T cell costimulatory multimeric binding molecules and uses thereof

Abstract

The present disclosure provides multimeric T cell engaging binding molecules comprising five, four, or two bivalent binding units that bind to a target antigen and a modified J chain, wherein the modified J chain comprises a J chain or a functional fragment or variant thereof, an anti-CD3 antigen-binding domain, and a heterologous polypeptide comprising a polypeptide agonist of a T cell costimulatory molecule. The T cell costimulatory molecule can be, for example, CD28 or 4-1BB. The present disclosure further provides multimeric binding molecules comprising five, four, or two bivalent binding units that bind to a target antigen and a modified J chain, wherein the modified J chain comprises a J chain or a functional fragment or variant thereof and a 4-1BB ligand (4-1BBL) trimer that can engage with and activate 4-1BB on a target cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 478,886, filed January 6, 2023, 63 / 498,948, filed April 28, 2023, 63 / 510,092, filed June 23, 2023, and 63 / 590,779, filed October 16, 2023, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on January 5, 2024, is named 043WO1-Sequence-Listing.xml, and is 281 kilobytes in size. [Background technology]

[0003] background T cell-engaging bispecific antibodies that can bind to tumor antigens and simultaneously target cytotoxic T cells, for example by binding to CD3, to promote tumor cell killing are useful and rapidly expanding tools in the treatment of various malignancies. See, e.g., Einsele, H., et al., Cancer 126:3192-3201 (2020). While significant progress has been made in T cell engager (TCE) therapeutics, there remains a need for improved therapeutic outcomes, particularly in solid tumor indications where the tumor microenvironment can pose a significant barrier to T cell infiltration. See, e.g., Lim, AR, et al., eLife 9:doi:10.7554 / eLife.55185 (2020).

[0004] T cell activation typically involves two signals: activation via engagement of an MHC antigen with the T cell receptor (TCR) (“signal 1”) and co-activation via costimulatory molecules (“signal 2”). Costimulatory molecules are receptors expressed on T cells that, when co-activated, can, for example, induce signal transduction to more fully activate the T cell upon T cell receptor engagement, thereby enhancing signal transduction and cytokine stimulation. This co-signaling can further regulate T cell differentiation, effector function, and survival. See, e.g., Baeuerle, P.A., and H. Wesche, Curr. Opin. Oncol. 34:552-558 (2022). Exemplary T cell costimulatory molecules include, but are not limited to, CD28 and 4-1BB. See, e.g., Jeong, S., and S.H. Park, Immune Netw. doi:10.4110 / in.2020.20.e3 (2020).

[0005] CD3 engagement by TCEs can substitute for traditional signal 1 but lacks signal 2 coactivation. Combining bispecific TCEs that engage CD3 with tumor-targeting bispecific antibodies that engage and coactivate T cell costimulatory molecules has shown promise in preclinical studies. See, for example, Skokos, D., et al., Sci. Transl. Med. 12(525):eaaw7888. doi:10.1126 / scitranslmed.aaw7888(2020).

[0006] Binding molecules, such as antibodies that can multimerize (e.g., IgA and IgM antibodies), have emerged as promising drug candidates, allowing for improved specificity, improved avidity, and the ability to bind to multiple targets, for example, in the fields of immuno-oncology and infectious diseases. See, for example, U.S. Patent Nos. 9,951,134, 9,938,347, 10,351,631, 10,400,038, 10,570,191, 10,604,559, 10,689,449, 10,787,520, 10,899,835, 11,555,075, 10,618,978, and 11,639,389; U.S. Patent Application Publication Nos. US2022 / 0106399, US2020 / 0392239, and US2022 / 0340676, the contents of which are incorporated herein by reference in their entireties. Bispecific IgM antibodies have demonstrated safety and efficacy as TCEs in preclinical studies. See, for example, U.S. Patent No. 10,787,520 (which describes, inter alia, a bispecific IgM antibody targeting CD20 and CD3 proteins), U.S. Patent Application Publication No. US2022 / 0289856 (which describes, inter alia, a bispecific IgM antibody targeting CD123 and CD3 proteins), and PCT Publication No. WO2023 / 150677A2 (which describes, inter alia, a bispecific IgM antibody targeting CD38 and CD3 proteins). In addition, the CD20xCD3 IgM bispecific antibody imvotamab (IGM-2323) has shown promise in early clinical studies. See, for example, Budde, E., et al., Blood 138(Suppl. 1):132doi:10.1182 / blood-2021-153355(2021).

[0007] There remains a need for safe TCEs with improved efficacy through both signal 1 and signal 2 T cell engagement. Summary of the Invention

[0008] overview Provided herein are multimeric binding molecules comprising five, four, or two bivalent binding units and a modified J chain, wherein each binding unit comprises two antibody heavy chains, each antibody heavy chain comprising an IgM or IgA heavy chain constant region, or a multimerized variant or fragment thereof, associated with an antigen-binding domain that specifically binds to a target antigen (a "target antigen-binding domain"); the modified J chain comprises (a) a J chain, or a functional fragment or variant thereof ("J"); (b) an anti-CD3 scFv ("C"); and (c) a heterologous polypeptide ("H"); the H comprises a polypeptide agonist of a T cell costimulatory molecule; and the J, C, and H are associated as a fusion protein. In some embodiments, the T cell costimulatory molecule comprises CD28 or 4-1BB. In some embodiments, the H comprises an anti-CD28 antibody or antigen-binding fragment thereof or a 4-1BB ligand ("4-1BBL") trimer.

[0009] In some embodiments, the modified J chain comprises, from N-terminal to C-terminal, CJH or HJC. In some embodiments, the C, J, and H are fused via amino acid linkers, which may be the same or different. In some embodiments, each linker independently consists of 5 to 25 amino acids. In some embodiments, each linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO:279), where n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGGSGGGGS (SEQ ID NO:11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGSGGGGSGGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0010] In some embodiments, H comprises an antigen-binding fragment of an anti-CD28 antibody. In some embodiments, the antigen-binding fragment of an anti-CD28 antibody comprises a single-chain Fv (scFv) fragment.

[0011] In some embodiments, the anti-CD28 scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 have the amino acid sequences: SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:133, SEQ ID NO:138, SEQ ID NO:164, and SEQ ID NO:165; SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:13 SEQ ID NO: 0, SEQ ID NO: 151, SEQ ID NO: 157, and SEQ ID NO: 171; SEQ ID NO: 98, SEQ ID NO: 116, SEQ ID NO: 135, SEQ ID NO: 147, SEQ ID NO: 155, and SEQ ID NO: 175; SEQ ID NO: 103, SEQ ID NO: 115, SEQ ID NO: 135, SEQ ID NO: 147, SEQ ID NO: 155, and SEQ ID NO: 175; SEQ ID NO: 96, SEQ ID NO: 110, SEQ ID NO: 125, SEQ ID NO: 147, SEQ ID NO: 155, and SEQ ID NO: 174; SEQ ID NO: 96, SEQ ID NO: 110, SEQ ID NO: 125, SEQ ID NO: 150, SEQ ID NO: 159, and SEQ ID NO: 176; SEQ ID NO: 100, SEQ ID NO: 120 , SEQ ID NO:127, SEQ ID NO:146, SEQ ID NO:153, and SEQ ID NO:172; SEQ ID NO:104, SEQ ID NO:123, SEQ ID NO:137, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:91, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:129, SEQ ID NO:139, SEQ ID NO:161, and SEQ ID NO:169; SEQ ID NO:97, SEQ ID NO:112, SEQ ID NO:124, SEQ ID NO:142, SEQ ID NO:163, and SEQ ID NO:166; SEQ ID NO:94 , SEQ ID NO:108, SEQ ID NO:132, SEQ ID NO:149, SEQ ID NO:160, and SEQ ID NO:177; SEQ ID NO:93, SEQ ID NO:109, SEQ ID NO:131, SEQ ID NO:148, SEQ ID NO:158, and SEQ ID NO:178; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170;SEQ ID NO:102, SEQ ID NO:114, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170; SEQ ID NO:102, SEQ ID NO:113, SEQ ID NO:136, SEQ ID NO:143, SEQ ID NO:162, and SEQ ID NO:170; SEQ ID NO:91, SEQ ID NO:119, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:92, SEQ ID NO:121, SEQ ID NO:134, SEQ ID NO:152, SEQ ID NO:156, and SEQ ID NO:167; or SEQ ID NO:92, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:141, SEQ ID NO:156, and SEQ ID NO:168.

[0012] In some embodiments, the VH and VL of the anti-CD28 scFv comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences SEQ ID NO:56 and SEQ ID NO:90, SEQ ID NO:57 and SEQ ID NO:86, SEQ ID NO:58 and SEQ ID NO:83, SEQ ID NO:59 and SEQ ID NO:83, SEQ ID NO:60 and SEQ ID NO:82, SEQ ID NO:60 and SEQ ID NO:87, SEQ ID NO:61 and SEQ ID NO:81, SEQ ID NO:62 and SEQ ID NO:87, SEQ ID NO:63 and SEQ ID NO:85, SEQ ID NO:64 and SEQ ID NO:76, SEQ ID NO:65 and SEQ ID NO:75, SEQ ID NO:66 and SEQ ID NO:89, SEQ ID NO:67 and SEQ ID NO:88, SEQ ID NO:68 and SEQ ID NO:82, SEQ ID NO:68 and SEQ ID NO:87, SEQ ID NO:69 and SEQ ID NO:78, SEQ ID NO:70 and SEQ ID NO:78, SEQ ID NO:71 and SEQ ID NO:80, SEQ ID NO:72 and SEQ ID NO:84, SEQ ID NO:73 and SEQ ID NO:77, or SEQ ID NO:74 and SEQ ID NO:79, respectively.

[0013] In some embodiments, the VH and VL are fused via an amino acid linker. In some embodiments, the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO:279), where n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGGSGGGGS (SEQ ID NO:11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGSGGGGSGGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0014] In some embodiments, the antigen-binding fragment of the antibody is an anti-CD28 single-domain heavy chain variable region (sdVH) or an anti-CD28 single-domain light chain variable region (sdVL). In some embodiments, the anti-CD28 sdVH comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO:183, SEQ ID NO:187, and SEQ ID NO:191; SEQ ID NO:184, SEQ ID NO:188, and SEQ ID NO:192; or SEQ ID NO:185, SEQ ID NO:189, and SEQ ID NO:193, or the anti-CD28 sdVL comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO:186, SEQ ID NO:190, and SEQ ID NO:194, respectively. In some embodiments, the anti-CD28 sdVH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or the anti-CD28 sdVL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 182.

[0015] In some embodiments, H comprises a 4-1BBL trimer. In some embodiments, the 4-1BBL trimer comprises three 4-1BBL monomers. In some embodiments, each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15 (where X is an integer between 50 and 99). In some embodiments, the soluble fragments of 4-1BBL each comprise amino acids 71 ​​to 254 of SEQ ID NO: 15 (SEQ ID NO: 197). In some embodiments, the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).

[0016] In some embodiments, the 4-1BBL monomers are fused via amino acid linkers, which may be the same or different. In some embodiments, each linker independently consists of 5 to 25 amino acids. In some embodiments, each linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO:279), where n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGGS (SEQ ID NO:11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGGSGGGGSGGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGSGS (SEQ ID NO:14). In some embodiments, H comprises the amino acid sequence: SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, or SEQ ID NO:201.

[0017] In some embodiments, C comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH of C comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL of C comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are selected from the amino acid sequences: SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:33; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:45; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:49, or SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55. In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of C comprise SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively.

[0018] In some embodiments, the VH and VL of C comprise amino acid sequences that are at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48. In some embodiments, C comprises the VH and VL amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 28, respectively.

[0019] In some embodiments, the VH and VL are fused via an amino acid linker. In some embodiments, the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO:279), where n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGGSGGGGS (SEQ ID NO:11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGSGGGGSGGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0020] In some embodiments, J comprises SEQ ID NO:7, or a functional fragment or variant thereof. In some embodiments, J comprises an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO:7). In some embodiments, J comprises the amino acid sequence SEQ ID NO:8 ("J*"). In some embodiments, the modified J chain comprises the amino acid sequence SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234. In some embodiments, the modified J chain comprises the amino acid sequence SEQ ID NO:213 or SEQ ID NO:214.

[0021] Also provided herein is a multimeric binding molecule comprising five, four, or two bivalent binding units and a modified J chain, wherein each binding unit comprises two antibody heavy chains, each antibody heavy chain comprising an IgM or IgA heavy chain constant region or a multimerized variant or fragment thereof, the modified J chain comprising a J chain or a functional fragment or variant thereof and a 4-1BBL trimer associated as a fusion protein, the 4-1BBL trimer comprising three 4-1BBL monomers, each comprising a soluble fragment of 4-1BBL.

[0022] In some embodiments, each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15 (where X is an integer between 50 and 99). In some embodiments, the soluble fragments of 4-1BBL each comprise amino acids 71 ​​to 254 of SEQ ID NO: 15 (SEQ ID NO: 197). In some embodiments, the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196). In some embodiments, the 4-1BBL monomers are fused via amino acid linkers, which may be the same or different. In some embodiments, each linker independently consists of 5 to 25 amino acids. In some embodiments, each linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO:279), where n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGGSGGGGS (SEQ ID NO:11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGGSGGGGGSGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14). In some embodiments, the 4-1BB trimer comprises the amino acid sequence: SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, or SEQ ID NO:201.

[0023] In some embodiments, the 4-1BBL trimer is fused to the N-terminus of the J chain, or fragment or variant thereof, or to the C-terminus of the J chain, or fragment or variant thereof, or copies of the 4-1BB trimer are fused to both the N-terminus and C-terminus of the J chain, or fragment or variant thereof. In some embodiments, the J chain, or functional fragment or variant thereof, comprises a mature human J chain, SEQ ID NO:7, or a functional fragment or variant thereof. In some embodiments, the J chain comprises an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO:7). In some embodiments, the J chain comprises the amino acid sequence: SEQ ID NO:8 ("J*"). In some embodiments, the modified J chain comprises the amino acid sequence: SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211.

[0024] In some embodiments, the multimeric binding molecule is a pentameric IgM antibody or IgM-like antibody comprising five bivalent binding units, each binding unit comprising two IgM heavy chain constant regions, or multimerized fragments or variants thereof, each comprising an IgM Cμ4 domain and an IgM tailpiece (μtp) domain. In some embodiments, each IgM heavy chain constant region, or multimerized fragments or variants thereof, further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof. In some embodiments, each IgM heavy chain constant region, or multimerized fragments or variants thereof, is a human IgM constant region. In some embodiments, each IgM heavy chain constant region comprises the amino acid sequence: SEQ ID NO:1, SEQ ID NO:2, or a multimerized variant or fragment thereof. In some embodiments, each IgM heavy chain constant region is a variant human IgM constant region comprising one or more single amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 1 or SEQ ID NO: 2, and the multimeric binding molecule has reduced CDC activity compared to a multimeric binding molecule in which each IgM heavy chain constant region comprises the amino acid sequence: SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, each variant human IgM constant region comprises an amino acid substitution corresponding to position L310 in SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P311 in SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 in SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position K315 in SEQ ID NO: 1 or SEQ ID NO: 2, or any combination thereof. In some embodiments, each IgM heavy chain constant region is a variant human IgM constant region comprising one or more single amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 1 or SEQ ID NO: 2, and the multimeric binding molecule, when administered to a subject animal, exhibits increased serum half-life compared to a multimeric binding molecule, administered in the same manner to the same animal species, wherein each IgM heavy chain constant region comprises the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, each variant IgM heavy chain constant region comprises a half-life-altering amino acid substitution at one or more amino acid positions corresponding to amino acids E345, S401, E402, or E403 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] In some embodiments, the multimeric binding molecule is a dimeric or tetrameric IgA antibody or IgA-like antibody, each comprising two or four bivalent IgA or IgA-like binding units, and each binding unit comprises two IgA heavy chain constant regions, or multimerized fragments or variants thereof, each comprising a Cα3 domain and an IgA tailpiece (αtp) domain. In some embodiments, each IgA heavy chain constant region, or multimerized fragments or variants thereof, further comprises a Cα1 domain, a Cα2 domain, an IgA hinge region, or any combination thereof. In some embodiments, each IgA heavy chain constant region, or multimerized fragments or variants thereof, is a human IgA constant region.

[0026] In some embodiments, each target antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), or a single-domain heavy chain variable region (sdVH). In some embodiments, each heavy chain comprises an sdVH fused N-terminally to a heavy chain constant region, or a multimerizing fragment or variant thereof. In some embodiments, each heavy chain comprises a VH fused N-terminally to a heavy chain constant region, or a multimerizing fragment or variant thereof. In some embodiments, each binding unit further comprises two antibody light chains, each comprising a VL fused N-terminally to a light chain constant region. In some embodiments, at least three, four, five, six, seven, eight, nine, or ten of the target antigen-binding domains are identical. In some embodiments, at least three, four, five, six, seven, eight, nine, or ten identical target antigen-binding domains specifically bind to a tumor-associated target antigen. In some embodiments, the tumor-associated target antigen is CD38, CD20, CD19, CD22, CD28, B-cell maturation antigen (BCMA), CD123, PD-L1, tumor-associated calcium transducer 2 (TROP-2), mesothelin, Mucl6, prostate-specific membrane antigen (PSMA), or six-transmembrane epithelial antigen of the prostate gland-1 (STEAP-1). In some embodiments, the tumor-associated target antigen is CD20. In some embodiments, the tumor-associated target antigen is mesothelin (MSLN). In some embodiments, the tumor-associated target antigen is CD38. In some embodiments, the tumor-associated target antigen is tumor-associated calcium transducer 2 (TROP-2). In some embodiments, the multimeric binding molecule conditionally activates T cells in the presence of the tumor-associated target antigen.

[0027] In some embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 identical target antigen-binding domains specifically bind to tumor-associated target antigens. In some embodiments, the target antigen is CD38, CD20, CD19, CD22, CD28, B-cell maturation antigen (BCMA), CD123, PD-L1, tumor-associated calcium signal transducer 2 (TROP-2), mesothelin, Muc16, prostate-specific membrane antigen (PSMA), or prostate six-transmembrane epithelial antigen-1 (STEAP-1). In some embodiments, the target antigen is CD20. In some embodiments, the tumor-associated target antigen is mesothelin (MSLN). In some embodiments, the tumor-associated target antigen is CD38. In some embodiments, the tumor-associated target antigen is tumor-associated calcium signal transducer 2 (TROP-2).

[0028] In some embodiments, the multimeric binding molecule conditionally activates T cells in the presence of a target antigen. In some embodiments, the multimeric binding molecule activates T cells after 7 days in the presence of a target antigen. In some embodiments, the multimeric binding molecule activates T cells after 10 days in the presence of a target antigen. In some embodiments, the multimeric binding molecule activates T cells after 14 days in the presence of a target antigen. In some embodiments, the multimeric binding molecule directs T cell-mediated killing of cells expressing a target antigen after 7 days in the presence of a target antigen. In some embodiments, the multimeric binding molecule directs T cell-mediated killing of cells expressing a target antigen after 10 days in the presence of a target antigen. In some embodiments, the multimeric binding molecule directs T cell-mediated killing of cells expressing a target antigen after 14 days in the presence of a target antigen. In some embodiments, the T cells are CD4-positive T cells. In some embodiments, the T cells are CD8-positive T cells.

[0029] Also provided herein are compositions comprising a multimeric binding molecule as disclosed herein and a pharmaceutically acceptable carrier.

[0030] Also provided herein is a polynucleotide comprising a nucleic acid sequence encoding a modified J chain of a multimeric binding molecule disclosed herein.

[0031] Also provided herein are vectors comprising the polynucleotides disclosed herein.

[0032] Also provided herein are compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a modified J chain of a multimeric binding molecule disclosed herein and a polynucleotide encoding a heavy chain of a multimeric binding molecule disclosed herein. In some embodiments, the composition further comprises a polynucleotide encoding a light chain of a multimeric binding molecule described herein.

[0033] Also provided herein are vectors comprising the compositions disclosed herein.

[0034] Also provided herein is a host cell comprising the composition disclosed herein or the vector disclosed herein, wherein the host cell is capable of expressing the multimeric binding molecules disclosed herein.

[0035] Also provided herein is a method for producing a multimeric binding molecule disclosed herein, comprising culturing a host cell disclosed herein and recovering the multimeric binding molecule.

[0036] Also provided herein are methods of treating cancer, comprising administering to a subject in need thereof a multimeric binding molecule disclosed herein.

[0037] Also provided herein is a multimeric binding molecule as disclosed herein for use in the treatment of cancer.

