Multi-specific constructs and uses thereof

Abstract

Bispecific and multispecific antibodies that target claudin 6 (CLDN6) and 4-1BB are provided. In the absence of cells expressing CLDN6, these antibodies can bind to 4-1BB, but cannot activate 4-1BB signaling. However, in the presence of cells expressing CLDN6, these antibodies can trigger CLDN6-dependent 4-1BB signaling, resulting in an effective immune response to tumor cells expressing CLDN6.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to international application No. PCT / CN2022 / 070870 filed on January 9, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to multispecific molecules, such as anti-CLDN6 / anti-4-1BB bispecific antibodies, and uses thereof, including the treatment of diseases or disorders. Background Art

[0004] Claudin is a class of proteins that form important components of cell tight junctions. Claudin-6 (CLDN6) is a four-spanning membrane protein involved in the formation of tight junctions. In normal adult tissues, CLDN6 mRNA and protein are absent. On the other hand, high CLDN6 transcript levels are frequently detected in a variety of human solid cancers (such as testicular cancer, ovarian cancer, uterine cancer, and lung adenocarcinoma). It is reported that, consistent with the mRNA level, CLDN6 protein is high and uniform in these human cancers.

[0005] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF receptor superfamily (TNFRSF) and is a co-stimulatory molecule that is expressed upon activation of immune cells (innate and adaptive immune cells). 4-1BB plays an important role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Many other studies have shown that activating 4-1BB enhances immune responses to eliminate tumors in mice. Therefore, 4-1BB is a promising target molecule in cancer immunology.

[0006] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. Summary of the invention

[0007] On the one hand, a multispecific construct is provided herein, comprising: a first antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB.

[0008] In some embodiments, a multispecific construct is provided herein, comprising a first antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, after the first antibody portion binds to the tumor antigen, the second antibody portion activates 4-1BB by at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times or 1000 times, including any range between these values. In some embodiments, in the absence of binding to a tumor antigen, the multispecific construct does activate 4-1BB signaling. In some embodiments, in the absence of binding to a tumor antigen, the second portion does not activate 4-1BB signaling.

[0009] In some embodiments, a multispecific construct is provided, comprising a) a first antibody portion that specifically binds to claudin-6 ("CLDN6"); and b) a second antibody portion that specifically binds to 4-1BB. In some embodiments, the first antibody portion comprises a full-length antibody comprising two heavy chains and two light chains.

[0010] Provided herein are multispecific constructs comprising: (1) a first antibody portion that specifically binds to claudin-6 ("CLDN6"); and (2) a second antibody portion that specifically binds to 4-1BB.

[0011] In some embodiments, the first antibody portion is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb. In some embodiments, the first antibody portion comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID No: 8. In some embodiments, the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises: (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) the VL comprises: (i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO:7 or a variant thereof, said variant comprising at least about 80% sequence identity with SEQ ID NO:7; and / or (2) the VL comprises the amino acid sequence of SEQ ID NO:8 or a variant thereof, said variant comprising at least about 80% sequence identity with SEQ ID NO:8. In some embodiments, the first antibody portion comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within the heavy chain variable region (VH), wherein the heavy chain variable region comprises the sequence shown in SEQ ID No: 18; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within the light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 19.In some embodiments, the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises: (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and (2) the VL comprises: (i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and (iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, the variant having at least about 80% sequence identity with SEQ ID NO: 18; and / or (2) the VL comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof, the variant having at least about 80% sequence identity with SEQ ID NO: 19. In some embodiments, binding of the first antibody portion to CLDN6 triggers activation of 4-1BB by the second antibody portion.

[0012] In some embodiments, the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the second antibody portion is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the second antibody portion is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomer variable antibody domain, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3, and the single monomer variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the sdAb comprises: (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the second antibody portion includes the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27.

[0013] In some embodiments, the multispecific construct is a bispecific antibody or bispecific binding fragment. In some embodiments, (1) the first antibody portion comprises: (a) HC-CDR1, HC-CDR2 and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7; (b) LC-CDR1, LC-CDR2 and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID No: 8, and (2) the second antibody portion comprises sdAb-CDR1, sdAb-CDR2 and sdAb CDR3, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain, the single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO: 27.

[0014] In some embodiments, a pharmaceutical composition is provided that comprises a multispecific construct described herein and a pharmaceutically acceptable carrier.

[0015] In some embodiments, a nucleic acid is provided that encodes a multispecific construct as described herein. In some embodiments, a vector is provided that comprises such a nucleic acid. In some embodiments, a host cell is provided that comprises such a nucleic acid or such a vector.

[0016] Also provided herein is a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific construct as described herein or a pharmaceutical composition as described herein. Also provided herein is the use of a multispecific construct as described herein in the preparation of a medicament for treating a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is cancer.

[0017] In some embodiments, a multispecific construct is provided, comprising: (1) a first antibody portion that specifically binds to a tumor antigen; and (2) a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, after the first antibody portion binds to the tumor antigen, the activation of 4-1BB by the second antibody portion is enhanced by at least 10 times. In some embodiments, the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the second antibody portion is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomer variable antibody domain, which comprises the amino acid sequences of CDR1, CDR2, and CDR3, respectively, and the single monomer variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the sdAb comprises: (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the tumor antigen is CLDN6.

[0018] Also provided herein are multispecific constructs comprising two heavy chain components and two light chain components, wherein: (a) each heavy chain component comprises the sequence shown in SEQ ID NO:28 and / or SEQ ID NO:44, and each light chain component comprises the sequence shown in SEQ ID NO:29; (b) each heavy chain component comprises the sequence shown in SEQ ID NO:30, and each light chain component comprises the sequence shown in SEQ ID NO:31; (c) each heavy chain component comprises the sequence shown in SEQ ID NO:32, and each light chain component comprises the sequence shown in SEQ ID NO:33; (d) each heavy chain component comprises the sequence shown in SEQ ID NO:34, and each light chain component comprises the sequence shown in SEQ ID NO:35; (e) each heavy chain component comprises the sequence shown in SEQ ID NO:36, and each light chain component comprises the sequence shown in SEQ ID NO:37; or (f) each heavy chain component comprises the sequence shown in SEQ ID NO:38, and each light chain component comprises the sequence shown in SEQ ID NO:39.

[0019] The present application provides kits comprising a multispecific construct described herein and / or a medicament described herein and a package insert or instructions for using the multispecific construct (or pharmaceutical composition) to treat a disease or disorder (eg, cancer).

[0020] It will be appreciated that one, some or all of the characteristics in the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A-1B The binding affinity of the CLDN6 x 4-1BB bispecific antibody (BsAb) disclosed herein to CLDN6 is illustrated.

[0022] Figure 2A-2C The binding affinity of the CLDN6 X 4-1BB BsAb disclosed herein to cells expressing CLDN6 is demonstrated.

[0023] Figure 3A-3D The binding affinity of the CLDN6 x 4-1BB BsAb disclosed herein to 4-1BB is illustrated.

[0024] Figure 4A-4B Binding of the CLDN6 x 4-1BB BsAb disclosed herein to soluble 4-1BB and cells expressing 4-1BB is demonstrated.

[0025] Figure 5A-5D CLDN6-dependent 4-1BB activation of the CLDN6 x 4-1BB BsAbs disclosed herein is demonstrated.

[0026] Figure 6A-6H Activation of PBMCs by the CLDN6 X 4-1BB BsAb disclosed herein is demonstrated.

[0027] Figure 7A-7B Tumor inhibition in hu4-1BB mice bearing CT26 murine colorectal cancer tumors following treatment with the CLDN6 X 4-1BB BsAb disclosed herein is demonstrated.

[0028] FIG. 8 illustrates liver function after CLDN6X4-1BB BsAb treatment of the present application.

