Multispecific constructs and methods thereof

Abstract

Bispecific and multispecific antibodies targeting claudin 6 (CLDN6) and 4-1BB are provided. These antibodies can bind to 4-1BB but cannot activate 4-1BB signaling in the absence of CLDN6-expressing cells. However, in the presence of CLDN6-expressing cells, these antibodies can induce CLDN6-dependent 4-1BB signaling, resulting in a strong immune response against CLDN6-expressing tumor cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 070870, filed January 9, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] The present application relates to multispecific molecules, such as anti-CLDN6 / anti-4-1BB bispecific antibodies, and uses thereof, including treating diseases or conditions. [Background technology]

[0003] Claudins are a family of proteins that form important components of tight cell junctions. Claudin-6 (CLDN6) is a four-transmembrane protein involved in the formation of tight junctions. CLDN6 mRNA and protein are absent in adult human normal tissues. On the other hand, high transcript levels of CLDN6 have been frequently detected in various human solid cancers, such as testicular, ovarian, uterine, and lung adenocarcinomas. Similar to mRNA levels, CLDN6 protein has been reported to be high and uniform in these human cancers.

[0004] 4-1BB (CD137, tumor necrosis factor superfamily 9) is a member of the TNF-receptor superfamily (TNFRSF) and a costimulatory molecule expressed after activation of immune cells, both 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 also shown that activation of 4-1BB enhances immune responses and eliminates tumors in mice. Thus, 4-1BB is suggested to be a promising target molecule in cancer immunology.

[0005] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entireties. Summary of the Invention

[0006] In one aspect, provided herein is a multispecific construct comprising a first antibody portion that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, where 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.

[0007] In some embodiments, provided herein is a multispecific construct comprising a first antibody moiety that specifically binds to a tumor antigen and a second antibody moiety that specifically binds to 4-1BB, where binding of the first antibody moiety to the tumor antigen triggers the second antibody moiety to activate 4-1BB. In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced by at least about any one of 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 (including any range between these values) after binding of the first antibody moiety to the tumor antigen. In some embodiments, the multispecific construct activates 4-1BB signaling without binding to the tumor antigen. In some embodiments, the second moiety does not activate 4-1BB signaling unless it binds to the tumor antigen.

[0008] In some embodiments, a multispecific construct is provided that comprises 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.

[0009] Provided herein is 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.

[0010] In some embodiments, the first antibody portion is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab')2, a scFv, and a sdAb. In some embodiments, the first antibody portion comprises (1) HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:7, and (2) LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the light chain variable region (VL) comprising the sequence set forth 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), where (a) the VH comprises (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and (b) the VL comprises (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 first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), where (1) the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising at least about 80% sequence identity to SEQ ID NO: 7, and / or (2) the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising at least about 80% sequence identity to SEQ ID NO: 8. In some embodiments, the first antibody portion comprises (1) HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 18, and (2) LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a light chain variable region (VL) comprising the sequence set forth 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), where (1) the VH comprises (i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and (2) the VL comprises (i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and (iii) an 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), where (1) the VH comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 18, and / or (2) the VL comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 19. In some embodiments, binding of the first antibody portion to CLDN6 triggers the second antibody portion to activate 4-1BB.

[0011] In some embodiments, the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the second antibody portion is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab')2, a scFv, and a sdAb. In some embodiments, the second antibody portion is a sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, each of which comprises the amino acid sequence of CDR1, CDR2, and CDR3 within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the sdAb comprises (1) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO:24; (2) an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25; and (3) an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, 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.

[0012] 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 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:7, and (b) LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:8, and (2) the second antibody portion comprises sdAb-CDR1, sdAb-CDR2, and sdAb CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27.

[0013] In some embodiments, a pharmaceutical composition is provided comprising a multispecific construct described herein and a pharma- ceutically acceptable carrier.

[0014] 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.

[0015] 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 described herein or a pharmaceutical composition described herein. Also provided herein is the use of a multispecific construct 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.

[0016] In some embodiments, a multispecific construct is provided that comprises (1) a first antibody moiety that specifically binds to a tumor antigen and (2) a second antibody moiety that specifically binds to 4-1BB, where binding of the first antibody moiety to the tumor antigen triggers the second antibody moiety to activate 4-1BB. In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced at least 10-fold after binding of the first antibody moiety to the tumor antigen. In some embodiments, the second antibody moiety specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the second antibody moiety is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 that comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain that comprises the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the sdAb comprises: (1) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the tumor antigen is CLDN6.

[0017] 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 set forth in SEQ ID NO:28 and / or SEQ ID NO:44 and each light chain component comprises the sequence set forth in SEQ ID NO:29; (b) each heavy chain component comprises the sequence set forth in SEQ ID NO:30 and each light chain component comprises the sequence set forth in SEQ ID NO:31; (c) each heavy chain component comprises the sequence set forth in SEQ ID NO:32 and each light chain component comprises the sequence set forth in SEQ ID NO:33; (d) each heavy chain component comprises the sequence set forth in SEQ ID NO:34 and each light chain component comprises the sequence set forth in SEQ ID NO:35; (e) each heavy chain component comprises the sequence set forth in SEQ ID NO:36 and each light chain component comprises the sequence set forth in SEQ ID NO:37; or (f) each heavy chain component comprises the sequence set forth in SEQ ID NO:38 and each light chain component comprises the sequence set forth in SEQ ID NO:39.

[0018] The application provides kits comprising a multispecific construct as described herein and / or a medicament as described herein, and a package insert or instructions for using the multispecific construct (or pharmaceutical composition) to treat a disease or condition (e.g., cancer).

[0019] It should be understood that one, some, or all of the features of the various embodiments described herein may 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 in the following detailed description. [Brief description of the drawings]

[0020] [Figure 1A] 1 shows the binding affinity of the CLDN6x4-1BB bispecific antibody (BsAb) disclosed herein to CLDN6. [Figure 1B] 1 shows the binding affinity of the CLDN6x4-1BB bispecific antibody (BsAb) disclosed herein to CLDN6.

[0021] [Figure 2A] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to CLDN6-expressing cells. [Figure 2B] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to CLDN6-expressing cells. [Figure 2C] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to CLDN6-expressing cells.

[0022] [Figure 3A] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to 4-1BB. [Figure 3B] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to 4-1BB. [Figure 3C] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to 4-1BB. [Figure 3D] 1 shows the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to 4-1BB.

[0023] [Figure 4A] 1 shows binding of the CLDN6x4-1BB BsAb disclosed herein to soluble 4-1BB and 4-1BB expressing cells. [Figure 4B] 1 shows binding of the CLDN6x4-1BB BsAb disclosed herein to soluble 4-1BB and 4-1BB expressing cells.