[0038] Also provided herein is the use of a multimeric binding molecule disclosed herein in the preparation of a medicament for the treatment of cancer. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows a schematic diagram of a costimulatory IgM T cell engager and how it may act at the immune synapse. [Figure 2-1] 1 shows exemplary IgM pentamers with modified human J chains fused to various moieties. A shows a modified human J chain fused to an anti-CD28 scFv at the N-terminus and an anti-CD3 scFv at the C-terminus. B shows a modified human J chain fused to an anti-CD28 scFv at the C-terminus and an anti-CD3 scFv at the N-terminus. C shows a modified human J chain fused to a 4-1BBL trimer at the N-terminus. D shows a modified human J chain fused to a 4-1BBL trimer at the C-terminus. [Figure 2-2] 1 shows exemplary IgM pentamers with modified human J chains fused to various moieties. E shows a modified human J chain fused to a 4-1BBL trimer at the C-terminus and an anti-CD3 scFv at the N-terminus. F shows a modified human J chain fused to a 4-1BBL trimer at the N-terminus and an anti-CD3 scFv at the C-terminus. G shows a modified human J chain fused to a 4-1BBL trimer at the N-terminus and a 4-1BBL trimer at the C-terminus. [Figure 3] 1 shows the amount of binding to 4-1BB+ cells of various concentrations of IgM antibodies bearing 4-1BBL modified J chains in various formats. [Figure 4A] Binding of various concentrations of A5 over time as measured by biolayer interferometry (BLI) is shown. [Figure 4B] Binding of various concentrations of A6 over time as measured by biolayer interferometry (BLI) is shown. [Figure 4C] Binding of various concentrations of A9 over time as measured by biolayer interferometry (BLI) is shown. [Figure 4D] Binding of various concentrations of D2 over time as measured by biolayer interferometry (BLI) is shown. [Figure 5A] Luminescence measured at concentrations A5, A6, A8, and A9 using the CD20 coating is shown. [Figure 5B]Luminescence measured at concentrations A5, A6, A8, and A9 without CD20 coating is shown. [Figure 5C] Shown is the luminescence (in RLU) measured at the highest concentrations tested of A5, A6, A8, A9, D1, D2 (with and without cross-linking), D3, and D4 (with and without cross-linking) in the presence or absence of CD20. [Figure 6A] Luminescence measured using CD20+ (Ramos) cells at concentrations of A1, A2, A3, A4, D1, and D2 is shown. [Figure 6B] Luminescence measured using CD20+ (Ramos) cells at concentrations of A5, A6, A8, A9, D1, and D2 is shown. [Figure 6C] Shown is the luminescence (RLU) measured at the highest tested concentrations of A1, A2, A3, A4, A5, A6, A8, A9, D1, and D2 in the presence of CD20+ cells (Ramos) or CD20- cells (OPM-2). [Figure 7A] Luminescence measured at concentrations A5, A6, A7, A8, A9, A10, A11, A12, D1, and D2 using low levels of CD20 (CA46) cells is shown. [Figure 7B] Luminescence measured at concentrations A5, A6, A7, A8, A9, A10, A11, A12, D1, and D2 using high levels of CD20 (DOHH2) cells is shown. [Figure 7C] Shown is the luminescence measured at the highest tested concentrations of A5, A6, A8, A9, A10, A11, A12, and D1 in the presence of cells expressing high levels of CD20 (DOHH2), cells expressing low levels of CD20 (CA46), or CD20- cells (OPM-2). [Figure 8] The amount of binding of B1, B2, B3, B4, D5, or D6 to 4-1BB as determined by ELISA is shown. [Figure 9A] Luminescence measured using mesothelin-negative cells at concentrations of B1, B2, B3, B4, D5, or D6 is shown. [Figure 9B]Luminescence measured using mesothelin-positive cells at concentrations of B1, B2, B3, B4, D5, or D6 is shown. [Figure 9C] Luminescence measured at the highest concentrations tested of B1, B2, B3, B4, D5, and D6 in the presence of mesothelin+ or mesothelin- cells is shown. [Figure 10A] Activation of CD4+ T cells by various concentrations of C4, C5, C6, and D7 after 24 hours is shown. [Figure 10B] Activation of CD4+ T cells by various concentrations of C4, C5, C6, and D7 after 48 hours is shown. [Figure 10C] Activation of CD8+ T cells by various concentrations of C4, C5, C6, and D7 after 24 hours is shown. [Figure 10D] Activation of CD8+ T cells by various concentrations of C4, C5, C6, and D7 after 48 hours is shown. [Figure 11] The amount of T cell dependent cytotoxicity (TDCC) against Ramos cells after 24 hours (A) or 48 hours (B) of co-culture with PBMC and C4, C5, C6, and D7 is shown. [Figure 12A] IL-2 concentrations after 24 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12B] IL-2 concentrations after 48 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12C] IL-6 concentrations after 24 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12D] IL-6 concentrations in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 after 48 hours are shown. [Figure 12E] IL-10 concentrations after 24 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12F] IL-10 concentrations after 48 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12G] TNFα concentrations after 24 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12H] TNFα concentrations after 48 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12I] IFNγ concentrations after 24 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 12J] IFNγ concentrations after 48 hours in supernatants from Ramos and PBMC cells treated with various concentrations of C4, C5, C6, and D7 are shown. [Figure 13A] Activation of CD4+ T cells in co-cultures of PBMC and H929_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 13B] Activation of CD4+ T cells in co-cultures of PBMC and OPM2_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 13C] Activation of CD4+ T cells in co-cultures of PBMC cells and MM1S_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 13D] Activation of CD4+ T cells in co-cultures of PBMC cells and RPMI8226_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 14A] Activation of CD8+ T cells in co-cultures of PBMC and H929_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 14B] Activation of CD8+ T cells in co-cultures of PBMC cells and OPM2_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 14C] Activation of CD8+ T cells in co-cultures of PBMC cells and MM1S_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 14D] Activation of CD8+ T cells in co-cultures of PBMC cells and RPMI8226_Luc / GFP cells by D18 or E1 after 48 hours is shown. [Figure 15A] The amount of TDCC relative to H929_Luc / GFP cells after 72 hours of co-culture with PBMC cells and D18 or E1 is shown. [Figure 15B] The amount of TDCC relative to OPM2_Luc / GFP cells after 72 hours of co-culture with PBMC cells and D18 or E1 is shown. [Figure 15C] The amount of TDCC relative to MM1S_Luc / GFP cells after 72 hours of co-culture with PBMC cells and D18 or E1 is shown. [Figure 15D] The amount of TDCC relative to RPMI8226_Luc / GFP cells after 72 hours of co-culture with PBMC cells and D18 or E1 is shown. [Figure 16A] IL-2 concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 16B] IL-2 concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 16C] IL-2 concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 16D] IL-2 concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 17A] IL-4 concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 17B] IL-4 concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 17C] IL-4 concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 17D] IL-4 concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 18A] IL-6 concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 18B] IL-6 concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 18C] IL-6 concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 18D] IL-6 concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 19A] IL-10 concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 19B] IL-10 concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 19C] IL-10 concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 19D]IL-10 concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 20A] TNFα concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells after 48 hours of treatment with D18 or E1 are shown. [Figure 20B] TNFα concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells are shown 48 hours after treatment with D18 or E1. [Figure 20C] TNFα concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 20D] TNFα concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 21A] IFN-gamma concentrations in supernatants from co-cultures of PBMC cells with H929_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 21B] IFN-gamma concentrations in supernatants from co-cultures of PBMC cells with OPM2_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 21C] IFN-gamma concentrations in supernatants from co-cultures of PBMC cells with MM1S_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 21D] IFN-gamma concentrations in supernatants from co-cultures of PBMC cells with RPMI8226_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown. [Figure 22A] Activation of CD4+ T cells in co-cultures of PBMC cells with Raji-luc-GFP cells by C6, C3, or D7 after 96 hours is shown. [Figure 22B]Activation of CD8+ T cells in co-cultures of PBMC cells and Raji-luc-GFP cells by C6, C3, or D7 after 96 hours is shown. [Figure 22C] The amount of TDCC on Ramos cells after 72 hours of co-culture with PBMC cells and C6, C3, or D7 is shown. [Figure 23] IL-2 (A) or IFNγ (B) concentrations in supernatants from co-cultures of PBMC cells with Raji-luc-GFP after 96 hours of treatment with C6, C3, or D7 are shown. [Figure 24A] Figure 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle; D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg. [Figure 24B] 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, C7 administered at 10 mg / kg, or C3 administered at 10 mg / kg. [Figure 24C] 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 1 mg / kg, C7 administered at 1 mg / kg, or C3 administered at 10 mg / kg. [Figure 24D] 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 0.1 mg / kg, C7 administered at 0.1 mg / kg, or C3 administered at 10 mg / kg. [Figure 25A] IL-2 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25B] 1 shows IL-10 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25C] 1 shows IFN-γ concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle; D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25D] 1 shows TNF-α concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle; D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25E] IL-4 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25F] IL-6 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25G]IL-8 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25H] 1 shows IL-1β concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25I] FIG. 1 shows IL-12p70 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 25J] 1 shows IL-13 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle; D7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; C7 administered at 10 mg / kg, 1 mg / kg, or 0.1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 19. [Figure 26] Shown are total infiltrating CD4+ T cells (A) or CD8+ T cells (B), normalized to tumor volume (mm3), detected in tumors harvested from human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 0.1 mg / kg, or C7 administered at 0.1 mg / kg. [Figure 27]Figure 1 shows T cell-dependent cellular cytotoxicity mediated by vehicle or incubation with antibodies D8, D9, D10, D7, or C6 at a concentration of 10 nM each at 3, 7, 10, and 14 days in a PBMC (effector) / Ramos cell (target) co-culture assay at effector to target ratios of 3:1 (A), 1:1 (B), or 1:3 (C). [Figure 28] Shown are CD4+ T cell levels mediated by vehicle or incubation with antibodies D8, D9, D10, D7, or C6 at a concentration of 10 nM each at 3, 7, 10, and 14 days in a PBMC (effector) / Ramos cell (target) co-culture assay at effector-to-target ratios of 3:1 (A), 1:1 (B), or 1:3 (C). [Figure 29] Shown are CD8+ T cell levels mediated by vehicle or incubation with antibodies D8, D9, D10, D7, or C6 at a concentration of 10 nM each at 3, 7, 10, and 14 days in a PBMC (effector) / Ramos cell (target) co-culture assay at effector-to-target ratios of 3:1 (A), 1:1 (B), or 1:3 (C). [Figure 30-1] AC show IL-2 (A), IL-4 (B), and IL-6 (C) levels mediated by vehicle or incubation with antibodies D8, D9, D10, D7, or C6 at a concentration of 10 nM each in a PBMC (effector) / Ramos cell (target) co-culture assay at effector-to-target ratios of 3:1, 1:1, or 1:3 at 72 hours (3 days). [Figure 30-2] D-F show IL-10 (D), TNFα ("TNFa", E), and interferon-gamma ("IFN-gamma", F) levels mediated by vehicle or incubation with antibodies D8, D9, D10, D7, or C6 at a concentration of 10 nM each in a PBMC (effector) / Ramos cell (target) co-culture assay at effector-to-target ratios of 3:1, 1:1, or 1:3 at 72 hours (3 days). [Figure 31] 2 is a survival plot showing the survival of tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, D7 administered at 10 mg / kg and 1 mg / kg; C7 administered at 10 mg / kg and 1 mg / kg; and C3 administered at 10 mg / kg, as described in Example 21. [Figure 32A] Figure 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control (administered at 10 mg / kg), C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. More specifically, tumor volume over time for each group (vehicle, control administered at 10 mg / kg, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg) is shown. [Figure 32B] Figure 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control (administered at 10 mg / kg), C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. More specifically, tumor volume over time is shown for the 10 mg / kg groups (control, C7, and D7). [Figure 32C] Figure 1 shows tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control (administered at 10 mg / kg), C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. More specifically, tumor volumes are shown for the 1 mg / kg C7 and D7 groups and the 10 mg / kg control group. [Figure 33] 2 is a plot showing the survival of tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, assessed over time in the Ramos human B-lymphocyte carcinoma xenograft model, as described in Example 22. [Figure 34A] IL-2 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. [Figure 34B] IL-4 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. [Figure 34C] 1 shows IL-10 concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. [Figure 34D] 1 shows IFN-γ concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. [Figure 34E] 1 shows TNF-α concentrations in serum collected from tumor-bearing human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control, C7 administered at 10 mg / kg and 1 mg / kg, and D7 administered at 10 mg / kg and 1 mg / kg, as described in Example 22. [Figure 35] FIG. 10 is a graph showing tumor volume over time in human PBMC-engrafted NSG-MHC I / II DKO mice treated with vehicle, control antibody, C7 and D7 administered at 10 mg / kg, as described in Example 23. [Figure 36A]1 shows the absolute cell counts of CD4 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 36B] 1 shows the absolute cell counts of CD4 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 36C] 1 shows the absolute cell counts of CD4 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 36D] 1 shows the absolute cell counts of CD4 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 36E] 1 shows the absolute cell counts of CD4 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 37A] 1 shows the absolute cell counts of CD8 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 37B] 1 shows the absolute cell counts of CD8 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 37C] 1 shows the absolute cell counts of CD8 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 37D]1 shows the absolute cell counts of CD8 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 37E] 1 shows the absolute cell counts of CD8 T cell subsets in peripheral blood measured at the end of the study and normalized to blood volume for the various treatment groups (vehicle, control, C7, and D7) described in Example 23. [Figure 38] Figure 1 shows T cell-dependent cytotoxicity (TDCC) over time as measured by real-time cell analysis (RTCA) for anti-mesothelin antibody constructs M2, M3, and M1 compared to no antibody and the isotype control D15 (5 nM each), tested using 50,000 peripheral blood mononuclear cells (PBMCs) and 15,000 OVCAR3 ovarian cancer cell line cells. [Figure 39] Figure 38 shows IL-2 (A), IL-4 (B), IL-6 (C), IL-10 (D), IFN-γ (E), and TNFα (F) expression at time 69 in the RTCA assay shown in Figure 38. [Figure 40] Figure 38 shows T cell counts and T cell activation at time 96 of the RTCA assay shown in Figure 38. A and B show CD4+ T cell counts and CD4+ T cell activation, respectively, and C and D show CD8+ T cell counts and CD8+ T cell activation, respectively. [Figure 41] Figure 1 shows T cell-dependent cytotoxicity over time as measured by real-time cell analysis (RTCA) of anti-TROP-2 antibody constructs T2, T3, and T1 compared to no antibody and isotype control D15 (5 nM each), tested using 50,000 peripheral blood mononuclear cells (PBMCs) and 15,000 OVCAR3 ovarian cancer cell line cells. [Figure 42] Figure 41 shows IL-2 (A), IL-4 (B), IL-6 (C), IL-10 (D), IFN-γ (E), and TNFα (F) expression at time 69 in the RTCA assay shown in Figure 41. [Figure 43]Figure 41 shows T cell counts and T cell activation at time 96 of the RTCA assay shown in Figure 41. A and B show CD4+ T cell counts and CD4+ T cell activation, respectively, and C and D show CD8+ T cell counts and CD8+ T cell activation, respectively. [Figure 44] FIG. 2 is a graph of percent cytotoxicity versus antibody concentration measured in a TDCC assay in which human PBMCs as effector cells (E) and TROP2-transduced HEK293 cells as target cells (T) were incubated with serial dilutions of T1 or T3, as described in Example 24. [Figure 45] FIG. 10 is a graph of percent cell viability versus antibody concentration measured in a TDCC assay in which human PBMCs as effector cells (E) and A431-Luc cells as target cells (T) were incubated with serial dilutions of antibody T3, T1, or D16, as determined by a luminescence-based viability assay, as described in Example 24. [Figure 46] FIG. 10 is a graph of percent cell viability versus antibody concentration measured in a TDCC assay in which human PBMCs as effector cells (E) and SKOV-3 cells as target cells (T) were incubated with serial dilutions of antibody T3, T1, or D16, as determined by a luminescence-based viability assay, as described in Example 24. [Figure 47] 2 is a graph of percent target cell killing (i.e., antibody-mediated redirected T cell killing against OVCAR3-Luc cells) versus antibody concentration for exemplary antibodies: D16, T3, T1, D17, M3, and M1, as determined in a TDCC assay as described in Example 24 and measured using a luciferase assay. [Figure 48] Figure 1 shows the cytokine release profiles for cytokines IL-2 (A), IL-4 (B), IL-6 (C), IL-10 (D), TNFα (E), and IFN-γ (F) mediated by test substances D16, T3, T1, D17, M3, and M1 at time 72 of the assay described in Example 24. [Figure 49]FIG. 10 is a graph of percent target cell killing (i.e., antibody-mediated redirected T cell killing against RERF-LC-KJ / CMV-Luc cells) versus antibody concentration for exemplary antibodies: D16, T3, and T1, as determined in a TDCC assay as described in Example 24 and measured using a luciferase assay. [Figure 50] Figure 1 shows the cytokine release profiles for cytokines IL-2 (A), IL-4 (B), IL-6 (C), IL-10 (D), TNFα (E), and IFN-γ (F) mediated by test articles D16, T3, and T1 at time 72 of the assay described in Example 24. [Figure 51] 1 is a plot showing tumor infiltrating lymphocyte (TIL) data (cells / gram tumor) of CD4+ and CD8+ TILs that infiltrated tumor tissue measured in a Ramos human B-lymphocyte carcinoma xenograft model study for the indicated treatment groups. [Figure 52A] Figure 26 is a plot of percent cell viability versus antibody concentration (log μg / mL) from the TDCC assay described in Example 25, in which the cytotoxicity of exemplary antibodies (T1 (anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody, half black diamonds) and T3 (anti-TROP-2 x anti-CD3 IgM, black circles), respectively) with and without CD28 costimulation was assessed in a TROP-2-expressing cell line (TROP-2-transduced HEK293T cells with high TROP-2 expression). [Figure 52B] Figure 2 is a plot of percent cell viability versus antibody concentration (log μg / mL) from the TDCC assay described in Example 25, in which the cytotoxicity of exemplary antibodies (T1 (anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody, half black diamonds) and T3 (anti-TROP-2 x anti-CD3 IgM, black circles), respectively) with and without CD28 costimulation was assessed in a TROP-2-expressing cell line (A431 melanoma cells with high TROP-2 expression). [Figure 52C]Figure 2 is a plot of percent cell viability versus antibody concentration (log μg / mL) from the TDCC assay described in Example 25, in which the cytotoxicity of exemplary antibodies (T1 (anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody, half black diamonds) and T3 (anti-TROP-2 x anti-CD3 IgM, black circles), respectively) with and without CD28 costimulation was assessed in a TROP-2-expressing cell line (SKOV-3 ovarian cancer cells with intermediate / low TROP-2 expression). [Figure 53A] Figure 1 is a graph of tumor volume (mm) versus time (days) in different antibody treatment groups (T1: TROP-2 x CD3 x CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2 x CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle evaluated in a humanized HEK293 / TROP-2 xenograft model as described in Example 26. Data for the 0.3 mg / kg treatment group are shown. [Figure 53B] Figure 1 is a graph of tumor volume (mm) versus time (days) in different antibody treatment groups (T1: TROP-2 x CD3 x CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2 x CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle evaluated in a humanized HEK293 / TROP-2 xenograft model as described in Example 26. Data for the 1 mg / kg treatment group are shown. [Figure 53C] Figure 1 is a graph of tumor volume (mm) versus time (days) in different antibody treatment groups (T1: TROP-2 x CD3 x CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2 x CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle evaluated in a humanized HEK293 / TROP-2 xenograft model as described in Example 26. Data for the 3 mg / kg treatment group are shown. [Figure 54A]FIG. 10 is a bar graph showing serum concentrations of IFN-γ for antibody treatment groups (T1: TROP-2×CD3×CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2×CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle, assessed using a humanized HEK293 / TROP-2 xenograft model as described in Example 26. [Figure 54B] FIG. 10 is a bar graph showing serum concentrations of IL-2 for antibody treatment groups (T1: TROP-2×CD3×CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2×CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle, assessed using a humanized HEK293 / TROP-2 xenograft model as described in Example 26. [Figure 54C] FIG. 10 is a bar graph showing serum concentrations of TNF-α for antibody treatment groups (T1: TROP-2×CD3×CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2×CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle, assessed using a humanized HEK293 / TROP-2 xenograft model as described in Example 26. [Figure 54D] FIG. 10 is a bar graph showing serum concentrations of IL-10 for antibody treatment groups (T1: TROP-2×CD3×CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2×CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle, assessed using a humanized HEK293 / TROP-2 xenograft model as described in Example 26. [Figure 55]1 is a bar graph showing the absolute numbers of anti-apoptotic CD8+ Bcl_xL T cells in mouse peripheral blood samples stained with a viability dye-containing antibody cocktail and analyzed by flow cytometry. Blood samples were collected from different antibody treatment groups (T1: TROP-2×CD3×CD28 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg; T3: TROP-2×CD3 IgM at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) and vehicle evaluated in a humanized HEK293 / TROP-2 xenograft model as described in Example 26. [Figure 56] FIG. 2 shows the results of a TDCC assay in which the T cell-dependent cytotoxicity of exemplary IgM antibodies with and without costimulatory activity (M1 (anti-mesothelin (MSLN) × anti-CD3 × anti-CD28 IgM antibody) and M3 (anti-mesothelin (MSLN) × anti-CD3 IgM antibody), respectively) against the gastric cancer cell line MKN45 was assessed by luminescence as described in Example 27. [Figure 57] Figure 10 is a graph of tumor volume (mm) versus time (days after MKN45 cell implantation) for different antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. A shows data for the 3 mg / kg treatment group, and B shows data for the 10 mg / kg treatment group. [Figure 58A] 1 is a bar graph showing serum concentrations of IFN-γ for antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. [Figure 58B]1 is a bar graph showing serum concentrations of IL-2 for antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. [Figure 58C] 1 is a bar graph showing serum concentrations of TNF-α for antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. [Figure 58D] 1 is a bar graph showing serum concentrations of IL-10 for antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. [Figure 59A] 2 is a bar graph showing the ability of various combinations of anti-CD28 and / or anti-CD3 IgM or IgG4 antibodies to activate T cells and induce cytokine release in the plate-based assay described in Example 29. Results are shown for one of two exemplary PBMC donors (PBMC Donor 1). The various antibodies and antibody combinations are detailed in Table 27. This figure shows CD4+ T cell activation. [Figure 59B]2 is a bar graph showing the ability of various combinations of anti-CD28 and / or anti-CD3 IgM or IgG4 antibodies to activate T cells and induce cytokine release in the plate-based assay described in Example 29. Results are shown for one of two exemplary PBMC donors (PBMC Donor 1). The various antibodies and antibody combinations are detailed in Table 27. This figure shows CD8+ T cell activation. [Figure 59C] 2 is a bar graph showing the ability of various combinations of anti-CD28 and / or anti-CD3 IgM or IgG4 antibodies to activate T cells and induce cytokine release in the plate-based assay described in Example 29. Results are shown for one of two exemplary PBMC donors (PBMC Donor 1). The various antibodies and antibody combinations are detailed in Table 27. The figure shows IL-2 expression. [Figure 60] 1A-B are bar graphs showing absolute intratumoral CD4+ T cell counts (A) and absolute intratumoral CD8+ T cell counts (B) at day 43 for antibody treatment groups: M1: anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7); M3: anti-mesothelin (MSLN) x anti-CD3 IgM antibody at doses of 3 mg / kg and 10 mg / kg (n=7), and vehicle (n=7), evaluated in a humanized MKN45 xenograft model as described in Example 28. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description definition As used herein, the term "a" or "an" entity refers to one or more of that entity. For example, "a binding molecule" is understood to refer to one or more binding molecules. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0041] Furthermore, as used herein, "and / or" refers to the specific disclosure of each of the two specified features or components, with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it includes "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0042] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, PS (ed.), 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, Lacki, JM (ed.) 5th ed., 2013, Academic Press, and Oxford Dictionary of Biochemistry and Molecular Biology, Cammack, R., et al., (eds.) 2nd ed., 2006, Oxford University Press provide those skilled in the art with a comprehensive dictionary of many of the terms used in this disclosure.

[0043] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various embodiment(s) of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0044] As used herein, the term "polypeptide" encompasses the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of any of these terms. The term "polypeptide" also refers to the product of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, and derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides can be produced in any manner, including chemical synthesis.

[0045] The size of the polypeptides disclosed herein can be about 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 20 or more amino acids, 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, 100 or more amino acids, 200 or more amino acids, 500 or more amino acids, 1,000 or more amino acids, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure but can adopt many different conformations are said to be unfolded. As used herein, the term glycoprotein refers to a protein bound to at least one carbohydrate moiety attached to the protein via an oxygen- or nitrogen-containing side chain of an amino acid (e.g., serine or asparagine). Asparagine (N)-linked glycans are described in more detail elsewhere in this disclosure.

[0046] An "isolated" polypeptide, or fragment, variant, or derivative thereof, is a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0047] As used herein, the term "non-naturally occurring polypeptide" or any grammatical variation thereof is a qualified definition that expressly excludes, but excludes only, forms of polypeptides that are or can be determined or interpreted by a judge, or administrative or judicial body, as "naturally occurring."

[0048] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the above polypeptides, and any combination thereof. As disclosed herein, the terms "fragment," "variant," "derivative," and "analog" include any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide (e.g., the property of specifically binding to an antigen). Polypeptide fragments include, for example, specific antibody fragments discussed elsewhere herein, as well as proteolytic and deletion fragments. Variants, e.g., polypeptide variants, include the fragments described above and also include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants may be non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been modified to exhibit additional features not found in the original polypeptide. Examples include fusion proteins. As used herein, a "derivative" of a polypeptide can refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as a "derivative" is a polypeptide containing one or more derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be substituted for proline, 5-hydroxylysine can be substituted for lysine, 3-methylhistidine can be substituted for histidine, homoserine can be substituted for serine, and ornithine can be substituted for lysine.

[0049] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a receptor or target (e.g., an epitope or antigenic determinant). As further described herein, a binding molecule can comprise one or more "binding domains," e.g., "antigen-binding domains," as described herein. Non-limiting examples of binding molecules are multimeric binding molecules, e.g., antibodies, e.g., IgM antibodies, IgM-like antibodies, IgA antibodies, or IgA-like antibodies, as described in detail herein, that retain antigen-specific binding. In certain embodiments, a "binding molecule" comprises a multimeric binding molecule, e.g., an IgM antibody, IgM-like antibody, IgA antibody, or IgA-like antibody, as described in detail herein.

[0050] As used herein, the terms "binding domain" or "antigen-binding domain" (which can be used interchangeably) refer to the region of a binding molecule, e.g., an antibody, that is necessary and sufficient to specifically bind to a target, e.g., an epitope, polypeptide, cell, or organ. For example, an "Fv," e.g., the heavy and light chain variable regions of an antibody, either as two separate polypeptide subunits or as a single chain, is a "binding domain." Other antigen-binding domains include, but are not limited to, single-domain heavy chain variable regions (sdVH) and single-domain light chain variable regions (sdVL) of antibodies, e.g., from camelid or shark species. Single-domain antigen-binding domains typically contain three complementarity-determining regions (CDRs). Antigen-binding domains can also be engineered; for example, six immunoglobulin complementarity-determining regions (CDRs) can be expressed within a fibronectin scaffold. A "binding molecule," e.g., an "antibody," as described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more "antigen-binding domains." An antigen-binding domain that specifically binds to a target antigen, e.g., a tumor-associated target antigen, is referred to herein as a "target antigen-binding domain."

[0051] The term "antibody" includes at least a heavy chain variable domain (e.g., sdVH from camelid species), at least a light chain variable domain (e.g., sdVL), or at least heavy and light chain variable domains. Basic immunoglobulin structure in vertebrate systems is relatively well understood. See, e.g., Greenfield, E.A. (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014. Unless otherwise specified, the term "antibody" encompasses anything from a small antigen-binding fragment of an antibody to a full-sized antibody (e.g., an IgG antibody containing two complete heavy chains and two complete light chains, an IgA antibody or IgA-like antibody which may contain four or eight complete heavy chains and four or eight complete light chains, as well as a J chain and / or secretory component, or an IgM antibody or IgM-like antibody which may contain ten complete heavy chains and ten complete light chains, as well as a J chain, or a functional fragment or variant thereof).

[0052] The term "immunoglobulin" encompasses a wide variety of biochemically distinguishable polypeptide classes. Immunoglobulin heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4 or α1-α2) within each. The nature of this chain determines the antibody's "isotype," as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present disclosure.

[0053] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can associate with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other; when an immunoglobulin is expressed, for example, by a hybridoma, a B cell, or a genetically engineered host cell, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of a particular antibody (e.g., an IgG antibody) comprises two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a "Y" structure; this structure is also referred to herein as the "H2L2 structure" or "binding unit."

[0054] The term "binding unit" is used herein to refer to a portion of a binding molecule, e.g., a portion of an antibody corresponding to a standard immunoglobulin structure that includes at least two heavy chains and may further include two light chains. In certain embodiments, for example, when the binding molecule is a bivalent IgG antibody or antigen-binding fragment thereof, the terms "binding molecule" and "binding unit" are equivalent. Such binding molecules are also referred to herein as "monomers." In other embodiments, for example, when the binding molecule is a "multimeric binding molecule," e.g., a dimeric or tetrameric IgA antibody or a pentameric IgM antibody, a dimeric, tetrameric, or pentameric IgA-like or IgM-like antibody, or a dimeric, tetrameric, or pentameric IgA- or IgM-derived binding molecule, the binding molecule includes two or more "binding units": two for an IgA dimer, four for an IgA tetramer, or five for an IgM pentamer, respectively. A binding unit need not comprise the heavy and light chains of a full-length antibody, but is typically bivalent, i.e., comprises two "antigen-binding domains," e.g., "target antigen-binding domains," as defined above. As used herein, certain binding molecules provided by the present disclosure are "pentamers" and comprise five bivalent binding units comprising an IgM constant region, or a multimerized fragment or variant thereof, and a J chain, or a functional fragment or variant thereof. Binding molecules, e.g., antibodies, comprising two or more, e.g., two, four, or five, binding units are referred to herein as "multimeric binding molecules."

[0055] The term "J chain" as used herein refers to the J chain of an IgM or IgA antibody of any animal species, including the mature human J chain, any functional fragment thereof, derivative thereof, and / or variant thereof, the amino acid sequence of which is provided as SEQ ID NO: 7. A variety of J chain variants and modified J chain derivatives are available, for example, in U.S. Pat. No. 10,899,835. Those skilled in the art will recognize that "functional fragment" or "functional variant" includes fragments and variants that are capable of associating with an IgM heavy chain constant region to form a pentameric IgM antibody.

[0056] The term "modified J chain" is used herein to refer to a derivative of a J chain polypeptide that includes a heterologous moiety, e.g., a heterologous polypeptide, e.g., a foreign antigen-binding domain or functional domain (e.g., a ligand that binds to an effector cell, e.g., a T cell receptor or an immunostimulatory molecule), introduced into or attached to the J chain sequence. Introduction can be accomplished by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety, or by attachment via a peptide or chemical linker. The term "modified human J chain" includes, but is not limited to, a native sequence human J chain comprising the amino acid sequence of SEQ ID NO: 7, or a functional fragment or functional variant thereof, that has been modified by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., a foreign binding domain. In certain embodiments, the heterologous moiety does not interfere with the efficient polymerization of IgM into pentamers or IgA into multimers, e.g., dimers or tetramers, and the binding of such polymers to targets. Exemplary modified J chains can be found, for example, in U.S. Pat. Nos. 9,951,134, 10,400,038, and 10,618,978, U.S. Patent Application Publication No. US-2019-0185570, and PCT Publication No. WO2021030688A1, each of which is incorporated by reference in its entirety.

[0057] The terms "valency," "bivalent," "multivalent," and grammatical equivalents refer to the number of binding domains, e.g., antigen-binding domains, e.g., target antigen-binding domains, in a given antibody or a given binding unit. Thus, the terms "bivalent," "tetravalent," and "hexavalent" with reference to a given antibody indicate the presence of two, four, and six antigen-binding domains, respectively. A typical pentameric IgM antibody, in which each binding unit is bivalent, has 10 or more valencies, e.g., target antigen-binding domains. Bivalent or multivalent antibodies can be monospecific, i.e., all of the antigen-binding domains are identical, or bispecific or multispecific, e.g., two or more antigen-binding domains are different, e.g., bind different epitopes on the same antigen, bind completely different antigens, and / or bind effector cells, e.g., T cells.

[0058] The term "epitope" includes any molecular determinant capable of specific binding to a binding molecule, such as the antigen-binding domain of an antibody. In certain embodiments, an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of a target bound by the antigen-binding domain of an antibody.

[0059] The term "target" is used in the broadest sense and includes substances that can be bound by binding molecules, such as multimeric binding molecules as provided herein. Targets can be, for example, polypeptides, nucleic acids, carbohydrates, lipids, or other molecules. Furthermore, a "target" can be, for example, a cell, organ, or organism that contains an epitope that can be bound by a binding molecule, such as a multimeric binding molecule as provided herein. As used herein, a "target antigen" is a target molecule, such as a polypeptide, nucleic acid, carbohydrate, lipid, or other molecule, that can be bound by a multimeric binding molecule as provided herein. In certain embodiments, a target antigen can appear on the surface of a cell, such as a tumor cell. A "tumor-specific antigen," as used herein, is a protein or other cell surface target antigen that is unique to tumor cells at least in later stages of the development of an organism. As used herein, a "tumor-associated target antigen" is a protein or other cell surface target antigen that is not necessarily unique to tumor cells, but is typically expressed in greater abundance and / or at higher density in tumor cells than in normal, healthy cells.

[0060] Both the light and heavy chains of binding molecules, such as multimeric binding molecules provided herein, are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In a standard antibody, the variable domains of both the variable light (VL) and variable heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant region domains of the light chain (CL) and the heavy chain (e.g., CH1, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as the constant region domain becomes more distal from the antigen-binding domain or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 (or CH4-μtp, for example, in the case of IgM) and CL domains comprise the carboxy termini of the heavy and light chains, respectively.

[0061] A "full-length IgM antibody heavy chain" is a polypeptide comprising, from N-terminal to C-terminal, an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CM1 or Cμ1), antibody heavy chain constant domain 2 (CM2 or Cμ2), antibody heavy chain constant domain 3 (CM3 or Cμ3), and antibody heavy chain constant domain 4 (CM4 or Cμ4), which may include a μ tailpiece.

[0062] As used herein, "IgM-like antibody" refers to a binding molecule comprising at least a multimerizing fragment or variant of an IgM heavy chain constant region that retains the ability to associate with, for example, a J chain to form a hexamer or pentamer. IgM-like antibodies typically comprise at least the Cμ4 and IgM tailpiece (μtp) domains of the IgM constant region, but may also comprise heavy chain constant region domains from other antibody isotypes, such as IgG, from the same or different species. IgM-like antibodies may also be antibody fragments lacking one or more constant region domains, so long as the IgM-like antibody is capable of multimerizing into hexamers and / or pentamers. Thus, an IgM-like antibody can be, for example, a hybrid IgM / IgG antibody or a "multimerizing fragment" of an IgM antibody.

[0063] A "full-length IgA antibody heavy chain" is a polypeptide comprising, from N-terminal to C-terminal, an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CA1 or Cα1), antibody heavy chain constant domain 2 (CA2 or Cα2), and antibody heavy chain constant domain 3 (CA3 or Cα3), which may include the α tailpiece.

[0064] As used herein, "IgA-like antibody" refers to a binding molecule comprising at least a multimerizing fragment or variant of an IgA heavy chain constant region that retains the ability to associate with, for example, a J chain to form dimers or tetramers. IgA-like antibodies typically comprise at least the Cα3 and IgA tailpiece (αtp) domains of the IgA constant region, but may also comprise heavy chain constant region domains from other antibody isotypes, such as IgG, from the same or different species. IgA-like antibodies may also be antibody fragments in which one or more constant region domains are deleted, so long as the IgA-like antibody is capable of multimerizing into dimers and / or tetramers. Thus, an IgA-like antibody can be, for example, a hybrid IgA / IgG antibody or a "multimerizing fragment" of an IgA antibody.

[0065] As described above, the variable region(s) enable a binding molecule, such as a multimeric binding molecule as provided herein, to selectively recognize and specifically bind to an epitope on a target antigen. For example, the VL and VH domains, sdVH domains, sdVL domains, or a subset of complementarity-determining regions (CDRs) of an antibody combine to form an antigen-binding domain. More specifically, the antigen-binding domain can be defined by six CDRs from the VH and VL chains, or three CDRs from each of the sdVH or sdVL chains. Certain antibodies form larger structures. For example, IgA can form molecules containing two or four H2L2 binding units and a J chain covalently linked via disulfide bonds, which can further associate with secretory component, and IgM can form pentameric molecules containing five H2L2 binding units and a J chain covalently linked via disulfide bonds.

[0066] The six "complementarity-determining regions" or "CDRs" present within the antigen-binding domain of an antibody (or three in the case of single-domain antibodies) are short, noncontiguous amino acid sequences that are specifically arranged to form the antigen-binding domain when the antibody assumes a three-dimensional configuration in an aqueous environment. The remaining amino acids within the antigen-binding domain, called the "framework" regions, exhibit less intermolecular variability. The framework regions primarily adopt a β-sheet conformation, while the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act as a scaffold that positions the CDRs in the correct orientation through interchain noncovalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface complementary to the epitope on the target antigen. This complementary surface promotes noncovalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and framework regions, respectively, have been defined in a variety of different ways, so that any given heavy or light chain variable region can be readily identified by one of ordinary skill in the art (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDapartment of Health and Human Services, (1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).