[0029] Figure 9A-9B Tumor inhibition in hu4-1BB mice bearing MC38 murine colon adenocarcinoma tumors following treatment with the CLDN6 X 4-1BB BsAb disclosed herein is demonstrated. DETAILED DESCRIPTION

[0030] definition

[0031] The term "antibody" is used in its broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0032] Full-length antibodies contain two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. The variable regions in the two chains generally contain three highly variable loops, called complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of antibodies and antigen-binding fragments disclosed herein can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are located between flanking extensions called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0033] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized bispecific antibody (ds diabody), a single-chain Fv (scFv), an scFv dimer (a bivalent bispecific antibody), a multispecific antibody formed by a portion of an antibody comprising one or more CDRs, a single domain antibody (e.g., a camelized single domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which a parent antibody or parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted to a framework region from one or more different human antibodies.

[0034] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0035] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev.Comp.Immunol., 27:55-77 (2003); and Honegger and Plückthun, J.Mol.Biol., 309:657-670 (2001) describe, wherein the definition includes overlap or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of an antibody or a transplanted antibody or its variants is intended to fall within the scope of the term as defined and used herein. The amino acid residues covering the CDR as defined in each of the references cited above are listed in Table 1 below, as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0036] Table 1: CDR Definition

[0037]

[0038] 1 The residue numbering follows the nomenclature of Kabat et al. (supra).

[0039] 2The residue numbering follows the nomenclature of Chothia et al. (supra).

[0040] 3 The residue numbering follows the nomenclature of MacCallum et al. (supra).

[0041] 4 The residue numbering follows the nomenclature of Lefranc et al. (supra).

[0042] 5 The residue numbering follows the nomenclature of Honegger and Plückthun (supra).

[0043] "Variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof, refers to the numbering system used in Kabat et al. (supra) for heavy chain variable domains or light chain variable domains for antibody compilation. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening of or insertion into the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat residue numbering can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence.

[0044] Unless otherwise indicated herein, the numbering of residues in the immunoglobulin heavy chain is as in the EU index numbering in Kabat et al. (supra), but with slight modifications. Briefly, we added 5 residues in the hypervariable loop before the heavy chain CDR1. "EU index numbering as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0045] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0046] The "humanized" form of non-human (e.g., rodent) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor hypervariable region (HVR) are replaced with residues from the hypervariable region of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) (donor antibody) with the desired antibody specificity, affinity, and ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may be included in residues that are not present in the receptor antibody or the donor antibody. These modifications are made in order to further improve antibody performance. Generally, a humanized antibody will substantially include at least one, usually all of two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs correspond to the FRs of human immunoglobulin sequences. The humanized antibody will optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0047] "Human antibodies" are antibodies having an amino acid sequence corresponding to an antibody produced by a human and / or that has been produced using any technique for producing human antibodies as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also useful for preparing human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 pages (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge but has had its endogenous loci disabled, such as an immunized xeno mouse (About XENOMOUSE TMFor human antibodies generated by human B cell hybridoma technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) for human antibodies generated by human B cell hybridoma technology.

[0048] "Percentage (%) of amino acid sequence identity" or "homology" for polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptides after alignment of the sequences, taking into account any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the aligned sequences. However, for the purposes of this article, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32 (5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5 (1): 113, 2004).

[0049] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of comparison positions multiplied by 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have a total of 50% homology. Generally, when comparing two sequences, the comparison is made to give the maximum homology.

[0050] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the C H 1. C H 2 and C H 3 domains (collectively referred to as C H ) and the CHL (or C L) domain.

[0051] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct classes, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0052] The "CH1 domain" (also called "C1" of the "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0053] The "hinge region" is generally defined as the region in IgG that corresponds to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bonds in the same position.

[0054] The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that carbohydrates can provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985).

[0055] The "CH3 domain" (also called a "C3" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (ie, from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 in IgG).

[0056] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. For example, the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with and without a mixture of antibodies with the K447 residue. Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0057] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an antibody that binds to an IgG antibody (gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Annu. Rev. Immunol. 15:203-234 (1997). ) FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0058] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0059] As used herein, a first antibody or fragment thereof "competes" for binding to a target antigen with a second antibody or fragment thereof in the presence of equimolar concentrations of the first antibody or fragment thereof, when the first antibody or fragment thereof inhibits the binding of the target antigen of the second antibody or fragment thereof by at least about 50% (e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. A high throughput process for "binning" antibodies based on cross-competition of the antibodies is described in PCT Publication No. WO 03 / 48731.

[0060] As used herein, the terms "specific binding," "specific recognition," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, that determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to the target with greater affinity, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the dissociation constant (K) of an antibody that specifically binds to a target is less than about 10% of the binding of the antibody to the target. D )≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may include, but need not necessarily be exclusive binding. The binding specificity of an antibody or antigen binding domain may be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM Testing and peptide scanning.

[0061] An "isolated" antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is not associated with all other components from its production environment.

[0062] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. Preferably, the isolated nucleic acid is not associated with all components associated with the environment in which it was produced. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from the form or setting in which it is found in nature. Thus, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that is naturally present in the cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0063] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to the sequence; or if a ribosome binding site is placed in a position that facilitates translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in the reading frame. However, enhancers do not have to be continuous. Connection is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used as a matter of routine practice.

[0064] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0065] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0066] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the initial transformed cell and the progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the initial transformed cell are included herein.

[0067] As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief of the disease (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses reducing the pathological consequences of cancer (e.g., tumor volume). The method of the present application contemplates any one or more of these aspects of treatment.

[0068] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0069] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, degree or likelihood of the characteristic. "Reduce" or "inhibit" refers to a decrease, reduction or cessation of an activity, function and / or amount compared to a reference activity, function and / or amount. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

[0070] The term "agonizing" or "agonize" refers to an increase or enhancement of any phenotypic characteristic or the incidence, degree or likelihood of the characteristic. "Increase" or "enhancement" refers to a decrease, reduction or cessation of activity, function and / or amount compared to a reference activity, function and / or amount. In certain embodiments, "increase" or "enhancement" refers to the ability to cause, for example, an overall increase of activity, function and / or amount by at least about 1 fold or more. In another embodiment, "increase" or "enhancement" refers to the ability to cause, for example, an overall increase of activity, function and / or amount by at least about 5 fold or more compared to a reference. In yet another embodiment, "increase" or "enhancement" refers to the ability to cause, for example, an overall increase of activity, function and / or amount by at least about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold or 1000 fold compared to a reference, including any ranges between these values, or greater than about 100 fold compared to a reference.

[0071] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. The reference can be obtained from a healthy and / or non-diseased sample. In some examples, the reference can be obtained from an untreated sample. In some examples, the reference is obtained from a non-diseased or untreated sample of an individual. In some examples, the reference is obtained from one or more healthy individuals who are not the individual or patient.

[0072] As used herein, "delaying the development of a disease" means postponing, hindering, slowing, slowing, stabilizing, inhibiting and / or delaying the development of a disease (e.g., cancer). The length of time for such a delay can vary, depending on the history of the disease and / or the individual being treated. It will be apparent to one skilled in the art that a substantial or significant delay actually encompasses prevention, in that the individual will not develop the disease. For example, the development of an advanced cancer (e.g., metastasis) can be delayed.

[0073] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed as having the disease.

[0074] As used herein, "inhibiting" a function or activity is reducing the function or activity when compared to conditions that are otherwise identical except for the condition or parameter of interest, or alternatively compared to another condition. For example, an antibody that inhibits tumor growth will reduce the growth rate of a tumor compared to the growth rate of the tumor in the absence of the antibody.

[0075] As used herein, "based on" includes assessing, determining or measuring the characteristics of an individual as described herein (and preferably, selecting an individual suitable for treatment). When the status of claudin-18 abnormality is "used as the basis" for selecting, assessing, measuring or determining a treatment method as described herein, the clinician uses the CLDN6 abnormality determined before and / or during treatment, and the obtained status (including the presence, absence, expression level, activity level and / or phosphorylation level of CLDN6) to assess any of the following: (a) the possible or expected suitability of the individual to initially receive treatment; (b) the possible or expected unsuitability of the individual to initially receive treatment; (c) the response to treatment; (d) the possible or expected suitability of the individual to continue to receive treatment; (e) the possible or expected unsuitability of the individual to continue to receive treatment; (f) to adjust the dose; or (g) to predict the possibility of clinical benefit.