[0024] [Figure 5A] 1 shows CLDN6-dependent 4-1BB activation of the CLDN6x4-1BB BsAb disclosed herein. [Figure 5B] 1 shows CLDN6-dependent 4-1BB activation of the CLDN6x4-1BB BsAb disclosed herein. [Figure 5C] 1 shows CLDN6-dependent 4-1BB activation of the CLDN6x4-1BB BsAb disclosed herein. [Figure 5D] 1 shows CLDN6-dependent 4-1BB activation of the CLDN6x4-1BB BsAb disclosed herein.

[0025] [Figure 6A] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6B] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6C] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6D] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6E]1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6F] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6G] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6H] 1 shows activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein.

[0026] [Figure 7A] 1 shows tumor inhibition in CT26 murine colon carcinoma tumor-bearing hu4-1BB mice following treatment with the CLDN6x4-1BB BsAb disclosed herein. [Figure 7B] 1 shows tumor inhibition in CT26 murine colon carcinoma tumor-bearing hu4-1BB mice following treatment with the CLDN6x4-1BB BsAb disclosed herein.

[0027] [Figure 8A] 1 shows liver function following treatment with the CLDN6x4-1BB BsAb of the present application. [Figure 8B] 1 shows liver function following treatment with the CLDN6x4-1BB BsAb of the present application.

[0028] [Figure 9A] 1 shows tumor inhibition in MC38 murine colon adenocarcinoma tumor-bearing hu4-1BB mice following treatment with the CLDN6x4-1BB BsAb disclosed herein. [Figure 9B] 1 shows tumor inhibition in MC38 murine colon adenocarcinoma tumor-bearing hu4-1BB mice following treatment with the CLDN6x4-1BB BsAb disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] definition The term "antibody" is used in its broadest sense and encompasses a variety of 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, so 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.

[0030] A full-length antibody comprises 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 may be referred to as "VH" and "VL", respectively. The variable regions in both chains usually contain three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are interposed between adjacent sections known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant regions of their heavy chains. 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. Some of the major antibody classes are divided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0031] The term "antigen-binding fragment" as used herein refers to an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single chain Fv (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, single domain antibody (e.g., camelized single domain antibody), nanobody, domain antibody, 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 as that bound by the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0032] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0033] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified 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 MPet al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and the definitions include overlapping or subsets of amino acid residues when compared against each other. However, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs as defined by each of the above cited references are set forth below in Table 1 for 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 hereby incorporated by reference in their entirety for use in this application and for possible inclusion in one or more claims herein. [Table 1]

[0034] The phrases "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody compositions in Kabat et al., supra. When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of the FR or hypervariable region (HVR) of the variable domain, or an insertion into the FR or HVR. For example, a heavy chain variable domain may contain one amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may also contain multiple inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody may be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0035] Unless otherwise indicated herein, the numbering of residues in immunoglobulin heavy chains is the EU index numbering according to Kabat et al., supra, with minor modifications. Briefly, five additional residues were added to the hypervariable loop before the heavy chain CDR1. "EU index according to Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0036] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0037] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region (Fc). 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).

[0038] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or an antibody made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be generated using a variety of 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). Human monoclonal antibodies can also be made using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (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 antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

[0039] "Percent (%) amino acid sequence identity" or "homology" with respect to the 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 polypeptide after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, for example, 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 evaluating alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent 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).

[0040] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of the two sequences compared are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of 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 positions compared, multiplied by 100. For example, if 6 out of 10 positions of two sequences are matched or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC are 50% homologous. Generally, the comparison is made when the two sequences are aligned to give maximum homology.

[0041] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant region domain is the C H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or C L ) domain.

[0042] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0043] The "CH1 domain" (also referred to as "C1" for "H1" domain) typically spans from about amino acid 118 to about amino acid 215 (EU numbering system).

[0044] The "hinge region" is generally defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds in the same positions.

[0045] The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically spans 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. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in the intact native IgG molecule. It has been speculated that the carbohydrates may provide an alternative for domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0046] The "CH3 domain" (also referred to as the "C3" domain) comprises the stretch from the C-terminal residue of the Fc region to the CH2 domain (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically to amino acid residue 446 or 447 of IgG).

[0047] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from amino acid residue position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by genetically engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0048] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, 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 de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those yet to be identified, are encompassed by the term "FcR" herein.

[0049] As used herein, the term "epitope" refers to the particular 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 in an antigen if they exhibit competitive binding to the antigen.

[0050] As used herein, a first antibody or fragment thereof "competes" for binding of a second antibody or fragment thereof to a target antigen if the first antibody or fragment thereof inhibits target antigen binding of the second antibody or fragment thereof by at least about 50% (such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof. And vice versa. A high throughput process for "binning" antibodies based on mutual competition is described in PCT Publication No. WO03 / 48731.

[0051] As used herein, the terms "specifically bind," "specifically recognize," 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 this target with greater affinity, avidity, more readily, and / or with a longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10%. -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M, or ≤ 10 -12 Dissociation constant of M (K D). In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not have to include, exclusive binding. Antibody or antigen binding domain binding specificity 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™ test, and peptide scan.

[0052] 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 free of association with all other components from its production environment.

[0053] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof 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 is produced. Preferably, an isolated nucleic acid is free of association with all components associated with the environment in which it is produced. An isolated nucleic acid molecule encoding a polypeptide and antibody described herein is present in a form or setting that is different from that in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide and antibody described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0054] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0055] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of inducing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0056] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0057] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0058] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For 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, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of this application contemplate any one or more of these aspects of treatment.

[0059] In the context of cancer, the term "treating" includes any or all of 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.

[0060] The term "inhibit" or "inhibiting" refers to the reduction or elimination of any phenotypic characteristic, or the reduction or elimination of the occurrence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, decrease, or prevention of an activity, function, and / or amount compared to a reference. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In yet another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0061] The term "agonizing" or "agonize" refers to an increase or enhancement of any phenotypic characteristic, or the occurrence, degree or likelihood of that characteristic. "Increasing" or "enhancing" refers to decreasing, reducing, or preventing an activity, function, and / or amount compared to a reference. In certain embodiments, "increasing" or "enhancing" refers to the ability to cause an overall increase in activity, function, and / or amount, for example, at least about 1-fold or more. In another embodiment, "increasing" or "enhancing" refers to the ability to cause an overall increase in activity, function, and / or amount, for example, at least about 5-fold or more compared to a reference. In yet another embodiment, "increasing" or "enhancing" refers to the ability to cause an overall increase in activity, function, and / or amount, for example, at least about any one of 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 range between these values), or more than about 100-fold compared to a reference.

[0062] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. The reference can be obtained from a healthy sample and / or a non-disease sample. In some examples, the reference can be obtained from an untreated sample. In some examples, the reference is obtained from a non-disease or untreated sample of an individual. In some examples, the reference is obtained from one or more healthy individuals that are not individuals or patients.