[0067] Where there is more than one definition of a term that is used and / or accepted within the art, the definition of the term as used herein includes all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These CDR regions are described, for example, by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids when comparing amino acids with each other. Other overlapping CDR definitions can be found, for example, in 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 MP et al., Dev. Comp. Immunol. 27:55-77 (2003), and Honegger and Plueckthun, J. Mol. Biol. 309:657-670 (2001), which are incorporated herein by reference in their entireties. Antibody variable domains can also be analyzed to identify variable region segments, including CDRs, using, for example, the IMGT information system (imgt.cines.fr / ) (IMGT® / V-Quest). (See, for example, Brochet et al., Nucl. Acids Res. 36:W503-508, 2008.) Application of any particular definition (or other definition known to those of skill in the art) is intended to be within the scope of the term as defined and used herein, unless otherwise indicated.The appropriate amino acids that encompass the "Kabat" and "Chothia" CDRs as defined by the above-cited references are set forth in Table 1 below for comparison. The exact amino acid numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which amino acids are included in a particular CDR given the amino acid sequence of the variable region of an antibody. Computer software packages that can identify CDRs in antibody variable regions according to various definitions may be readily available, including abYsis (abysis.org, last accessed January 5, 2023).

[0068] [Table 1]

[0069] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, consecutive numbering is used for all amino acid sequences in this disclosure unless the use of the Kabat numbering system is explicitly stated.

[0070] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al., "Tabulation and Analysis of Amino Acid and Nucleic Acid Sequences of Precursors, V-Regions, C-Regions, J Chain, T Cell Receptors for Antigen, T Cell Surface Antigens, β-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, α-2 Macroglobulins, and Other Related Proteins," US Department of Health and Human Services (1991). The IgM constant region can be numbered consecutively (i.e., amino acid #1 starts with the first amino acid of the constant region) or by using the Kabat numbering system. A comparison of the sequential numbering of the two alleles of the human IgM constant region (presented herein as SEQ ID NO: 1 (allele IGHM*03) and SEQ ID NO: 2 (allele IGHM*04)) with the numbering according to the Kabat system is set forth below. Underlined amino acid residues are not considered in the Kabat system (the double underlined "X" below can be serine (S) (SEQ ID NO: 1) or glycine (G) (SEQ ID NO: 2)). TIFF2026502759000002.tif105156

[0071] Antibodies include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFvs), single-chain antibodies, disulfide-linked Fvs (sdFvs), fragments containing either the VL or VH domains, and fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0072] "Specifically bind" means that a binding molecule, such as an antibody, binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule, such as an antibody, "specifically binds" to an epitope if it binds to that epitope more readily via its antigen-binding domain than it binds to a random, unrelated epitope. The term "specificity" is used herein to refer to the relative affinity with which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" can be considered to have higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" binds to epitope "C" with higher specificity than to related epitope "D."

[0073] As used herein, the term "affinity" refers to the measure of the strength of binding between one or more antigen-binding domains (e.g., binding domains of immunoglobulin molecules) and an individual epitope. See, for example, Greenfield, EA (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. Avidity is related to both the affinity of individual antigen-binding domains within the population for a particular epitope and the valency of the immunoglobulin and antigen. For example, the interaction between a bivalent monoclonal antibody and a target antigen with a highly repetitive epitope structure (e.g., a polymer) would be a high-avidity interaction. The interaction between a bivalent monoclonal antibody and a receptor present at high density on a cell surface would also be a high-avidity interaction.

[0074] Binding molecules, such as the multimeric binding molecules provided herein, can also be described or specified in terms of their cross-reactivity.As used herein, the term " cross-reactivity " refers to the ability of a binding molecule, such as an antibody specific to a certain antigen, to react with a second antigen, and is a measure of the relationship between two different antigenic substances.Therefore, an antibody is cross-reactive when it binds to an epitope other than the epitope that triggers its formation.Cross-reactive epitopes contain many of the same complementary structural features as the triggering epitope, and in some cases can match better than the original epitope.

[0075] "Antigen-binding antibody fragments," including single-chain antibodies or other antigen-binding domains, may be present alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. Also included are antigen-binding fragments that can include any combination of variable region(s) and one or more of hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. The antibody can be from any animal origin, including fish, birds, and mammals. The antibody can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable region can be of condricthoid origin (e.g., from shark). As used herein, a "human" antibody includes antibodies having a human immunoglobulin amino acid sequence and includes antibodies isolated from a human immunoglobulin library or from animals transgenic for one or more human immunoglobulins, which may or may not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 by Kucherlapati et al. According to embodiments of the present disclosure, multimeric binding molecules, such as IgM antibodies described herein, can include antigen-binding fragments of the antibody, e.g., scFv fragments, so long as the IgM antibody is capable of forming multimers, e.g., hexamers or pentamers, and IgA antibodies as provided herein can include antigen-binding fragments of the antibody, e.g., scFv fragments, so long as the IgA antibody is capable of forming multimers, e.g., dimers or tetramers.

[0076] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from an immunoglobulin heavy chain. A binding molecule (e.g., an antibody comprising a heavy chain subunit) can include a VH domain, a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, a tailpiece, e.g., an IgM tailpiece, or any variant or fragment thereof. For example, a binding molecule (e.g., an antibody) can include, but is not limited to, a VH domain plus a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, a binding molecule, e.g., an antibody, can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, an IgM tailpiece, and a J chain. Furthermore, binding molecules, e.g., antibodies, may lack all or part of certain constant region portions, e.g., CH2 domains. Those skilled in the art will understand that such domains (e.g., heavy chain subunits) may be modified to differ in amino acid sequence from the original immunoglobulin molecule. According to embodiments of the present disclosure, multimeric binding molecules, e.g., pentameric IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, or dimeric or tetrameric IgA antibodies, IgA-like antibodies, or other IgA-derived binding molecules, as provided herein, contain a sufficient portion of the IgM or IgA heavy chain constant region to enable the pentameric IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules, or dimeric or tetrameric IgA antibodies, IgA-like antibodies, or other IgA-derived binding molecules to form multimers, e.g., pentamers, tetramers, or dimers. As used herein, such heavy chain constant region fragments include "multimerization fragments."

[0077] As used herein, the term "light chain subunit" includes an amino acid sequence derived from an immunoglobulin light chain. A light chain subunit contains at least a VL domain and may further contain a CL (e.g., a CK or Cλ) domain.

[0078] A binding molecule (e.g., an antibody) can be described or specified in terms of the epitope(s) or portion(s) of the target, e.g., target antigen, that the binding molecule recognizes or specifically binds. The portion of the target antigen that specifically interacts with the target antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target antigen can contain a single epitope or at least two epitopes, and can contain any number of epitopes depending on the size, conformation, and type of antigen.

[0079] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified) is obtained from a second species. In some embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is human.

[0080] The term "multispecific antibody" or "bispecific antibody" refers to an antibody that has antigen-binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules in addition to the canonical antibody structure can be constructed with two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Stroehlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Bispecific antibodies can also be diabodies.

[0081] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain, constant region, and / or J chain have been altered by at least partial deletion, addition, or substitution of one or more amino acids. In certain embodiments, entire CDRs from an antibody with known specificity can be grafted into the framework regions of a heterologous antibody. The replacement CDRs can be from an antibody of the same class, or even subclass, as the antibody from which the framework region is derived, although the CDRs can also be from an antibody of a different class, e.g., an antibody from a different species. Engineered antibodies in which one or more "donor" CDRs from a non-human antibody with known specificity are grafted into human heavy or light chain framework regions are referred to herein as "humanized antibodies." In certain embodiments, not all CDRs are replaced with complete CDRs from the donor variable region, yet the antigen-binding capacity of the donor can be transferred to the variable domain of the recipient. This is well within the skill of one in the art and, with routine experimentation, will result in functional engineered or humanized antibodies in light of the teachings set forth in, for example, U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

[0082] As used herein, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or any combination of these techniques).

[0083] As used herein, the terms "linked," "fused," or "fusion," or other grammatical equivalents, can be used interchangeably. These terms refer to the linking of two or more elements or components together by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the linking of two or more polynucleotide open reading frames (ORFs) to form a longer, contiguous ORF in a manner that maintains the translational reading frame of the original ORFs. A recombinant fusion protein is thus a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs (segments that are not normally linked in nature). Although the reading frame is thus made contiguous throughout the fused segments, the segments can be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding CDRs of immunoglobulin variable regions can be fused in-frame but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a contiguous polypeptide.

[0084] In the context of a polypeptide, a "linear sequence" or "sequence" refers to the order of amino acids in a polypeptide in the direction from the amino terminus to the carboxyl terminus, with adjacent amino acids in the sequence being contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino terminal" or "N-terminal" to another portion of the polypeptide is the portion that occurs earlier in the contiguous polypeptide chain. Similarly, a portion of a polypeptide that is "carboxy terminal" or "C-terminal" to another portion of the polypeptide is the portion that occurs later in the contiguous polypeptide chain. For example, in a typical antibody, the variable domain is "N-terminal" to the constant region, and the constant region is "C-terminal" to the variable domain.

[0085] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, such as a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. This includes, but is not limited to, gene transcription into RNA (e.g., messenger RNA (mRNA)) and translation of such mRNA into polypeptide(s). When the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene results in a "gene product." As used herein, a gene product can be either a nucleic acid (e.g., a messenger RNA resulting from transcription of a gene) or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation) or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, etc.).

[0086] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by uncontrolled cell growth. Cancer can be classified, for example, as a solid tumor or solid malignancy, or a blood cancer or malignancy. Either type can migrate to distant sites as metastasis. Solid tumors can be classified, for example, as a sarcoma, carcinoma, melanoma, or metastases thereof.

[0087] The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation, such as cancer. "Tumor" and "neoplasm," as used herein, refer to any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0088] The terms "metastasis," "metasitases," "metastatic," and other grammatical synonyms, as used herein, refer to cancer cells that spread or migrate from their site of origin (e.g., a primary tumor) to other areas of the body and give rise to similar cancerous lesions at the new location. A "metastatic" or "metastatic" cell is one that loses adhesive contacts with neighboring cells and migrates from the primary site of disease via the bloodstream or lymph to invade nearby body structures. These terms also refer to the process of metastasis, including, but not limited to, detachment of cancer cells from the primary tumor, intravasation of tumor cells into the circulation, survival and migration of cancer cells to distant sites, adhesion and extravasation from the circulation to new sites, and microcolony formation at distant sites, as well as tumor growth and development at distant sites.

[0089] Examples of such solid tumors can include, for example, squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, renal cell carcinoma, ductal carcinoma, soft tissue sarcoma, osteosarcoma, melanoma, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, stomach cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, esophageal cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, any metastasis thereof, or any combination thereof.

[0090] Examples of blood cancers or hematological malignancies include, but are not limited to, leukemia, lymphoma, myeloma, acute myeloid leukemia (AML), chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, any metastasis thereof, or any combination thereof.

[0091] In certain embodiments, cancers treatable via the methods provided herein include, but are not limited to, sarcoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, esophageal cancer, gastric cancer, kidney cancer, liver cancer, bladder cancer, colorectal cancer, and pancreatic cancer.

[0092] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat" a disease or disorder in a subject, e.g., a human. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; slow or stop cancer cell division, inhibit or slow the growth of tumor size; inhibit, e.g., inhibit, slow, prevent, stop, delay, or reverse, the invasion of cancer cells into peripheral organs, including the spread of cancer to soft tissue and bone; inhibit, e.g., inhibit, slow, prevent, reduce, stop, delay, or reverse, tumor metastasis; inhibit, e.g., inhibit, slow, prevent, stop, delay, or reverse, tumor growth; relieve to some extent one or more symptoms associated with cancer, reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0093] Terms such as "treat" or "treatment" or "treating" or "alleviate" or "alleviating" refer to therapeutic measures that cure, slow, relieve the symptoms, reduce the severity of the symptoms, and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as "prevent," "prevention," "avoid," "prevent," "prophylactic," and the like refer to prophylactic or preventative measures that prevent the onset of an undiagnosed targeted pathological condition or disorder. Thus, a "person in need of treatment" can include someone who already has the disorder.

[0094] A subject is successfully "treated" according to the methods of the present disclosure if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; or a slowing or reversal of tumor growth; inhibition (e.g., suppression, prevention, slowing, reduction, delay, or reversal) of cancer cell invasion into peripheral organs, including metastasis (e.g., the spread of cancer to soft tissue and bone); inhibition (e.g., suppression, slowing, prevention, reduction, reversal, delay, or absence) of tumor metastasis; inhibition (e.g., suppression, slowing, prevention, reduction, reversal, delay, or absence) of tumor growth; a reduction in one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; an improvement in quality of life; or any combination of effects. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, a reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), a delay or slowing of disease progression, an improvement or remission of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment can include those already with the condition or disorder.

[0095] "Subject" or "individual" or "animal" or "patient" or "mammal" means any mammalian subject. In certain embodiments, a subject is one for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0096] As used herein, the term "subjects who would benefit from a therapy" refers to a subset of subjects among all candidate subjects who would benefit from the administration of a given therapeutic agent, e.g., a binding molecule such as an antibody that comprises one or more antigen-binding domains. Such binding molecules (e.g., antibodies) can be used, for example, for diagnostic procedures and / or for the treatment or prevention of disease.

[0097] As used herein, the term "serum half-life" or "plasma half-life" refers to the time (e.g., minutes, hours, or days) it takes for the serum or plasma concentration of a drug, e.g., a binding molecule such as an antibody described herein, to decrease by 50% after administration. Two half-lives can be described: the alpha half-life, α half-life, or t1 / 2α, which is the rate of decline in plasma concentration due to the process of redistribution of the drug from a central compartment (e.g., blood in the case of intravenous delivery) to a peripheral compartment (e.g., tissue or organ), and the beta half-life, β half-life, or t1 / 2β, which is the rate of decline due to excretion or metabolic processes.

[0098] As used herein, the term "area under the plasma drug concentration-time curve" or "AUC" reflects the actual body exposure to a drug after administration of a dose of the drug and is expressed in mg*h / L. This area under the curve can be measured, for example, from time 0 (t0) to infinity (∞) and depends on the rate of elimination of the drug from the body and the administered dose.

[0099] As used herein, the term "mean residence time" or "MRT" refers to the average length of time that a drug remains in the body.

[0100] As used herein, "pharmaceutically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable, e.g., that the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients preferably meet required toxicity and manufacturing testing standards and / or are described in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0101] A "pharmaceutically acceptable salt" is a salt that retains at least some of the biological activity of the free (non-salt) compound and can be administered to an individual as a drug or pharmaceutical. Examples of such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, and the like; and (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, and the like. Acceptable inorganic bases that can be used to prepare salts include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Pharmaceutically acceptable salts can be prepared in situ during the manufacturing process or by separately reacting the purified compound in free acid or free base form, respectively, with a suitable organic or inorganic base or acid and isolating the salt so formed during subsequent purification.

[0102] The term "excipient," as used herein, refers to an inactive or inert substance that can be used in making a drug or pharmaceutical formulation as an active ingredient. The term excipient can encompass a variety of substances, including, but not limited to, binders, disintegrants, coatings, compression / encapsulation aids, creams or lotions, lubricants, solutions for parenteral administration, chewable tablet materials, sweeteners or flavoring agents, suspending / gelling agents, or any substance used as a wet granulation agent. Examples of binders include carbomer, povidone, xanthan gum, etc.; examples of coating agents include cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, enteric coating, etc.; examples of compression / encapsulation aids include calcium carbonate, dextrose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrous or monohydrate; optionally combined with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; examples of disintegrants include croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; examples of creams or lotions include cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, enteric coating, etc.; examples of encapsulation aids include calcium carbonate, dextrose, fructose dc (dc = "directly compressible"), honey dc, lactose (anhydrous or monohydrate; optionally combined with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; examples of disintegrants include croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; and examples of creams or lotions include cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, enteric coating, etc. Examples of suitable lubricants include maltodextrin and carrageenan; lubricants include magnesium stearate, stearic acid, sodium stearyl fumarate; chewable tableting agents include dextrose, fructose dc, lactose (monohydrate, optionally combined with aspartame or cellulose); suspending / gelling agents include carrageenan, sodium starch glycolate, xanthan gum; sweeteners include aspartame, dextrose, fructose dc, sorbitol, sucrose dc; and wet granulating agents include calcium carbonate, maltodextrin, and microcrystalline cellulose.

[0103] IgM antibodies and IgM-like antibodies IgM is the first immunoglobulin produced by B cells in response to stimulation by an antigen. Naturally occurring IgM is naturally present in serum at approximately 1.5 mg / ml and has a half-life of approximately 5 days. IgM is typically a pentameric or hexameric molecule and therefore can contain five or six binding units. An IgM binding unit typically contains two light chains and two heavy chains. The IgG heavy chain constant region contains three heavy chain constant regions (CH1, CH2, and CH3), while the IgM heavy (μ) chain constant region further contains a fourth constant domain (CH4) and includes a C-terminal IgM tailpiece. A human IgM constant region typically comprises the amino acid sequence SEQ ID NO:1 (e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1, identical to allele IGHM*03) or SEQ ID NO:2 (e.g., GenBank Accession No. sp|P01871.4, identical to allele IGHM*04). The human Cμ1 region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO:1 or SEQ ID NO:2, the human Cμ2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO:1 or SEQ ID NO:2, the human Cμ3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO:1 or SEQ ID NO:2, the Cμ4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO:1 or SEQ ID NO:2, and μtp ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO:1 or SEQ ID NO:2.

[0104] Other forms and alleles of the human IgM constant region with minor sequence variations include, but are not limited to, GenBank Accession Nos. CAB37838.1 and pir||MHHU. Amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO:1 or SEQ ID NO:2, as described and claimed elsewhere in this disclosure, can also be incorporated into alternative human IgM sequences, as well as IgM constant region amino acid sequences of other species.

[0105] The five IgM binding units can complex with an additional small polypeptide chain (J chain), or a functional fragment or variant thereof, to form a pentameric IgM antibody, as discussed elsewhere herein. The precursor form of the human J chain is presented as SEQ ID NO:6. The signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO:6, and the mature human J chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO:6. The mature human J chain comprises the amino acid sequence: SEQ ID NO:7.

[0106] Exemplary variants and modified J chains are provided elsewhere herein. Without a J chain, IgM or IgM-like antibodies typically assemble into hexamers consisting of up to 12 target antigen-binding domains. With a J chain, IgM or IgM-like antibodies typically assemble into pentamers containing up to 10 antigen-binding domains, and pentamers containing more than 10 antigen-binding domains when the J chain is a modified J chain containing one or more heterologous polypeptides containing additional antigen-binding domain(s) capable of binding to effector cells, e.g., T cells. Assembly of five or six IgM binding units into pentameric or hexameric IgM or IgM-like antibodies is believed to involve Cμ4 and the IgM tailpiece domain. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Thus, a pentameric or hexameric IgM antibody typically comprises at least the Cμ4 and μtp domains. A "multimerizing fragment" of an IgM heavy chain constant region therefore comprises at least the Cμ4 and μtp domains. The IgM heavy chain constant region may additionally comprise a Cμ3 domain or fragment thereof, a Cμ2 domain or fragment thereof, a Cμ1 domain or fragment thereof, and / or other IgM heavy chain domains. In certain embodiments, an IgM antibody as provided herein can comprise a complete IgM heavy (μ) chain constant domain, e.g., as provided herein, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, derivative, or analog thereof.

[0107] In certain embodiments, the present disclosure provides a pentameric binding molecule, e.g., an IgM antibody, comprising ten IgM-derived heavy chains and a modified J chain, each of which comprises an IgM heavy chain constant region associated with a target antigen-binding domain, i.e., an antigen-binding domain that specifically binds to a target antigen, e.g., a tumor-associated target antigen. Furthermore, IgM-based binding molecules, e.g., IgM antibodies, can consistently form pentameric oligomers, resulting in more homogeneous expression products. Excellent complement fixation may also be an advantageous effector function of IgM-based antibodies.

[0108] In certain embodiments, the two IgM heavy chain constant regions contained in each binding unit are human heavy chain constant regions. In some embodiments, the heavy chain is glycosylated. In some embodiments, the heavy chain can be mutated to affect glycosylation. See, for example, U.S. Patent Application Publication No. US2022 / 0306760A1, the entire contents of which are incorporated herein by reference.

[0109] When the IgM antibodies provided herein are pentamers, the IgM antibodies further comprise a modified J chain or variant thereof, as described elsewhere herein, that further comprises one or more heterologous moieties attached to the J chain. In certain embodiments, the J chain can be mutated to affect, e.g., improve, the serum half-life of the IgM antibodies provided herein, as discussed elsewhere in this disclosure. In certain embodiments, the J chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.

[0110] An IgM heavy chain constant region can comprise one or more of a Cμ1 domain or a fragment or variant thereof, a Cμ2 domain or a fragment or variant thereof, a Cμ3 domain or a fragment or variant thereof, and / or a Cμ4 domain or a fragment or variant thereof, provided that the constant region performs the desired function in an IgM antibody, e.g., can associate with a second IgM constant region to form a binding unit comprising one, two, or more target antigen-binding domains, and / or associate with other binding units (and, in the case of a pentamer, a J chain) to form a hexamer or pentamer. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a Cμ4 domain or a fragment or variant thereof, an IgM tailpiece (μtp) or a fragment or variant thereof, or a combination of a Cμ4 domain and a μtp or a fragment or variant thereof. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof in an individual binding unit each further comprise a Cμ3 domain or fragment or variant thereof, a Cμ2 domain or fragment or variant thereof, a Cμ1 domain or fragment or variant thereof, or any combination thereof.

[0111] In certain embodiments, each of the two IgM heavy chain constant regions in a given binding unit is associated with a target antigen-binding domain, e.g., the Fv portion of an antibody, e.g., the VH and VL of a human or mouse antibody, and the VL can be associated with a light chain constant region. In certain embodiments, multimeric binding molecules as provided herein comprise at least three, e.g., three, four, five, six, seven, eight, nine, or ten, target antigen-binding domains that specifically bind to tumor-specific or tumor-associated target antigens.

[0112] In some embodiments, a binding unit of an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule comprises two light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains, each comprising a VL located amino-terminal to an immunoglobulin light chain constant region.

[0113] IgA antibodies and IgA-like antibodies IgA plays an important role in mucosal immunity and accounts for approximately 15% of all immunoglobulins produced. IgA antibodies can be monomers, dimers, or tetramers, each containing one, two, or four IgA binding units. An IgA binding unit contains two IgA heavy chains and may further contain two light chains. The IgA heavy chain constant region contains three heavy chain constant domains (Cα1, Cα2, and Cα3), including the hinge region between Cα1 and Cα2 and the C-terminal IgA tailpiece. There are two subtypes of human IgA: IgA1 and IgA2. The human IgA1 constant region typically contains the amino acid sequence SEQ ID NO: 3. The human Cα1 domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 3, the human IgA1 hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 3, the human Cα3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 3, and the αtp extends from about amino acid 331 to about amino acid 352 of SEQ ID NO: 3. The human IgA2 constant region typically comprises the amino acid sequence SEQ ID NO: 4. The human Cα1 domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO:4, the human IgA2 hinge region extends from about amino acid 102 to about amino acid 111 of SEQ ID NO:4, the human Cα2 domain extends from about amino acid 113 to about amino acid 206 of SEQ ID NO:4, the human Cα3 domain extends from about amino acid 215 to about amino acid 317 of SEQ ID NO:4, and the αtp extends from about amino acid 318 to about amino acid 340 of SEQ ID NO:4.

[0114] Two or four IgA binding units can complex with two additional polypeptide chains, a J chain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8) and a secretory component (precursor, SEQ ID NO: 5; mature: amino acids 19-603 of SEQ ID NO: 5) to form secretory IgA (sIgA) antibodies. Assembly of IgA binding units into dimeric or tetrameric sIgA antibodies is believed to involve the Cα3 and αtp domains (collectively referred to herein as Cα3-tp domains). Thus, dimeric or tetrameric sIgA antibodies provided in this disclosure typically contain an IgA constant region comprising at least the Cα3 and αtp domains.

[0115] The IgA heavy chain constant region can additionally comprise a Cα2 domain or fragment thereof, an IgA hinge region, a Cα1 domain or fragment thereof, and / or other IgA heavy chain domains. In certain embodiments, an IgA antibody or IgA-like binding molecule as provided herein can comprise the complete IgA heavy (α) chain constant region (e.g., SEQ ID NO: 3 or SEQ ID NO: 4), or a variant thereof. In some embodiments, each IgA heavy chain constant region or multimerization fragment thereof is a human IgA constant region.

[0116] In some embodiments, a binding unit of an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprises two light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains, each comprising a VL located amino-terminal to an immunoglobulin light chain constant region.

[0117] In some embodiments, the present disclosure provides dimeric or tetrameric binding molecules, such as IgA antibodies or IgA-like antibodies having two or four IgA "binding units," that can specifically bind to a target antigen, e.g., a tumor-specific or tumor-associated target antigen. The use of IgA-based binding molecules may, for example, enable greater tissue distribution of the binding molecules provided herein. Mucosal distribution may be beneficial for certain cancers, such as lung cancer, gastric cancer, ovarian cancer, colorectal cancer, or squamous cell carcinoma. Similarly, tetrameric or dimeric IgA antibodies or IgA-like antibodies as provided herein may have binding characteristics or biological activity that are distinguishable from binding molecules containing five binding units, e.g., pentameric IgM antibodies. For example, dimeric binding molecules are smaller and may achieve better tissue penetration, for example, into certain solid tumors.

[0118] In certain embodiments, the present disclosure provides dimeric or tetrameric IgA or IgA-like antibodies comprising two or four bivalent binding units and a modified J chain, wherein each binding unit comprises two IgA heavy chain constant regions or multimerized fragments thereof. In certain embodiments, the two IgA heavy chain constant regions are human heavy chain constant regions.

[0119] An IgA heavy chain constant region can comprise one or more of a Cα1 domain, an IgA hinge region, a Cα2 domain, a Cα3 domain, and / or an αtp, provided that the constant region is capable of performing the desired function in the binding molecule, e.g., associating with a light chain constant region to facilitate the formation of a target antigen-binding domain or associating with another IgA binding unit to form a dimeric binding molecule. In certain embodiments, the two IgA heavy chain constant regions or fragments thereof within an individual binding unit each comprise a Cα3 domain or multimerization fragment thereof, a tailpiece (αtp) or multimerization fragment thereof, or any combination of a Cα3 domain, tp, or multimerization fragments thereof. In certain embodiments, the two IgA heavy chain constant regions or fragments thereof within an individual binding unit each further comprise a Cα2 domain or fragment thereof, a Cα1 domain or fragment thereof, an IgA hinge region, or a Cα1 domain or fragment thereof, an IgA hinge region, and a Cα2 domain or fragment thereof.

[0120] In certain embodiments, each of the two IgA heavy chain constant regions in a given binding unit is associated with a target antigen-binding domain, e.g., the Fv portion of an antibody, e.g., the VH and VL of a human or murine antibody, and the VL can be associated with a light chain constant region. In certain embodiments, a multimeric binding molecule as provided herein comprises at least three target antigen-binding domains capable of specifically binding to a target antigen, e.g., a tumor-associated or tumor-specific target antigen.

[0121] J chain and its functional fragments or variants Certain multimeric binding molecules provided herein comprise a modified J chain, or a functional fragment or variant thereof. In certain embodiments, the multimeric binding molecules provided herein are pentameric and comprise a modified J chain, or a functional fragment or variant thereof. In certain embodiments, the binding molecules provided herein are dimeric or tetrameric and comprise a modified J chain, or a functional fragment or variant thereof. In some embodiments, the dimeric, tetrameric, or pentameric binding molecules comprise a modified J chain comprising a naturally occurring J chain sequence, such as a mature human J chain sequence (e.g., SEQ ID NO: 7). Alternatively, in some embodiments, the dimeric, tetrameric, or pentameric binding molecules comprise a modified J chain comprising a variant J chain sequence, such as a variant sequence described herein, with reduced glycosylation and / or reduced binding to polymeric Ig receptors (e.g., pIgR, Fc alpha-mu receptor (FcαμR), or Fc mu receptor (FcμR)). See, e.g., U.S. Pat. No. 10,899,835 (incorporated herein by reference in its entirety). In some embodiments, the modified J chain of a dimeric, tetrameric, or pentameric binding molecule can comprise a functional fragment of a naturally occurring or variant J chain. Those skilled in the art will understand that a "functional fragment" or "functional variant" in this context includes fragments and variants thereof that can associate with binding units, e.g., IgM or IgA heavy chain constant regions, to form pentameric IgM antibodies, IgM-like antibodies, or IgM-derived binding molecules, or dimeric or tetrameric IgA antibodies, IgA-like antibodies, or IgA-derived binding molecules, and / or that can associate with certain immunoglobulin receptors, e.g., pIgR.