[0076] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0077] It is understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0078] Reference herein to "about" a value or parameter includes (and describes) variations directed to that value or parameter itself. For example, description referring to "about X" includes description of "X".

[0079] As used herein, reference to "not" a value or parameter generally means and describes "different from" a value or parameter. For example, a method is not used to treat type X cancer means that the method is used to treat cancers other than type X.

[0080] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0081] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0082] Antibody Binding Affinity

[0083] The binding specificity of the antibody portion of the multispecific constructs described herein can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM Testing and peptide scanning.

[0084] In some embodiments, the dissociation constant K D To measure binding affinity. The dissociation constant can be determined by any analytical technique known in the art, including biochemical or biophysical techniques, such as fluorescence activated cell sorting (FACS), flow cytometry, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), biolayer interferometry (BioLayer interferometry) (see, e.g., ForteBio's Octet system), mesoscale discovery assay (meso scalediscover assay) (see, e.g., MSD-SET), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped flow analysis, and colorimetric or fluorescent protein melting analysis; or cell binding assays.

[0085] In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is D About 10 -7 M to about 10 -12 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 - 11 M, about 10 -11 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M, or about 10 -7 M to about 10 -9M. In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is D Stronger than about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Any of M. In some embodiments, the target antigen (eg, CLDN6 or 4-1BB) is a human antigen.

[0086] In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is on About 10 3 M -1 s -1 to about 10 8 M -1 s -1 , about 10 3 M -1 s -1 to about 10 4 M -1 s -1 , about 10 4 M -1 s -1 to about 10 5 M -1 s -1 , about 10 5 M -1 s -1 to about 10 6 M -1 s -1 , about 10 6 M -1 s -1 to about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 to about 10 8 M -1 s -1 In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is on About 10 3 M -1 s -1 to about 10 5 M -1 s -1 , about 10 4 M -1 s-1 to about 10 6 M -1 s -1 , about 10 5 M -1 s -1 to about 10 7 M -1 s -1 , about 10 6 M -1 s -1 to about 10 8 M -1 s -1 , about 10 4 M -1 s -1 to about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is on No more than about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 In some embodiments, the target antigen (eg, CLDN6 or 4-1BB) is a human antigen.

[0087] In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is off About 1s -1 to about 10 -6 s -1 , about 1s -1 to about 10 -2 s-1, about 10 -2 s -1 to about 10 -3 s -1, about 10 -3 s -1 to about 10 -4 s -1 , about 10 -4 s -1 to about 10 -5 s -1 , about 10 -5 s -1 to about 10 -6 s -1 , about 1S -1 to about 10 -5 s -1 , about 10 -2 s -1 to about 10 -6 s -1 , about 10 -3 s -1 to about 10 -6 s -1 , about 10 -4 s -1 to about 10 -6 s -1 , about 10 -2 s -1 to about 10 -5 s -1 , or about 10 -3 s -1 to about 10 -5 s -1 In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., CLDN6 or 4-1BB) is off At least about 1s -1 , 10 -2 s -1 , 10 - 3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 In some embodiments, the target antigen (eg, CLDN6 or 4-1BB) is a human antigen.

[0088] Chimeric or humanized antibodies

[0089] In some embodiments, one or more antibody portions of the multispecific constructs of the present application are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, a chimeric antibody is a "class switching" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0090] In some embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibodies. Generally, humanized antibodies include one or more variable domains, wherein HVR (e.g., CDR) (or a portion thereof) is derived from non-human antibodies, and FR (or a portion thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0091] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "directed selection" method of FR shuffling).

[0092] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies, a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)). 1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0093] Human Antibodies

[0094] In some embodiments, one or more antibody portions of the multispecific construct of the present application are human antibodies (referred to as human domain antibodies, or human DAbs). Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001), Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008) and Chen, Mol. Immunol. 47 (4): 912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0095] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or it is present outside the chromosome or randomly incorporated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patents 6,075,181 and 6,150,584, which describe XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes KM Technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0096] Human antibodies (e.g., human DAbs) can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (see, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0097] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The following describes techniques for selecting human antibodies from antibody libraries.

[0098] Substitution, insertion, and deletion variants

[0099] In some embodiments, antibody variants comprising one or more amino acid replacements are included in the multispecific constructs described herein. Sites of interest for replacement mutagenesis include HVR (or CDR) and FR. Conservative replacements are shown under the "preferred replacements" heading of Table 2. More substantial changes are provided under the "exemplary replacements" heading of Table 2, and are further described below with respect to amino acid side chain categories. Amino acid replacements can be introduced into the antibodies of interest, and the products can be screened to obtain desired activities, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0100] Table 2. Amino acid substitutions

[0101] Original residue Exemplary Replacements Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0102] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0103] Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0104] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, relative to the parent antibody, certain biological properties of the resulting variant selected for further study will be modified (e.g., improved) (e.g., increased affinity, reduced immunogenicity) and / or certain biological properties of the parent antibody will be substantially retained. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0105] Changes (e.g., substitutions) can be made in HVR, for example, changes to improve antibody affinity. Such changes can be made in HVR "hotspots", i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or in SDR (a-CDR), and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation achieved by constructing a secondary library and reselecting therefrom has been described in, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A second library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0106] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside HVR "hot spots" or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR is unchanged, or contains no more than one, two or three amino acid substitutions.

[0107] As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, a residue or a group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further replacements can be introduced at amino acid positions that show functional sensitivity to the initial replacement. Alternatively or additionally, the contact points between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as replacement candidates. Variants can be screened to determine whether they contain desired properties.

[0108] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide, which increases the serum half-life of the antibody.

[0109] Glycosylation variants

[0110] In some embodiments, one or more antibody portions of the multispecific constructs of the present application are altered to increase or decrease the extent to which the construct is glycosylated. By altering the amino acid sequence to produce or remove one or more glycosylation sites, the addition or deletion of glycosylation sites of the antibody can be conveniently achieved.

[0111] When the antibody portion comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are generally attached to the Fc region C by N-bonds. H 2 domains. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose connected to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibody portion can be modified to produce antibody variants with certain improved properties.

[0112] In some embodiments, the antibody portion has a carbohydrate structure that lacks fucose attached to the Fc region (directly or indirectly). For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of sugar structures (e.g., complexes, hybrids, and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0113] In some embodiments, the antibody portion has an oligosaccharide that is cut into two, for example, a biantennary oligosaccharide connected to the Fc region of the antibody is cut into two by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide connected to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0114] Fc region variants

[0115] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody portion, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0116] In some embodiments, the Fc fragment has some but not all effector functions, making it a desired candidate for use in applications where the half-life of the antibody portion in vivo is important, but certain effector functions (such as complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity), but retains FcRn binding ability. The main cells that mediate ADCC, namely NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of molecules of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0117] Antibodies with reduced effector function include those with substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056), wherein the amino acid numbering is according to the EU index. See, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. Such Fc mutants include Fc mutants with substitutions in two or more of amino acid positions 265, 269, 270, 297, and 327 (wherein the amino acid numbering is according to the EU index), including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581), wherein the amino acid numbering is according to the EU index.

[0118] Certain antibody variants with improved or reduced binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0119] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises a L234A mutation and / or a L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising a S228P, F234A, and / or L235A mutation.

[0120] In some embodiments, the antibody portion comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0121] In some embodiments, alterations are made in the Fc region that result in altered (ie, improved or reduced) CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0122] In some embodiments, the antibody partial variant comprises a variant Fc region comprising one or more amino acid substitutions that change the half-life and / or change the binding to the neonatal Fc receptor (FcRn). The neonatal Fc receptor (FcRn) is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), and antibodies with increased half-life and improved binding to FcRn are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region in which there are one or more substitutions that change the binding of the Fc region to FcRn. Such Fc variants include those variants (U.S. Pat. No. 7,371,826) having substitutions (e.g., substitutions of Fc region residue 434) at one or more Fc region residues.