[0063] As used herein, "delaying the onset of disease" means to postpone, prevent, slow down, retard, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0064] "Preventing" as used herein includes providing prophylaxis with respect to the onset or recurrence of a disease in an individual who may have a predisposition to the disease, but has not yet been diagnosed with the disease.

[0065] As used herein, "inhibiting" a function or activity refers to a decrease in function or activity as compared to conditions that are otherwise the same under the condition or parameter of interest, or as compared to another condition, e.g., an antibody that inhibits tumor growth reduces the rate of tumor growth as compared to the rate of tumor growth in the absence of the antibody.

[0066] As used herein, "based on" includes evaluating, determining, or measuring the characteristics of an individual described herein (and preferably selecting an individual suitable for treatment). When the status of claudin-18 abnormality is "used as the basis" of the method of selecting, evaluating, measuring, or determining a treatment described herein, the CLDN6 abnormality determined before and / or during treatment, and the resulting status (including the presence, absence, expression level, activity level, and / or phosphorylation level of CLDN6) are used by the clinician in assessing any of the following: (a) the likelihood or likelihood that the individual is suitable for initially receiving treatment(s); (b) the likelihood or likelihood that the individual is not suitable for initially receiving treatment(s); (c) responsiveness to treatment; (d) the likelihood or likelihood that the individual is suitable for continued treatment(s); (e) the likelihood or likelihood that the individual is not suitable for continued treatment(s); (f) dosage adjustment; or (g) predicting the likelihood of clinical benefit.

[0067] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, an individual is a human.

[0068] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0069] Reference herein to "about" a value or parameter includes (and describes) the variation on that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0070] As used herein, a reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0071] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0072] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the content clearly dictates otherwise.

[0073] Antibody binding affinity The binding specificity of the antibody moieties of the multispecific constructs described herein can be determined experimentally by methods known in the art, including, but not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ test and peptide scan.

[0074] In some embodiments, the binding affinity is determined by the dissociation constant K D 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 (see, e.g., the Octet System by ForteBio), mesoscale discover 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.

[0075] In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is D is about 10-7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M, or about 10 -7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, the target antigen (e.g., CLDN6 or 4-1BB) is a human antigen.

[0076] In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 103 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is on is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 5 M -1 s-1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is on is 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 (e.g., CLDN6 or 4-1BB) is a human antigen.

[0077] In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is off takes about 1s -1 ~about 10 -6 s -1 , about 1s -1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1, about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 , or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., CLDN6 or 4-1BB) is off is 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 (e.g., CLDN6 or 4-1BB) is a human antigen.

[0078] Chimeric or humanized antibodies In some embodiments, one or more of the antibody moieties of the multispecific construct of the present application is a chimeric antibody. 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, the chimeric antibody comprises a non-human variable region (e.g., a variable region of mouse origin) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0079] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues of a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0080] Humanized antibodies and methods for making humanized antibodies are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 78,087,409; 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 a "guided selection" approach to FR shuffling).

[0081] Human framework regions that may be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular light chain variable region or heavy chain variable region subgroup (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (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 obtained from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Immunol. 272:10678-10684 (1997)). al., J. Biol. Chem. 271:22611-22618 (1996)).

[0082] Human antibodies In some embodiments, one or more of the antibody moieties of the multispecific constructs of the present application are human antibodies (also known as human domain antibodies, or human DAbs). Human antibodies can be produced using a variety of 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.

[0083] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes K-MMOUSE® technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0084] Human antibodies (e.g., human DAbs) can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have also been described (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 via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing 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).

[0085] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from an antibody library are described below.

[0086] Substitution, insertion, and deletion variants In some embodiments, antibody variants comprising one or more amino acid substitutions are included in the multispecific constructs described herein. Sites of interest for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are shown in Table 2 under the heading of "exemplary substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 2]

[0087] Amino acids can be classified into groups based on 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 that influence chain directionality: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0088] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0089] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have an alteration (e.g., improvement) in a certain biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained a certain biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0090] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made to "hot spots" of HVRs, i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting from the secondary libraries is described, for example, in 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 genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This 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. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Often, CDR-H3 and CDR-L3 in particular are targeted.

[0091] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such modifications do not substantially reduce the antigen-binding ability of the antibody. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such modifications may be outside the "hot spots" or CDRs of the HVRs. In some embodiments of the variant VHH sequences provided above, each HVR may be unmodified or contain no more than one, two, or three amino acid substitutions.

[0092] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or 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 there is an effect on antibody-antigen interaction. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, contact points between the antibody and antigen are identified from a crystal structure of an antigen-antibody complex. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0093] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusing an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0094] Glycosylation variants In some embodiments, one or more of the antibody moieties of the multispecific construct of the present application are modified to increase or decrease the degree of glycosylation of the construct. Addition or removal of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0095] If the antibody moiety comprises an Fc region, the carbohydrate attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are generally located at the C of the Fc region. H The oligosaccharide is N-linked to Asn297 in the 2 domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can contain a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody moiety can be made to generate antibody variants with improved certain properties.

[0096] In some embodiments, the antibody moiety has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose can be determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycan structures (e.g., complex, mixed and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO2008 / 077546. Asn297 refers to an asparagine residue located near position 297 (Fc region residues in EU numbering) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294-300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 01321 40; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 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, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the alpha-1,6-fucosyltransferase gene FUT8 has been knocked out (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).

[0097] In some embodiments, the antibody moiety has bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants comprising at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0098] Fc region variants In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody moiety, 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) that comprises an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0099] In some embodiments, Fc fragments possess some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody portion is important, but certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas 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 to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 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 assay methods can be employed (see, e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, Calif.; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 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 et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0100] Antibodies with reduced effector function include antibodies containing substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (wherein amino acid numbering is according to the EU index). See, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Such Fc variants include Fc variants containing substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 (wherein amino acid numbering is according to the EU index), including so-called "DANA" Fc variants in which residues 265 and 297 are replaced by alanine (U.S. Patent No. 7,332,581) (wherein amino acid numbering is according to the EU index).

[0101] Certain antibody variants with improved or reduced binding to FcRs have also been 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)).

[0102] 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 a L235A mutation.

[0103] In some embodiments, the antibody portion comprises an Fc region comprising one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (residues according to EU numbering).

[0104] In some embodiments, modifications are made to the Fc region that result in altered (i.e., either improved or decreased) C1q 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).

