226, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 227; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 249, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 250, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 251; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 253, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 254, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 255; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 256, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 257, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 258; aa. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 260, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 261, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 262; bb. CDRH1 having the amino acid sequence shown in SEQ ID NO: 263, CDRH2 having the amino acid sequence shown in SEQ ID NO: 264, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 265; cc. the amino acid sequence shown in SEQ ID NO: 281; dd. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 63; ee. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182; ff. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189; gg. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 196; hh. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 203; ii. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 210; jj. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 217; kk. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 224; ll. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 231; mm. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 238; nn. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 245; oo. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 252; pp. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 259; or qq. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:

266.

16. The method according to any one of the preceding claims, wherein ABD3 comprises: a. A heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 64, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 65, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 66; b. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 67, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 68, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 69; or c. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:

70.

17. The method according to any one of the preceding claims, wherein the multispecific binding agent comprises: a. An antigen binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 7, an antigen binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; b. An antigen binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 14, an antigen binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; c. An antigen binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 21, an antigen binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; d. An antigen binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 28, an antigen binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; e. An antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 35, an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; f. An antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 42, an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; g. An antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 49, an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 70; or h. An antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 56, an antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

70.

18. The method according to any one of the preceding claims, wherein the multispecific binding agent comprises: a. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 7, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; b. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 14, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; c. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 21, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; d. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 28, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; e. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 35, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; f. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 42, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; g. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 49, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO: 70; or h. An antigen-binding domain 1 (ABD1) comprising the amino acid sequence shown in SEQ ID NO: 56, an antigen-binding domain 2 (ABD2) comprising the amino acid sequence shown in SEQ ID NO: 63, and an antigen-binding domain 3 (ABD3) comprising the amino acid sequence shown in SEQ ID NO:

70.

19. The method according to any one of claims 13 to 18, wherein the polypeptide chain forms a dimer via a dimerization domain and / or a hinge region, and the dimerization domain comprises a CH3 domain and optionally a CH2 domain.

20. The method according to any one of claims 13 to 19, wherein each of the polypeptide chains independently comprises an amino acid sequence of Formula III in an N-terminal to C-terminal manner X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Wherein Ab a1 , Ab d1 or Ab d2 represents one of antigen binding domains 1 (ABD1), Ab a1 , Ab d1 or Ab d2 represents one of antigen binding domains 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 represents one of antigen binding domains 3 (ABD3); wherein X or Y independently is present or absent and comprises an amino acid sequence; wherein L b1 and L c1 and L c2 each independently comprises one or more linkers; and wherein DD represents a dimerization domain.

21. The method according to any one of claims 13 to 19, wherein each of the polypeptide chains independently comprises an amino acid sequence of Formula III in an N-terminal to C-terminal manner X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Among them, Ab a1 represents the antigen-binding domain 1 (ABD1), Ab d1 represents the antigen-binding domain 2 (ABD2), and Ab d2 represents the antigen-binding domain 3 (ABD3); wherein X or Y independently is present or absent and comprises an amino acid sequence; wherein L b1 and L c1 and L c2 each independently includes one or more linkers; and wherein DD represents a dimerization domain.

22. The method according to any one of claims 19 to 21, wherein the dimerization domain is the CH2-CH3 domain of a native human antibody heavy chain.

23. The method according to any one of claims 191 to 22, wherein the two polypeptide chains are the same and assemble to form a homodimer.

24. The method according to any one of claims 19 to 22, wherein the two polypeptide chains are different and assemble to form a heterodimer.

25. The method according to any one of the preceding claims, wherein the multispecific binder comprises two identical polypeptide chains, and wherein each said polypeptide chain comprises: a. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:77; b. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:78; c. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:79; d. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:80; e. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:81; f. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:82; g. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:83; h. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:85; or i. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

86.

26. The method according to any one of the preceding claims, wherein the multispecific binder is humanized.

27. The method according to any one of claims 13 to 26, wherein the polypeptide chain comprises amino acid substitutions, additions or deletions outside the complementarity determining regions.

28. The method according to any one of the preceding claims, wherein the multispecific binder comprises two polypeptide chains, each polypeptide chain comprising respectively: a. the amino acid sequence shown in SEQ ID NO:77; b. the amino acid sequence shown in SEQ ID NO:78; c. the amino acid sequence shown in SEQ ID NO:79; d. the amino acid sequence shown in SEQ ID NO:80; e. the amino acid sequence shown in SEQ ID NO:81; f. the amino acid sequence shown in SEQ ID NO:82; g. the amino acid sequence shown in SEQ ID NO:83; h. the amino acid sequence shown in SEQ ID NO:85; or i. the amino acid sequence shown in SEQ ID NO:

86.