[0122] As provided herein, the J chain is modified, e.g., by the introduction of a heterologous moiety or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of the binding molecule to assemble and bind to its binding target(s). See U.S. Patent Nos. 9,951,134, 10,400,038, 10,618,978, and 11,639,389, each of which is incorporated by reference in its entirety.

[0123] Thus, multimeric binding molecules provided herein, including IgA, IgA-like, IgM, or IgM-like antibodies described elsewhere herein, include modified J chains or functional fragments or variants thereof that include a heterologous moiety, e.g., a heterologous polypeptide, attached, e.g., fused or chemically conjugated, to the J chain or fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused to the N-terminus of the J chain or functional fragment or variant thereof, or to the C-terminus of the J chain or functional fragment or variant thereof. In certain embodiments, the heterologous moiety, e.g., a heterologous polypeptide, is attached to both the N-terminus and C-terminus of the J chain or functional fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused internally within the J chain or functional fragment or variant thereof. In some embodiments, the heterologous polypeptide can be introduced into the J chain at or near a glycosylation site. In some embodiments, the heterologous polypeptide can be introduced into the J chain within about 10 amino acid residues from the C-terminus or within about 10 amino acids from the N-terminus. In certain embodiments, a heterologous polypeptide can be introduced into the mature human J chain of SEQ ID NO: 7 between cysteine ​​residues 92 and 101 of SEQ ID NO: 7, or an equivalent position in the J chain sequence, e.g., a J chain variant or a functional fragment of a J chain. In further embodiments, a heterologous polypeptide can be introduced into the mature human J chain of SEQ ID NO: 7 at or near a glycosylation site. In further embodiments, a heterologous polypeptide can be introduced into the mature human J chain of SEQ ID NO: 7 within about 10 amino acid residues of the C-terminus or within about 10 amino acids of the N-terminus.

[0124] In certain embodiments, the heterologous moiety can be a peptide or polypeptide sequence fused in-frame to the J chain or chemically conjugated to a J chain or a fragment or variant thereof. In certain embodiments, the heterologous polypeptide is fused to the J chain or a functional fragment thereof via an amino acid linker. Any suitable linker can be used, for example, the amino acid linker can comprise at least 5 amino acids, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, or more amino acids. In certain embodiments, the amino acid linker comprises at least 5 amino acids but no more than 25 amino acids. In certain embodiments, the amino acid linker can consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In certain embodiments, the amino acid linker can comprise the amino acid sequence (GGGGS) n (SEQ ID NO: 279), where n is an integer from 2 to 5. In certain embodiments, the amino acid linker consists of GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGSGGGGSGGGGGS (SEQ ID NO: 11), GGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12), GGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 13), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0125] In certain embodiments, the heterologous moiety can be a chemical moiety conjugated to the J chain. Heterologous moieties to be attached to the J chain can include, but are not limited to, binding moieties such as antibodies or antigen-binding fragments thereof, e.g., single-chain Fv (scFv) molecules, cytokines such as IL-2 or IL-15 (see, e.g., PCT Publication No. WO2021030688A1, which is incorporated herein by reference in its entirety), binding molecules such as human serum albumin (HSA) or stabilizing peptides capable of increasing the half-life of HSA-binding molecules, or heterologous chemical moieties such as polymers or cytotoxins. In certain embodiments, described in more detail elsewhere herein, the heterologous moiety includes a polypeptide agonist of a T cell costimulatory molecule. In certain embodiments, the modified J chain of a multimeric binding molecule as provided herein comprises two heterologous moieties: an anti-CD3 scFv and a polypeptide agonist of a T cell costimulatory molecule. In certain embodiments, the T cell costimulatory molecule is CD28. In certain embodiments, the T cell costimulatory molecule is 4-1BB. In certain embodiments, the polypeptide agonist of the T cell costimulatory molecule is an anti-CD28 antibody or an antigen-binding fragment thereof. In certain embodiments, the polypeptide agonist of the T cell costimulatory molecule is a 4-1BB ligand (4-1BBL) trimer.

[0126] In some embodiments, the modified J chain comprises an antigen-binding domain. In certain embodiments, the antigen-binding domain associated with the modified J chain is an antibody or antigen-binding fragment thereof. In certain embodiments, the antigen-binding domain can be, for example, an scFv antigen-binding domain or single-chain antigen-binding domain (sdVH) derived from a camelid antibody or a cartilaginous fish antibody, or an scFv antigen-binding domain or single-chain antigen-binding domain (sdVL) derived from an antibody light chain. In certain embodiments, the antigen-binding domain binds to T cells, e.g., cytotoxic T cells.

[0127] The antigen-binding domain can be introduced into the J chain at any position that allows the antigen-binding domain to bind to its antigen without interfering with the function of the J chain or the function of the associated multimeric binding molecule, e.g., a pentameric IgM or dimeric IgA antibody, including, but not limited to, at or near the C-terminus, at or near the N-terminus, or at an internal site accessible based on the three-dimensional structure of the J chain.

[0128] Variant J chains conferring increased serum half-life In certain embodiments, the modified J chain of a multimeric binding molecule as provided herein comprises a functional variant J chain that contains one or more single amino acid substitutions, deletions, or insertions compared to a reference J chain that is identical to the variant J chain except for one or more single amino acid substitutions, deletions, or insertions. For example, certain amino acid substitutions, deletions, or insertions can result in an IgM-derived multimeric binding molecule that exhibits an increased serum half-life when administered to a subject animal compared to a reference IgM-derived binding molecule that is identical except for one or more single amino acid substitutions, deletions, or insertions in the variant-modified J chain and that is administered to the same animal species using the same method. In certain embodiments, the variant-modified J chain can contain one, two, three, or four single amino acid substitutions, deletions, or insertions compared to the reference J chain.

[0129] In certain embodiments, the modified J chain comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7). An "amino acid corresponding to amino acid Y102 of the mature wild-type human J chain" refers to an amino acid in the J chain sequence that is homologous to Y102 in the human J chain. See, e.g., U.S. Pat. No. 10,899,835, incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 7 is conserved in the J chain amino acid sequences of at least 43 other species. See Figure 4 of U.S. Pat. No. 9,951,134, incorporated herein by reference. Certain mutations at the position corresponding to Y102 in SEQ ID NO: 7 can inhibit binding of certain immunoglobulin receptors, e.g., human or mouse Fcαμ receptors, mouse Fcμ receptors, and / or human or mouse polymeric Ig receptors (pIgRs), to IgM pentamers containing the variant J chain.

[0130] A multimeric binding molecule comprising a modified J chain having a mutation at the amino acid corresponding to Y102 of SEQ ID NO:7 has an improved serum half-life when administered to an animal over a corresponding multimeric binding molecule that is identical except for the substitution and administered to the same species in the same manner. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO:7 can be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO:7 can be substituted with alanine (A), serine (S), or arginine (R). In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO:7 can be substituted with alanine. In certain embodiments, the modified J chain, or functional fragment or variant thereof, comprises a variant human J chain, referred to herein as "J*," comprising the amino acid sequence: SEQ ID NO:8.

[0131] Wild-type J chains typically contain one N-linked glycosylation site. In certain embodiments, modified J chains, including variant J chains or functional fragments thereof, of multimeric binding molecules as provided herein contain a mutation within the asparagine (N)-linked glycosylation motif N-X1-S / T, e.g., starting at an amino acid position corresponding to amino acid 49 (motif N6) of mature human J chain (SEQ ID NO: 7) or J* (SEQ ID NO: 8), where N is asparagine, X1 is any amino acid except proline, and S / T are serine or threonine, and the mutation prevents glycosylation at that motif. As shown in U.S. Pat. No. 10,899,835, a mutation that prevents glycosylation at this site can result in multimeric binding molecules as provided herein that exhibit an increased serum half-life when administered to a subject animal compared to a reference multimeric binding molecule that is identical except for the mutation(s) that prevent glycosylation in the variant J chain and that is administered to the same animal species in the same manner.

[0132] For example, in certain embodiments, a modified J chain comprising a variant J chain or functional fragment thereof of a pentameric IgM-derived or dimeric IgA-derived binding molecule as provided herein can comprise an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO:7 or SEQ ID NO:8, with the proviso that the amino acid corresponding to S51 is not substituted with threonine (T), or the variant J chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO:7 or SEQ ID NO:8. In certain embodiments, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S), or aspartic acid (D). In certain embodiments, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 can be substituted with alanine (A). In another embodiment, the position corresponding to N49 of SEQ ID NO:7 or SEQ ID NO:8 can be substituted with aspartic acid (D).

[0133] Variant IgM constant region The IgM heavy chain constant region of the multimeric binding molecules provided herein can be engineered to confer certain desired properties to the multimeric binding molecules provided herein. For example, in certain embodiments, the IgM heavy chain constant region can be engineered to confer an enhanced serum half-life to the multimeric binding molecules provided herein. Exemplary IgM heavy chain constant region mutations that can increase the serum half-life of IgM-derived binding molecules are disclosed in U.S. Patent No. 10,899,835, which is incorporated herein by reference in its entirety. For example, a variant IgM heavy chain constant region of an IgM antibody, IgM-like antibody, or IgM-derived binding molecule provided herein can contain amino acid substitutions at positions corresponding to amino acids S401, E402, E403, R344, and / or E345 of the wild-type human IgM constant region (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). "Amino acids corresponding to amino acids S401, E402, E403, R344, and / or E345 of a wild-type human IgM constant region" means amino acids in the sequence of an IgM constant region of any species that are homologous to S401, E402, E403, R344, and / or E345 in the human IgM constant region. In certain embodiments, amino acids corresponding to S401, E402, E403, R344, and / or E345 of SEQ ID NO: 1 or SEQ ID NO: 2 can be substituted with any amino acid, for example, alanine.

[0134] In certain embodiments, IgM antibodies, IgM-like antibodies, or other IgM-derived binding molecules as provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity against cells in the presence of complement compared to a reference IgM antibody, IgM-like antibody, or other IgM-derived binding molecule comprising a corresponding reference human IgM constant region that is identical except for mutations that confer reduced CDC activity. These CDC mutations can be combined with any of the mutations that confer increased serum half-life as provided herein. A "corresponding reference human IgM constant region" refers to a human IgM constant region that is identical to the variant IgM constant region except for the modification(s) in the constant region that affect CDC activity. In certain embodiments, the variant human IgM constant region contains one or more amino acid substitutions, e.g., in the Cμ3 domain, compared to a wild-type human IgM constant region, as described, for example, in U.S. Patent No. 11,401,337, incorporated herein by reference in its entirety. Assays for measuring CDC are well known to those of skill in the art, and exemplary assays are described, for example, in US Pat. No. 11,401,337.

[0135] In certain embodiments, a variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution at positions L310, P311, P313, and / or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04) corresponding to the wild-type human IgM constant region. In certain embodiments, a variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2 corresponding to the wild-type human IgM constant region. In other embodiments, a variant IgM constant region as provided herein contains an amino acid substitution at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2 corresponding to the wild-type human IgM constant region. In other embodiments, a variant IgM constant region as provided herein contains a combination of substitutions at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and position P313 of SEQ ID NO: 1 or SEQ ID NO: 2 corresponding to the wild-type human IgM constant region. These proline residues can be independently substituted with any amino acid, for example, alanine, serine, or glycine. In certain embodiments, a variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2, corresponding to the wild-type human IgM constant region. The lysine residues can be independently substituted with any amino acid, for example, alanine, serine, glycine, or aspartic acid. In certain embodiments, a variant human IgM constant region that confers reduced CDC activity comprises an amino acid substitution at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2, corresponding to the wild-type human IgM constant region, for example, with aspartic acid.

[0136] Human and certain non-human primate IgM constant regions typically contain five naturally occurring asparagine (N)-linked glycosylation motifs or sites. As used herein, an "N-linked glycosylation motif" comprises or consists of the amino acid sequence N-X1-S / T, where N is asparagine, X1 is any amino acid except proline (P), and S / T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. The N-linked glycosylation motifs occur in the human IgM heavy chain constant region of SEQ ID NO: 1 or SEQ ID NO: 2, starting at positions 46 ("N1"), 209 ("N2"), 272 ("N3"), 279 ("N4"), and 440 ("N5"). These five motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the five are conserved in mouse IgM heavy chain constant regions. Thus, in some embodiments, the IgM heavy chain constant region of a multimeric binding molecule as provided herein comprises five N-linked glycosylation motifs: N1, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., N1, N2, and N3) on each IgM heavy chain constant region are occupied by complex glycans.

[0137] In certain embodiments, at least one, at least two, at least three, or at least four of the N-X1-S / T motifs may contain an amino acid insertion, deletion, or substitution that prevents glycosylation of the motif. In certain embodiments, the IgM-derived multimeric binding molecule may contain an amino acid insertion, deletion, or substitution in motif N1, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs N1, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation of the motif. In some embodiments, the IgM constant region contains two or more substitutions compared to the wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). See, for example, US2022 / 036760, which is incorporated herein by reference in its entirety.

[0138] Multimeric binding molecules that induce T cell costimulation The present disclosure provides T cell engaging multimeric binding molecules, e.g., IgM, IgM-like, IgA, and IgA-like antibodies, that include two, four, or ten binding units, each comprising a total of three, four, five, six, seven, eight, nine, or ten target antigen-binding domains, i.e., antigen-binding domains that specifically bind to a target antigen, e.g., a tumor-associated or tumor-specific target antigen, and a modified J chain, which target T cells and activate them through two signals, i.e., "signal 1," which targets CD3, and "signal 2," which targets a T cell costimulatory molecule. Targeting of signals 1 and 2 is achieved, for example, by dual T cell engagement via a "bidentate" modified J chain that includes a J chain or a functional fragment or variant thereof, an scFv that specifically binds to CD3, and a polypeptide agonist of a T cell costimulatory molecule. The T cell costimulatory molecule can be, for example, CD28 or 4-1BB, and the polypeptide agonist can be, for example, an scFv of an anti-CD28 agonist antibody or a trimer of the receptor-binding portion of 4-1BB ligand (4-1BBL). A schematic diagram of an exemplary IgM antibody comprising a bidentate modified J chain and how it can function to enhance T cell activation is shown in Figure 1. Multimeric binding molecules as provided herein allow for dual engagement of T cells while maintaining the benefits of full avidity for targeting a desired target antigen, e.g., a tumor-associated antigen. This enhanced T cell activation can enhance the ability of the multimeric binding molecule to promote tumor cell killing by T cells, increase the number and types of T cells available to kill tumor cells, and, in some embodiments, enhance T cell-mediated killing in settings where T cell numbers are normally low, e.g., solid tumors.

[0139] Costimulatory molecules are receptors expressed on T cells that, when co-activated, can, for example, induce signal transduction to more fully activate T cells upon T cell receptor (TCR) engagement, thereby enhancing signal transduction and cytokine stimulation. This co-signaling can further regulate T cell differentiation, effector function, and survival. See, e.g., Baeuerle, PA, and H. Wesche, Curr. Opin. Oncol. 34:552-558 (2022). Exemplary T cell costimulatory molecules include, but are not limited to, CD28 and 4-1BB. See, e.g., Jeong, S., and SH. Park, Immune Netw. doi:10.4110 / in.2020.20.e3 (2020).

[0140] CD28 is expressed on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells and interacts with various ligands, including CD80 and CD86. Engagement of CD28 with its ligands promotes T cell stimulation and cytokine expression. See Esensten, JH, et al., Immunity 44:973-988 (2016). Human CD28, a type 1 membrane protein, is presented as SEQ ID NO:236. The signal peptide of human CD28 extends from amino acid 1 to about amino acid 18 of SEQ ID NO:236. The extracellular domain of human CD28 extends from about amino acid 19 to about amino acid 152 of SEQ ID NO:236. The transmembrane domain of human CD28 extends from about amino acid 153 to about amino acid 179 of SEQ ID NO:236. The cytoplasmic domain of human CD28 extends from about amino acid 180 to amino acid 220 of SEQ ID NO:236.

[0141] 4-1BB, also known as CD137 or TNFRSF9, is a tumor necrosis factor superfamily receptor (TNFSFR) expressed on activated T cells, for example. 4-1BB is expressed on both activated CD4+ and CD8+ T cells (Bartkowiak, T, and MA Curran, Front Oncol. 5: doi:10.3389 / fonc.2015.00117 (2015); Vinay, DS, and BS Kwon, Mol Cancer Ther. 11: doi:10.1158 / 1535-7163.MCT-11-0677 (2012)). Upon activation, CD8+ effector T cells upregulate 4-1BB expression, and 4-1BB signaling promotes survival, T cell proliferation, and enhanced effector function (Bartkowiak, T, and MA Curran, Front Oncol. 5:doi:10.3389 / fonc.2015.00117(2015)). Interaction with its trimeric ligand (4-1BBL, TNFSF9) expressed on activated antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), results in costimulatory proliferation, survival, and enhanced effector function in CD8+ effector T cells (Moran, AE, et al., Curr Opin Immunol. 25:10.1016 / j.coi.2013.01.004(2013)). Human 4-1BB, a type 1 membrane protein, is presented as SEQ ID NO: 195. The signal peptide of human 4-1BB extends from about amino acid 1 to about amino acid 23 of SEQ ID NO: 195. The extracellular domain of human 4-1BB extends from about amino acid 24 to about amino acid 186 of SEQ ID NO: 195. The transmembrane domain of human 4-1BB extends from about amino acid 187 to about amino acid 213 of SEQ ID NO: 195. The cytoplasmic domain of human 4-1BB extends from about amino acid 214 to amino acid 255 of SEQ ID NO: 195.

[0142] The ligand for 4-1BB (4-1BBL) is a type 2 transmembrane glycoprotein receptor found on antigen-presenting cells (APCs). Gramaglia et al., Eur. J. Immunol. 30(2):392-402, 2000. 4-1BBL is a high-affinity ligand for 4-1BB. Chin et al., Nature Comm. 9:4679, 2018. 4-1BBL induces proliferation of activated peripheral blood T cells and may be involved in activation-induced cell death (AICD). Vinay et al., BMB Rep. 47(3):122-129, 2014. Furthermore, 4-1BBL provides T cell costimulatory signals for survival, proliferation, and differentiation and may be involved in cognate interactions between T cells and B cells / macrophages. Eun et al., J. Immunol. 194(1):134-141, 2015. Therefore, 4-1BBL is thought to be involved in cancer, infectious diseases, and autoimmune diseases. Vinay et al., Mol. Cancer Therap. 11(5):1062-1070, 2012. Human 4-1BBL, a type 2 membrane protein, is presented as SEQ ID NO: 15. The cytoplasmic domain of human 4-1BBL extends from amino acid 1 to about amino acid 28 of SEQ ID NO: 15. The transmembrane domain of human 4-1BBL extends from about amino acid 29 to about amino acid 49 of SEQ ID NO: 15. The extracellular domain of human 4-1BBL extends from about amino acid 50 to amino acid 254 of SEQ ID NO: 15.

[0143] The 4-1BB / 4-1BBL complex typically consists of three 4-1BB monomers bound to a trimeric 4-1BBL (Won et al., J. Biol. Chem. 285:9202-9210 (2010); Rabu et al., J. Biol. Chem. 280(50):41472-41481 (2005)). Each 4-1BB monomer binds to two 4-1BBLs via a cysteine-rich domain (CRD) (Bitra et al., J. Biol. Chem. 293(4):1317-1329 (2018)).

[0144] Exemplary T cell engaging multimeric binding molecules provided by the present disclosure comprise a modified J chain, the modified J chain comprising (a) a J chain or a functional fragment or variant thereof ("J"), (b) an anti-CD3 scFv ("C"), and (c) a heterologous polypeptide ("H"), wherein H comprises a polypeptide agonist of a T cell costimulatory molecule, and the J, C, and H are associated, from the N-terminus to the C-terminus, as a fusion protein, e.g., a CJH or HJC. In some embodiments, the T cell costimulatory molecule comprises CD28, and the agonist is, e.g., an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the T cell costimulatory molecule comprises 4-1BB, and the agonist is, e.g., a 4-1BB ligand (4-1BBL) trimer, as described elsewhere herein.

[0145] In some embodiments, C and J are fused via an amino acid linker. In some embodiments, H and J are fused via an amino acid linker. If a linker is present between C and J and between H and J, the linkers can be the same or different. In some embodiments, one or both of the individual linkers is 5-25 amino acids long, e.g., the amino acid sequence (GGGGS). n(wherein n is an integer between 1 and 5 (SEQ ID NO:280), an integer between 2 and 5 (SEQ ID NO:279), or an integer between 2 and 3 (SEQ ID NO:281). Exemplary linkers include GGGGS (SEQ ID NO:9), GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGGS (SEQ ID NO:11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:12), GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:13), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, one or both of the individual linkers consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, one or both of the individual linkers consists of GGGGSGGGGSGGGGS (SEQ ID NO:11). In some embodiments, one or both of the individual linkers consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0146] In some embodiments, the T cell costimulatory molecule comprises CD28, and the costimulatory molecule agonist comprises an anti-CD28 antibody or antigen-binding fragment thereof, e.g., a single-chain Fv (scFv) fragment. In some embodiments, the anti-CD28 antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, e.g., the VHCDR1, VHCDR2, and VHCDR3 sequences set forth in Table 3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, e.g., the VLCDR1, VLCDR2, and VLCDR3 sequences set forth in Table 4.

[0147] In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are, respectively, the amino acid sequences: SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:133, SEQ ID NO:138, SEQ ID NO:164, and SEQ ID NO:165; SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:130, SEQ ID NO:151, SEQ ID NO:157, and SEQ ID NO:171; SEQ ID NO:98, SEQ ID NO:116, SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:135 , SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:100, SEQ ID NO:120, SEQ ID NO:127, SEQ ID NO:146, SEQ ID NO:153, and SEQ ID NO:172; SEQ ID NO:104, SEQ ID NO:123, SEQ ID NO:137, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:91, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:129, SEQ ID NO:139, SEQ ID NO:161, and SEQ ID NO:169; SEQ ID NO:97, SEQ ID NO:112, SEQ ID NO:124, SEQ ID NO:142, SEQ ID NO:163, and SEQ ID NO:166; SEQ ID NO:94, SEQ ID NO:108, SEQ ID NO:132, SEQ ID NO:149, SEQ ID NO:160, and SEQ ID NO:177; SEQ ID NO:93, SEQ ID NO:109, SEQ ID NO:131, SEQ ID NO:148, SEQ ID NO:158, and SEQ ID NO:178; SEQ ID NO:99, SEQ ID NO: Sequence number 117, SEQ ID NO:128, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170; SEQ ID NO:102, SEQ ID NO:114, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170; SEQ ID NO:102, SEQ ID NO:113, SEQ ID NO:136, SEQ ID NO:143, SEQ ID NO:162, and SEQ ID NO:170;SEQ ID NO:91, SEQ ID NO:119, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:92, SEQ ID NO:121, SEQ ID NO:134, SEQ ID NO:152, SEQ ID NO:156, and SEQ ID NO:167; or SEQ ID NO:92, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:141, SEQ ID NO:156, and SEQ ID NO:168.

[0148] In some embodiments, a VH comprising three VH CDRs as described above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VH amino acid sequence set forth in Table 2. In some embodiments, a VL comprising three VL CDRs as described above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VL amino acid sequence set forth in Table 2. In some embodiments, the VH comprises a VH amino acid sequence set forth in Table 2, and the VL comprises a VL amino acid sequence set forth in Table 2. In some embodiments, the VH and VL comprising such VH and VL CDRs comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences of SEQ ID NO:56 and SEQ ID NO:90, SEQ ID NO:57 and SEQ ID NO:86, SEQ ID NO:58 and SEQ ID NO:83, SEQ ID NO:59 and SEQ ID NO:83, SEQ ID NO:60 and SEQ ID NO:82, SEQ ID NO:60 and SEQ ID NO:87, SEQ ID NO:61 and SEQ ID NO:81, SEQ ID NO:62 and SEQ ID NO:87, SEQ ID NO:63 and SEQ ID NO:85, SEQ ID NO:64 and SEQ ID NO:76, SEQ ID NO:65 and SEQ ID NO:75, SEQ ID NO:66 and SEQ ID NO:89, SEQ ID NO:67 and SEQ ID NO:88, SEQ ID NO:68 and SEQ ID NO:82, SEQ ID NO:68 and SEQ ID NO:87, SEQ ID NO:69 and SEQ ID NO:78, SEQ ID NO:70 and SEQ ID NO:78, SEQ ID NO:71 and SEQ ID NO:80, SEQ ID NO:72 and SEQ ID NO:84, SEQ ID NO:73 and SEQ ID NO:77, or SEQ ID NO:74 and SEQ ID NO:79, respectively.

[0149] In some embodiments, the anti-CD28 antibody or antigen-binding fragment of the antibody is an anti-CD28 single-domain heavy chain variable region (sdVH) or an anti-CD28 single-domain light chain variable region (sdVL). In some embodiments, the sdVH or sdVL comprises the complementarity determining regions CDR1, CDR2, and CDR3 set forth in Table 6. In some embodiments, the anti-CD28 sdVH comprises the complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO:183, SEQ ID NO:187, and SEQ ID NO:191; SEQ ID NO:184, SEQ ID NO:188, and SEQ ID NO:192; or SEQ ID NO:185, SEQ ID NO:189, and SEQ ID NO:193, respectively. In some embodiments, the anti-CD28 sdVL comprises complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 186, SEQ ID NO: 190, and SEQ ID NO: 194, respectively.

[0150] In some embodiments, the anti-CD28 sdVH or sdVL comprising the above CDRs comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence set forth in Table 5. In some embodiments, the anti-CD28 sdVH or sdVL comprising the above CDRs comprises an amino acid sequence set forth in Table 5. In some embodiments, the anti-CD28 sdVH comprising the above CDRs comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or the anti-CD28 sdVL comprising the above CDRs comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 182. In some embodiments, the anti-CD28 sdVH comprises SEQ ID NO:179, SEQ ID NO:180, or SEQ ID NO:181, or the anti-CD28 sdVL comprises SEQ ID NO:182.

[0151] In some embodiments, an anti-CD3 scFv ("C") comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH of C comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, e.g., the VHCDR1, VHCDR2, and VHCDR3 sequences set forth in Table 9, and the VL of C comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, e.g., the VLCDR1, VLCDR2, and VLCDR3 sequences set forth in Table 10.

[0152] In some embodiments, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of C comprise the amino acid sequences: SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:33; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:45; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:49, or SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.

[0153] In some embodiments, the VH of C comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VH sequence set forth in Table 8, and the VL of C comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VL sequence set forth in Table 8. In some embodiments, the VH and VL of C comprise amino acid sequences at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48, respectively.

[0154] In some embodiments, the VH and VL of the anti-CD28 antibody or antigen-binding fragment thereof are fused via an amino acid linker. In some embodiments, the VH and VL of the anti-CD3 antibody or antigen-binding fragment thereof are fused via an amino acid linker. In some embodiments, the linker is fused via an amino acid linker consisting of 5 to 25 amino acids, for example, the amino acid sequence (GGGGS). n (wherein n is an integer between 1 and 5 (SEQ ID NO:280), an integer between 2 and 5 (SEQ ID NO:279), or an integer between 2 and 3 (SEQ ID NO:281). Exemplary linkers include GGGGS (SEQ ID NO:9), GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGGS (SEQ ID NO:11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:12), GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:13), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, the linker consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker consists of GGGGSGGGGSGGGGGS (SEQ ID NO:11). In some embodiments, the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0155] In some embodiments, the modified J chain comprises SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, or SEQ ID NO:234.

[0156] Multimeric binding molecules expressing 4-1BBL trimers The present disclosure further provides multimeric binding molecules, e.g., IgM, IgM-like, IgA, and IgA-like binding molecules, comprising two, four, or ten binding units, each comprising a total of three, four, five, six, seven, eight, nine, or ten target antigen-binding domains, i.e., antigen-binding domains that specifically bind to a target antigen, e.g., a tumor-associated or tumor-specific target antigen, and a modified J chain, which activate T cells by engaging the T cell costimulatory molecule 4-1BB via the expression of a trimer of the receptor-binding portion of 4-1BB ligand (4-1BBL) on the modified J chain. Human 4-1BB and 4-1BBL are described above. Multimeric binding molecules as provided herein enable T cell engagement and stimulation via activating 4-1BB while maintaining the full avidity advantage of targeting a desired target antigen, e.g., a tumor-associated antigen.

[0157] In some embodiments, the modified J chain comprises a 4-1BBL trimer comprising three 4-1BBL monomers. In some embodiments, each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X-254 of SEQ ID NO: 15 (where X is an integer between 50 and 99, e.g., 58 or 71). In some embodiments, the 4-1BBL trimer comprises SEQ ID NO: 196 or SEQ ID NO: 197.