[0105] In some embodiments, the antibody partial variant comprises a variant Fc region that comprises one or more amino acid substitutions that modify the half-life and / or alter binding to the fetal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to the fetal Fc receptor (FcRn), responsible for transport of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region that comprises one or more substitutions that modify binding of the Fc region to FcRn. Such Fc variants include those that comprise substitutions at one or more of the Fc region residues, for example, substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0106] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0107] Cysteine ​​Engineered Antibody Variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of one or more antibody moieties of the multispecific constructs herein are replaced with cysteine ​​residues. In specific embodiments, the replaced residues are present at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, 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) of the heavy chain Fc region. Cysteine ​​engineered antibody moieties can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0108] Anti-CLDN6 antibody part The anti-CLDN6 antibody portion of the multispecific constructs described in this application includes any antibody portion that specifically binds to claudin-6 ("CLDN6").

[0109] Claudin 6 (CLDN6) Claudins are a family of tight junction membrane proteins expressed in epithelia and endothelium that form paracellular barriers and pores that determine the permeability of tight junctions. Claudins are recognized as important regulators in cancer development, progression, and metastasis, and play distinct roles in various cancers due to differences in tissue-dependent expression patterns (Tabaries et al.(2017).“The role of claudins in cancer metastasis.” Oncogene 36,1176-1190). Claudin-6 (CLDN6), a member of the claudin family, functions as a tight junction molecule that plays an important role in cell-cell adhesion in epithelial or endothelial cell sheets. CLDN6 encodes a four-transmembrane protein with a size of 220 amino acids and a molecular weight of 23,292 Da. CLDN6 has been identified as an origin of cell adhesion signaling involved in the control of nuclear receptor activity by targeting molecules of the nuclear receptor superfamily and managing 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 cancer (Zhang et al. (2021) Front. Cell. Dev. Biol. 9: 726656). In some embodiments, the CLDN6 is human CLDN6 ("hCLDN6"). In some embodiments, the hCLDN6 comprises the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof (e.g., a post-translational modification variant and / or a conformational variant).

[0110] Exemplary Anti-CLDN6 Antibody Moieties In some embodiments, the anti-CLDN6 antibody portion comprises, for binding to CLDN6, a) a heavy chain variable region (V H ) and the light chain variable region (V L ), where: a) V Hcomprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and b) V L competes for binding to CLDN6 with an antibody portion 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 comprises: a) a heavy chain variable region (V H ) and the light chain variable region (V L ), where: a) V H comprises i) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) V L competes with an antibody portion comprising i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0111] The anti-CLDN6 antibody moiety described in the present application includes any antibody moiety that specifically binds to CLDN6. In some embodiments, the anti-CLDN6 antibody moiety of the present application includes a) an HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of CDR1, CDR2, and CDR3, respectively, in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:7, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to the sequence set forth in SEQ ID NO:7; and b) an HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of CDR1, CDR2, and CDR3, respectively, in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:8, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to the sequence set forth in SEQ ID NO:8. and LC-CDR1, LC-CDR2, and LC-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising a variant thereof having sequence identity of at least one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising CDR1, CDR2, and CDR3 in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:7 and CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:8.

[0112] In some embodiments, the anti-CLDN6 antibody portion comprises a) an HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of CDR1, CDR2, and CDR3, respectively, in a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 18, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to the sequence set forth in SEQ ID NO: 18; and b) an HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of CDR1, CDR2, and CDR3, respectively, in a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90% and LC-CDR1, LC-CDR2, and LC-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising a variant thereof having sequence identity of at least one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising CDR1, CDR2, and CDR3 in the heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 18 and CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 19.

[0113] 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) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about three (e.g., about one, two, three, or any of) amino acid substitutions in the HC-CDR1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about three (e.g., about one, two, three, or any of) amino acid substitutions in the HC-CDR2, and iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about three (e.g., about one, two, three, or any of) amino acid substitutions in the HC-CDR3. and b) the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR1, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR2, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR3.

[0114] 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) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about three (e.g., about one, two, three, or any of) amino acid substitutions in HC-CDR1, ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about three (e.g., about one, two, three, or any of) 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 comprising up to about three (e.g., about one, two, three, or any of) amino acid substitutions in HC-CDR3. and b) the VL comprises i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR1, ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR2, and iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR3.

[0115] 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:8. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising a VH comprising the sequence set forth in SEQ ID NO:7 and a VL comprising the sequence set forth in SEQ ID NO:8.

[0116] 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO: 19. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising a VH comprising the sequence set forth in SEQ ID NO: 18 and a VL comprising the sequence set forth in SEQ ID NO: 19.

[0117] The anti-CLDN6 antibody portion of the present application may be in any suitable format known in the art. In some embodiments, the anti-CLDN6 antibody portion may be selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab')2, a scFv, and a sdAb.

[0118] 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 the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof. In some embodiments, the Fc fragment is an IgG1 or IgG4 Fc fragment.

[0119] 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 substitutions selected from the group consisting of N297A, N297Q, N297G, or L235E. In some embodiments, the Fc fragment is an IgG1 fragment comprising one or more substitutions selected from the group consisting of 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 sequence set forth in SEQ ID NO:42.

[0120] 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:11. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising two heavy chains each comprising SEQ ID NO:9 and / or SEQ ID NO:42, and two light chains each comprising SEQ ID NO:11.

[0121] 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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 having at least about 80% (e.g., including at least about any one of 80%, 85%, 87%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:11. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising two heavy chains each comprising SEQ ID NO: 10 and / or SEQ ID NO: 43, and two light chains each comprising SEQ ID NO: 11.

[0122] 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 comprising at least about 80% (e.g., at least about any 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 comprising at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:22. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising two heavy chains each comprising SEQ ID NO:20 and two light chains each comprising SEQ ID NO:22.

[0123] 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 having at least about 80% (e.g., at least about any 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 having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:22. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising two heavy chains each comprising SEQ ID NO:21 and two light chains each comprising SEQ ID NO:22.

[0124] Anti-4-1BB antibody part The anti-4-1BB antibody portion of the multispecific construct described in this 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 that contains four extracellular cysteine-rich domains ("CRDs", i.e., CRD1, CDR2, CRD3, and CRD4), followed by a short transmembrane domain and a C-terminal cytoplasmic region. The CRD2 and CRD3 of h4-1BB interact with the ligand 4-1BBL (Bitra et al. (2018) J Biol Chem. 293(26):9958-9969). In contrast to other TNFRs, h4-1BB exists as a disulfide-linked dimer, and dimerization is thought to occur through an unpaired cysteine ​​(Cys121) in the CRD4 of h4-1BB. In some embodiments, h4-1BB comprises a sequence set forth in SEQ ID NO: 41 or a variant thereof (e.g., a post-translational modification variant and / or a conformational variant). In some embodiments, the anti-4-1BB antibody portion binds to the CRD3 / CRD4 region of 4-1BB.

[0125] The anti-4-1BB antibody moiety may be in any suitable format known in the art. In some embodiments, the anti-4-1BB antibody moiety is selected from the group consisting of a full-length antibody, a Fab, a Fab', a F(ab')2, a scFv, and a sdAb. In some embodiments, the anti-4-1BB antibody moiety comprises a single domain antibody that binds to 4-1BB.