29. The method according to any one of the preceding claims, wherein the multispecific binder or the antigen-binding domain of the multispecific binder specifically binds to human DR2.

30. The method according to any one of the preceding claims, wherein the multispecific binder or the antigen-binding domain of the multispecific binder specifically binds to human PD-1.

31. The method according to any one of the preceding claims, wherein the multispecific binder or the antigen-binding domain of the multispecific binder specifically binds to human CD47.

32. The method according to any one of the preceding claims, wherein the multispecific binder is conjugated to a therapeutic moiety, a detectable moiety or a protein that allows for an extended half-life or attached to a nanoparticle.

33. A binder comprising one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises: a. a heavy-chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 176, a heavy-chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 177, and a heavy-chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 178; b. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 179, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 180, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 181; c. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 183, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 184, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 185; d. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 186, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 187, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 188; e. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 190, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 191, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 192; f. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 193, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 194, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 195; g. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 197, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 198, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 199; h. CDRH1 having the amino acid sequence shown in SEQ ID NO: 200, CDRH2 having the amino acid sequence shown in SEQ ID NO: 201, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 202; i. CDRH1 having the amino acid sequence shown in SEQ ID NO: 204, CDRH2 having the amino acid sequence shown in SEQ ID NO: 205, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 206; j. CDRH1 having the amino acid sequence shown in SEQ ID NO: 207, CDRH2 having the amino acid sequence shown in SEQ ID NO: 208, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 209; k. CDRH1 having the amino acid sequence shown in SEQ ID NO: 211, CDRH2 having the amino acid sequence shown in SEQ ID NO: 212, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 213; l. CDRH1 having the amino acid sequence shown in SEQ ID NO: 214, CDRH2 having the amino acid sequence shown in SEQ ID NO: 215, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 216; m. CDRH1 having the amino acid sequence shown in SEQ ID NO: 218, CDRH2 having the amino acid sequence shown in SEQ ID NO: 219, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 220; n. CDRH1 having the amino acid sequence shown in SEQ ID NO: 221, CDRH2 having the amino acid sequence shown in SEQ ID NO: 222, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 223; o. CDRH1 having the amino acid sequence shown in SEQ ID NO: 225, CDRH2 having the amino acid sequence shown in SEQ ID NO: 226, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 227; p. CDRH1 having the amino acid sequence shown in SEQ ID NO: 228, CDRH2 having the amino acid sequence shown in SEQ ID NO: 229, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 230; q. CDRH1 having the amino acid sequence shown in SEQ ID NO: 232, CDRH2 having the amino acid sequence shown in SEQ ID NO: 233, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 234; r. CDRH1 having the amino acid sequence shown in SEQ ID NO: 235, CDRH2 having the amino acid sequence shown in SEQ ID NO: 236, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 237; s. CDRH1 having the amino acid sequence shown in SEQ ID NO: 239, CDRH2 having the amino acid sequence shown in SEQ ID NO: 240, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 241; t. CDRH1 having the amino acid sequence shown in SEQ ID NO: 242, CDRH2 having the amino acid sequence shown in SEQ ID NO: 243, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 244; u. CDRH1 having the amino acid sequence shown in SEQ ID NO: 246, CDRH2 having the amino acid sequence shown in SEQ ID NO: 247, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 248; v. CDRH1 having the amino acid sequence shown in SEQ ID NO: 249, CDRH2 having the amino acid sequence shown in SEQ ID NO: 250, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 251; w. CDRH1 having the amino acid sequence shown in SEQ ID NO: 253, CDRH2 having the amino acid sequence shown in SEQ ID NO: 254, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 255; x. CDRH1 having the amino acid sequence shown in SEQ ID NO: 256, CDRH2 having the amino acid sequence shown in SEQ ID NO: 257, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 258; y. CDRH1 having the amino acid sequence shown in SEQ ID NO: 260, CDRH2 having the amino acid sequence shown in SEQ ID NO: 261, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 262; z. CDRH1 having the amino acid sequence shown in SEQ ID NO: 263, CDRH2 having the amino acid sequence shown in SEQ ID NO: 264, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 265; aa. The amino acid sequence shown in SEQ ID NO: 281; bb. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 182; cc. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 189; dd. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 196; ee. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 203; ff. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 210; gg. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 217; hh. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 224; ii. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 231; jj. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 238; kk. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 245; ll. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 252; mm. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 259; or nn. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO:

266.