[0123] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0124] Cysteine ​​engineered antibody variants

[0125] In some embodiments, it may be desirable to produce a cysteine ​​engineered antibody portion, such as a "thioMAb," in which one or more residues of one or more antibody portions in the multispecific constructs herein are replaced with cysteine ​​residues. In a specific embodiment, the replacement residue occurs at an accessible site of the antibody. By replacing these residues with cysteine, the reactive thiol group is positioned at an accessible site of the antibody, and can be used to conjugate the antibody with other moieties (such as a drug moiety or a linker-drug moiety) to produce an immunoconjugate, as further described herein. In some embodiments, any one or more of the following residues can be replaced with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibody portions can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0126] Anti-CLDN6 antibody part

[0127] The anti-CLDN6 antibody portion of the multispecific constructs described herein includes any antibody portion that specifically binds to claudin-6 ("CLDN6").

[0128] Claudin 6 (CLDN6)

[0129] Claudin is a class of tight junction membrane proteins, which are expressed in epithelial cells and endothelial cells and form paracellular barriers and pores, which determine tight junction permeability. Claudin has been recognized as a key regulator of cancer occurrence, progression and metastasis, and plays different roles in various cancers according to its different tissue-dependent expression patterns (Tabaries et al. (2017). "The role of claudins in cancer metastasis." Oncogene 36, 1176-1190). Claudin-6 (CLDN6) is a member of the claudin family and a tight junction molecule that plays a vital role in intercellular adhesion in epithelial or endothelial cell layers. It encodes a tetraspanin with a size of 220 amino acids and a molecular weight of 23292Da. CLDN6 has been identified as the origin of cell adhesion signaling, and participates in the regulation of nuclear receptor activity by targeting molecules of the nuclear receptor superfamily and regulating their gene expression (Sugimoto et al. (2019). "Cell adhesion signals regulate the nuclear receptor activity." Proc. Natl. Acad. Sci. USA 116, 24600-24609). CLDN6 appears to be significantly upregulated in 20 types of human cancers (Zhang et al. (2021) Front. Cell. Dev. Biol. 9: 726656). In some embodiments, CLDN6 is human CLDN6 ("hCLDN6"). In some embodiments, hCLDN6 comprises the amino acid sequence shown in SEQ ID NO: 40 or a variant thereof (e.g., a post-translationally modified variant and / or a conformational variant).

[0130] Exemplary anti-CLDN6 antibody portions

[0131] In some embodiments, the anti-CLDN6 antibody portion comprises a) a heavy chain variable region (V H ) and light chain variable region (V L ) wherein the antibody partially competes for binding to CLDN6, wherein: a) the V H comprising: i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and b) the V LThe invention relates to an antibody comprising: i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CLDN6 antibody portion is combined with a heavy chain variable region (V H ) and light chain variable region (V L ) wherein the antibody partially competes for binding to CLDN6, wherein: a) the V H comprising: i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and b) the V L comprising: i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0132] The anti-CLDN6 antibody portions described herein include any antibody portion that specifically binds to CLDN6. In some embodiments, the anti-CLDN6 antibody portion of the present application comprises: a) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity with the sequence shown in SEQ ID NO: 7; and b) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID NO: 8 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater than 99%) sequence identity with the sequence shown in SEQ ID NO: 8. The sequence shown in NO: 8 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises CDR1, CDR2 and CDR3 in the heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7, and comprises CDR1, CDR2 and CDR3 in the light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID NO: 8.

[0133] In some embodiments, the anti-CLDN6 antibody portion comprises: a) HC-CDR1, HC-CDR2, and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 18, or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to the sequence shown in SEQ ID NO: 18; and b) LC-CDR1, LC-CDR2, and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID NO: 19, or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to the sequence shown in SEQ ID NO: 19. The sequence shown in NO: 19 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises CDR1, CDR2 and CDR3 in the heavy chain variable region (VH), wherein the heavy chain variable region comprises the sequence shown in SEQ ID No: 18, and comprises CDR1, CDR2 and CDR3 in the light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 19.

[0134] In some embodiments, the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises: i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1, ii) a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR2, and iii) a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR3; and b) the VL comprises: i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1; NO:4 or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR1, ii) a LC-CDR2 or a variant thereof comprising the amino acid sequence of SEQ ID NO:5, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR2, and iii) a LC-CDR3 or a variant thereof comprising the amino acid sequence of SEQ ID NO:6, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR3.

[0135] In some embodiments, the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises: i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1, ii) a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR2, and iii) a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR3; and b) the VL comprises: i) a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1. NO: 15 or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR1, ii) a LC-CDR2 or a variant thereof comprising the amino acid sequence of SEQ ID NO: 16, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR2, and iii) a LC-CDR3 or a variant thereof comprising the amino acid sequence of SEQ ID NO: 17, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR3.

[0136] In some embodiments, the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 7; and / or the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 8. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises a VH and a VL, wherein the VH comprises the sequence shown in SEQ ID No: 7 and the VL comprises the sequence shown in SEQ ID NO: 8.

[0137] In some embodiments, the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 18; and / or the VL comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 19. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises a VH and a VL, wherein the VH comprises the sequence shown in SEQ ID No: 18 and the VL comprises the sequence shown in SEQ ID NO: 19.

[0138] The anti-CLDN6 antibody portion of the present application may be in any suitable form known in the art. In some embodiments, the anti-CLDN6 antibody portion may be selected from a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb.

[0139] In some embodiments, the anti-CLDN6 antibody portion comprises a full-length antibody comprising two heavy chains and two light chains. In some embodiments, the full-length antibody has an Fc fragment selected from Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and mixtures thereof. In some embodiments, the Fc fragment is selected from Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and mixtures thereof. In some embodiments, the Fc fragment is an IgG1 or IgG4 Fc fragment.

[0140] In some embodiments, the Fc fragment has reduced FcγR binding affinity compared to wild-type Fc. In some embodiments, the Fc fragment comprises one or more replacements selected from N297A, N297Q, N297G or L235E. In some embodiments, the Fc fragment is an IgG1 fragment comprising one or more replacements selected from N297A, N297Q, N297G, L235E and / or L234A / L235A. In some embodiments, the Fc fragment is an IgG1 fragment comprising N297A. In some embodiments, the Fc fragment is an IgG4 fragment comprising N297A, N297Q, N297G, L235E and / or F234A / L235A. In some embodiments, the Fc fragment is an IgG4 fragment comprising N297A. In some embodiments, the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 42.

[0141] In some embodiments, the anti-CLDN6 antibody portion comprises a full-length antibody comprising two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 42, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 9 or SEQ ID NO: 42; and / or the light chain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 9 or SEQ ID NO: 42; ID NO: 11 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 9 and / or SEQ ID NO: 42, and each light chain comprising SEQ ID NO: 11.

[0142] In some embodiments, the anti-CLDN6 antibody portion comprises a full-length antibody comprising two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 43, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 43; and / or the light chain comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof, which has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 43; ID NO: 11 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 10 and / or SEQ ID NO: 43, and each light chain comprising SEQ ID NO: 11.

[0143] In some embodiments, the anti-CLDN6 antibody portion comprises a full-length antibody comprising two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 20, or a variant thereof, which comprises at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 20; and / or the light chain comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof, which comprises at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 22. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: which comprises two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 20 and each light chain comprising SEQ ID NO: 22.

[0144] In some embodiments, the anti-CLDN6 antibody portion comprises a full-length antibody comprising two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 21, or a variant thereof, which has at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 21; and / or the light chain comprises the amino acid sequence of SEQ ID NO: 22, or a variant thereof, which has at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 22. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: which comprises two heavy chains and two light chains, each heavy chain comprising SEQ ID NO: 21, and each light chain comprising SEQ ID NO: 22.