[0126] Exemplary Anti-4-1BB Antibody Moieties In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3 within a single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., including at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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., the same) as the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO: 27.

[0127] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO:24, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; b) an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and c) an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR3.

[0128] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO:24, an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26.

[0129] 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 having at least about 80% (e.g., at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more 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., the same) as the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO: 27.

[0130] Multispecific constructs In one aspect, provided herein is a multispecific construct comprising a first antibody portion that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, where 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.

[0131] In some embodiments, provided herein is a multispecific construct comprising a first antibody moiety that specifically binds to a tumor antigen and a second antibody moiety that specifically binds to 4-1BB, where binding of the first antibody moiety to the tumor antigen triggers the second antibody moiety to activate 4-1BB. In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced by at least about any one of 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 (including any range between these values) after binding of the first antibody moiety to the tumor antigen. In some embodiments, the multispecific construct activates 4-1BB signaling without binding to the tumor antigen. In some embodiments, the second moiety does not activate 4-1BB signaling unless it binds to the tumor antigen.

[0132] In some embodiments of the multispecific construct of the present application, the second antibody moiety is an sdAb. In some embodiments, the sdAb 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 comprising the amino acid sequence set forth in SEQ ID NO:27.

[0133] In some embodiments, the sdAb comprises an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR3.

[0134] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO:27, or a variant thereof having at least about 80% (e.g., including at least about any of 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 sdAb for 4-1BB (e.g., human 4-1BB) is comparable (e.g., the same) as the affinity of an sdAb comprising SEQ ID NO:27.

[0135] The tumor antigen to which the first antibody portion specifically binds can be any suitable tumor antigen known in the art. In some embodiments, the tumor antigen is CLDN6.

[0136] In some embodiments, the present 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 described herein.

[0137] In some embodiments of the multispecific constructs of the present application, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises (1) a CDR1 sequence within a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO:7, or a variant thereof having at least about 80% (e.g., including at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:7. and (2) LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:8, or a variant thereof having at least about 80% (e.g., including at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:8. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising CDR-1, CDR-2 and CDR-3 of the VH set forth in SEQ ID NO:7 and CDR-1, CDR-2 and CDR-3 of the VL set forth 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0138] In some embodiments, a 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 comprising up to about three (such as about one, two, three, or any other) amino acid substitutions in the HC-CDR1, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about three (such as about one, two, three, or any other) amino acid substitutions in the HC-CDR2; and (iii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 3. or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in HC-CDR3, and the VL comprises (i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR1; (ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR2; and (iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about three (such as about one, two or three) 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0139] In some embodiments, a 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 VH that is at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 17 and / or the VL comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO: 8. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising a VH set forth in SEQ ID NO: 7 and a VL set forth 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0140] In some embodiments, a 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) a CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR19, CDR110, CDR19, CDR111, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19 ... and (2) LC-CDR1, LC-CDR2, and LC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in the light chain variable region (VL) that comprises the sequence set forth in SEQ ID NO:19, or a variant thereof having at least about 80% (e.g., including at least about any of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO:19. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising CDR-1, CDR-2 and CDR-3 of the VH set forth in SEQ ID NO: 18 and CDR-1, CDR-2 and CDR-3 of the VL set forth 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0141] In some embodiments, a 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 comprising up to about three (such as about one, two, three, or any other) amino acid substitutions in the HC-CDR1, (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about three (such as about one, two, three, or any other) amino acid substitutions in the HC-CDR2, and (iii) an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14. or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in HC-CDR3, and the VL comprises (i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR1; (ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in LC-CDR2; and (iii) an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about three (such as about one, two or three) 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0142] In some embodiments of the multispecific constructs of the application, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) that include an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion includes a VH that is at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 200%, 201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 300%, 300, 300, 310, 311%, 312%, 313%, 314%, 315%, 316%, 317%, 318%, 319%, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 36 and / or the VL comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more than 99%) sequence identity to SEQ ID NO: 19. In some embodiments, the affinity of such an anti-CLDN6 antibody portion for CLDN6 (e.g., human CLDN6) is comparable (e.g., the same) as the affinity of an anti-CLDN6 antibody portion comprising a VH set forth in SEQ ID NO: 18 and a VL set forth 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0143] In some embodiments of the multispecific constructs of the present application, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-CLDN6 antibody portion comprises, a VH comprising the amino acid sequence of SEQ ID NO: 18, and / or a VL comprising 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 comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about three (such as about one, two or three) 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 having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity (including any range between these values)).

[0144] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb.

[0145] In some embodiments, a 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, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27, or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one 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). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (eg, human 4-1BB) is comparable (eg, the same) as the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO:27.

[0146] In some embodiments, a 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, wherein the sdAb comprises an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (such as about one, two or three) amino acid substitutions in sdAb-CDR3.

[0147] 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, the sdAb comprising 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 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity (including any range between these values)). 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., the same) as the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO:27.

[0148] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that includes an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion includes (1) a heavy chain variable region (VH) that includes amino acid sequences of CDR1, CDR2, and CDR3, respectively, that include the sequence set forth 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, in a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:8, wherein the anti-4-1BB antibody portion comprises an sdAb, the sdAb comprising sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27.

[0149] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that includes an antibody (e.g., a single domain antibody) that binds to 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion includes (1) a heavy chain variable region (VH) that includes amino acid sequences of CDR1, CDR2, and CDR3, respectively, that include the sequence set forth in SEQ ID NO:18. and (2) LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:19, wherein the anti-4-1BB antibody portion comprises an sdAb, the sdAb comprising sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27.

[0150] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-CLDN6 antibody portion that comprises an antibody (e.g., a full-length antibody) that specifically binds CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that comprises an antibody (e.g., a single domain antibody) that binds 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: a VH comprises the amino acid sequence of SEQ ID NO:7, or a variant thereof having at least about 80% sequence identity to SEQ ID NO:7 (e.g., at least about any one 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)) and / or a VL comprises the amino acid sequence of SEQ ID NO: The anti-4-1BB antibody portion comprises an amino acid sequence of SEQ ID NO:8 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:8 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity (including any ranges between these values)), and the anti-4-1BB antibody portion comprises an sdAb, the sdAb comprising an amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity (including any ranges between these values)). In some embodiments, the affinity of such a multispecific construct (e.g., a bispecific antibody) for CLDN6 and 4-1BB is comparable (e.g., the same) as the affinity of a multispecific construct comprising an anti-CLDN6 antibody portion comprising a full-length antibody comprising the VH set forth in SEQ ID NO:7 and the VL set forth in SEQ ID NO:8, and an anti-4-1BB antibody portion comprising a single domain antibody comprising the sequence set forth in SEQ ID NO:27.