34. The binder according to claim 33, wherein the binder further comprises one or more antigen-binding domains 1 (ABD1), and the antigen-binding domain 1 comprises; a. A heavy-chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 1, a heavy-chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 2, and a heavy-chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 3; b. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 4, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 5, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 6; c. A CDRH1 having the amino acid sequence shown in SEQ ID NO: 8, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 9, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 10; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 11, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 13; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 29, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 31; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 39, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 41; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence shown in SEQ ID NO: 46, CDRH2 having the amino acid sequence shown in SEQ ID NO: 47, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence shown in SEQ ID NO: 50, CDRH2 having the amino acid sequence shown in SEQ ID NO: 51, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 52; p. CDRH1 having the amino acid sequence shown in SEQ ID NO: 53, CDRH2 having the amino acid sequence shown in SEQ ID NO: 54, and CDRH3 having the amino acid sequence shown in SEQ ID NO: 55; q. The amino acid sequence shown in SEQ ID NO: 280; r. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 7; s. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 14; t. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 21; u. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 28; v. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 35; w. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 42; x. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO: 49; or y. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:

56.

35. The binder according to claim 36 or 37, wherein the binder further comprises one or more antigen-binding domains 3 (ABD3), and wherein the antigen-binding domain 3 comprises: a. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 64, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 65, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 66; b. a CDRH1 having the amino acid sequence shown in SEQ ID NO: 67, a CDRH2 having the amino acid sequence shown in SEQ ID NO: 68, and a CDRH3 having the amino acid sequence shown in SEQ ID NO: 69; or c. an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence shown in SEQ ID NO:

70.

36. The binder according to any one of claims 33 to 35, wherein the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, and each polypeptide chain comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

37. The binder according to claim 36, wherein the polypeptide chains form a dimer via a dimerization domain and / or a hinge region, and the dimerization domain comprises a CH3 domain and optionally a CH2 domain.

38. The binder according to claim 36 or 37, wherein each of the polypeptide chains independently comprises, in an N-terminal to C-terminal manner, the amino acid sequence of formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Wherein Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 1 (ABD1), Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 3 (ABD3); wherein X or Y is independently present or absent and comprises an amino acid sequence; wherein L b1 and L c1 and L c2 each independently includes one or more linkers; and wherein DD represents a dimerization domain.

39. The binder according to claim 36 or 37, wherein each of the polypeptide chains independently comprises, in an N-terminal to C-terminal manner, the amino acid sequence of formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Among them, Ab a1 represents the antigen-binding domain 1 (ABD1), Ab d1 represents the antigen-binding domain 2 (ABD2), and Ab d2 represents the antigen-binding domain 3 (ABD3); wherein X or Y is independently present or absent and comprises an amino acid sequence, wherein L b1 and L c1 and L c2 each independently includes one or more linkers; and wherein DD represents a dimerization domain.

40. The binder according to any one of claims 36 to 39, wherein the dimerization domain is the CH2-CH3 domain of a native human antibody heavy chain.

41. The binder according to any one of claims 36 to 40, wherein the two polypeptide chains are identical and assemble to form a homodimer.

42. The binder according to any one of claims 36 to 40, wherein the two polypeptide chains are identical and assemble to form a heterodimer.

43. A pharmaceutical composition comprising the binder according to any one of claims 36 to 42.

44. A nucleic acid molecule encoding an antigen-binding domain comprising the amino acid sequence shown in any one of SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 or 266, wherein the nucleic acid sequence is at least 50% identical to the nucleic acid sequence shown in any one of SEQ ID NO: 141 - 150 or 267 - 279.

45. The nucleic acid molecule according to claim 44, comprising a nucleic acid sequence that is at least 50% identical to any one of SEQ ID NO: 151 - 164.

46. The nucleic acid molecule according to claim 44 or 45, wherein the nucleic acid sequence encodes a binder having the amino acid sequence shown in any one of SEQ ID NO: 71 - 75.

47. The nucleic acid molecule according to claim 44 or 45, wherein the nucleic acid sequence encodes a binder having an amino acid sequence shown in any one of SEQ ID NOs: 77-83 or 85-86.

48. The nucleic acid molecule according to any one of claims 44 to 47, wherein the nucleic acid sequence is codon-optimized.

49. A vector comprising the nucleic acid molecule according to any one of claims 44 to 48.

50. A method for preparing a multispecific binder, the method comprising transforming a cell with an expression vector comprising the nucleic acid molecule according to any one of claims 44 to 48.

51. The method according to claim 50, which comprises isolating and / or purifying the multispecific binder.

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