[0145] Anti-4-1BB antibody part

[0146] The anti-4-1BB antibody portion of the multispecific construct described in the present application includes any antibody portion that specifically binds to 4-1BB. In some embodiments, 4-1BB is human 4-1BB ("h4-1BB"). H4-1BB is a type I transmembrane receptor with four cysteine-rich extracellular domains ("CRD", i.e., CRD1, CDR2, CRD3, and CRD4), followed by a short transmembrane domain and a C-terminal cytoplasmic region. CRD2 and CRD3 of h4-1BB interact with ligand 4-1BBL (Bitra et al. (2018) J Biol Chem. 293 (26): 9958-9969). Compared with other TNFRs, h4-1BB exists in the form of a disulfide-bonded dimer, and dimerization may be achieved by unpaired cysteine ​​(Cys 121 ) occurs. In some embodiments, h4-1BB comprises the sequence shown in SEQ ID NO: 41 or a variant thereof (e.g., a post-translationally modified variant and / or a conformational variant). In some embodiments, the anti-4-1BB antibody portion binds to the CRD3 / CRD4 region of 4-1BB.

[0147] The anti-4-1BB antibody portion can be any suitable form known in the art. In some embodiments, the anti-4-1BB antibody portion is selected from a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb. In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody bound to 4-1BB.

[0148] Exemplary Anti-4-1BB Antibody Portions

[0149] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomeric variable antibody domain, comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, the single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to the sequence set forth in SEQ ID NO: 27. In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable (e.g., identical) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO: 27.

[0150] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) a sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; b) a sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and c) a sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.

[0151] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) a sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; a sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0152] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to the sequence shown in SEQ ID NO: 27. In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., identical to) the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO: 27.

[0153] Multispecific constructs

[0154] On the one hand, a multispecific construct is provided herein, comprising: a first antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB.

[0155] In some embodiments, a multispecific construct is provided herein, comprising a first antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, after the first antibody portion binds to the tumor antigen, the second antibody portion activates 4-1BB by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, or 1000 times, including any range between these values. In some embodiments, in the absence of binding to a tumor antigen, the multispecific construct does activate 4-1BB signaling. In some embodiments, in the absence of binding to a tumor antigen, the second portion does not activate 4-1BB signaling.

[0156] In some embodiments of the multispecific constructs of the present application, the second antibody portion is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomeric variable antibody domain, which comprises the amino acid sequences of CDR1, CDR2, and CDR3, respectively, and the single monomeric variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27.

[0157] In some embodiments, the sdAb comprises: a sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; a sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and a sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.

[0158] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater than 99%) sequence identity to the sequence shown in SEQ ID NO: 27. In some embodiments, the affinity of this sdAb for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., identical to) the affinity of the sdAb comprising SEQ ID NO: 27.

[0159] The tumor antigen that specifically binds to the first antibody portion can be any suitable tumor antigen known in the art. In some embodiments, the tumor antigen is CLDN6.

[0160] In some embodiments, the application provides a multispecific construct that binds to both CLDN6 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion. The anti-CLDN6 antibody portion and the anti-4-1BB antibody portion can be any of those antibody portions described herein.

[0161] In some embodiments of the multispecific construct of the present application, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises: (1) HC-CDR1, HC-CDR2, and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7 or a variant thereof, the variant being the same as SEQ ID No: 8. NO:7 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity; (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within a light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID No:8 or a variant thereof, wherein the variant has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity with SEQ ID NO:8. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: which comprises CDR-1, CDR-2 and CDR-3 of the VH shown in SEQ ID NO:7, and CDR-1, CDR-2 and CDR-3 of the VL shown in SEQ ID NO:8. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0162] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, and 3) amino acid substitutions in HC-CDR1, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, and 3) amino acid substitutions in HC-CDR2; and (iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, and 3) amino acid substitutions in HC-CDR3; and the VL comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, and 3) amino acid substitutions in HC-CDR1; NO:4 or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR1; (ii) an LC-CDR2 or a variant thereof comprising the amino acid sequence of SEQ ID NO:5, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR2; and (iii) an LC-CDR3 or a variant thereof comprising the amino acid sequence of SEQ ID NO:6, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0163] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises: the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity to SEQ ID NO: 7; and / or the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater than 99%) sequence identity to SEQ ID NO: 8. NO: 8 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises the VH shown in SEQ ID NO: 7, and the VL shown in SEQ ID NO: 8. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0164] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises: (1) HC-CDR1, HC-CDR2, and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within the heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 18 or a variant thereof, the variant being the same as SEQ ID No: 20. NO:18 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity; (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within a light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID No:19 or a variant thereof, wherein the variant has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity to SEQ ID NO:19. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: which comprises CDR-1, CDR-2 and CDR-3 of VH shown in SEQ ID NO: 18, and CDR-1, CDR-2 and CDR-3 of VL shown in SEQ ID NO: 19. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0165] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR2, and (iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR3; and the VL comprises: (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in HC-CDR1 NO: 15 or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR1; (ii) an LC-CDR2 or a variant thereof comprising the amino acid sequence of SEQ ID NO: 16, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR2; and (iii) an LC-CDR3 or a variant thereof comprising the amino acid sequence of SEQ ID NO: 17, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in LC-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0166] In some embodiments of the multispecific construct of the present application, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises: the VH comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, the variant having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity with SEQ ID NO: 18; and / or the VL comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof, the variant having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater) sequence identity with SEQ ID NO: 19. NO: 19 has at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than 99%) sequence identity. In some embodiments, the affinity of this anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable to (e.g., the same as) the affinity of the following anti-CLDN6 antibody portion: it comprises the VH shown in SEQ ID NO: 18 and the VL shown in SEQ ID NO: 19. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0167] In some embodiments of the multispecific construct of the present application, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises: the VH comprises the amino acid sequence of SEQ ID NO: 18; and / or the VL comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein the variant comprises up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises: an sdAb comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 27.

[0168] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion comprises an sdAb.

[0169] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomeric variable antibody domain, which comprises the amino acid sequences of CDR1, CDR2, and CDR3, respectively, and the single monomeric variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27, or a variant thereof, wherein the variant has at least about 80% sequence identity (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) to SEQ ID NO: 27. In some embodiments, the affinity of the anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27.

[0170] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, and the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof, the variant comprising up to about 3 (e.g., any one of about 1, 2, or 3) amino acid substitutions in sdAb-CDR3.

[0171] In some embodiments, the multispecific construct described herein is a multispecific construct (eg, a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, and the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (eg, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity with SEQ ID NO: 27. In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (eg, human 4-1BB) is comparable (eg, identical) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO: 27.

[0172] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), and the anti-4-1BB antibody portion comprises an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: (1) HC-CDR1, HC-CDR2, and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 7; and (2) LC-CDR1, LC-CDR2, and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID No: 8. No: the sequence shown in 8; and the anti-4-1BB antibody portion comprises sdAb, and the sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 within a single monomer variable antibody domain, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3, and the single monomer variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27.

[0173] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), and the anti-4-1BB antibody portion comprises an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: (1) HC-CDR1, HC-CDR2, and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a heavy chain variable region (VH), the heavy chain variable region comprising the sequence shown in SEQ ID No: 18; and (2) LC-CDR1, LC-CDR2, and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a light chain variable region (VL), the light chain variable region comprising the sequence shown in SEQ ID No: 18. No: the sequence shown in 19; and the anti-4-1BB antibody portion comprises sdAb, and the sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 within a single monomer variable antibody domain, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3, and the single monomer variable antibody domain comprises the amino acid sequence shown in SEQ ID NO: 27.

[0174] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), and the anti-4-1BB antibody portion comprises an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 7; and / or the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 8. NO: 8 has at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity; and the anti-4-1BB antibody portion comprises an sdAb, and the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity with SEQ ID NO: 27. In some embodiments, the affinity of such a multispecific construct (e.g., a bispecific antibody) for CLDN6 and 4-1BB is comparable to (e.g., the same as) the affinity of a multispecific construct comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises a full-length antibody comprising the VH shown in SEQ ID NO: 7 and the VL shown in SEQ ID NO: 8, and the anti-4-1BB antibody portion comprises a single domain antibody comprising the sequence shown in SEQ ID NO: 27.