[0151] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-CLDN6 antibody portion that comprises an antibody (e.g., a full-length antibody) that specifically binds CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that comprises an antibody (e.g., a single domain antibody) that binds 4-1BB (e.g., human 4-1BB), wherein the anti-CLDN6 antibody portion comprises: a VH comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 18 (e.g., at least about any one 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)); and / or a VL comprises the amino acid sequence of SEQ ID NO: The anti-4-1BB antibody portion comprises an amino acid sequence of SEQ ID NO:19 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:19 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity (including any ranges between these values)), and the anti-4-1BB antibody portion comprises an sdAb, the sdAb comprising an amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity (including any ranges between these values)). In some embodiments, the affinity of such a multispecific construct (e.g., a bispecific antibody) for CLDN6 and 4-1BB is comparable (e.g., the same) as the affinity of a multispecific construct comprising an anti-CLDN6 antibody portion comprising a full-length antibody comprising the VH set forth in SEQ ID NO: 18 and the VL set forth in SEQ ID NO: 19, and an anti-4-1BB antibody portion comprising a single domain antibody comprising the sequence set forth in SEQ ID NO: 27.

[0152] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that specifically binds to 4-1BB, where the anti-4-1BB antibody portion is fused to the N-terminus of one or both of the heavy 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 that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion that specifically binds to 4-1BB, where the anti-4-1BB antibody portion is fused to the C-terminus of one or both of the heavy chains of the anti-CLDN antibody (e.g., a full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that includes an anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion that specifically binds to 4-1BB, where the anti-4-1BB antibody portion is fused to the N-terminus of one or both of the 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 that includes an anti-CLDN6 antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion is fused to the C-terminus of one or both of the light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody).

[0153] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 (e.g., human CLDN6) and an anti-4-1BB antibody portion that includes a single domain antibody that binds to 4-1BB (e.g., human 4-1BB), where the single domain antibody is fused to the N-terminus of one or both of the 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 that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion that includes a single domain antibody that binds to 4-1BB, where the single domain antibody is fused to the C-terminus of one or both of the 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 that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion that includes a single domain antibody that binds to 4-1BB, where the single domain antibody is fused to the N-terminus of one or both of the 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 that includes an anti-CLDN6 antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to CLDN6 and an anti-4-1BB antibody portion that includes a single domain antibody that binds to 4-1BB, where the single domain antibody is fused to the C-terminus of one or both of the light chains of the anti-CLDN6 antibody (e.g., a full-length anti-CLDN6 antibody).

[0154] In some embodiments, the anti-4-1BB antibody portion is fused to the anti-CLDN6 antibody portion via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker has a length of about 4 to about 50 amino acids. In some embodiments, the linker is selected from the group consisting of (GS)n, (GGGS)n, (GGGGS)n, and (GSGGS)n. In some embodiments, n is 0 to 8. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGSGGGGGS.

[0155] In some embodiments, provided herein are multispecific constructs comprising a heavy chain component and a light chain component, wherein the heavy chain component has an amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 44, or 45, or at least about 80% sequence identity (e.g., at least about any one of 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% to SEQ ID NO: 28, 30, 32, 34, 36, 38, 44, or 45 (which and / or the light chain comprises an amino acid sequence of SEQ ID NO: 29, 31, 33, 35, 37, 39, or a variant thereof having at least about 80% sequence identity (e.g., at least about any one 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: 29, 31, 33, 35, 37, 39.

[0156] In some embodiments, provided herein are multispecific constructs 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: 29, 31, 33, 35, 37, 39.

[0157] In some embodiments, a multispecific construct is provided comprising two heavy chain components and two light chain components, wherein (a) each heavy chain component comprises the sequence set forth in SEQ ID NO:28 and / or 44 and each light chain component comprises the sequence set forth in SEQ ID NO:29; (b) each heavy chain component comprises the sequence set forth in SEQ ID NO:30 and / or SEQ ID NO:45 and each light chain component comprises the sequence set forth in SEQ ID NO:31; (c) each heavy chain component comprises the sequence set forth in SEQ ID NO:32 and each light chain component comprises the sequence set forth in SEQ ID NO:33; (d) each heavy chain component comprises the sequence set forth in SEQ ID NO:34 and each light chain component comprises the sequence set forth in SEQ ID NO:35; (e) each heavy chain component comprises the sequence set forth in SEQ ID NO:36 and each light chain component comprises the sequence set forth in SEQ ID NO:37; or (f) each heavy chain component comprises the sequence set forth in SEQ ID NO:38 and each light chain component comprises the sequence set forth in SEQ ID NO:39.

[0158] In some embodiments, provided herein is a multispecific construct comprising a first antibody moiety that specifically binds to CLDN6 and a second antibody moiety that specifically binds to 4-1BB, where binding of the first antibody moiety to CLDN6 triggers the second antibody moiety to activate 4-1BB. In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced at least 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 after binding of the first antibody moiety to CLDN6. In some embodiments, the multispecific construct does not activate 4-1BB signaling unless it binds to CLDN6. In some embodiments, the second moiety activates 4-1BB signaling without binding to CLDN6.

[0159] 4-1BB signaling activation is the expected mechanism for agonist antibodies such as utomirumab (PF-05082566) and urelumab (BMS-663513). However, some anti-4-1BB moieties of the antibodies of the present disclosure do not necessarily have such activity. Indeed, in some embodiments, the anti-4-1BB moiety of the antibody is preferably unable to independently activate 4-1BB in the absence of CLDN6 binding. Interestingly, as experimental examples show, when the anti-CLDN6 moiety binds to the CLDN6 protein on cells, such CLDN6 binding can induce activation of 4-1BB signaling.

[0160] Compared with known anti-4-1BB agonist antibodies, which generally have dose-limiting on-target hepatotoxicity, the antibodies of the present disclosure are contemplated to be much safer. In tissues such as the liver that do not express CLDN6 under healthy conditions, the antibodies of the present disclosure are not expected to induce cytotoxic immune responses, since they cannot activate 4-1BB signaling. In contrast, in tumor tissues that express and / or are accessible to CLDN6, the antibodies can initiate a strong immune response against tumor cells. Thus, unlike anti-4-1BB antibodies currently in clinical development that suffer from on-target / intrinsic toxicity, the antibodies of the present disclosure may be both potent and safe in cancer therapy.

[0161] nucleic acid Nucleic acid molecules encoding the multispecific constructs or various antibody portions described herein are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) is provided that encodes one or more polypeptides of the multispecific constructs or various antibody portions. In some embodiments, a nucleic acid (or set of nucleic acids) is provided that encodes a multispecific construct (e.g., an anti-CLDN6 / anti-4-1BB bispecific antibody) or a polypeptide portion thereof.