[0158] In some embodiments, the C-terminus of the first 4-1BBL monomer is fused to the N-terminus of the second 4-1BBL monomer via an amino acid linker. In some embodiments, the C-terminus of the second 4-1BBL monomer is fused to the N-terminus of the third 4-1BBL monomer via an amino acid linker. If linkers are present between the first and second 4-1BBL monomers and between the second and third 4-1BBL monomers, the linkers may be the same or different. In some embodiments, one or both of the individual linkers is 5 to 25 amino acids long, e.g., the amino acid sequence (GGGGS). n(wherein n is an integer between 1 and 5 (SEQ ID NO:280), an integer between 2 and 5 (SEQ ID NO:279), or an integer between 2 and 3 (SEQ ID NO:281). Exemplary linkers include GGGGS (SEQ ID NO:9), GGGGSGGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGGS (SEQ ID NO:11), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:12), GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:13), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14), or GGGGSGGGGSGGGG (SEQ ID NO:235). In some embodiments, one or both of the individual linkers consists of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, one or both of the individual linkers consists of GGGGSGGGGSGGGGS (SEQ ID NO:11). In some embodiments, one or both of the individual linkers consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO:14).

[0159] In some embodiments, the 4-1BBL trimer comprises SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, or SEQ ID NO:201.

[0160] In some embodiments, the modified J chain comprises a sequence set forth in Table 7. In some embodiments, the modified J chain comprises SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, or SEQ ID NO:214.

[0161] [Table 2] TIFF2026502759000004.tif223164TIFF2026502759000005.tif213164TIFF2026502759000006.tif224164TIFF2026502759000007.tif55163

[0162] [Table 3] TIFF2026502759000009.tif57156

[0163] Table 4

[0164] Table 5

[0165] Table 6

[0166] Table 7 TIFF2026502759000014.tif170158TIFF2026502759000015.tif191159TIFF20265027590 00016.tif113158TIFF2026502759000017.tif201159TIFF2026502759000018.tif201159 TIFF2026502759000019.tif201159TIFF2026502759000020.tif196159TIFF20265027590 00021.tif185159TIFF2026502759000022.tif196159TIFF2026502759000023.tif144158

[0167] Table 8 TIFF2026502759000025.tif89164

[0168] Table 9

[0169] [Table 10]

[0170] While those skilled in the art can contemplate a variety of different multimeric binding molecules based on this disclosure and are therefore encompassed by this disclosure, in certain embodiments, binding molecules as described above are provided in which each binding unit comprises two IgA or IgM heavy chains, each comprising a VH positioned amino terminal to an IgA or IgM constant region or a multimerized fragment thereof, and two immunoglobulin light chains, each comprising a VL positioned amino terminal to an immunoglobulin light chain constant region.

[0171] Furthermore, in certain embodiments, at least one binding unit of a binding molecule, or at least two, at least three, at least four, at least five, or at least six binding units of a binding molecule, comprises two of the target antigen-binding domains described above. In certain embodiments, the two target antigen-binding domains in at least one binding unit of a binding molecule, or in at least two, at least three, at least four, at least five, or at least six binding units of a binding molecule, can be different or identical to each other.

[0172] In certain embodiments, two IgA or IgM heavy chains within at least one binding unit of a binding molecule, or at least two, at least three, at least four, at least five, or at least six binding units of a binding molecule, are identical.

[0173] In certain embodiments, the two light chains in at least one binding unit of a binding molecule, or in at least two, at least three, at least four, at least five, or at least six binding units of a binding molecule, are identical. In certain embodiments, the two identical light chains in at least one binding unit, or in at least two, at least three, at least four, at least five, or at least six binding units of a binding molecule, are kappa light chains, e.g., human kappa light chains, or lambda light chains, e.g., human lambda light chains.

[0174] Tumor-associated target antigens In certain embodiments, the present disclosure provides multimeric binding molecules, e.g., IgM, IgM-like, IgA, and IgA-like binding molecules, comprising two, four, or ten binding units that together comprise three, four, five, six, seven, eight, nine, or ten "target antigen-binding domains," i.e., antigen-binding domains that specifically bind to a target antigen, e.g., a tumor-associated or tumor-specific target antigen. In certain embodiments, the target antigen is a tumor-specific antigen, i.e., a target antigen that is expressed primarily only on tumor or cancer cells, or that may be expressed only at undetectable levels on normal, healthy adult cells. In certain embodiments, the target is a tumor-associated antigen, i.e., a target antigen that is expressed on both healthy and cancerous cells, but at a much higher density on cancerous cells than on normal, healthy cells. Exemplary tumor-specific and tumor-associated antigens include, but are not limited to, CD38, CD20, CD19, CD22, CD28, B-cell maturation antigen (BCMA), CD123, PD-L1, tumor-associated calcium transducer 2 (TROP-2), mesothelin, Muc16, prostate-specific membrane antigen (PSMA), or six-transmembrane epithelial antigen of the prostate-1 (STEAP-1).

[0175] In some embodiments, the tumor-associated target antigen is CD20. CD20 (UniProt P11836) is a 36-kDa non-glycosylated, four-transmembrane protein (MS4A1 gene product) that is exclusively expressed in B lymphocytes and more than 90% of B lymphocytic lymphomas. CD20 is expressed at the late pre-B cell stage and is upregulated in most normal and malignant B-lineage cells before being downregulated in terminally differentiated plasma cells. This B lymphocyte surface molecule is involved in the development and differentiation of B cells into plasma cells. Exemplary binding domains that bind to CD20 are provided, for example, in U.S. Pat. No. 10,787,520, the entire contents of which are incorporated herein by reference.

[0176] In some embodiments, the tumor-associated target antigen is mesothelin (MSLN). Mesothelin is a 71-kDa cell surface GPI-linked glycoprotein (UniProt Q13421) that has limited normal tissue distribution (in mesothelial cells of the peritoneum, pericardium, and pleura) but is overexpressed in a wide variety of solid tumors. See, for example, Ordonez, NGAm. J. Surg. Pathol. 27:1418-1428 (2003). Various mesothelin-targeted therapies are in preclinical and clinical development. Target antigen-binding domains that specifically bind to MSLN are described, for example, in U.S. Pat. No. 5,489,525, U.S. Patent Application Publication No. US2017 / 0029502A1, and U.S. Pat. Nos. 10,851,175B2 and 8,435,494B2, which are incorporated herein by reference in their entireties. In certain embodiments, a target antigen-binding domain that specifically binds to MSLN comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the amino acid sequences SEQ ID NO:256 and SEQ ID NO:257, SEQ ID NO:258 and SEQ ID NO:259, SEQ ID NO:260 and SEQ ID NO:261, SEQ ID NO:262 and SEQ ID NO:263, or SEQ ID NO:264 and SEQ ID NO:265 (Table 25). In certain embodiments, the VH and VL comprise the amino acid sequences SEQ ID NO:262 and SEQ ID NO:263, respectively.

[0177] In some embodiments, the tumor-associated target antigen is CD38. CD38 (UniProt P28907) is a type II transmembrane glycoprotein that is expressed at relatively low levels in normal myeloid and lymphoid cells but is overexpressed in various hematological malignancies, including chronic lymphocytic leukemia (CLL), multiple myeloma (MM), Hodgkin's lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), and peripheral T-cell lymphoma (PTCL), as well as various solid tumors, including prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, and liver cancer, and is therefore considered a potential target for directed therapeutics (Martin et al., Cells 8:1522 (2019)). Daratumumab and isatuximab, monoclonal antibodies that specifically bind to CD38, have been approved for the treatment of multiple myeloma. Exemplary binding domains and multimeric binding molecules that specifically bind to CD38 are described, for example, in International Publication No. WO2023 / 150677A2, which is incorporated herein by reference in its entirety.

[0178] In some embodiments, the tumor-associated target antigen is tumor-associated calcium signal transducer 2 (TROP-2). TROP-2 (UniProt P09758) is a glycoprotein highly expressed in various epithelial cancers, including thyroid cancer, lung cancer, head and neck cancer, gastric cancer, kidney cancer, uterine cancer, colorectal cancer, oral cancer, pancreatic cancer, urothelial cancer, prostate cancer, breast cancer, cervical cancer, endometrial cancer, and ovarian cancer. Lombardi, P. et al., Cancers 15:1744 (2023). Accordingly, drugs targeting TROP-2, including monoclonal antibodies, are commercially available or are in preclinical and clinical development. Id. For example, the anti-TROP-2 antibody-drug conjugate sacituzumab govitecan (TRODELVY®) was recently approved for the treatment of some patients with triple-negative breast cancer or metastatic urothelial cancer. Target antigen-binding domains that specifically bind to TROP-2 are described, for example, in International Publication No. WO1998 / 012227A1, U.S. Patent No. 9,849,176B2, U.S. Patent Application Publication No. US2012 / 0237518A1, U.S. Patent No. 9,850,312B2, U.S. Patent Application Publication No. US2008 / 0213267A1, and U.S. Patent No. 9,399,074B2, which are incorporated by reference in their entireties. In certain embodiments, a target antigen-binding domain that specifically binds to TROP-2 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise the amino acid sequences SEQ ID NO:244 and SEQ ID NO:245, SEQ ID NO:246 and SEQ ID NO:247, SEQ ID NO:248 and SEQ ID NO:249, SEQ ID NO:250 and SEQ ID NO:251, SEQ ID NO:252 and SEQ ID NO:253, or SEQ ID NO:254 and SEQ ID NO:255 (Table 24). In certain embodiments, the VH and VL comprise the amino acid sequences SEQ ID NO:248 and SEQ ID NO:249, respectively.

[0179] In some embodiments, multimeric binding molecules as provided herein conditionally activate T cells in the presence of tumor-associated or tumor-specific target antigens. More specifically, in certain embodiments, multimeric binding molecules as provided herein activate T cells only in the presence of tumor-associated or tumor-specific target antigens. In certain embodiments, multimeric binding molecules as provided herein provide efficient T cell activation with moderate cytokine release, even when the T cell costimulatory molecule is CD28 and the anti-CD28 binding domain on the J chain has superagonist activity. In certain embodiments, multimeric binding molecules as provided herein can improve the ability of cytotoxic T lymphocytes (CTLs) to infiltrate the tumor microenvironment of solid tumors and kill tumor cells. Those skilled in the art will understand that tumor-specific and tumor-associated target antigens appear under several different names in the literature, but these well-known therapeutic targets can be readily identified using databases available online, such as EXPASY.org.

[0180] How to use The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include carcinomas, including, but not limited to, adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of such cancers include osteosarcoma, chondrosarcoma, fibrosarcoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as liver cancer and hepatoma, bladder cancer, breast cancer (including hormone-mediated breast cancer, see, e.g., Innes et al. Br. J. Cancer. 94:1057-1065 (2006)) and triple-negative breast cancer (TNBC), colon cancer, colorectal cancer, endometrial carcinoma, myeloma (e.g., multiple myeloma), salivary gland cancer, kidney cancer such as renal cell carcinoma and Wilms' tumor, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, various types of head and neck cancer including, but not limited to, squamous cell carcinoma, and cancers of mucinous origin, e.g., mucinous ovarian cancer, cholangiocarcinoma (liver), and papillary renal carcinoma. Mucosal distribution, such as that provided by IgA-based binding molecules as provided herein, may be beneficial for certain cancers, e.g., lung cancer, ovarian cancer, colorectal cancer, or squamous cell carcinoma.

[0181] The effective dose of the composition for treating cancer varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. In certain embodiments, the treatment methods provided herein can provide improved safety in that the composition exhibits greater cytotoxicity (e.g., induces apoptosis to a greater extent) to cancer cells than to non-cancer cells, such as normal human liver cells. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0182] The compositions of the present disclosure can be administered by any suitable method, for example, parenterally, intracerebroventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0183] The subject to which treatment is administered can be any animal, for example a mammal, in need of treatment, and in certain embodiments, the subject is a human subject.

[0184] In its simplest form, the preparation administered to a subject comprises a multimeric binding molecule, e.g., a dimeric, tetrameric, or pentameric antibody as provided herein, administered in a conventional dosage form.

[0185] Multimeric binding molecules as provided herein can be administered by any suitable method, such as via IV infusion, as described elsewhere herein. In certain embodiments, multimeric binding molecules as provided herein are introduced into a tumor or in the vicinity of tumor cells.

[0186] All types of tumors are potentially treatable by this approach, including, but not limited to, cancers of the breast, lung, pancreas, ovary, kidney, colon, and bladder, as well as melanoma, sarcoma, and lymphoma. Mucosal distribution may be beneficial for certain cancers, such as lung, ovarian, colorectal, or squamous cell carcinoma.

[0187] Therefore, in some embodiments, the method provided herein is a method for inhibiting, delaying, or reducing malignant cell growth in a subject with cancer, wherein the cancer is a blood cancer or solid tumor.In some embodiments, the cancer is a blood cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, any metastasis thereof, or any combination thereof.In some embodiments, the cancer is a solid tumor, such as bladder cancer, colorectal cancer, sarcoma (e.g., fibrosarcoma), gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), or pancreatic cancer.

[0188] Pharmaceutical compositions and methods of administration Methods for preparing and administering multimeric binding molecules, such as dimeric or pentameric binding molecules as provided herein, to subjects in need thereof are known or readily determined in light of the present disclosure. Routes of administration of multimeric binding molecules can be, for example, oral, parenteral, by inhalation, or topical. As used herein, the term "parenteral" includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While these administration forms are contemplated as preferred forms, another example of an administration form is an injectable solution, particularly a solution for intravenous or intraarterial injection or infusion. Suitable pharmaceutical compositions can include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin).

[0189] As discussed herein, multimeric binding molecules, such as dimeric or pentameric binding molecules provided herein, can be administered in a pharmaceutically effective amount for in vivo treatment of cancers expressing a target antigen, where one or more target antigen-binding domains of the multimeric binding molecule bind to the target antigen, e.g., a tumor-specific or tumor-associated target antigen. In this regard, it will be understood that the disclosed binding molecules and compounds can be formulated to facilitate administration and promote stability of the active agent. Thus, pharmaceutical compositions can include a pharmaceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of a multimeric binding molecule, such as a dimeric or pentameric binding molecule provided herein, refers to an amount sufficient to achieve effective binding to the target and achieve a therapeutic benefit. A pharmaceutically effective amount of a cancer therapy, such as that provided herein, refers to an amount sufficient to achieve a therapeutic benefit. Suitable formulations are described in Remington: The Science and Practice of Pharmacy (Elsevier Science) 23rd ed. (2020).

[0190] In accordance with the scope of the present disclosure, the multimeric binding molecules provided herein can be administered to a subject in need of treatment in an amount sufficient to produce a therapeutic effect. The multimeric binding molecules provided herein can be administered to a subject in a conventional dosage form prepared by combining the multimeric binding molecules of the present disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and nature of the pharmaceutically acceptable carrier or diluent can be determined by the amount of active ingredient to be combined, the route of administration, and other well-known variables.

[0191] The term "therapeutically effective dose or amount" or "effective amount" refers to the amount of multimeric binding molecule that, when administered, results in a beneficial therapeutic response for the treatment of a patient with cancer.

[0192] The amount of the administered multimeric binding molecule can be easily determined by those skilled in the art without undue experimentation, taking into account the present disclosure.Factors that affect the mode of administration and the respective amounts of the multimeric binding molecule include, but are not limited to, the severity of the disease, the course of the disease, and the age, height, weight, health status, and physical condition of the individual receiving therapy.Similarly, the amount of the administered multimeric binding molecule can depend on the mode of administration and whether the subject receives a single dose or multiple doses of the agent.

[0193] Further embodiments Embodiment 1. 1. A multimeric binding molecule comprising 5, 4, or 2 bivalent binding units and a modified J chain, each said binding unit comprises two antibody heavy chains; each said antibody heavy chain IgM or IgA heavy chain constant regions or multimerized variants or fragments thereof, each associated with a target antigen binding domain Including, the modified J chain comprises: (a) a J chain or a functional fragment or variant thereof (“J”); (b) an anti-CD3 scFv (“C”); and (c) a heterologous polypeptide (“H”); H comprises a polypeptide agonist of a T cell costimulatory molecule; the J, the C, and the H are associated as a fusion protein; The multimeric binding molecule.

[0194] Embodiment 2. 2. The multimeric binding molecule of embodiment 1, wherein said T cell costimulatory molecule comprises CD28 or 4-1BB.

[0195] Embodiment 3. 3. The multimeric binding molecule of embodiment 1 or 2, wherein H comprises an anti-CD28 antibody or antigen-binding fragment thereof or a 4-1BB ligand ("4-1BBL") trimer.

[0196] Embodiment 4. 4. The multimeric binding molecule according to any one of embodiments 1 to 3, wherein the modified J chain comprises, from the N-terminus to the C-terminus, CJH or HJC.

[0197] Embodiment 5. 5. The multimeric binding molecule of embodiment 4, wherein said C, said J, and said H are fused via amino acid linkers which may be the same or different.

[0198] Embodiment 6. The multimeric binding molecule of embodiment 5, wherein each said linker independently consists of 5 to 25 amino acids.

[0199] Embodiment 7. 7. The multimeric binding molecule of embodiment 6, wherein each linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279) (wherein n is an integer from 2 to 5), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0200] Embodiment 8. 8. The multimeric binding molecule of embodiment 7, wherein said linker consists of GGGGSGGGGS (SEQ ID NO: 10).

[0201] Embodiment 9. 8. The multimeric binding molecule of embodiment 7, wherein said linker consists of GGGGSGGGSGGGGS (SEQ ID NO: 11).

[0202] Embodiment 10. 8. The multimeric binding molecule of embodiment 7, wherein said linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).

[0203] Embodiment 11. The multimeric binding molecule of any one of embodiments 3 to 10, wherein H comprises an antigen-binding fragment of an anti-CD28 antibody.

[0204] Embodiment 12. The multimeric binding molecule of embodiment 11, wherein the antigen-binding fragment of the anti-CD28 antibody comprises a single-chain Fv (scFv) fragment.

[0205] Embodiment 13. The anti-CD28 scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, and the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 have the amino acid sequences: SEQ ID NO: 100, SEQ ID NO: 120, SEQ ID NO: 127, SEQ ID NO: 146, SEQ ID NO: 153, and SEQ ID NO: 172; SEQ ID NO: 95; Sequence number 105, SEQ ID NO:133, SEQ ID NO:138, SEQ ID NO:164, and SEQ ID NO:165; SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:130, SEQ ID NO:151, SEQ ID NO:157, and SEQ ID NO:171; SEQ ID NO:98, SEQ ID NO:116, SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:96 , SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:104, SEQ ID NO:123, SEQ ID NO:137, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:91, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:129, SEQ ID NO:139, SEQ ID NO:161, and SEQ ID NO:169; SEQ ID NO:97, SEQ ID NO:112, SEQ ID NO:124, SEQ ID NO:142, SEQ ID NO:163, and SEQ ID NO:166; SEQ ID NO:94, SEQ ID NO:108, SEQ ID NO:132, SEQ ID NO:149, SEQ ID NO:160, and SEQ ID NO:177; SEQ ID NO:93, SEQ ID NO:109, SEQ ID NO:131, SEQ ID NO:148, SEQ ID NO:158, and SEQ ID NO:178; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170;The multimeric binding molecule of embodiment 12, comprising SEQ ID NO: 102, SEQ ID NO: 114, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 162, and SEQ ID NO: 170; SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 136, SEQ ID NO: 143, SEQ ID NO: 162, and SEQ ID NO: 170; SEQ ID NO: 91, SEQ ID NO: 119, SEQ ID NO: 126, SEQ ID NO: 146, SEQ ID NO: 154, and SEQ ID NO: 173; SEQ ID NO: 92, SEQ ID NO: 121, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 156, and SEQ ID NO: 167; or SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 134, SEQ ID NO: 141, SEQ ID NO: 156, and SEQ ID NO: 168.

[0206] Embodiment 14. 14. The multimeric binding molecule of embodiment 13, wherein the VH and VL of the anti-CD28 scFv comprise amino acid sequences at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences: SEQ ID NO:61 and SEQ ID NO:81, SEQ ID NO:56 and SEQ ID NO:90, SEQ ID NO:57 and SEQ ID NO:86, SEQ ID NO:58 and SEQ ID NO:83, SEQ ID NO:59 and SEQ ID NO:83, SEQ ID NO:60 and SEQ ID NO:82, SEQ ID NO:60 and SEQ ID NO:87, SEQ ID NO:62 and SEQ ID NO:87, SEQ ID NO:63 and SEQ ID NO:85, SEQ ID NO:64 and SEQ ID NO:76, SEQ ID NO:65 and SEQ ID NO:75, SEQ ID NO:66 and SEQ ID NO:89, SEQ ID NO:67 and SEQ ID NO:88, SEQ ID NO:68 and SEQ ID NO:82, SEQ ID NO:68 and SEQ ID NO:87, SEQ ID NO:69 and SEQ ID NO:78, SEQ ID NO:70 and SEQ ID NO:78, SEQ ID NO:71 and SEQ ID NO:80, SEQ ID NO:72 and SEQ ID NO:84, SEQ ID NO:73 and SEQ ID NO:77, or SEQ ID NO:74 and SEQ ID NO:79, respectively.

[0207] Embodiment 15. The multimeric binding molecule of embodiment 13 or 14, wherein said VH and said VL are fused via an amino acid linker.

[0208] Embodiment 16. The multimeric binding molecule of embodiment 15, wherein the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279) (wherein n is an integer from 2 to 5), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0209] Embodiment 17. 16. The multimeric binding molecule of embodiment 15, wherein said linker consists of GGGGSGGGGS (SEQ ID NO: 10).

[0210] Embodiment 18. 16. The multimeric binding molecule of embodiment 15, wherein said linker consists of GGGGSGGGSGGGGS (SEQ ID NO: 11).

[0211] Embodiment 19. 16. The multimeric binding molecule of embodiment 15, wherein said linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).

[0212] Embodiment 20. 12. The multimeric binding molecule of embodiment 11, wherein the antigen-binding fragment of the antibody is an anti-CD28 single domain heavy chain variable region (sdVH) or an anti-CD28 single domain light chain variable region (sdVL).

[0213] Embodiment 21. 21. The multimeric binding molecule of embodiment 20, wherein the anti-CD28 sdVH comprises complementarity determining regions CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 183, SEQ ID NO: 187, and SEQ ID NO: 191; SEQ ID NO: 184, SEQ ID NO: 188, and SEQ ID NO: 192; or SEQ ID NO: 185, SEQ ID NO: 189, and SEQ ID NO: 193; or the anti-CD28 sdVL comprises complementarity determining regions CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 comprise the amino acid sequences SEQ ID NO: 186, SEQ ID NO: 190, and SEQ ID NO: 194, respectively.

[0214] Embodiment 22. 22. The multimeric binding molecule of embodiment 21, wherein the anti-CD28 sdVH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or the anti-CD28 sdVL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 182.

[0215] Embodiment 23. 11. The multimeric binding molecule of any one of embodiments 3 to 10, wherein said H comprises a 4-1BBL trimer.

[0216] Embodiment 24. 24. The multimeric binding molecule of embodiment 23, wherein said 4-1BBL trimer comprises three 4-1BBL monomers.

[0217] Embodiment 25. 25. The multimeric binding molecule of embodiment 24, wherein each said 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15 (wherein X is an integer between 50 and 99).

[0218] Embodiment 26. 26. The multimeric binding molecule of embodiment 25, wherein the soluble fragments of 4-1BBL each comprise amino acids 71 ​​to 254 of SEQ ID NO: 15 (SEQ ID NO: 197).

[0219] Embodiment 27. 27. The multimeric binding molecule of embodiment 25 or 26, wherein said soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).

[0220] Embodiment 28. 28. The multimeric binding molecule of any one of embodiments 24 to 27, wherein said 4-1BBL monomers are fused via amino acid linkers, which may be the same or different.

[0221] Embodiment 29. 29. The multimeric binding molecule of embodiment 28, wherein each said linker independently consists of 5 to 25 amino acids.

[0222] Embodiment 30. 30. The multimeric binding molecule of embodiment 29, wherein each said linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279) (wherein n is an integer from 2 to 5), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0223] Embodiment 31. 31. The multimeric binding molecule of embodiment 30, wherein said linker consists of GGGGSGGGGS (SEQ ID NO: 10).

[0224] Embodiment 32. 31. The multimeric binding molecule of embodiment 30, wherein said linker consists of GGGGSGGGSGGGGS (SEQ ID NO: 11).

[0225] Embodiment 33. 31. The multimeric binding molecule of embodiment 30, wherein said linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).

[0226] Embodiment 34. 34. The multimeric binding molecule of any one of embodiments 23 to 33, wherein H comprises the amino acid sequence: SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, or SEQ ID NO: 201.

[0227] Embodiment 35. The C comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the C comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, the VL of the C comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, and the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 are the amino acid sequences: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO: 35, SEQ ID NO:43, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:45; SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:49, or SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.

[0228] Embodiment 36. The multimeric binding molecule of embodiment 35, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of C comprise SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[0229] Embodiment 37. The multimeric binding molecule of embodiment 35, wherein the VH and VL of C comprise amino acid sequences that are at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48, respectively.

[0230] Embodiment 38. 38. The multimeric binding molecule of embodiment 37, wherein C comprises the VH and VL amino acid sequences SEQ ID NO: 24 and SEQ ID NO: 28, respectively.

[0231] Embodiment 39. The multimeric binding molecule of any one of embodiments 35 to 38, wherein said VH and said VL are fused via an amino acid linker.

[0232] Embodiment 40. 40. The multimeric binding molecule of embodiment 39, wherein the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279) (wherein n is an integer from 2 to 5), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGSGGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0233] Embodiment 41. 41. The multimeric binding molecule of embodiment 40, wherein said linker consists of GGGGSGGGGS (SEQ ID NO: 10).

[0234] Embodiment 42. 41. The multimeric binding molecule of embodiment 40, wherein said linker consists of GGGGSGGGSGGGGS (SEQ ID NO: 11).

[0235] Embodiment 43. 41. The multimeric binding molecule of embodiment 40, wherein said linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).

[0236] Embodiment 44. 44. The multimeric binding molecule of any one of embodiments 1 to 43, wherein J comprises SEQ ID NO: 7 or a functional fragment or variant thereof.

[0237] Embodiment 45. 45. The multimeric binding molecule of any one of embodiments 1 to 44, wherein the J comprises a variant J chain having an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7).

[0238] Embodiment 46. 46. ​​The multimeric binding molecule of any one of embodiments 1 to 45, wherein J comprises the amino acid sequence SEQ ID NO: 8 ("J*").

[0239] Embodiment 47. 47. The multimeric binding molecule of any one of embodiments 1 to 22 or 35 to 46, wherein the modified J chain comprises the amino acid sequence: SEQ ID NO: 233, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 234.

[0240] Embodiment 48. 47. The multimeric binding molecule of any one of embodiments 1 to 10 or 23 to 46, wherein the modified J chain comprises the amino acid sequence: SEQ ID NO: 213 or SEQ ID NO: 214.

[0241] Embodiment 49. 1. A multimeric binding molecule comprising 5, 4, or 2 bivalent binding units and a modified J chain, each said binding unit comprises two antibody heavy chains; each said antibody heavy chain IgM or IgA heavy chain constant regions or multimerized variants or fragments thereof, each associated with a target antigen binding domain Including, the modified J chain comprises a J chain, or a functional fragment or variant thereof, and a 4-1BBL trimer associated as a fusion protein; the 4-1BBL trimer is Three 4-1BBL monomers, each containing a soluble fragment of 4-1BBL Including, The multimeric binding molecule.

[0242] Embodiment 50. 50. The multimeric binding molecule of embodiment 49, wherein each said 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15 (wherein X is an integer between 50 and 99).

[0243] Embodiment 51. The multimeric binding molecule of embodiment 50, wherein said soluble fragments of 4-1BBL each comprise amino acids 71 ​​to 254 of SEQ ID NO: 15 (SEQ ID NO: 197).

[0244] Embodiment 52. 52. The multimeric binding molecule of embodiment 50 or 51, wherein said soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).

[0245] Embodiment 53. 53. The multimeric binding molecule of any one of embodiments 49 to 52, wherein said 4-1BBL monomers are fused via amino acid linkers, which may be the same or different.

[0246] Embodiment 54. 54. The multimeric binding molecule of embodiment 53, wherein each said linker independently consists of 5 to 25 amino acids.

[0247] Embodiment 55. 55. The multimeric binding molecule of embodiment 54, wherein each said linker independently consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279) (wherein n is an integer from 2 to 5), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).

[0248] Embodiment 56. 56. The multimeric binding molecule of embodiment 55, wherein said linker consists of GGGGSGGGGS (SEQ ID NO: 10).

[0249] Embodiment 57. 56. The multimeric binding molecule of embodiment 55, wherein said linker consists of GGGGSGGGSGGGGS (SEQ ID NO: 11).

[0250] Embodiment 58. 56. The multimeric binding molecule of embodiment 55, wherein said linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).

[0251] Embodiment 59. 59. The multimeric binding molecule of any one of embodiments 49 to 58, wherein the 4-1BB trimer comprises the amino acid sequence: SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, or SEQ ID NO: 201.