[0175] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), and the anti-4-1BB antibody portion comprises an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: the VH comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 18; and / or the VL comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 19. NO: 19 has at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity; and the anti-4-1BB antibody portion comprises an sdAb, and the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof, the variant having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity with SEQ ID NO: 27. In some embodiments, the affinity of such a multispecific construct (e.g., a bispecific antibody) for CLDN6 and 4-1BB is comparable to (e.g., the same as) the affinity of a multispecific construct comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises a full-length antibody comprising the VH shown in SEQ ID NO: 18 and the VL shown in SEQ ID NO: 19, and the anti-4-1BB antibody portion comprises a single domain antibody comprising the sequence shown in SEQ ID NO: 27.

[0176] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), the anti-4-1BB antibody portion specifically binds to 4-1BB, wherein the anti-4-1BB antibody portion is fused to the N-terminus of one or two heavy chains of the anti-CLDN6 antibody (e.g., full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, the anti-4-1BB antibody portion specifically binds to 4-1BB, wherein the anti-4-1BB antibody portion is fused to the C-terminus of one or two heavy chains of the anti-CLDN antibody (e.g., full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, the anti-4-1BB antibody portion specifically binds to 4-1BB, wherein the anti-4-1BB antibody portion is fused to the N-terminus of one or two light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, wherein the anti-4-1BB antibody portion is fused to the C-terminus of one or two light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody).

[0177] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6), and the anti-4-1BB antibody portion comprises a single domain antibody that binds to 4-1BB (e.g., human 4-1BB), wherein the single domain antibody is fused to the N-terminus of one or two heavy chains of the anti-CLDN antibody (e.g., full-length anti-CLDN antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, and the anti-4-1BB antibody portion comprises a single domain antibody that binds to 4-1BB, wherein the single domain antibody is fused to the C-terminus of one or two heavy chains of the anti-CLDN antibody (e.g., full-length anti-CLDN antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, the anti-4-1BB antibody portion comprising a single domain antibody that binds to 4-1BB, wherein the single domain antibody is fused to the N-terminus of one or two light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided, comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, the anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6, the anti-4-1BB antibody portion comprising a single domain antibody that binds to 4-1BB, wherein the single domain antibody is fused to the C-terminus of one or two light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody).

[0178] In some embodiments, the anti-4-1BB antibody portion is fused to the anti-CLDN6 antibody portion via a joint. In some embodiments, the joint is a peptide joint. In some embodiments, the length of the joint is about four to about fifty amino acids. In some embodiments, the joint is selected from (GS)n, (GGGS)n, (GGGGS)n and (GSGGS)n. In some embodiments, n is 0-8. In some embodiments, the joint comprises the amino acid sequence of GGGGSGGGGSGGGGS.

[0179] In some embodiments, provided herein are multispecific constructs comprising a heavy chain component and a light chain component, wherein the heavy chain component comprises an amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 44 or 45, or a variant thereof, which has at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: 28, 30, 32, 34, 36, 38, 44 or 45; and / or the light chain comprises an amino acid sequence of SEQ ID NO: 29, 31, 33, 35, 37, 39, or a variant thereof, which has at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity to SEQ ID NO: NO: 29, 31, 33, 35, 37, 39 have at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values) sequence identity.

[0180] In some embodiments, provided herein is a multispecific construct comprising a heavy chain component and a light chain component, wherein the heavy chain component comprises the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 44 or 45; and / or the light chain comprises the amino acid sequence of SEQ ID NO: SEQ ID NO: 29, 31, 33, 35, 37, 39.

[0181] In some embodiments, a multispecific construct is provided, which comprises two heavy chain components and two light chain components, wherein: (a) each heavy chain component comprises the sequence shown in SEQ ID NO:28 and / or 44, and each light chain component comprises the sequence shown in SEQ ID NO:29; (b) each heavy chain component comprises the sequence shown in SEQ ID NO:30 and / or SEQ ID NO:45, and each light chain component comprises the sequence shown in SEQ ID NO:31; (c) each heavy chain component comprises the sequence shown in SEQ ID NO:32, and each light chain component comprises the sequence shown in SEQ ID NO:33; (d) each heavy chain component comprises the sequence shown in SEQ ID NO:34, and each light chain component comprises the sequence shown in SEQ ID NO:35; (e) each heavy chain component comprises the sequence shown in SEQ ID NO:36, and each light chain component comprises the sequence shown in SEQ ID NO:37; or (f) each heavy chain component comprises the sequence shown in SEQ ID NO:38, and each light chain component comprises the sequence shown in SEQ ID NO:39.

[0182] In some embodiments, a multispecific construct is provided herein, comprising a first antibody portion that specifically binds to CLDN6; and a second antibody portion that specifically binds to 4-1BB, wherein the binding of the first antibody portion to CLDN6 triggers the second antibody portion to activate 4-1BB. In some embodiments, after the first antibody portion binds to CLDN6, the second antibody portion activates 4-1BB by at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times or 1000 times. In some embodiments, the multispecific construct does not activate 4-1BB signaling without binding to CLDN6. In some embodiments, the second portion does activate 4-1BB signaling without binding to CLDN6.

[0183] Activation of 4-1BB signaling is an expected mechanism for agonist antibodies such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB portion of some antibodies disclosed herein does not require such activity. In fact, in some embodiments, preferably in the absence of CLDN6 binding, the anti-4-1BB portion of the present antibody cannot independently activate 4-1BB. As shown in the experimental examples, interestingly, when the anti-CLDN6 portion binds to the CLDN6 protein on the cell, this CLDN6 binding can trigger 4-1BB signaling activation.

[0184] Known anti-4-1BB agonist antibodies are generally associated with dose-limiting on-target hepatotoxicity, and compared with these antibodies, the antibodies disclosed herein are considered to be far safer. In tissues (such as the liver), where CLDN6 is not expressed under healthy conditions, it is expected that the antibodies disclosed herein will not trigger a cytotoxic immune response because they will not activate 4-1BB signaling. In tumor tissues expressing and / or accessible to CLDN6, by contrast, the present antibodies can initiate a potent immune response to tumor cells. Therefore, unlike those anti-4-1BB antibodies currently being developed clinically with on-target / intrinsic toxicity, the antibodies disclosed herein can be both effective and safe in treating cancer.

[0185] Nucleic Acids

[0186] Also contemplated are nucleic acid molecules encoding multispecific constructs or various antibody portions as described herein. In some embodiments, nucleic acids (or a set of nucleic acids) encoding one or more polypeptides or various antibody portions of a multispecific construct are provided. In some embodiments, nucleic acids (or a set of nucleic acids) encoding a multispecific construct (e.g., anti-CLDN6 / anti-4-1BB bispecific antibody) or a polypeptide portion thereof are provided.

[0187] Also contemplated herein are isolated host cells comprising a multispecific construct (e.g., an anti-CLDN6 / anti-4-1BB bispecific antibody), a nucleic acid encoding a polypeptide component of a multispecific construct, or a vector comprising a nucleic acid encoding a polypeptide component of a multispecific construct (e.g., an anti-CLDN6 / anti-4-1BB bispecific antibody) described herein.

[0188] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to nucleic acid sequences under at least moderately stringent hybridization conditions, wherein the nucleic acid sequences encode multispecific constructs (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies) or various antibody portions described herein.

[0189] The present application also provides a vector into which the nucleic acid of the present application is inserted.

[0190] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0191] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Generally, suitable vectors contain a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0192] Treatment

[0193] Also provided herein is a method for treating a disease or illness of an individual. The method includes applying a multispecific construct as described herein (e.g., anti-CLDN6 / anti-4-1BB bispecific antibody) to an individual (e.g., mammal, such as a human). In some embodiments, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.

[0194] In some embodiments of the methods, the disease or condition is a proliferative disorder. In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cancer is a solid tumor, melanoma, renal cancer, ovarian cancer, colorectal cancer, squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer, or non-Hodgkin lymphoma (NHL).

[0195] Compositions, kits and articles of manufacture

[0196] Also provided herein is a composition (such as a formulation) comprising any of the multispecific constructs described herein (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies), a nucleic acid encoding any of the multispecific constructs or a portion thereof, a vector comprising a nucleic acid encoding one of the multispecific constructs, or a host cell comprising the nucleic acid or the vector.