[0162] Also contemplated herein is an isolated host cell comprising a multispecific construct described herein (e.g., an anti-CLDN6 / anti-4-1BB bispecific antibody), a nucleic acid(s) 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).

[0163] The present application also includes variants of these nucleic acid sequences, for example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to the nucleic acid sequences encoding the multispecific constructs (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies) or various antibody portions described herein.

[0164] The present application also provides a vector into which a nucleic acid of the present application is inserted.

[0165] Nucleic acids can be cloned into many types of vectors. For example, the 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.

[0166] Furthermore, the expression vector can be provided to cells in the form of a viral vector.Viral vector technology is well known in the art and 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 useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (see, for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0167] Treatment method Also provided herein are methods of treating a disease or condition in an individual. The methods include administering a multispecific construct (e.g., an anti-CLDN6 / anti-4-1BB bispecific antibody) described herein to an individual (e.g., a mammal, such as a human). In some embodiments, the individual is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, a laboratory animal, etc.

[0168] In some embodiments of the method, 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, colon cancer, squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer, or non-Hodgkin's lymphoma (NHL).

[0169] Compositions, kits and articles of manufacture Also provided herein are compositions (such as formulations) comprising any one 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 vector.

[0170] Suitable formulations of the multispecific constructs described herein (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies) can be obtained by mixing the multispecific constructs having the desired purity with optional pharma- ceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0171] Kits comprising any one of the multispecific constructs described herein (e.g., anti-CLDN6 / anti-4-1BB bispecific antibodies) are also provided. The kits may be useful for any of the methods of treatment described herein.

[0172] The kits of the present application are in suitable packaging, including but not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic pouches), etc. The kits may optionally include additional components, such as buffers and instructional information.

[0173] Thus, the application also provides an article of manufacture. The article of manufacture 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, jars, flexible packaging, and the like. Generally, the container holds the composition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0174] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0175] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. The following examples are intended to be purely illustrative of the present application, and therefore should not be construed as limiting the present application in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0176] Example 1: Generation of CLDN6x4-1BB bispecific antibody Exemplary CLDN6x4-1BB bispecific antibodies shown in Table 3 below were designed and generated. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0177] Example 2. Antigen binding activity of CLDN6x4-1BB BsAb 2.1 Binding affinity of CLDN6×4-1BB BsAb to CLDN6 The binding affinity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT (prepared in Example 1) to human CLDN6 was measured by surface plasmon resonance (SPR). As shown in Figures 1A and 1B, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bound to CLDN6 virus-like particles (VLPs) at 2.36 x 10 -9 M and 1.61×10 -9 K of M D was bonded with.

[0178] A CHO-K1 cell line stably expressing human CLDN6 (CHO-K1-CLDN6) was prepared to evaluate the binding ability of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB 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 min at 4°C. Then, phycoerythrin (PE)-conjugated anti-human IgG antibody was added after washing, and the cells were incubated for another 30 min at 4°C. The mean fluorescence intensity (MFI) of PE was evaluated by FACS. As shown in Figure 2A, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bound to CLDN6-expressing cells in a concentration-dependent manner.

[0179] OVCAR3 and OV90 are human ovarian cancer cell lines with endogenous CLDN6 expression. As shown in Figure 2B and Figure 2C, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT were able to bind to OVCAR3 and OV90. Overall, the binding affinity of the BsAb containing the anti-CLDN6-1 antibody moiety to human CLDN6 is comparable to that of the parent CLDN6-1 antibody to human CLDN6. The binding signal correlated well with the CLDN6 expression level on the surface of OVCAR3 and OV90 cells.

[0180] 2.2 Binding affinity of CLDN6×4-1BB BsAb to 4-1BB The binding affinity of CLDN6-1×4-1BB NA and CLDN6-1×4-1BB WT to human 4-1BB was measured by SPR. As shown in Figures 3A to 3D, CLDN6-1×4-1BBNA and CLDN6-1×4-1BB WT had a binding affinity of 1.64×10 -8 M and 1.57×10 -8 K of M D The affinity of the parent anti-4-1BB sdAb antibody conjugated to the IgG1 Fc fragment (4-1BB sdAb-Fc) for 4-1BB was measured in parallel and found to be 4.393 × 10 -9 K of M D This suggested that the affinity of the 4-1BB antibody portion of BsAb for 4-1BB was equivalent to that of the 4-1BB sdAb-Fc for 4-1BB.

[0181] The binding of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to soluble recombinant human 4-1BB was analyzed by ELISA. As shown in Figure 4A, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bound to recombinant human 4-1BB in a concentration-dependent manner with EC50 of 0.129 nM and 0.078 nM, respectively. Such EC50 was comparable to that of 4-1BB sdAb-Fc. In addition, the binding of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to 4-1BB-expressing HEK293 cells was evaluated by FACS. As shown in Figure 4B, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT were able to bind to 4-1BB with EC50 of 0.406 nM and 0.347 nM, respectively, which was comparable to that of 4-1BB sdAb-Fc.

[0182] Example 3. Functional activity of CLDN6-1x4-1BB BsAb 3.1 Cell line-based functional characterization of CLDN6×4-1BB BsAb To test the ability of CLDN6x4-1BB bispecific antibody to activate 4-1BB signaling, GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line stably expressing 4-1BB and NFκB luciferase reporter was used as effector cells and CLDN6 expressing cells (CHO-K1 CLDN6, OVCAR3 or OV90) were used as target cells. RKO colon cancer cells, which do not express CLDN6, were used as negative control.

[0183] Briefly, GloResponse™ NFκB-luc2 / 4-1BB Jurkat cells (5.0 × 10 per well) were cultured in 100 wells. 4 The cell density was 5.0 × 10 in a white 96-well plate. 4The antibody was serially diluted and added to the plate. After 6 hours of incubation at 37°C, the luminescence was measured. As shown in Figures 5A-5D, urelumab induced 4-1BB activation regardless of CLDN6 expression, whereas the 4-1BB sdAb-Fc of the present application had no agonist activity under the same experimental setting, despite being able to bind to 4-1BB. Similarly, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT also induced NFκB activity in the presence of all CLDN6-expressing target cells, regardless of the expression level of CLDN6. In contrast, when RKO cells, which do not express CLDN6, were used as target cells, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT showed significantly lower 4-1BB activation compared to urelumab, as shown in Figure 5D.

[0184] 3.2 CLDN6×4-1BB BsAb stimulates immune responses in human peripheral blood mononuclear cells (PBMCs) Pre-activated human PBMCs were co-cultured with CLDN6-expressing cells or RKO cells at an effector-to-target (E:T) ratio of 10:1. Different concentrations of antibodies 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.