[0252] Embodiment 60. 59. The multimeric binding molecule of any one of embodiments 49 to 58, wherein the 4-1BBL trimer is fused to the N-terminus of the J chain, or a fragment or variant thereof, or is fused to the C-terminus of the J chain, or a fragment or variant thereof, or wherein copies of the 4-1BB trimer are fused to both the N-terminus and the C-terminus of the J chain, or a fragment or variant thereof.

[0253] Embodiment 61. 61. The multimeric binding molecule of any one of embodiments 49 to 60, wherein the J chain or functional fragment or variant thereof comprises a mature human J chain, SEQ ID NO: 7, or a functional fragment or variant thereof.

[0254] Embodiment 62. 62. The multimeric binding molecule of any one of embodiments 49 to 61, wherein the J comprises a variant J chain having an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7).

[0255] Embodiment 63. 63. The multimeric binding molecule of any one of embodiments 49 to 62, wherein J comprises the amino acid sequence SEQ ID NO: 8 ("J*").

[0256] Embodiment 64. 64. The multimeric binding molecule of any one of embodiments 49 to 63, wherein the modified J chain comprises the amino acid sequences: SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211.

[0257] Embodiment 65. an IgM or IgM-like antibody comprising five bivalent binding units; Each of the coupling units is Two IgM heavy chain constant regions, or multimerized fragments or variants thereof, each comprising an IgM Cμ4 domain and an IgM tailpiece domain. Including, 65. The multimeric binding molecule of any one of embodiments 1 to 64.

[0258] Embodiment 66. 66. The multimeric binding molecule of embodiment 65, wherein each of said IgM heavy chain constant regions or multimerized fragments or variants thereof further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof.

[0259] Embodiment 67. 67. The multimeric binding molecule of embodiment 65 or 66, wherein said IgM heavy chain constant region or multimerized fragment or variant thereof is a human IgM constant region.

[0260] Embodiment 68. 68. The multimeric binding molecule of embodiment 67, wherein each said IgM heavy chain constant region comprises the amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 2, or a multimerized variant or fragment thereof.

[0261] Embodiment 69. 69. The multimeric binding molecule of embodiment 67 or 68, wherein each of said IgM heavy chain constant regions is a variant human IgM constant region comprising one or more single amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein said multimeric binding molecule has reduced CDC activity compared to a multimeric binding molecule in which each IgM heavy chain constant region comprises the amino acid sequence: SEQ ID NO: 1 or SEQ ID NO: 2.

[0262] Embodiment 70. 70. The multimeric binding molecule of embodiment 69, wherein each said variant human IgM constant region comprises an amino acid substitution corresponding to position L310 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position K315 of SEQ ID NO: 1 or SEQ ID NO: 2, or any combination thereof.

[0263] Embodiment 71. 71. The multimeric binding molecule of any one of embodiments 67 to 70, wherein each IgM heavy chain constant region is a variant human IgM constant region comprising one or more single amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the multimeric binding molecule, when administered to a subject animal, exhibits an increased serum half-life compared to a multimeric binding molecule comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, administered in the same manner to the same animal species.

[0264] Embodiment 72. 72. The multimeric binding molecule of embodiment 71, wherein each said variant IgM heavy chain constant region comprises a half-life-altering amino acid substitution at one or more amino acid positions corresponding to amino acid E345, S401, E402, or E403 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0265] Embodiment 73. 65. The multimeric binding molecule of any one of embodiments 1 to 64, which is a dimeric or tetrameric IgA antibody or IgA-like antibody comprising two or four bivalent binding units, each of said binding units comprising two IgA heavy chain constant regions or multimerized fragments or variants thereof, each comprising a Cα3 domain and an IgA tailpiece (αtp) domain.

[0266] Embodiment 74. 74. The multimeric binding molecule of embodiment 73, wherein the IgA heavy chain constant region or multimerized fragment or variant thereof further comprises a Cα1 domain, a Cα2 domain, an IgA hinge region, or any combination thereof, respectively.

[0267] Embodiment 75. 75. The multimeric binding molecule of embodiment 73 or 74, wherein each said IgA heavy chain constant region or multimerized fragment or variant thereof is a human IgA constant region.

[0268] Embodiment 76. 76. The multimeric binding molecule of any one of embodiments 1 to 75, wherein each of said target antigen-binding domains comprises a heavy chain variable region (VH) and a light chain variable region (VL), or a single-domain heavy chain variable region (sdVH).

[0269] Embodiment 77. 77. The multimeric binding molecule of embodiment 76, wherein each of said heavy chains comprises, from N- to C-terminus, an sdVH fused to said heavy chain constant region, or a multimerized fragment or variant thereof.

[0270] Embodiment 78. 77. The multimeric binding molecule of embodiment 76, wherein each said heavy chain comprises, from N- to C-terminus, a VH fused to said heavy chain constant region, or a multimerizing fragment or variant thereof.

[0271] Embodiment 79. 79. The multimeric binding molecule of embodiment 78, wherein each of said binding units further comprises two antibody light chains, each of said antibody light chains comprising, from N- to C-terminus, a VL fused to a light chain constant region.

[0272] Embodiment 80. 80. The multimeric binding molecule of any one of embodiments 1 to 79, wherein at least 3, 4, 5, 6, 7, 8, 9, or 10 of said target antigen binding domains are identical.

[0273] Embodiment 81. 81. The multimeric binding molecule of embodiment 80, wherein said at least 3, 4, 5, 6, 7, 8, 9, or 10 identical target antigen binding domains specifically bind to a tumor target antigen.

[0274] Embodiment 82. 82. The multimeric binding molecule of embodiment 81, wherein the tumor target antigen is CD38, CD20, CD19, CD22, CD28, B-cell maturation antigen (BCMA), CD123, PD-L1, tumor-associated calcium transducer and activator of receptor 2 (TROP-2), mesothelin, Muc16, prostate-specific membrane antigen (PSMA), or six-transmembrane epithelial antigen of the prostate (STEAP-1).

[0275] Embodiment 83. 83. The multimeric binding molecule of embodiment 82, wherein said tumor target antigen is CD20.

[0276] Embodiment 84. 83. The multimeric binding molecule of embodiment 82, wherein said tumor target antigen is CD38.

[0277] Embodiment 85. 83. The multimeric binding molecule of embodiment 82, wherein said tumor target antigen is mesothelin.

[0278] Embodiment 86. 83. The multimeric binding molecule of embodiment 82, wherein said tumor target antigen is TROP-2.

[0279] Embodiment 87. 87. The multimeric binding molecule of any one of embodiments 81 to 86, which conditionally activates T cells in the presence of said target antigen.

[0280] Embodiment 88. The multimeric binding molecule of any one of embodiments 81 to 87, wherein the T cells are activated after 7 days in the presence of said target antigen.

[0281] Embodiment 89. 89. The multimeric binding molecule of embodiment 88, which activates T cells after 10 days in the presence of said target antigen.

[0282] Embodiment 90. 90. The multimeric binding molecule of embodiment 89, which activates T cells after 14 days in the presence of said target antigen.

[0283] Embodiment 91. 91. The multimeric binding molecule of any one of embodiments 81 to 90, which, in the presence of said target antigen, leads to T cell-mediated killing of cells expressing said target antigen after 7 days.

[0284] Embodiment 92. 92. The multimeric binding molecule of embodiment 91, which, in the presence of said target antigen, leads to T cell-mediated killing of cells expressing said target antigen after 10 days.

[0285] Embodiment 93. 93. The multimeric binding molecule of embodiment 92, which, in the presence of said target antigen, leads to T cell-mediated killing of cells expressing said target antigen after 14 days.

[0286] 94. The multimeric binding molecule of any one of embodiments 87 to 93, wherein said T cells are CD4 positive T cells.

[0287] 95. The multimeric binding molecule of any one of embodiments 87 to 93, wherein said T cells are CD8-positive T cells.

[0288] 96. A composition comprising a multimeric binding molecule according to any one of embodiments 1 to 95 and a pharmaceutically acceptable carrier.

[0289] 97. A polynucleotide comprising a nucleic acid sequence encoding the modified J chain of the multimeric binding molecule of any one of embodiments 1 to 95.

[0290] Embodiment 98. A vector comprising the polynucleotide of embodiment 97.

[0291] Embodiment 99. A composition comprising the polynucleotide of embodiment 97 and a polynucleotide encoding the heavy chain of the multimeric binding molecule of any one of embodiments 1 to 95.

[0292] Embodiment 100. The composition of embodiment 99, further comprising a polynucleotide encoding the light chain of the multimeric binding molecule of any one of embodiments 1 to 95.

[0293] Embodiment 101. A vector comprising the composition of embodiment 99 or 100.

[0294] Embodiment 102. A host cell comprising a composition according to embodiment 99 or 100 or a vector according to embodiment 101, said host cell being capable of expressing a multimeric binding molecule according to any one of embodiments 1 to 95.

[0295] Embodiment 103. A method for producing a multimeric binding molecule according to any one of embodiments 1 to 95, comprising culturing a host cell according to embodiment 102 and recovering the multimeric binding molecule.

[0296] Embodiment 104. A method of treating cancer, comprising administering to a subject in need thereof a multimeric binding molecule of any one of embodiments 1 to 95.

[0297] Embodiment 105. 96. The multimeric binding molecule of any one of embodiments 1 to 95 for use in the treatment of cancer.

[0298] Embodiment 106. 96. Use of a multimeric binding molecule according to any one of embodiments 1 to 95 in the preparation of a medicament for the treatment of cancer.

[0299] The present disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art and are explained fully in the literature. For example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), DNGlover and BD Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4, Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press), Mullis et al. U.S. Patent No. 4,683,195, Hames and Higgins, eds. Press), Freshney(2016)Culture Of Animal Cells,7th Edition (Wiley-Blackwell), Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985), Perbal (1988) A Practical Guide To Molecular Cloning;2d Edition (Wiley-Interscience), Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory), SCSee Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science), Methods in Enzymology, Vols. 151-155 (Academic Press, Inc., N.Y.), Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Weir and Blackwell, eds., and Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0300] General principles of antibody engineering are described, for example, in Strohl, W.R., and L.M. Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are described, for example, in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of immunology are described, for example, in Abbas and Lichtman (2017), Cellular and Molecular Immunology 9th Edition (Elsevier). Additionally, standard immunological methods known in the art can be obtained, for example, according to Current Protocols in Immunology (Wiley Online Library), Wild, D. (2013), The Immunoassay Handbook 4th Edition (Elsevier Science), Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press), and Ossipow and Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

[0301] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

[0302] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0303] Example 1: Antibody construction To generate various IgM antibodies, modified J chains were prepared containing 4-1BBL trimers bearing three copies of either the "58" fragment (SEQ ID NO: 196) or the "71" fragment (SEQ ID NO: 197) of 4-1BBL at the N- and / or C-termini of a J chain containing a Y-to-A amino acid substitution at position 102 ("Y102A" or "J*"; the amino acid sequence of the variant is presented as SEQ ID NO: 8), which improves the serum half-life of IgM pentamers containing the J chain variant. See U.S. Patent No. 10,899,835. The 4-1BBL monomers were connected by 10- or 15-amino acid-long linkers (SEQ ID NOs: 10 and 11, respectively), and the trimers were also connected to the J chain by these linkers. Some molecules also contain an anti-CD3 scFv (SEQ ID NO: 219). The generated IgM antibodies contain target antigen-binding domains that bind to exemplary tumor-specific or tumor-associated target antigens, CD20 or mesothelin. A list of the molecules produced, including 4-1BBL, is shown in Table 11 ("MSLN" is mesothelin).

[0304] [Table 11]

[0305] Additional IgM antibodies were generated with modified J chains comprising one of four anti-CD28 scFvs: W (VH: SEQ ID NO: 69, VL: SEQ ID NO: 78, scFv: SEQ ID NO: 284), X (VH: SEQ ID NO: 58, VL: SEQ ID NO: 83, scFv: SEQ ID NO: 225), Y (VH: SEQ ID NO: 59, VL: SEQ ID NO: 83, scFv: SEQ ID NO: 226), or Z (VH: SEQ ID NO: 61, VL: SEQ ID NO: 81, scFv: SEQ ID NO: 227), and optionally an anti-CD3 scFv (SEQ ID NO: 219). A list of the generated molecules comprising anti-CD28 scFv is shown in Table 12.

[0306] [Table 12]

[0307] Schematic diagrams of exemplary IgM antibodies that have been generated and contemplated are shown in Figures 2A-2G.

[0308] The constructs were generated according to standard cloning protocols. Briefly, the heavy chain variable region (VH) and light chain variable region (VL) of exemplary target antigen binding domains were cloned into an IgM format containing a modified J chain. The resulting constructs were expressed in Expi293 cells and purified according to the method described in Keyt et al., Antibodies: 9: 53, doi: 10.3390 / antib9040053 (2020).

[0309] Additional control molecules, also produced as described above or purchased commercially, are shown in Table 13.

[0310] [Table 13]

[0311] Example 2: Binding to 4-1BB+ cells To determine the binding capacity of IgM antibodies containing 4-1BBL modified J chain constructs, a flow cytometry-based binding assay was performed.

[0312] Human 4-1BB+293T cells (Crown Bioscience #C2012) were dissociated using enzyme-free cell dissociation buffer (Gibco #13150-016) and washed twice with staining buffer (BD Pharmingen™ #554656). Binding assays were performed in 96-well V-bottom plates at a cell density of 30,000 cells / well.

[0313] IgM antibodies A1-A6 and A8-A9 containing 4-1BBL-modified J chains, as described in Example 1, were tested along with negative control D1. Antibody concentrations were adjusted to 10 nM in staining buffer and added to cells to give a final volume of 100 μL / well. The assay plate was then incubated on ice for 1 hour, and the cells were washed three times with staining buffer. 1:1,000 diluted mouse anti-human kappa-AF647 (SouthernBiotech #9230-31) and goat anti-human Fc-AF647 (SouthernBiotech #2014-31) secondary antibodies were added to all antibody wells at 100 μL / well. The assay plate was then incubated on ice for 45 minutes, and the cells were washed three times with staining buffer. A 1:1000 diluted 7-aminoactinomycin D (7-AAD) viability dye was then added to the cells, and fluorescence was analyzed using an INTELLICYT® IQUE® flow cytometer. Data were plotted as antibody concentration (x-axis) and relative fluorescence as a percentage of viable cells (y-axis) using GraphPad Prism8 software.

[0314] FIG. 3 shows that IgM antibodies with 4-1BBL-modified J chains in various formats bound to 4-1BB+ cells, whereas the negative control antibody D1 (without 4-1BBL) did not bind to 4-1BB+ cells.

[0315] Example 3: Biolayer Interferometry Affinity Measurement Assay The binding affinity and avidity of 4-1BBL contained in antibodies A5, A6, or A10, or 4-1BBL trimer D2 fused to Fc (Acrobiosystems #41L-H5269), were determined by biolayer interferometry (BLI) on an Octet-384 (Sartorius / ForteBio, NY, USA) using an Anti-Penta-His biosensor (Sartorius / ForteBio catalog number 18-5120). PBST (1x PBS + 1% BSA + 0.05% Tween-20) buffer was used as the reagent diluent and sensor hydration buffer. The experiment followed a six-step sequential assay at 24°C. First, the biosensor was hydrated for 10 minutes. Samples and buffer were applied to a 384-well plate. After a 30-second initial baseline, the sensor was filled with 10 nM 4-1BB-Fc with AVITAG™ for 240 seconds, and then the sensor was transferred to buffer for a 30-second baseline. Various concentrations of antibody or 4-1BB-Fc fusion were then allowed to associate in PBST for 240 seconds and dissociate for 500 seconds. Results were analyzed using a global fit model using ForteBio Data Analysis Software 9.0. D The values ​​are shown in Table 14. The binding over time for various concentrations of A5, A6, A9, and D2 is shown in Figures 4A-4D, respectively.

[0316] [Table 14]

[0317] Example 4: Luciferase reporter assay using CD20 coating To determine the ability of 4-1BBL trimers on the J chain of IgM antibodies to stimulate 4-1BB in the presence or absence of an IgM antibody target, a series of luciferase reporter assays were performed in the presence or absence of CD20. Antibodies A5, A6, A8, and A9 described in Example 1 were tested along with control molecules D1 (anti-CD20 IgM without 4-1BBL on the J* chain), D2 (4-1BBL trimer fused to Fc) with or without anti-Fc bridges, D3 (anti-4-1BB IgM antibody), and D4 (anti-4-1BB IgG antibody) with or without anti-Fc bridges.

[0318] For assays performed in the presence of CD20, 96-well opaque white plates were coated with 100 μl / well of 50 nM TrxA-tagged human CD20 (Acrobio# CD0-H5143, LOT 2113-7BNR2-GC) in phosphate-buffered saline (PBS), incubated at 37°C for 1.5 hours, and then removed with shaking.

[0319] Adherent cell line 293T / huCD137+ cells (Crown Bio#C2012 lot# 160331-IGM042020) were cultured in DMEM+10% FBS+1 μg / ml puromycin and dissociated using trypsin-free cell dissociation buffer. The viable cell density was 6.0 × 10 5 The cell concentration was adjusted to 30,000 cells / ml per well. 50 μl of cells per well were then added to both CD20 coated and uncoated plates.

[0320] 50 μL of serial dilutions of DMEM + 10% FBS + 1 μg / ml puromycin were added per well to A5, A6, A8, A9, D1, D2, and D3. In the crosslinking conditions, 10 μg / mL of anti-human Fc antibody was also added to the cells.

[0321] Jurkat human 4-1BB positive luciferase reporter cells (Promega #JA2351) were thawed at 37°C for 2 minutes and washed once in complete RPMI-1640 + 10% heat-inactivated FBS buffer. Jurkat cell density was 6.25 x 10 5 Adjusted to viable cells / mL. 20 μL of 4-1BB+ reporter cells were added to the wells.

[0322] Plates were incubated at 37°C in a 5% CO2 incubator for 16 hours. 100 μL of BIO-GLO™ (Promega #G7940) was added directly to the cells and incubated at room temperature for 10 minutes. Luminescence (RLU) was measured using a Perkin Elmer ENVISION® reader. Graphs were plotted using GraphPad Prism8 software with antibody concentration on the x-axis and RLU on the y-axis.

[0323] The luminescence measured at each concentration of A5, A6, A8, and A9 with and without CD20 coating is shown in Figures 5A and 5B, respectively. Figure 5C shows the luminescence measured at the highest tested concentrations of A5, A6, A8, A9, D1, D2 (with and without cross-linking), D3, and D4 (with and without cross-linking) in the presence or absence of CD20. In the presence of CD20, anti-CD20 IgM antibodies containing 4-1BBL trimers on the J chain (A5, A6, A8, and A9) exhibited greater agonistic activity than anti-4-1BB IgM antibody (D3), anti-4-1BB IgG antibody (D4) with or without cross-linking, 4-1BBL trimers fused to Fc with or without cross-linking (D2), or anti-CD20 IgM without 4-1BBL trimers (D1). However, in the absence of CD20, anti-CD20 IgM antibodies containing 4-1BBL trimers on the J chain exhibited less agonistic activity than 4-1BBL trimers fused to Fc with or without cross-linking (D2) or anti-4-1BB IgM antibodies (D3).

[0324] Example 5: Luciferase reporter assay using CD20-expressing cells Jurkat human 4-1BB positive luciferase reporter cells (Promega #JA2351) were thawed at 37°C for 2 minutes and washed once in complete assay buffer (RPMI-1640 + 10% heat-inactivated FBS buffer). Jurkat cell density was 6.25 x 10 5 The cell density of CD20+ target cells (Ramos cells) and CD20- target cells (OPM-2) was adjusted to 7.5 x 10 viable cells / mL in assay buffer. 5 Adjusted to viable cells / mL.

[0325] Test molecules A1, A2, A3, A4, A5, A6, A8, A9, D1, or D2 were diluted to 20 nM and then serially diluted in assay buffer. 10 μL of test molecule was added to a 384-well opaque plate, followed by 10 μL of CD20+ and CD20- cells. The wells were incubated at room temperature for 15 minutes. Finally, 20 μL of 4-1BB+ reporter cells were added to the wells, and the wells were incubated at 37°C for 16 hours. 20 μL of BIO-GLO™ Luciferase Reagent (Promega #G7941) was added per well to each well, and luminescence (RLU) was read on an ENVISION® (Perkin Elmer 2103). Graphs were plotted using GraphPad Prism8 software, with antibody concentration on the x-axis and RLU on the y-axis.

[0326] The luminescence measured at concentrations of A1, A2, A3, A4, A5, A6, A8, A9, D1, and D2 using CD20+ (Ramos) cells is shown in Figures 6A (A1, A2, A3, A4, D1, and D2) and 6B (A5, A6, A8, A9, D1, and D2). Figure 6C shows the luminescence measured at the highest concentrations tested of A1, A2, A3, A4, A5, A6, A8, A9, D1, and D2 in the presence of CD20+ cells (Ramos) or CD20- cells (OPM-2).

[0327] In the presence of CD20-expressing cells, anti-CD20 IgM antibodies containing 4-1BBL trimers on the J chain exhibited agonistic activity. However, in the presence of cells that do not express CD20, anti-CD20 IgM antibodies containing 4-1BBL trimers on the J chain did not exhibit agonistic activity. The 4-1BBL trimer-Fc fusion antibody, D2, exhibited agonistic activity regardless of CD20 expression.

[0328] Example 6: Luciferase reporter assay using CD20-expressing cells The assay described in Example 5 was repeated using cells expressing high levels of CD20 (DOHH2), cells expressing low levels of CD20 (CA46), and cells not expressing CD20 (OPM-2). In addition to antibodies A1, A2, A3, A4, A5, A6, A8, A9, D1, and D2 tested in Example 5, antibodies A7 and A10 (each with a 4-1BBL trimer at both the N- and C-termini) and A11 and A12 (each with a 4-1BBL trimer at one terminus and an anti-CD3 scFv at the other) were evaluated.

[0329] The luminescence measured at concentrations of A5, A6, A7, A8, A9, A10, A11, A12, D1, and D2 using low-level CD20 (CA46) cells is shown in Figure 7A, and the luminescence measured at concentrations of A5, A6, A7, A8, A9, A10, A11, A12, D1, and D2 using high-level CD20 (DOHH2) cells is shown in Figure 7B. Figure 7C shows the luminescence measured at the highest concentrations tested of A5, A6, A8, A9, A10, A11, A12, and D1 in the presence of cells expressing high levels of CD20 (DOHH2), cells expressing low levels of CD20 (CA46), or CD20- cells (OPM-2).

[0330] All anti-CD20 IgM antibodies containing the 4-1BBL trimer on the J chain exhibited agonistic activity dependent on the CD20 level. Antibodies with two copies of the 4-1BBL trimer exhibited higher levels of agonistic activity, and antibodies with both anti-CD3 scFv and the 4-1BBL trimer exhibited the highest levels of agonistic activity.

[0331] Example 7: Antibody binding measured by ELISA Six IgM antibodies that bind to mesothelin, B1, B2, B3, B4, D5, and D6, were generated as described in Example 1. B1–B4 contain a 4-1BBBL trimer on the J chain, D5 contains an unmodified J chain, and D6 contains an anti-CD3 scFv on the J chain. Binding to 4-1BB was then measured in an ELISA assay as follows: 96-well white polystyrene ELISA plates (Pierce 15042) were coated with 100 μL per well of 1.0 μg / mL recombinant target 4-1BB overnight at 4°C. The plates were then washed five times with 0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, 100 μL of serial dilutions of B1, B2, B3, B4, D5, or D6 were added to the wells and incubated at room temperature for 2 hours. Plates were then washed 10 times and incubated with HRP-conjugated mouse anti-human kappa (Southern Biotech, 9230-05; diluted 1:6000 in 2% BSA-PBS) for 30 minutes. After 10 final washes with 0.05% PBS-Tween, plates were read using SuperSignal chemiluminescent substrate (ThermoFisher, 37070). Luminescence data were collected on an ENVISION® plate reader (PerkinElmer) and analyzed using a four-parameter logistic model in GraphPad Prism. Binding to 4-1BB is shown in Figure 8. Antibodies B1-B4 bound to 4-1BB, whereas D5 and D6 did not.

[0332] Example 8: Luciferase reporter assay using mesothelin-expressing cells HEK-293T cells or mesothelin-expressing HEK-293T cells were cultured at 1.5 × 10 in complete assay buffer (RPMI-1640 + 10% heat-inactivated FBS). 5 The concentration was adjusted to viable cells / mL. 20 μL of mesothelin+ and mesothelin- cells were added to each well of a 384-well opaque plate and incubated overnight at 37°C with 5% CO2 to allow the cells to attach to the plate.

[0333] The next day, test molecules B1, B2, B3, B4, D5, or D6 were diluted to 20 nM and then serially diluted in assay buffer. Jurkat human 4-1BB positive luciferase reporter cells (Promega #JA2351) were thawed at 37°C for 2 minutes and washed once in assay buffer. Jurkat cell density was 1.5 x 10 6 The concentration was adjusted to viable cells / mL. 10 μL of test molecules were added to a 384-well opaque plate. The plate was incubated at 37°C for 15 minutes. Finally, 10 μL of 4-1BB+ reporter cells were added to the wells, and the wells were incubated at 37°C for 24 hours. 20 μL per well of BIO-GLO™ Luciferase Reagent (Promega #G7941) was added to each well, and luminescence (RLU) was read on an ENVISION® (Perkin Elmer 2103). Graphs were plotted using GraphPad Prism8 software, with antibody concentration on the x-axis and RLU on the y-axis.

[0334] Figure 9A shows the luminescence measured at each concentration of B1, B2, B3, B4, D5, or D6 using mesothelin- cells, and Figure 9B shows the luminescence measured at each concentration of B1, B2, B3, B4, D5, or D6 using mesothelin+ cells. Figure 9C shows the luminescence measured at the highest concentrations of B1, B2, B3, B4, D5, and D6 tested in the presence of mesothelin+ or mesothelin- cells.

[0335] In the presence of mesothelin-expressing cells, anti-mesothelin IgM antibodies containing 4-1BBL trimers on the J chain exhibited agonistic activity, with IgM antibodies containing 4-1BBL at the N-terminus exhibiting greater agonistic activity than IgM antibodies containing 4-1BBL at the C-terminus. However, in the presence of cells that do not express mesothelin, anti-mesothelin IgM antibodies containing 4-1BBL trimers at the C-terminus of the J chain did not exhibit agonistic activity. Although anti-mesothelin IgM antibodies containing 4-1BBL trimers at the N-terminus of the J chain exhibited agonistic activity in the presence of cells that do not express mesothelin, the agonistic activity was significantly less than that in the presence of mesothelin.

[0336] Example 9: T cell activation The ability of antibodies containing J chains, including anti-CD28 scFv and anti-CD3 scFv, to enhance T cell activation was assessed as follows: 100 × 10 3 Human PBMCs were cultured for 24 or 48 hours in the presence of serial dilutions of C4, C5, C6, and D7 (described in Example 1) in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS). Cells were stained for FACS analysis using the following staining panel: anti-CD19 FITC, anti-CD3 PerCP-Cy5.5, anti-CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend, and the fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14). CD4+ T cell activation at various concentrations after 24 and 48 hours is shown in Figures 10A-10B, respectively. CD8+ T cell activation at various concentrations after 24 and 48 hours is shown in Figures 10C-10D, respectively. Combination of CD28 and CD3 stimulation (antibodies C4, C5, and C6) resulted in greater T cell activation than CD3 stimulation alone (antibody D7).

[0337] Generally, the exemplary IgM antibodies having a J chain comprising anti-CD3 x anti-CD28 showed increased binding to T cells when compared to IgM antibodies having only anti-CD3 or only anti-CD28 on the J chain, suggesting dual engagement of T cells. The exemplary IgM antibodies having dual signal engagement had improved T cell activation compared to the exemplary IgM antibodies having only signal 1 (anti-CD3), whereas no or minimal T cell activation was observed in the absence of signal 1 engagement or in the absence of target cells.

[0338] Example 10: T cell-induced cytotoxicity The ability of antibodies containing J chains, including anti-CD28 scFv and anti-CD3 scFv, to enhance T cell-induced cytotoxicity (TDCC) was assessed as follows: 100 × 103 Human PBMCs were cocultured with Ramos cells in the presence of serial dilutions of C4, C5, C6, and D7 (described in Example 1) in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS) for 24 or 48 hours. 50 μL of supernatant was removed and frozen at -80°C for subsequent cytokine release analysis. Cells were stained for FACS analysis using the following staining panel: anti-CD19 FITC, anti-CD3 PerCP-Cy5.5, anti-CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend, and the fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14). The number of viable CD19+ B cells in each well was compared with the counting beads added to calculate the percentage killing. Data were then analyzed and plotted using GraphPad Prism. The dose-response curves after 24 and 48 hours are shown in Figures 11A-11B, respectively. The combination of CD28 and CD3 stimulation (antibodies C4, C5, and C6) resulted in greater TDCC activity than CD3 stimulation alone (antibody D7).