[0197] Suitable formulations of the multispecific constructs described herein (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies) can be obtained by mixing the multispecific construct having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)).

[0198] Also provided is a kit comprising any of the multispecific constructs described herein (eg, anti-CLDN6 / anti-4-1BB bispecific antibodies). The kit can be used in any of the methods of treatment described herein.

[0199] The kit of the present application adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, cans, flexible packaging (such as sealed Mylar or plastic bags), etc. The kit can optionally provide additional components, such as buffer and explanatory information.

[0200] Therefore, the present application also provides articles. The articles may include a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, cans, flexible packages, etc. Generally, the container contains the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle).

[0201] Those skilled in the art will recognize that several embodiments may be present within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but certainly should not be construed as limiting its scope in any way.

[0202] Example

[0203] The proposal of following examples is to provide how to make and use complete disclosure and description of the present invention to those of ordinary skill in the art, and is not intended to limit the scope of the inventor's invention, nor is it intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the used numerals (such as amount, temperature, etc.), but some experimental errors and deviations should be considered. The following examples are intended to be only examples of the present application, and therefore should not be considered to limit the present application in any way. The following examples and detailed description are for illustration only, not limitation.

[0204] Example 1: Preparation of CLDN6 x 4-1BB bispecific antibody

[0205] Exemplary CLDN6 x 4-1BB bispecific antibodies shown in Table 3 below were designed and prepared.

[0206] Table 3. Exemplary CLDN6 x 4-1BB bispecific antibodies

[0207]

[0208]

[0209]

[0210] Example 2. Antigen Binding Activity of CLDN6 X 4-1BB BsAb

[0211] 2.1 Binding affinity of CLDN6 x 4-1BB BsAb to CLDN6

[0212] The binding affinity of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT (prepared as described in Example 1) to human CLDN6 was detected by surface plasmon resonance (SPR). Figure 1A and Figure 1B As shown, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT bind to CLDN6 virus-like particles (VLPs), K D They are 2.36×10 -9 M and 1.61×10 - 9 M.

[0213] A CHO-K1 cell line stably expressing human CLDN6 (CHO-K1-CLDN6) was prepared to evaluate the binding ability of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT to CLDN6. The parental CLDN6-1 antibody was used as a control. Briefly, CHO-K1-CLDN6 cells were incubated with different concentrations of BsAb in FACS buffer for 30 minutes at 4°C. Then, phycoerythrin (PE)-conjugated anti-human IgG antibody was added after washing, and the cells were further incubated at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE was evaluated by FACS. Figure 2A As shown, both CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT bound to cells expressing CLDN6 in a concentration-dependent manner.

[0214] OVCAR3 and OV90 are human ovarian cancer cell lines with endogenous CLDN6 expression. Figure 2B and Figure 2CAs shown, both CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT were able to bind to OVCAR3 and OV90. Overall, the binding affinity of the BsAb containing the anti-CLDN6-1 antibody portion to human CLDN6 was comparable to that of the parental CLDN6-1 antibody to human CLDN6. The binding signal was closely correlated with the expression level of CLDN6 on the surface of OVCAR3 and OV90 cells.

[0215] 2.2 Binding affinity of CLDN6 x 4-1BB BsAb to 4-1BB

[0216] The binding affinity of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT to human 4-1BB was detected by SPR. Figure 3A-3D As shown, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT bind to monomeric human 4-1BB, K D They are 1.64×10 -8 M and 1.57×10 -8 M. The affinity of the parent anti-4-1BB sdAb antibody conjugated to the IgG1 Fc fragment (4-1BB sdAb-Fc) to 4-1BB was also tested, and it was found that K D is 4.393×10 -9 M, indicating that the affinity of the 4-1BB antibody part in the BsAb for 4-1BB is comparable to that of the 4-1BB sdAb-Fc for 4-1BB.

[0217] The binding of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT to soluble recombinant human 4-1BB was analyzed by ELISA. Figure 4A As shown, both CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT bind to recombinant human 4-1BB in a concentration-dependent manner with EC50s of 0.129 nM and 0.078 nM, respectively. Such EC50s are comparable to the EC50 of 4-1BB sdAb-Fc. In addition, the binding of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT to HEK293 cells expressing 4-1BB was evaluated by FACS. Figure 4B As shown, both CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT were able to bind to 4-1BB with EC50s of 0.406 nM and 0.347 nM, respectively. Such EC50s are comparable to the EC50 of 4-1BB sdAb-Fc.

[0218] Example 3. Functional activity of CLDN6-1 x 4-1BB BsAb

[0219] 3.1 Cell line-based functional characterization of CLDN6 X 4-1BB BsAb

[0220] To test the ability of the CLDN6 x 4-1BB bispecific antibody to activate 4-1BB signaling, a GloResponse TM NFκB-luc2 / 4-1BB Jurkat cell line was used as effector cells, and cells expressing CLDN6 (CHO-K1 CLDN6, OVCAR3 or OV90) were used as target cells. RKO colon cancer cells that do not express CLDN6 were used as negative controls.

[0221] Briefly, GloResponse TM NFκB-luc2 / 4-1BB Jurkat cells (density 5.0x10 per well) 4 cells) and 5.0x10 4 Target cells were mixed in a white 96-well plate. Antibodies were serially diluted and added to the plate. Luminescence was measured after incubation at 37°C for 6 hours. FIG. 5A to FIG. 5D As shown, regardless of CLDN6 expression, urerulumab induced 4-1BB activation, while the 4-1BB sdAb-Fc of the present application, despite being able to bind to 4-1BB, had no agonist activity under the same experimental setting. Similarly, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT induced NFκB activity in the presence of all target cells expressing CLDN6, regardless of CLDN6 expression levels. In contrast, when RKO cells that do not express CLDN6 were used as target cells, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT resulted in significantly lower 4-1BB activation compared to urerulumab, as shown in Table 1. Figure 5D shown.

[0222] 3.2 CLDN6 X 4-1BB BsAb promotes the immune response of human peripheral blood mononuclear cells (PBMCs)

[0223] Preactivated human PBMCs were co-cultured with cells expressing CLDN6 or RKO at an effector-to-target (E:T) ratio of 10: 1. Antibodies at different concentrations were added to the mixed cultures. After 48 hours, the levels of IL-2 or IFNγ in the culture medium were measured using a homogeneous HTRF assay.

[0224] like Figure 6A-6F As shown, CLDN6-1 X 4-1BB NA and CLDN6-1 X 4-1BB WT stimulated IL-2 and IFNγ production when PBMCs were co-cultured with target cells expressing CLDN6. However, in the presence of RKO that does not express CLDN6, CLDN6-1 X 4-1BB NA and CLDN6-1 X 4-1BB WT did not stimulate PBMCs to produce IL-2 or IFNγ, as shown in Figure 2. Figure 6G and Figure 6H As shown, the activity of CLDN6-1 X 4-1BB NA and CLDN6-1 X 4-1BB WT is dependent on the presence of tumor antigen. In contrast, 4-1BB sdAb-Fc was inactive in this assay.

[0225] Example 4. CLDN6-1 x 4-1BB BsAb inhibits tumor growth

[0226] 4.1CT26 syngeneic mouse colorectal cancer model

[0227] CT26 is an N-nitroso-N-methylurethane (NNMU)-induced undifferentiated colon cancer cell line established from BALB / c mice with invasive colon cancer. CT26 cells endogenously expressing CLDN6 were subcutaneously implanted into BALB / c humanized 4-1BB mice. When tumors grew to an average of 100 mm 3 Mice were treated intraperitoneally with (a) human IgG, (b) CLDN6-1 x 4-1BB NA (2 mg / kg), (c) CLDN6-1 x 4-1BB WT (2 mg / kg), or (d) a combination of parental CLDN6-1 antibody and 4-1BB sdAb-Fc (1.8 mg / kg and 0.7 mg / kg). Treatments were administered twice per week for a total of 6 doses. Tumor growth was monitored by volume measurement. Fig. 7A and Figure 7B As shown, both CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1BB NA exhibited antitumor activity, with CLDN6-1 x 4-1BB WT achieving even stronger activity with a tumor growth inhibition (TGI) of 75%.