[0185] As shown in Figures 6A-6F, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT stimulated IL-2 and IFNγ production when PBMCs were cocultured with CLDN6-expressing target cells. However, as shown in Figures 6G and 6H, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT did not stimulate IL-2 or IFNγ production from PBMCs in the presence of RKO, which does not express CLDN6. This suggested that the activity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT was dependent on the presence of tumor antigen. In contrast, 4-1BB sdAb-Fc was inactive in this assay.

[0186] Example 4. Tumor growth inhibition by CLDN6-1x4-1BB BsAb 4.1 CT26 syngeneic mouse colon cancer model CT26 is an N-nitroso-N-methylurethane-(NNMU)-induced undifferentiated colon cancer cell line established from BALB / c mice with advanced colon cancer. CT26 cells, which endogenously express CLDN6, were implanted subcutaneously into BALB / c humanized 4-1BB mice. Tumors grew to an average of 100 mm 3 Once tumors had grown to 100% tumor size, mice were treated intraperitoneally with (a) human IgG, (b) CLDN6-1x4-1BB NA (2 mg / kg), (c) CLDN6-1x4-1BB WT (2 mg / kg), or (d) the parental CLDN6-1 antibody in combination with 4-1BB sdAb-Fc (1.8 mg / kg and 0.7 mg / kg). Treatments were administered twice weekly for a total of six times. Tumor growth was monitored by volumetric measurement. As shown in Figure 7A and Figure 7B, both CLDN6-1x4-1BB WT and CLDN6-1x4-1BB NA exhibited antitumor activity, but CLDN6-1x4-1BB WT showed even stronger activity with 75% tumor growth inhibition (TGI).

[0187] 4.2 MC38 syngeneic mouse colon adenocarcinoma model 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. Tumors grew to an average of 100 mm 3 Once tumors had grown to 100% tumor size, mice 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 a week for a total of three times. See FIG. 9A. Tumor growth was monitored by volumetric measurement. As shown in FIG. 9B, CLDN6-1×4-1BB NA exhibited significant antitumor activity, while treatment with CLDN6-1×4-1BB WT achieved greater tumor growth inhibition at the same dose, leading to complete tumor regression at the higher dose.

[0188] Example 5. Hepatotoxicity assessment of CLDN6x4-1BB BsAb A major concern with t4-1BB agonist antibody therapy is dose-limiting hepatotoxicity, as observed in the clinical development of urelumab. The most common adverse events were elevated alanine transaminase (ALT), elevated aspartate aminotransferase (AST), and fatigue. Therefore, we further evaluated the hepatotoxicity of CLDN6-1×4-1BB WT and CLDN6-1×4-1BB NA.

[0189] Briefly, blood samples were taken from hu4-1BB mice after treatment with CLDN6-1×4-1BB WT or CLDN6-1×4-1BB NA at different doses twice a week for ALT and AST measurement. As shown in Figure 8, no significant elevation of ALT and AST was observed, suggesting little risk of liver toxicity commonly induced by other 4-1BB agonist antibodies.

[0190] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods that are functionally equivalent are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Therefore, the present disclosure is intended to cover the modifications and variations of the present disclosure, provided they fall within the scope of the appended claims and their equivalents.

[0191] 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.

[0192] The present invention has been described in terms of specific embodiments that are recognized or proposed by the inventors to include preferred modes for carrying out the invention. In light of this disclosure, it will be apparent to those skilled in the art that numerous modifications and variations can be made to the specific embodiments exemplified without departing from the intended scope of the invention. For example, codon redundancy allows modifications to be made to the underlying DNA sequence without affecting the protein sequence. Furthermore, biological functional equivalence considerations allow modifications to be made to the protein structure without affecting the type or amount of biological action. All such modifications are intended to be included within the scope of the appended claims. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

Claims

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

2. The first antibody portion and the second antibody portion may individually be a full-length antibody, Fab, Fab', F(ab') ２ , scFv, and sdAb. (a) the first antibody portion is (1) HC-CDR1, HC-CDR2, and HC-CDR3, each of which contains the amino acid sequences of CDR1, CDR2, and CDR3 in a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 7; (2) LC-CDR1, LC-CDR2, and LC-CDR3, each containing the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 8; Contains, or (b) the first antibody portion is (1) HC-CDR1, HC-CDR2, and HC-CDR3, each of which contains the amino acid sequences of CDR1, CDR2, and CDR3 in a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 18; (2) LC-CDR1, LC-CDR2, and LC-CDR3, each containing the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 19; Including, The multispecific construct of claim 1.

4. (1) The first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH is (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) comprises an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (b) the VL is (i) 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) comprises an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (2) the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), (a) the VH is (i) HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (b) the VL is (i) an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; (ii) an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (iii) The multispecific construct of claim 1, comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

17. (a) the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising at least about 80% sequence identity to SEQ ID NO: 7; and / or (2) the VL comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising at least about 80% sequence identity to SEQ ID NO: 8; or (b) the first antibody portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 18; and / or (2) The VL comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 19; The multispecific construct of claim 1.

6. Binding of the first antibody portion to CLDN6 induces the second antibody portion to activate 4-1BB, and / or the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB; The multispecific construct of claim 1.

7. the second antibody portion is an sdAb; Preferably, the sdAb 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 comprising the amino acid sequence set forth in SEQ ID NO: 27; More preferably, the sdAb (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) an 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 Including, Even more preferably, 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. The multispecific construct of claim 2.

8. (1) the first antibody portion is (a) HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, in a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 7; (b) a light chain variable region (VL) comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, comprising the sequence set forth in SEQ ID NO:8; (2) the second antibody portion comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3 within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27; The multispecific construct of claim 1.

9. A pharmaceutical composition comprising a multispecific construct according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A nucleic acid encoding a multispecific construct according to any one of claims 1 to 8.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10 or a vector comprising said nucleic acid.

13. 9. A composition comprising the multispecific construct of any one of claims 1 to 8, or a pharmaceutical composition comprising the multispecific construct of any one of claims 1 to 8 and a pharmaceutically acceptable carrier, for treating a disease or condition in a subject in need thereof.

14. The composition of claim 13 , wherein the disease or condition is cancer.

15. 10. Use of a multispecific construct according to any one of claims 1 to 8 in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

16. (1) a first antibody portion that specifically binds to a tumor antigen; (2) a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion to the tumor antigen induces the second antibody portion to activate 4-1BB.

17. activation of 4-1BB by the second antibody moiety is enhanced by at least 10-fold after binding of the first antibody moiety to the tumor antigen; and / or the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB; 17. The multispecific construct of claim 16.

18. the second antibody portion is an sdAb; Preferably, the sdAb 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 comprising the amino acid sequence set forth in SEQ ID NO: 27; More preferably, the sdAb (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) an 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 Including, 17. The multispecific construct of claim 16.

19. 17. The multispecific construct of claim 16, wherein the tumor antigen is CLDN6.

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

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