[0339] Example 11: Cytokine Release Supernatants from the TDCC assay obtained in Example 10 were assayed for a panel of cytokines, including IL-2, IL-6, IL-10, TNFα, and IFNγ, using the Cytometric Bead Array Human Th1-2 Cytokine Kit II (BD, 551809) according to the manufacturer's protocol, except for using smaller volumes to adapt the plate-based assay. Results were then analyzed using GraphPad Prism. Cytokine concentrations in supernatants from PBMCs treated with various concentrations of antibody after 24 and 48 hours are shown in Figures 12A and 12B for IL-2, 12C and 12D for IL-6, 12E and 12F for IL-10, 12G and 12H for TNFα, and 12I and 12J for IFNγ. Enhanced TDCC and T cell activation correlated with cytokine responses.

[0340] Example 12: Construction of further antibodies Additional IgM pentameric antibodies listed in Table 15 were generated according to the methods described in Example 1. IgM-based anti-CD38 antibodies are disclosed in PCT Publication No. WO2023150677A2, which is incorporated by reference herein in its entirety. The VH and VL sequences are listed in Table 26.

[0341] [Table 15]

[0342] Example 13: T cell activation The ability of the exemplary anti-CD38 IgM antibody E1 described in Example 12 to enhance T cell activation compared to the anti-CD38 IgM antibody D18 described in Example 12 was assessed as follows: 100×10 3Human PBMCs were cocultured with H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, or RPMI8226_Luc / GFP cells in the presence of serial dilutions of E1 and D18 in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS) for 48 h. Cells were stained for FACS analysis with the following staining panel: anti-CD19 FITC, anti-CD3 PerCP-Cy5.5, anti-CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend using the fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14). Activation of CD4+ T cells cocultured with H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, or RPMI8226_Luc / GFP cells at various concentrations of E1 and D18 after 48 hours is shown in Figures 13A-13D, respectively. Activation of CD8+ T cells cocultured with H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, or RPMI8226_Luc / GFP cells at various concentrations of E1 and D18 after 72 hours is shown in Figures 14A-14D, respectively. Combination of CD28 and CD3 stimulation (E1) resulted in greater T cell activation than CD3 stimulation alone (D18).

[0343] Example 14: T cell-induced cytotoxicity The ability of the exemplary anti-CD38 IgM antibody E1, described in Example 12, to enhance T cell-induced cytotoxicity (TDCC) compared to the anti-CD38 IgM antibody D18, also described in Example 12, was assessed as follows: 10x10 3 Human PBMCs were cultured at 10 × 10 in the presence of serial dilutions of E1 and D18 in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS). 3The cells were co-cultured with H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, or RPMI8226_Luc / GFP cells for 72 hours. Fifty microliters of supernatant was removed and frozen at -80°C for subsequent cytokine release analysis. Cells were stained for FACS analysis using the following staining panel: anti-CD3 PerCP-Cy5.5, anti-CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend using the fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14). The number of viable GFP+ target cells in each well was compared with the number of counting beads added to calculate the percentage killing. Data were then analyzed and plotted using GraphPad Prism. The percentage killing of H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, or RPMI8226_Luc / GFP cells after 72 hours at various doses is shown in Figures 15A-15D, respectively. The combination of CD28 and CD3 stimulation (E1) resulted in greater TDCC activity than CD3 stimulation alone (D18).

[0344] Example 15: Cytokine Release Supernatants from the TDCC assay obtained in Example 14 were assayed for a panel of cytokines including IL-2, IL-4, IL-6, IL-10, TNFα, and IFNγ using the Cytometric Bead Array Human Th1-2 Cytokine Kit II (BD, 551809) according to the manufacturer's protocol, except that smaller volumes were used to accommodate the plate-based assay. Results were then analyzed using GraphPad Prism. Cytokine concentrations in supernatants from cocultures of PBMCs with H929_Luc / GFP, OPM2_Luc / GFP, MM1S_Luc / GFP, and RPMI8226_Luc / GFP cells 48 hours after treatment with D18 or E1 are shown in Figures 16A-16D for IL-2, 17A-17D for IL-4, 18A-18D for IL-6, 19A-19D for IL-10, 20A-20D for TNFα, and 21A-21D for IFNγ. Enhanced TDCC and T cell activation correlated with cytokine responses.

[0345] Example 16: T cell activation The ability of the exemplary anti-CD20 x anti-CD3 x anti-CD28 IgM antibody C6 described in Example 1 to enhance T cell activation compared to the anti-CD20 x anti-CD3 IgM antibody D7 described in Example 1 and the anti-CD20 x anti-CD28 IgM antibody C3 described in Example 1 was evaluated as follows. 3Human PBMCs were cocultured with Raji-Luc-GFP cells in the presence of serial dilutions of C6, D7, or C3 in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS) for 96 hours. Cells were stained for FACS analysis with the following antibody panel: anti-CD3-BV421, anti-CD8-BV650, anti-CD25-BV785, and anti-CD4-BV605 (Biolegend) using the fixable viability dye Aqua (Biolegend #423102). The activation of CD4+ or CD8+ T cells cocultured with Raji-Luc-GFP cells at various concentrations of C6, D7, or C3 after 96 hours is shown in Figures 22A-22B, respectively. The combination of CD28 and CD3 stimulation (C6) resulted in greater T cell activation than CD3 (D7) or CD28 (C3) stimulation alone.

[0346] Example 17: T cell-induced cytotoxicity The ability of the exemplary anti-CD20 x anti-CD3 x anti-CD28 IgM antibody C6 described in Example 1 to enhance T cell-induced cytotoxicity (TDCC) compared to the anti-CD20 x anti-CD3 IgM antibody D7 described in Example 1 and the anti-CD20 x anti-CD28 IgM antibody C3 described in Example 1 was evaluated as follows: 50 x 10 3 Human PBMCs were cocultured with Raji-Luc-GFP cells in the presence of serial dilutions of C6, D7, or C3 in a total volume of 200 μL of culture medium (RPMI-1640 + 10% heat-inactivated FBS) for 96 hours. 100 μL of supernatant was removed and frozen at -80°C for subsequent cytokine release analysis. Cells were stained with the fixable viability dye Aqua (Biolegend #423102). The number of viable Raji-luc-GFP cells (gated GFP + Aqua - ) in each well was compared with the antibody-untreated group to calculate the percentage killing. Data were then plotted using GraphPad Prism. The percentage killing of Raji-Luc-GFP cells at various concentrations after 96 hours is shown in Figure 22C. The combination of CD28 and CD3 stimulation (C6) resulted in greater TDCC activity than CD3 (D7) or CD28 (C3) stimulation alone.

[0347] Example 18: Cytokine Release Supernatants from the TDCC assay obtained in Example 17 were assayed for cytokines, including IL-2 and IFN-γ, using the Cytometric Bead Array Human Th1-2 Cytokine Kit II (BD, 551809) according to the manufacturer's protocol, except that smaller volumes were used to adapt the plate-based assay. Results were then plotted using GraphPad Prism. Cytokine IL-2 and IFN-γ concentrations in supernatants from cocultures of PBMC cells with Raji-Luc-GFP cells 96 hours after treatment with C6, D7, or C3 are shown in Figures 23A and 23B, respectively. Enhanced TDCC and T cell activation correlated with cytokine responses.

[0348] Example 19: In vivo activity The anti-tumor activity of exemplary IgM antibodies: C3 (anti-CD20 x anti-CD28 IgM antibody described in Example 1), C7 (anti-CD20 x anti-CD3 x anti-CD28 IgM antibody described in Example 1), and D7 (anti-CD20 x anti-CD3 IgM antibody described in Example 1) was evaluated in the Ramos human B lymphoma xenograft model. NSG-MHC I / II DKO mice were injected with 10 7 On day 0, mice were engrafted with 2 × 10 human PBMCs. 6 Ramos cells were implanted subcutaneously. Two days later, animals received six doses every other day (q2d x 6) or 12 doses every other day (q2d x 12) intravenously, as shown in Table 16. Tumor size was measured two to three times weekly. Whole blood samples were collected 4 days after PBMC engraftment, 4 hours after the first dose, 4 hours after the sixth dose, and between the sixth and twelfth doses. In addition, tumors, spleens, and blood were collected at the end of the study.

[0349] [Table 16]

[0350] Graphs of tumor volume versus days after Ramos implantation (i.e., time in days) for various treatment groups up to day 23 are shown in Figures 24A-D. Tumor volume over time for each group is shown in Figure 24A, tumor volume over time for the 10 mg / kg groups (Groups 3, 4, and 7) is shown in Figure 24B, and tumor volumes for the 1 mg / kg C7 and D7 groups and the 10 mg / kg C3 group are shown in Figure 24C. Tumor volumes for the 0.1 mg / kg C7 and D7 groups and the 10 mg / kg C3 group are shown in Figure 24D. As shown in Figure 24A, tumor volumes were smaller in the 1 mg / kg and 10 mg / kg C7 (anti-CD20 x anti-CD3 x anti-CD28) treatment groups than in any of the other treatment groups. As shown in Figure 24B, tumor volume reached 0 mm on day 23, particularly for Group 4 (C7 administered at 10 mg / kg). 3 It was.

[0351] Table 17 provides a summary of the number of tumor growth inhibited (TGI) and tumor-free (TF) mice in the various treatment groups, determined on day 16 (the last study day when all mice in the vehicle group were alive).

[0352] [Table 17]

[0353] As shown by the data in Table 17, for the treatment groups evaluated, the greatest percent tumor growth inhibition was observed in the 10 mg / kg C7 and 1 mg / kg C7 treatment groups with anti-CD20 x anti-CD3 x anti-CD28 IgM antibodies (Groups 4 and 5, respectively). The next highest percent tumor growth inhibition was observed in Group 7 (D7, anti-CD20 x anti-CD3 IgM antibodies administered at 10 mg / kg).

[0354] Serum samples collected from each treatment group were analyzed for cytokine IL-2, IL-10, IFN-γ, TNF-α, IL-4, IL-6, IL-8, IL-1β, IL-12p70, and IL-13 concentrations 4 hours after the first dose using an MSD multiplex cytokine assay kit (Meso Scale Discovery) according to the manufacturer's protocol. Results were then plotted using GraphPad Prism. IL-2, IL-10, IFN-γ, TNF-α, IL-4, IL-6, IL-8, IL-1β, IL-12p70, and IL-13 serum concentrations are shown in Figures 25A-25J, respectively. At each dose level, C7 (anti-CD20 × anti-CD3 × anti-CD28) stimulated greater IL-2, IL-10, IFN-γ, TNF-α, IL-6, and IL-13 release than the same dose of D7 (anti-CD20 × anti-CD3) and greater release than 10 mg / kg of C3 (anti-CD20 × anti-CD28). Referring to the serum IL-4 levels shown in Figure 25E, at 10 mg / kg, C3 stimulated greater IL-4 release than the same dose of C7 or D7; at the 1 mg / kg dose, C7 stimulated greater IL-4 release than D7; and at the 0.1 mg / kg dose, D7 stimulated greater IL-4 release than C7. Referring to the serum IL-8 levels shown in Figure 25G, at a dose of 10 mg / kg, C7 stimulated IL-8 to a greater extent than either D7 or C3, and at doses of 1 mg / kg and 0.1 mg / kg, D7 stimulated IL-8 to a greater extent than C7. As shown in Figure 25H, at a dose of 10 mg / kg, D7 stimulated IL-1β to a greater extent than either C7 or C3, and at both doses of 1 mg / kg and 0.1 mg / kg, D7 stimulated IL-1β to a greater extent than C7. Referring to Figure 25I, at a dose of 10 mg / kg, C7 stimulated IL-12p70 to a greater extent than either D7 or C3. Similarly, at a dose of 1 mg / kg, C7 stimulated IL-12p70 to a greater extent than D7. In contrast, at a dose of 0.1 mg / kg, D7 stimulated IL-12p70 to a slightly greater extent than C7.

[0355] The number of T cells at the tumor site for each treatment was assessed as follows. Tumors were harvested, and cells were stained for flow cytometry analysis using the following antibody staining panel: anti-mouse CD45-BV785, anti-human CD45-APC / Cy7, anti-human CD8-PerCP-Cy5.5, and anti-human CD4-AF700 (Biolegend) using the fixable viability dye BV510. The number of infiltrating CD4+ or CD8+ T cells normalized to tumor volume at the 0.1 mg / kg dose is shown in Figures 26A and 26B, respectively. C7 (anti-CD20 × anti-CD3 × anti-CD28) increased both CD4+ and CD8+ T cells at the tumor site to a greater extent than both vehicle and D7 (anti-CD20 × anti-CD3).

[0356] Example 20: Long term T cell dependent cellular cytotoxicity (TDCC), T cell proliferation and cytokine release Long-term T cell-dependent cellular cytotoxicity (TDCC), T cell proliferation and cytokine release were assessed in an in vitro model using human PBMCs and Ramos tumor cells.

[0357] PBMCs and CD20-expressing target tumor cells (Ramos) were washed and resuspended in culture medium (RPMI1640 + 10% heat-inactivated FBS). Coculture assays were set up in 96-well U-bottom tissue culture plates containing 200 μL of culture medium per well at various PBMC to Ramos cell ratios, either without antibody or in the presence of antibodies D8, D9, D10, D7, or C6 (described in Example 1), all at a concentration of 10 nM. Three different effector (PBMC) to target (Ramos cell) ratios were tested. For a 3:1 ratio, 3 × 10 cells per well were used. 4 1 x 10 PBMCs and 1 x 10 4 Seed 1 x 10 Ramos cells per well for a 1:1 ratio. 4 1 x 10 PBMCs and 1 x 10 4 Seed 1 x 10 Ramos cells per well for a 1:3 ratio. 4 PBMCs and 3 x 10 4Ramos cells were seeded onto the plates. The plates were then placed in a humidified incubator at 37°C with 5% CO2. At 3, 7, 10, and 14 days, the plates were removed, and the cell mixture was resuspended in each well by pipetting. 100 μL of the resuspended cell culture mix was removed from each well and transferred to a new tissue culture plate, supplemented with 100 μL of fresh medium containing the same antibody concentrations, for continued culture at 37°C and 5% CO2. The cell culture mix from the original tissue culture plate was used to assay TDCC and CD4+ and CD8+ T cell proliferation at each time point, as well as cytokine analysis at 3 days (72 hours).

[0358] The cell culture mix to be assayed was centrifuged at 300 g for 5 minutes, and a 50 μL supernatant sample was collected from each well and frozen at -80°C for subsequent cytokine analysis. Five thousand counting beads (Life Technologies, catalog number C36950) were then added to each well and mixed with the cells by pipetting. The cell and counting bead mixture was centrifuged again, and the cell and counting bead mixture was resuspended in a multicolor flow antibody cocktail to stain for human CD3, CD4, CD8, CD25, and CD19. A fixable viability dye (EFLUOR® 780, Thermo Fisher Scientific, catalog number 65-0865-14) was also included in the staining cocktail for viability staining. After a 30-minute incubation at 4°C, samples were washed with 200 μL of FACS stain buffer (BD Biosciences, catalog number 554656) and then resuspended in 25 μL of FACS stain buffer for flow cytometry analysis using an INTELLICYT® IQUE® Screener PLUS (Sartorius) and INTELLICYT® FORECYT® software (Sartorius).

[0359] For TDCC and T cell proliferation analysis, target cells (Ramos and primary B cells) were identified by CD19 staining, and T cell subsets were identified by CD3, CD4, CD8, and CD25 staining results from FACS analysis. The number of live target cells and T cell subsets in each well was calculated by comparing the number of target cell events with the number of counting bead events. The cell number was then divided by the volume of the cell culture sample to obtain the cell density in the culture, which was monitored over multiple time points to evaluate the long-term cytotoxicity and T cell proliferation effects of various test substances. TDCC results at three different effector-to-target ratios are shown in Figures 27A-C, respectively. CD4 T cell proliferation results at three different effector-to-target ratios are shown in Figures 28A-C, respectively. CD8 T cell proliferation results at three different effector-to-target ratios are shown in Figures 29A-C, respectively.

[0360] To measure cytokine release mediated by test substances, supernatant samples from the 3-day (72-hour) time point were thawed and analyzed by flow cytometry using the BD Biosciences Human Th1 / Th2 Cytokine Cytometry Bead Array (CBA) Kit II (Cat. No. 551809) according to the manufacturer's recommendations, except that a smaller culture volume was used to accommodate the plate-based format. Flow cytometry analysis was performed on an INTELLICYT® IQUE® Screener PLUS, and data were analyzed using INTELLICYT® FORECYT® software, including standard curve fitting and cytokine concentration calculation. The levels of IL-2, IL-4, IL-6, IL-10, TNFα, and IFN-γ at each of the three different effector-to-target ratios are shown in Figures 30A–F, respectively.

[0361] Example 21: In vivo efficacy - survival studies The anti-tumor activity of exemplary IgM antibodies: C3 (anti-CD20 x anti-CD28 IgM antibody described in Example 1), C7 (anti-CD20 x anti-CD3 x anti-CD28 IgM antibody described in Example 1), and D7 (anti-CD20 x anti-CD3 IgM antibody described in Example 1) was evaluated in the Ramos human B lymphocytic carcinoma xenograft model by examining overall survival. NSG-MHC I / II DKO mice were injected with 10 7 On day 0, mice were engrafted with 2 × 10 human PBMCs. 6 Ramos cells were implanted subcutaneously. Two days later, animals were intravenously dosed six times every other day (q2d x 6) or 12 times every other day (q2d x 12) as shown in Table 18. The low-dose group received a total of 12 doses, while the other groups received six doses. Tumors with a size of 2000 mm 3 Mice were euthanized when they reached 0.05. Median overall survival in days for each treatment group is shown in Table 18. Survival over time for each treatment group was determined using the Kaplan-Meier method, and the resulting survival curves are shown in Figure 31.

[0362] As shown in Table 18, at both the 10 mg / kg and 1 mg / kg doses, mice treated with C7 had significantly longer median overall survival than mice treated with C3 or D7, or D7, respectively. At the 10 mg / kg dose, the median overall survival of mice treated with C7 was approximately 2 times that of mice treated with C3 and approximately 1.6 times that of mice treated with D7, and at the 1 mg / kg dose, the median overall survival of mice treated with C7 was approximately 1.5 times that of mice treated with D7.

[0363] [Table 18]

[0364] Example 22: In vivo efficacy In a further preclinical efficacy study, the anti-tumor activity of exemplary IgM antibodies C7 (an anti-CD20 x anti-CD3 x anti-CD28 IgM antibody described in Example 1), D7 (an anti-CD20 x anti-CD3 IgM antibody described in Example 1), and the isotype control antibody D15 were evaluated in the Ramos human B lymphoma xenograft model. NSG-MHC I / II DKO mice were injected with 10 7 On day 0, mice were engrafted with 2 × 10 human PBMCs. 6 Ramos cells were implanted subcutaneously. Two days later, animals were intravenously dosed twice weekly for a total of 12 doses (biw x 12) as shown in Table 19. Tumor size was measured two to three times weekly. Whole blood samples were collected 4 days after PBMC engraftment, 4 hours after the first dose, 4 hours after the sixth dose, and between the sixth and twelfth doses. In addition, tumors, spleens, and blood were collected at the end of the study.

[0365] [Table 19]

[0366] Graphs of tumor volume versus days after Ramos implantation (i.e., time in days) for each treatment group are shown in Figures 32A-C. Tumor volume over time for each group is shown in Figure 32A, tumor volume over time for the 10 mg / kg treatment groups (Groups 2, 3, and 5) is shown in Figure 32B, and tumor volumes for the 1 mg / kg C7 and D7 treatment groups and the 10 mg / kg control group are shown in Figure 32C. Tumor volume over time was smaller for the C7 (anti-CD20 × anti-CD3 × anti-CD28) treatment group at both the 1 mg / kg and 10 mg / kg doses than for any other treatment group.

[0367] Table 20 provides a summary of the number of tumor growth inhibited (TGI) and tumor-free (TF) mice in the various treatment groups, determined on day 16 (the last study day when all mice in the vehicle group were alive). As shown in Table 20, in this preclinical model and experiment, antibody C7 demonstrated superior anti-tumor efficacy when compared to an equivalent dose of antibody D7.

[0368] [Table 20]

[0369] Median overall survival (OS) in days for each treatment group is shown in Table 21. Survival over time for each of the treatment groups was determined using the Kaplan-Meier method, and the resulting survival curves are shown in Figure 33.

[0370] [Table 21]

[0371] As shown in Table 21, at both the 10 mg / kg and 1 mg / kg doses, mice treated with C7 had a longer median overall survival than mice treated with D7, and mice treated with C7 or D7 demonstrated extended survival times over both the vehicle and control groups. At the end of the study, all mice in the C7 (10 mg / kg) treatment group were alive, so a median overall survival value could not be calculated, and 5 of 8 mice were tumor-free at day 34.

[0372] Serum samples collected from each treatment group were analyzed for cytokine IL-2, IL-4, IL-10, IFN-γ, and TNF-α concentrations 4 hours after the first dose using an MSD multiplex cytokine assay kit (Meso Scale Discovery) according to the manufacturer's protocol. Results were then plotted using GraphPad Prism. IL-2, IL-4, IL-10, IFN-γ, and TNF-α serum concentrations (pg / mL) are shown in Figures 34A–34E, respectively. In this experiment, C7 (anti-CD20 × anti-CD3 × anti-CD28) stimulated greater IL-2 release than D7 (anti-CD20 × anti-CD3) at both the 10 mg / kg and 1 mg / kg doses. For the other cytokines investigated, IL-4, IL-10, IFN-γ, and TNF-α, C7 stimulated greater cytokine release than D7 at the 10 mg / kg dose, but at lower doses, D7 stimulated greater or nearly equal (IL-10) cytokine release than C7.

[0373] Example 23: Pharmacodynamic (PD) studies Pharmacodynamic studies were performed using the Ramos human B-lymphoma xenograft model. NSG-MHC I / II DKO mice were administered 10 mg of ribozyme-3 (RI) at day -14. 7 On day 0, mice were engrafted with 2 × 10 human PBMCs. 6 Ramos cells were subcutaneously implanted. On day 3, whole blood samples were collected to confirm CD3 engraftment. Antibody dosing was initiated when tumors were 100-150 mm 3 (typically 100mm 3Treatment was initiated on day 5 or 6, when the tumor reached a volume of approximately 1000 mg / kg (approximately 1000 mg / kg). Mice were treated with vehicle (Group 1), control antibody D15 from Table 13, in which the IgM pentamer binds to an unrelated non-mammalian target (Group 2), exemplary antibody C7 (anti-CD20 x anti-CD3 x anti-CD28 IgM antibody described in Example 1; Group 3), or exemplary antibody D7 (anti-CD20 x anti-CD3 IgM antibody described in Example 1; Group 4). Mice were dosed twice weekly for a total of three doses (biw x 3). Tumor size and body weight were measured three times weekly throughout the study. Whole blood samples were collected 24 hours after the third dose. At the end of the study, tumors, spleens, and peripheral blood were collected for flow cytometry and immunohistochemistry analysis.

[0374] [Table 22]

[0375] The tumor volumes over time for the various treatment groups (groups 1, 2, 3, and 4) are shown in Figure 35. Tumor volumes over time were smaller in the C7 (anti-CD20 x anti-CD3 x anti-CD28) and D7 (CD20 x CD3) treatment groups compared to the vehicle and control groups. At day 14, the tumor volume in the group of mice treated with antibody C7 (group 3) was smaller than the tumor volume in the group of mice treated with antibody D7 (group 4).

[0376] Collected peripheral blood was incubated with RBC lysis buffer (Thermo Fisher Scientific) and washed with Pharmingen Stain™ buffer (BD Biosciences). Cells were stained for FACS analysis using the following antibody staining panel: anti-Bcl_xL-APC (Cell Signaling), anti-human CD45-APC / Cy7 (Biolegend), anti-mouse CD45-BV785, anti-CCR7 FITC, anti-CD4-AF700, anti-CD8-PerCp / Cy5.5, anti-PD1-BV605, anti-CD45PRO-PE / Cy7, anti-K167-BV421, and anti-granzyme b-PE. Live / dead cells were stained using the ZOMBIE AQUA™ Fixable Viability Kit (Biolegend). The absolute numbers of CD4+ and CD8+ T cell subsets in peripheral blood, normalized to blood volume, are shown in Figures 36A-E and 37A-E, respectively.

[0377] Mice were sacrificed after three doses, and tumor samples were collected and analyzed by flow cytometry analysis and IHC staining using immunohistochemistry to assess the presence of tumor-infiltrating T cells. The results of flow cytometry analysis are shown in Figure 51, which shows cells per gram of tumor for the treatment groups described above. As shown, T cell infiltration into tumors was significantly enhanced with the exemplary costimulatory CD20xCD3xCD28 IgM antibody C7.

[0378] For immunohistochemistry, formalin-fixed, paraffin-embedded (FFPE) sections from mice were stained with anti-CD3 and anti-CD8 to identify tumor-infiltrating T cells. An exemplary costimulatory IgM, C7 (CD20 × CD3 × CD28), resulted in an increase in tumor-infiltrating T cells compared with the bispecific IgM, D7 (CD20 × CD3), whereas the nontargeting control IgM, D15, with a CD3 × CD28 J chain, and the vehicle control had no effect (data not shown).

[0379] Example 24: Construction and characterization of antibodies targeting solid tumors Additional IgM pentameric antibodies listed in Table 23 were produced according to the methods described in Example 1.

[0380] [Table 23]

[0381] [Table 24] TIFF2026502759000042.tif100164

[0382] [Table 25]

[0383] T-cell dependent cytotoxicity: The T-cell dependent cytotoxicity (TDCC) activity of various solid tumor targeting constructs shown in Table 23 against the ovarian cancer cell line OVCAR3 (ATCC Catalog No. HTB-161) (as well as against additional cell lines described below, including HEK293T / TROP2, A431, and SKOV-3) was assessed by real-time cell analysis or luminescence as follows.

[0384] TDCC activity was assessed using the XCELLIGENCE™ Real-Time Cell Analysis Instrument (RTCA). This instrument utilizes microelectrodes fused to the bottom of a specialized 96-well E-Plate to measure the impedance of electron flow caused by the presence of adherent cells (represented by the normalized cell index calculated by the XCELLIGENCE™ software). Adherent target tumor cells were added to the 96-well E-Plate at a specific seeding density. The E-Plate was placed in the RTCA in a humidified cell incubator at 37°C with 5% CO2 for 24 hours to allow the target tumor cells to settle and adhere to the bottom of the plate. Effector cells (PBMCs or T cells) and test substances were then added to the wells, after which culture and real-time cell index monitoring were resumed. Cell index data were exported and analyzed using GraphPad Prism. In some cases, supernatant samples were collected at specific time points for cytokine analysis using the BD Biosciences Human Th1 / Th2 Cytokine Cytometry Bead Array (CBA) Kit II (Cat. No. 551809). At the end of the co-culture assay, T cell activation can be assessed by harvesting cell samples from the E-Plates for flow cytometric analysis of various biomarkers (CD3, CD4, CD8 and CD25).

[0385] The results for the mesothelin-targeted constructs are shown in Figure 38, and the results for the TROP-2-targeted constructs are shown in Figure 41. Constructs M1 and T1, which have costimulatory activity (binding to CD2 and CD28), showed slightly improved activity over constructs M3 and T3, which bind only to CD3, while constructs M2 and T2, which bind only to tumor targets and CD28, showed activity comparable to the no-antibody and isotype control constructs.

[0386] Cytokine Release: To measure cytokine release mediated by test substances, supernatant samples from the 72-hour timepoint of the RTCA assay shown in Figures 38 and 41 were thawed and analyzed by flow cytometry using the BD Biosciences Human Th1 / Th2 Cytokine Cytometry Bead Array (CBA) Kit II (Cat. No. 551809) according to the manufacturer's recommendations, except that a smaller culture volume was used to accommodate the plate-based format. Flow cytometry analysis was performed on an INTELLICYT® IQUE® Screener PLUS, and data were analyzed using INTELLICYT® IQUE FORECYT® software, including standard curve fitting and cytokine concentration calculation. Levels of IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNFα are shown in Figures 39A-39F for the anti-mesothelin constructs, respectively, and in Figures 42A-42F for the anti-TROP-2 constructs, respectively.

[0387] T cell activation: The number of T cells and the degree of T cell activation for both CD4+ and CD8+ T cell subsets at 96 hours in the RTCA assay shown in Figures 38 and 41 were measured as follows. Cells from each well were collected by pipetting multiple times and transferred to a new U-bottom 96-well plate. Cells were stained for FACS analysis using the following staining panel: anti-CD3 PerCP-Cy5.5, anti-CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend, and the fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14). The number of different T cell subsets in each well was compared with the added counting beads to calculate the number of T cells, and the T cell activation status was assessed based on the CD25 staining results. Data were then analyzed and plotted using GraphPad Prism. The results for the mesothelin-targeted constructs are shown in Figures 40A-40D, and the results for the TROP-2-targeted constructs are shown in Figures 43A-43D. Constructs M1 and T1, which have costimulatory activity (binding to both CD3 and CD28), showed slightly improved T cell numbers and activation compared with constructs M3 and T3, which bind only to CD3, while constructs M2 and T2, which bind only to tumor targets and CD28, showed activity comparable to the no-antibody and isotype control constructs.