[0228] 4.2 MC38 syngeneic mouse colorectal adenocarcinoma model

[0229] The MC38 tumorigenic epithelial cell line was isolated from mice with colon adenocarcinoma. MC38 cells engineered to express human CLDN6 were implanted subcutaneously into C57BL / 6 humanized 4-1BB mice. When tumors grew to an average of 100 mm 3Mice were treated intraperitoneally with (a) vehicle (control), (b) CLDN6-1×4-1BB NA (1.5 mg / kg), (c) CLDN6-1×4-1BB WT (1.5 mg / kg), or (d) CLDN6-1×4-1BB WT (4.5 mg / kg). Treatments were administered once per week for a total of 3 doses. Fig. 9A Tumor growth was monitored by volume measurement. Fig. 9B As shown, CLDN6-1 x 4-1BB NA exhibited significant antitumor activity, while CLDN6-1 x 4-1BB WT treatment achieved more significant tumor growth inhibition at the same dose and led to complete tumor regression at a higher dose.

[0230] Example 5. Evaluation of hepatotoxicity of CLDN6 X 4-1BB BsAb

[0231] As observed in the clinical development of urerulumab, the main problem of t4-1BB agonist antibody therapy is dose-limiting hepatotoxicity. The most common adverse events were elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), and fatigue. Therefore, the hepatotoxicity of CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1BB NA was further evaluated.

[0232] Briefly, blood samples were collected from hu4-1BB mice for ALT and AST measurements after twice weekly treatment with different doses of CLDN6-1 x 4-1BB WT or CLDN6-1 x 4-1BB NA. As shown in Figure 8, no significant increase in ALT and AST was observed, indicating a minimal risk of hepatotoxicity, which is commonly induced by other 4-1BB agonist antibodies.

[0233] The present disclosure is not limited in scope by the specific embodiments described, which are intended to be a single illustration of the various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0234] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0235] The present invention has been described by the specific embodiments found or proposed by the inventors to include preferred modes for practicing the present invention. It will be appreciated by those skilled in the art that, in view of the present disclosure, many modifications and changes may be made to the specific embodiments of the examples without departing from the intended scope of the present invention. For example, due to codon redundancy, the DNA sequence constituting the basis may be changed without affecting the protein sequence. In addition, due to considerations of biological functional equivalence, the protein structure may be changed without affecting the type or amount of the biological action. All such modifications are intended to be included within the scope of the appended claims.

[0236] Summary table of amino acid sequences

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

Claims

1. A multispecific construct comprising: (1) a first antibody portion that specifically binds to claudin-6 ("CLDN6"); and (2) A second antibody portion that specifically binds to 4-1BB.

2. The multispecific construct of claim 0, wherein the first antibody portion is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab')2, scFv and sdAb.

3. The multispecific construct of claim 2 or 3, wherein the first antibody portion comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within a heavy chain variable region (VH), wherein the heavy chain variable region comprises the sequence shown in SEQ ID No: 7; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID No:

8.

4. The multispecific construct of any one of claims 0-0, wherein the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises: (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and; (b) the VL comprises: (i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

5. The multispecific construct of any one of claims 0-0, wherein the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof, wherein the variant has at least about 80% sequence identity with SEQ ID NO: 7; and / or (2) The VL comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with SEQ ID NO:

8.

6. The multispecific construct of any one of claims 0-2, wherein the first antibody portion comprises: (1) HC-CDR1, HC-CDR2 and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 within a heavy chain variable region (VH), wherein the heavy chain variable region comprises the sequence shown in SEQ ID No: 18; and (2) LC-CDR1, LC-CDR2 and LC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID NO:

19.

7. The multispecific construct of any one of claims 0-2 and 6, wherein the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) The VH comprises: (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and; (2) The VL comprises: (i) LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and (iii) LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

8. The multispecific construct of any one of claims 1-2 and 6-7, wherein the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant has at least about 80% sequence identity with SEQ ID NO: 18; and / or (2) The VL comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof, wherein the variant has at least about 80% sequence identity with SEQ ID NO:

19.

9. The multispecific construct of any one of claims 0-8, wherein binding of the first antibody portion to CLDN6 triggers activation of 4-1BB by the second antibody portion.

10. The multispecific construct of claim 9, wherein the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB.

11. The multispecific construct of any one of claims 0-10, wherein the second antibody portion is selected from the group consisting of a full length antibody, Fab, Fab', F(ab')2, scFv and sdAb.

12. The multispecific construct of claim 11, wherein the second antibody portion is a sdAb.

13. The multispecific construct of claim 12, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 within a single monomeric variable antibody domain, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, and the single monomeric variable antibody domain comprises the amino acid sequence shown in SEQ ID NO:

27.

14. The multispecific construct of claim 12, wherein the sdAb comprises: (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

15. The multispecific construct of any one of claims 0-14, wherein the second antibody portion comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:

27.

16. The multispecific construct of any one of claims 0-15, wherein the multispecific construct is a bispecific antibody or a bispecific binding fragment.

17. The multispecific construct of any one of claims 0-5 and 9-16, wherein: (1) The first antibody portion comprises: (a) HC-CDR1, HC-CDR2 and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a heavy chain variable region (VH), wherein the heavy chain variable region comprises the sequence shown in SEQ ID No: 7; (b) LC-CDR1, LC-CDR2 and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a light chain variable region (VL), wherein the light chain variable region comprises the sequence shown in SEQ ID No: 8, and (2) The second antibody portion comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 within a single monomeric variable antibody domain, which respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3, and the single monomeric variable antibody domain comprises the amino acid sequence shown in SEQ ID NO:

27.

18. A pharmaceutical composition comprising the multispecific construct of any one of claims 0-17, and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding the multispecific construct of any one of claims 0-17.

20. A vector comprising the nucleic acid of claim 19.

21. A host cell comprising the nucleic acid of claim 19 or the vector of claim 20.

22. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of the multispecific construct of any one of claims 0-17 or the pharmaceutical composition of claim 18.

23. The method of claim 22, wherein the disease or condition is cancer.

24. Use of the multispecific construct of any one of claims 0-17 in the preparation of a medicament for treating a disease or disorder in a subject in need thereof.

25. A multispecific construct comprising: (1) a first antibody portion that specifically binds to a tumor antigen; and (2) a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB.

26. The multispecific construct of claim 0, wherein after the first antibody portion binds to the tumor antigen, the second antibody portion enhances activation of 4-1BB by at least 10-fold.

27. The multispecific construct of claim 24 or 25, wherein the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB.

28. The multispecific construct of any one of claims 24-27, wherein the second antibody portion is an sdAb.

29. The multispecific construct of claim 0, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 within a single monomeric variable antibody domain, comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, the single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:

27.

30. The multispecific construct of claim 0, wherein the sdAb comprises: (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:

26.

31. The multispecific construct of any one of claims 0-0, wherein the tumor antigen is CLDN6.

32. A multispecific construct comprising two heavy chain components and two light chain components, wherein: (a) each heavy chain component comprises the sequence shown in SEQ ID NO: 28, and each light chain component comprises the sequence shown in SEQ ID NO: 29; (b) each heavy chain component comprises the sequence shown in SEQ ID NO:30, and each light chain component comprises the sequence shown in SEQ ID NO:31; (c) each heavy chain component comprises the sequence shown in SEQ ID NO:32, and each light chain component comprises the sequence shown in SEQ ID NO:33; (d) each heavy chain component comprises the sequence shown in SEQ ID NO:34, and each light chain component comprises the sequence shown in SEQ ID NO:35; (e) each heavy chain component comprises the sequence shown in SEQ ID NO:36, and each light chain component comprises the sequence shown in SEQ ID NO:37; or (f) each heavy chain component comprises the sequence shown in SEQ ID NO:38, and each light chain component comprises the sequence shown in SEQ ID NO:39.

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