[0388] T cell-dependent cytotoxicity: Antibody-mediated redirected T cell killing of HEK293 / TROP-2 cells (generated in-house) was measured in a TDCC assay in which human PBMCs as effector cells (E) and TROP2-transduced HEK293 cells as target cells (T) were incubated with serial dilutions of the indicated antibodies at a 3:1 E:T ratio in 96-well plates in a humidified incubator at 37°C for 72 hours. After incubation, cell viability was assessed by RTCA using impedance as described above. Percent cytotoxicity after antibody treatment was calculated by normalization to the cell index of the non-antibody-treated group, and the results are shown in Figure 44. The IC50 values ​​determined for antibodies T1 and T3 were 0.04792 and 0.3422, respectively. Data represent two independent donors.

[0389] The T cell-dependent cytotoxicity (TDCC) activity of various solid tumor targeting constructs listed in Table 23 against the epidermoid carcinoma cell line A431 (American Type Culture Collection, ATCC catalog number CRL-1555-LUC2) was assessed by luminescence as follows. Antibody-mediated redirected T cell killing of A431-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and A431-Luc cells as target cells (T) were incubated at a 1:1 E:T ratio with serial dilutions of the indicated antibodies in a 96-well plate in a humidified incubator at 37°C for 96 hours at 5% CO2. After incubation, a luminescence-based viability assay was used to determine viable cells as described below. The percent cell survival after antibody treatment with T3, T1, or D16 was calculated by normalizing to the viable cells in the non-antibody-treated group. Data represent three independent donors. The results are shown in Figure 45. The IC50 values ​​determined for antibodies T3, T1, and D16 were 0.04448, 0.01560, and 0.001432, respectively.

[0390] Luciferase-tagged adherent target tumor cells were added to a 96-well cell culture plate at a specific seeding density and cultured for 24 hours in a humidified cell incubator at 37°C under 5% CO2 to allow the target tumor cells to settle and adhere to the bottom. Effector cells (PBMCs or T cells) and test substances were then added to the wells, and the culture was resumed. After incubation, the viability of the luciferase-tagged tumor cells was determined by luminescence readout using the Promega BIO-GLO™ Luciferase Assay System (Cat. No. G7940), and the data were analyzed using GraphPad Prism.

[0391] The T cell-dependent cytotoxicity (TDCC) activity of various solid tumor targeting constructs shown in Table 23 against the ovarian adenocarcinoma cell line SKOV-3 (BPS Bioscience, Catalog No. 7842) was assessed by luminescence as follows. Antibody-mediated redirected T cell killing against SKOV3-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and SKOV3-Luc cells as target cells (T) were incubated at an E:T ratio of 3:1 with serial dilutions of the indicated antibodies in a 96-well plate in a humidified incubator at 37°C for 120 hours. After incubation, viable cells were determined using the luminescence-based viability assay described above. The percent cell survival after treatment with antibodies T3, T1, or D16 was calculated by normalizing to the viable cells in the no-antibody treatment group. Data represent three independent donors. The results are shown in Figure 46.

[0392] Further T cell dependent cytotoxicity (TDCC) activity evaluation and analysis was performed against the ovarian cancer cell line OVCAR3 (ATCC Catalog No. HTB-161) of various exemplary solid tumor targeting constructs (D16, T3, T1, D17, M3, and M1) shown in Table 23. Further cytokine release measurements were also performed.

[0393] Antibody-mediated redirected T cell killing of OVCAR3 cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and OVCAR3-Luc cells as target cells (T) were incubated at an E:T ratio of 1:3 with serial dilutions of antibodies D16, T3, T1, D17, M3, and M1 in 96-well plates in a humidified 5% CO2 incubator at 37°C for 120 hours (5 days). After incubation, luciferase-tagged tumor cell viability was determined by luminescence readout using the Promega BIO-GLO™ Luciferase Assay System (Cat. No. G7940), and data were analyzed using GraphPad Prism. Results are graphically presented in Figure 47, which shows percent target cell killing versus antibody concentration for antibodies D16, T3, T1, D17, M3, and M1.

[0394] Cytokine release: Test article-mediated cytokine release was determined using the BD Biosciences Human Th1 / Th2 Cytokine Cytometric Bead Array (CBA) Kit II, as described above, using supernatant samples at 72 hours (3 days). Test article-mediated levels of IL-2, IL-4, IL-6, IL-10, TNFα (also referred to herein as TNF), and IFN-γ are shown in Figures 48A-48F, respectively.

[0395] T cell-dependent cytotoxicity: Furthermore, antibody-mediated redirected T cell killing of RERF cells, i.e., RERF-LC-KJ / CMV-Luc cells or RERF-Luc (JCRB Cell Bank, catalog no. JCRB1558), was measured in a TDCC assay in which human PBMCs as effector cells (E) and RERF cells as target cells (T) were incubated at a 1:1 E:T ratio with serial dilutions of antibodies D16, T3, and T1 in 96-well plates in a humidified 5% CO2 incubator at 37°C for 120 hours (5 days). After incubation, the viability of luciferase-tagged tumor cells was determined by luminescence readout using the Promega BIO-GLO™ Luciferase Assay System (catalog no. G7940), and data were analyzed using GraphPad Prism. The results are shown graphically in Figure 49.

[0396] Cytokine release: Test article-mediated cytokine release was determined as described above using supernatant samples at 72 hours (3 days). Test article-mediated levels of IL-2, IL-4, IL-6, IL-10, TNF, and IFN-γ are shown in Figures 50A-50F, respectively.

[0397] Example 25: T cell-dependent cytotoxicity assay using high, medium and low TROP-2 expressing cell lines Antibody-mediated redirected T cell killing by exemplary IgM antibodies, T1 (anti-TROP-2 × anti-CD3 × anti-CD28 IgM antibody) and T3 (anti-TROP-2 × anti-CD3 IgM), with or without CD28 costimulation, respectively, was evaluated in a T cell-dependent cellular cytotoxicity (TDCC) assay. Human PBMCs as effector cells (E) were cocultured with the following high, medium, or low TROP-2 expressing cells as target cells (T): TROP-2-transduced HEK293T cells with high TROP-2 expression, A431 melanoma cells with high TROP-2 expression, and SKOV-3 ovarian cancer cells with medium / low TROP-2 expression, at E:T ratios of 3:1, 1:1, and 3:1, respectively, in the presence of serial dilutions of T1 or T3 antibody. Incubation was carried out at 37°C in a humidified incubator with 5% CO for 3–4 days. After incubation, cell viability was assessed by RTCA using impedance. Percent survival after antibody treatment was calculated by normalization to the viable cells in the "no antibody" treatment group. A plot of percent cell survival versus antibody concentration (log, μg / mL) is shown in Figure 52A (HEK293 / TROP-2 高 Target cells, E:T=3.1), Figure 52B (A431 TROP-2高 target cells, E:T=1:1), and Fig. 52C (SCOV-3 TROP-2低 The plots show the effect of CD28 co-stimulation on target cells (E:T = 3:1). As shown in each of the three plots, enhanced cytotoxicity by an exemplary IgM antibody with CD28 co-stimulation (T1, anti-TROP-2 x anti-CD3 x anti-CD28) was observed in TROP-2-transduced HEK293T cells with high TROP-2 expression, A431 melanoma cells with high TROP-2 expression, and SKOV-3 ovarian cancer cells with intermediate / low TROP-2 expression, when compared with an exemplary IgM antibody without CD28 co-stimulation (T3, anti-TROP-2 x anti-CD3 IgM).

[0398] Example 26: In vivo studies: antitumor activity, cytokine release, and antiapoptotic T cell numbers In a preclinical efficacy study, the anti-tumor activity of exemplary IgM antibodies T1 (anti-TROP-2 × anti-CD3 × anti-CD28 IgM antibody) and T3 (anti-TROP-2 × anti-CD3 IgM) was evaluated in a HEK293T / TROP-2 xenograft model. NSG-MHCI / II DKO mice were injected with 10 7 On day 0, mice were engrafted with 10 human PBMCs. 7 TROP-2-transduced HEK293T cells were implanted subcutaneously. Animals received 12 doses (biw x 12) of antibody T1 or antibody T3 at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, or vehicle (1 x PBS) (see Table 26), administered intravenously twice weekly starting on day 3. For the 3 mg / kg treatment group, antibody dosing was stopped after 7 doses to compare the sustained effects of treatment with an exemplary anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody and an exemplary anti-TROP-2 x anti-CD3 IgM antibody (lacking the CD28 costimulatory signal).

[0399] [Table 26]

[0400] Graphs of tumor volume over time for the various treatment groups are shown in Figures 53A (0.3 mg / kg group), 53B (1 mg / kg group), and 53C (3 mg / kg group). As shown in all three graphs, treatment with an IgM antibody such as T1 (with CD28 costimulation) resulted in enhanced in vivo anti-tumor activity compared to the exemplary IgM antibody T3 (lacking CD28 costimulation), particularly when evaluated at lower doses, for example, in a humanized HEK293 / TROP-2 xenograft model.

[0401] Serum samples from each treatment group were collected 4 hours after the first dose and analyzed for the cytokines IFN-γ, IL-2, TNF-α, and IL-10 using an MSD multiplex cytokine assay kit (Meso Scale Discovery) according to the manufacturer's protocol. Results were then plotted using GraphPad Prism. Serum concentrations of IFN-γ, IL-2, TNF-α, and IL-10 are shown in Figures 54A-54D, respectively. A dose-dependent increase in the levels of released cytokines interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 was observed in the T1 IgM antibody-treated group, i.e., after treatment with the exemplary TROP-2×CD3×CD28 IgM antibody (i.e., with CD28 costimulation).

[0402] Mouse peripheral blood samples were collected on day 12 (24 hours before the fourth dose). Blood samples were processed and stained for flow cytometry analysis using an antibody cocktail containing ZOMBIE AQUA™ BV510 viability dye, anti-human CD45 (clone 2D1, APC / Cy7), anti-mouse CD45 (clone 30F-11, AF700), anti-CD4 (clone OKT4, BV785), anti-CD8 (clone SK1, PerCp / Cy5.5), and anti-Bcl_xL (clone 54H6, APC), purchased from either Biolegend or Cell Signaling Technology. Samples were then analyzed by flow cytometry. Increased absolute cell numbers of anti-apoptotic Bcl_xL-positive CD8 (Figure 55) and CD4 T (data not shown) cells were observed in peripheral blood samples from treatment groups treated with the exemplary IgM antibody T1:TROP-2xCD3xCD28 IgM (with CD28 costimulation) compared to the T3 (TROP-2xCD3 IgM) and vehicle-treated groups.

[0403] Example 27: T cell dependent cytotoxicity assay The T cell-dependent cytotoxicity (TDCC) activity of the following exemplary IgM antibodies, shown in Table 23, with and without costimulatory activity: M1 (anti-mesothelin (MSLN) × anti-CD3 × anti-CD28 IgM antibody) and M3 (anti-mesothelin (MSLN) × anti-CD3 IgM antibody), respectively, against the gastric cancer cell line MKN45 was assessed by luminescence as follows. Antibody-mediated redirected T cell killing of MKN45-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and MKN45-Luc cells as target cells (T) were incubated at an E:T ratio of 3:1 with serial dilutions of the exemplary antibodies M1 and M3 in a 96-well plate in a humidified incubator at 5% CO for 4 days at 37°C. After incubation, viable cells were determined using the luminescence-based viability assay described above. The percent cell survival after antibody treatment was calculated by normalization to the viable cells in the "no antibody" treatment group. Data are representative of three independent donors. The results are shown graphically in Figure 56. As shown in the figure, improved cytotoxicity was observed in treating MKN45 cells with exemplary antibody M1 (with CD28 costimulation) over antibody M3 (lacking CD28 costimulation) in this exemplary TDCC assay.

[0404] Example 28: In vivo studies: antitumor activity and cytokine release In a preclinical study, the anti-tumor activity of exemplary IgM antibodies with and without costimulatory activity, M1 (anti-mesothelin (MSLN) x anti-CD3 x anti-CD28 IgM antibody) and M3 (anti-mesothelin (MSLN) x anti-CD3 IgM antibody), respectively, shown in Table 23, was evaluated in a humanized MKN45 gastric cancer xenograft model. NSG-MHCI / II DKO mice were injected with 10 7 On day 0, mice were engrafted with 10 human PBMCs. 7MKN45 cells were subcutaneously implanted. Animals (n = 7 per group) were intravenously administered antibody M1, antibody M3, or vehicle (1x PBS) at antibody doses of 3 mg / kg or 10 mg / kg twice weekly, starting on day 3, for a total of 12 doses (biw x 12). Results are shown graphically in Figures 57A (3 mg / kg dose) and 57B (10 mg / kg dose) as mean tumor volume versus time (days after MKN45 cell implantation). As shown in Figures 57A and 57B, treatment with an IgM antibody such as M1 (anti-mesothelin (MSLN) x anti-CD3 x anti-CD28, i.e., with CD28 costimulation) resulted in enhanced in vivo antitumor activity compared to the exemplary IgM antibody M3 (anti-mesothelin (MSLN) x anti-CD3, i.e., lacking CD28 costimulation), for example, when evaluated in a humanized MKN45 gastric cancer xenograft model.

[0405] Cytokine release: Serum samples from each treatment group were collected 4 hours after the first dose and analyzed for the cytokines IFN-γ, IL-2, TNF-α, and IL-10 using an MSD multiplex cytokine assay kit (Meso Scale Discovery) according to the manufacturer's protocol. The results were then plotted using GraphPad Prism. Serum concentrations of IFN-γ, IL-2, TNF-α, and IL-10 are shown in Figures 58A, B, C, and D, respectively. A dose-dependent increase in the levels of released cytokines interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 was observed in the M1 IgM antibody-treated group, i.e., after treatment with the exemplary MSLNxCD3xCD28 IgM antibody (i.e., with an IgM antibody with CD28 costimulation).

[0406] Intratumoral T cell counts: Mouse tumor samples were harvested on day 43 (termination). Tumor samples were processed and stained with an antibody cocktail containing a viability dye (ZOMBIE AQUA™ BV510), anti-human CD45 (clone 2D1, APC / Cy7), anti-mouse CD45 (clone 30F-11, AF700), anti-CD4 (clone OKT4, BV785), and anti-CD8 (clone SK1, PerCp / Cy5.5). All reagents were purchased from Biolegend. Samples were then subjected to flow cytometry and gated for absolute CD4+ T cell counts (Figure 60A) and absolute CD8+ T cell counts (Figure 60B). Increased absolute cell counts of both CD4 and CD8 T cells were observed in tumors following higher doses of CD28 costimulation.

[0407] Example 29: Comparison of various IgG and IgM anti-CD28 anti-CD3 antibody combinations on non-targeted T cell activation and cytokine release The ability of various combinations of anti-CD28 and / or anti-CD3 IgM or IgG4 antibodies to activate T cells and induce cytokine release was evaluated using a plate-based assay by the following method. See, e.g., Stebbings, R., et al. J Immunol 179:3325-3331 (2007). This experiment compares two anti-CD28 binding domains, namely, anti-CD28 Z (VH and VL amino acid sequences of SEQ ID NOs: 61 and 81, respectively) and anti-CD28 binding domain W (VH and VL amino acid sequences of SEQ ID NOs: 69 and 78, respectively), which has known potent superagonist activity. The constructs listed in Table 27, either alone or in combination, were bound to a solid phase and contacted with human PBMCs from two different donors in a plate-based in vitro assay. PBMCs were assayed for T cell activation, and the resulting culture supernatants were assayed for cytokine release.

[0408] [Table 27]

[0409] Various antibody constructs (each at 100 μg / mL in 25 μL of phosphate-buffered saline (PBS)) were dispensed singly or in combination into wells of a 96-well U-bottom plate. Each condition was set up in triplicate or quadruplicate. To immobilize the antibodies to the wells, the plates were left uncovered in a ventilated biological safety cabinet to air-dry overnight. The plates were then washed once with 200 μL of PBS and once more with 200 μL of culture medium (RPMI1640 + 10% heat-inactivated FBS). Frozen human PBMCs from two different donors were thawed in a 37°C water bath, washed with culture medium, and then resuspended in culture medium at 0.25 × 10 6 Resuspended human PBMCs (200 µL, 5 x 10 cells / mL) 4 Cells (containing 1000 cells) were dispensed into each well of the antibody-coated, washed plate. The plate was then incubated at 37°C under 5% CO2 and 95% humidity for 72 hours. Cell pellets and culture supernatants were collected. IL-2 levels in the cell culture supernatant samples were analyzed using a Human Th1 / Th2 Cytokine Cytometry Bead Array (CBA) Kit II (BD, Cat. No. 551809). IL-2 levels measured for one exemplary PBMC donor are shown in Figure 59C. Cultured PBMC pellets were stained with fluorescently labeled antibodies against CD3, CD4, CD8, and CD25 for flow analysis to assess the T cell activation status of CD4 and CD8 T cell populations (CD25 positive percentage). T cell activation of PBMCs from the same exemplary PBMC donor is shown in Figure 59A (CD4+ T cells) and Figure 59B (CD8+ T cells).

[0410] Anti-CD28 W (D28), an IgG4 antibody, potently activated T cells and induced strong IL-2 release even in the absence of CD3 stimulation, whereas anti-CD28 Z (D27), an IgG4 antibody, only weakly activated T cells in the absence of CD3 stimulation and did not induce significant IL-2 release. Both anti-CD28 W IgG4 antibody (D28) and anti-CD28 Z IgG4 antibody (D27) combined with anti-CD3 AA as an IgG4 antibody (D25 + D28 or D25 + D27), or both anti-CD28 W IgG4 antibody (D28) and anti-CD28 Z IgG4 antibody (D27) combined with anti-CD3 AA ScFv-expressing antiviral IgM isotype control with a J chain (D20 + D28 or D20 + D27), demonstrated potent T cell activation and high IL-2 release, in some cases exceeding the quantitation limit of the cytokine readout assay (5000 pg / mL IL-2). Neither anti-CD28 W nor anti-CD28 Z expressed on the J chain of the antiviral isotype control IgM (D22 and D21, respectively) demonstrated significant T cell activation or cytokine release in the absence of CD3 stimulation. Furthermore, cytokine release from D22 and D21 remained low even in the presence of separate anti-CD3 IgG4 molecules (D25 + D21 or D25 + D22) or separate antiviral IgM isotype control molecules expressing anti-CD3 on the J chain (D20 + D21 or D20 + D22). Finally, efficient T cell activation and moderate IL-2 release were observed with trispecific antiviral IgM isotype control molecules coexpressing both anti-CD28 Z or anti-CD28 W and anti-CD3 AA on the J chain (D15 and D23, respectively).

[0411] [Table 28]

[0412] [Table 29] TIFF2026502759000048.tif208157TIFF2026502759000049.tif218157TIFF2026502759000050.tif224156TIFF2026502759000051.tif204158TIFF2026502759000052.tif183157TIFF2026502759000053.tif204157TIFF2026502759000054.tif222158TIFF2026502759000055.tif223158TIFF2026502759000056.tif208157TIFF2026502759000057.tif191157TIFF2026502759000058.tif118158TIFF2026502759000059.tif203158TIFF2026502759000060.tif218157TIFF2026502759000061.tif191157TIFF2026502759000062.tif236164

Claims

1. 1. A multimeric binding molecule comprising five, four, or two bivalent binding units and a modified J chain, each said binding unit comprises two antibody heavy chains; each said antibody heavy chain IgM or IgA heavy chain constant regions or multimerized variants or fragments thereof, each associated with a target antigen-binding domain Including, the modified J chain comprises: (a) a J chain or a functional fragment or variant thereof (“J”); (b) an anti-CD3 scFv (“C”); and (c) a heterologous polypeptide (“H”); H comprises a polypeptide agonist of a T cell costimulatory molecule; the J, the C, and the H are associated as a fusion protein; The multimeric binding molecule.

2. The multimeric binding molecule of claim 1, wherein the T cell costimulatory molecule comprises CD28 or 4-1BB.

3. 3. The multimeric binding molecule of claim 2, wherein H comprises an anti-CD28 antibody or antigen-binding fragment thereof or a 4-1BB ligand ("4-1BBL") trimer.

4. The multimeric binding molecule of claim 1, wherein the modified J chain comprises, from the N-terminus to the C-terminus, CJH or HJC.

5. The multimeric binding molecule of claim 4, wherein the C, the J, and the H are fused via amino acid linkers that may be the same or different.

6. The multimeric binding molecule of claim 3, wherein H comprises an antigen-binding fragment of an anti-CD28 antibody.

7. The antigen-binding fragment of the anti-CD28 antibody is a single-chain Fv (scFv) fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, and the VL comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, and the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 have the amino acid sequences: SEQ ID NO:100, SEQ ID NO:120, SEQ ID NO:127, SEQ ID NO:146, and SEQ ID NO:15, respectively. SEQ ID NO:3, and SEQ ID NO:172; SEQ ID NO:95, SEQ ID NO:105, SEQ ID NO:133, SEQ ID NO:138, SEQ ID NO:164, and SEQ ID NO:165; SEQ ID NO:98, SEQ ID NO:106, SEQ ID NO:130, SEQ ID NO:151, SEQ ID NO:157, and SEQ ID NO:171; SEQ ID NO:98, SEQ ID NO:116, SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:135, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:175; SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:125, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:104, SEQ ID NO:123, SEQ ID NO:137, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:91, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:146, SEQ ID NO:154, and SEQ ID NO:173; SEQ ID NO:101, SEQ ID NO:111, SEQ ID NO:129, SEQ ID NO:139, SEQ ID NO:161, and SEQ ID NO:169; SEQ ID NO:97, SEQ ID NO:112, SEQ ID NO:124, SEQ ID NO:142, SEQ ID NO:163, and SEQ ID NO: No. 166; SEQ ID NO:94, SEQ ID NO:108, SEQ ID NO:132, SEQ ID NO:149, SEQ ID NO:160, and SEQ ID NO:177; SEQ ID NO:93, SEQ ID NO:109, SEQ ID NO:131, SEQ ID NO:148, SEQ ID NO:158, and SEQ ID NO:178; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:147, SEQ ID NO:155, and SEQ ID NO:174; SEQ ID NO:99, SEQ ID NO:117, SEQ ID NO:128, SEQ ID NO:150, SEQ ID NO:159, and SEQ ID NO:176; SEQ ID NO:102, SEQ ID NO:107, SEQ ID NO:136, SEQ ID NO:140, SEQ ID NO:162, and SEQ ID NO:170;7. The multimeric binding molecule of claim 6, comprising: SEQ ID NO: 102, SEQ ID NO: 114, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 162, and SEQ ID NO: 170; SEQ ID NO: 102, SEQ ID NO: 113, SEQ ID NO: 136, SEQ ID NO: 143, SEQ ID NO: 162, and SEQ ID NO: 170; SEQ ID NO: 91, SEQ ID NO: 119, SEQ ID NO: 126, SEQ ID NO: 146, SEQ ID NO: 154, and SEQ ID NO: 173; SEQ ID NO: 92, SEQ ID NO: 121, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 156, and SEQ ID NO: 167; or SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 134, SEQ ID NO: 141, SEQ ID NO: 156, and SEQ ID NO:

168.

8. The multimeric binding molecule of claim 7, wherein the VH and VL of the anti-CD28 scFv comprise amino acid sequences at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences: SEQ ID NOs:61 and 81, SEQ ID NOs:56 and 90, SEQ ID NOs:57 and 86, SEQ ID NOs:58 and 83, SEQ ID NOs:59 and 83, SEQ ID NOs:60 and 82, SEQ ID NOs:60 and 87, SEQ ID NOs:62 and 87, SEQ ID NOs:63 and 85, SEQ ID NOs:64 and 76, SEQ ID NOs:65 and 75, SEQ ID NOs:66 and 89, SEQ ID NOs:67 and 88, SEQ ID NOs:68 and 82, SEQ ID NOs:68 and 87, SEQ ID NOs:69 and 78, SEQ ID NOs:70 and 78, SEQ ID NOs:71 and 80, SEQ ID NOs:72 and 84, SEQ ID NOs:73 and 77, or SEQ ID NOs:74 and 79, respectively.

9. The multimeric binding molecule of claim 7, wherein the VH and the VL are fused via an amino acid linker.

10. The C comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the C comprises VH complementarity determining regions VHCDR1, VHCDR2, and VHCDR3, the VL of the C comprises VL complementarity determining regions VLCDR1, VLCDR2, and VLCDR3, and the VHCDR1, the VHCDR2, the VHCDR3, the VLCDR1, the VLCDR2, and the VLCDR3 are the amino acid sequences: SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31; SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:2 1, SEQ ID NO: 22, and SEQ ID NO: 23; SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 33; SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41; SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 45; SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 49, or SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO:

55.

11. The multimeric binding molecule of claim 10, wherein the VH and VL of C comprise amino acid sequences that are at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:28; SEQ ID NO:16 and SEQ ID NO:20; SEQ ID NO:24 and SEQ ID NO:32; SEQ ID NO:34 and SEQ ID NO:38; SEQ ID NO:42 and SEQ ID NO:44; or SEQ ID NO:46 and SEQ ID NO:48, respectively.

12. The multimeric binding molecule of claim 10, wherein the VH and the VL are fused via an amino acid linker.

13. The linker has the amino acid sequence (GGGGS) ｎ (wherein n is an integer from 2 to 5) (SEQ ID NO: 279), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235). The multimeric binding molecule of any one of claims 5, 9, or 12, comprising:

14. The multimeric binding molecule of claim 1 , wherein J comprises SEQ ID NO: 7 or a functional fragment or variant thereof.

15. 15. The multimeric binding molecule of claim 14, wherein the J comprises a variant J chain having an alanine substitution at an amino acid position corresponding to amino acid Y102 of a mature wild-type human J chain (SEQ ID NO: 7).

16. 16. The multimeric binding molecule of claim 15, wherein J comprises the amino acid sequence: SEQ ID NO: 8 ("J*").

17. The multimeric binding molecule of claim 16, wherein the modified J chain comprises the amino acid sequence: SEQ ID NO: 233, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO:

234.

18. the multimeric binding molecule is an IgM antibody comprising five bivalent binding units; each said bivalent binding unit Two IgM heavy chain constant regions, or multimerized fragments or variants thereof, each comprising an IgM Cμ4 domain and an IgM tailpiece domain. Including, The multimeric binding molecule of claim 1 .

19. 19. The multimeric binding molecule of claim 18, wherein each of the IgM heavy chain constant regions or multimerized fragments or variants thereof further comprises a Cμ1 domain, a Cμ2 domain, a Cμ3 domain, or any combination thereof.

20. The multimeric binding molecule of claim 18, wherein each of the IgM heavy chain constant regions or multimerized fragments or variants thereof is a human IgM constant region comprising the amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 2, or a multimerized variant or fragment thereof.

21. The multimeric binding molecule of any one of claims 1 to 20, wherein each of the target antigen-binding domains is identical and comprises a heavy chain variable region (VH) and a light chain variable region (VL), or a single-domain heavy chain variable region (sdVH).

22. 22. The multimeric binding molecule of claim 21, wherein each of the heavy chains comprises, from N- to C-terminus, the VH fused to the heavy chain constant region or a multimerized fragment or variant thereof.

23. 23. The multimeric binding molecule of claim 22, wherein each of the binding units further comprises two antibody light chains, each of the antibody light chains comprising, from the N-terminus to the C-terminus, the VL fused to a light chain constant region.

24. The multimeric binding molecule of any one of claims 1 to 20, wherein the target antigen is CD38, CD20, CD19, CD22, CD28, B-cell maturation antigen (BCMA), CD123, PD-L1, tumor-associated calcium transducer 2 (TROP-2), mesothelin, Mucl6, prostate-specific membrane antigen (PSMA), or six-transmembrane epithelial antigen of the prostate (STEAP-1).

25. The multimeric binding molecule of any one of claims 1 to 20, which conditionally activates T cells in the presence of the target antigen.

26. The multimeric binding molecule of claim 25, wherein the T cell is a CD4-positive T cell.

27. The multimeric binding molecule of claim 25, wherein the T cell is a CD8-positive T cell.

28. A composition comprising the multimeric binding molecule of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

29. A polynucleotide comprising a nucleic acid sequence encoding the modified J chain and a nucleic acid sequence encoding the antibody heavy chain of the multimeric binding molecule of any one of claims 1 to 20.

30. A vector comprising the polynucleotide of claim 29.

31. 31. A host cell comprising the vector of claim 30, wherein the host cell is capable of expressing the multimeric binding molecule.

32. A method for producing a multimeric binding molecule according to any one of claims 1 to 27, comprising culturing a host cell according to claim 31 and recovering the multimeric binding molecule.

33. A method for treating cancer, comprising administering to a subject in need thereof a multimeric binding molecule of any one of claims 1 to 20.

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