ANTI-ROID ANTIBODY CONSTRUCTIONS

Description

ANTI-ROID ANTIBODY CONSTRUCTIONS Divided from patent application no. BR 11 2020 021269 5, filed on April 18, 2020. 1. CROSS-REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 659,635 filed April 18, 2018, the disclosure of which is incorporated by reference in the present invention in its entirety. 2. LISTING OF SEQUENCES

[0002] This request incorporates, by reference, a sequence listing submitted with this request as an ASCII text file, titled 14529-001228_SEQ_LISTING.txt, created on April 15, 2019, with a size of 336,627 bytes. 3. HISTORY

[0003] The design and therapeutic use of multispecific antibodies—antibody-derived proteins designed to recognize multiple targets—is an area of ​​intensive research. Multispecific antibodies offer the promise of greater therapeutic control than is routinely provided by monospecific monoclonal antibodies. For example, multispecific antibodies can be designed to provide greater target specificity than monospecific antibodies, reducing the untargeted effects associated with many antibody therapies, particularly antibody-based immunotherapies. Multispecific antibodies also offer the promise of therapeutic strategies that are not possible Petition 870260031312, dated 02 / 04 / 2026, page 10 / 417 2 / 285 with monospecific antibodies, such as synergistic targeting of multi-cell receptors, especially in immunotherapy. One such immunotherapy is the use of bispecific antibodies to recruit and redirect T cells to target and kill specific populations of tumor cells through bispecific binding of a T cell marker and a tumor cell marker. For example, targeting a B-cell lymphoma using CD3xCD19 bispecific antibodies, such as Cd3xCD19 BiTE blinatumomab (Blincyto), is described in North American Publication No. 2006 / 0193852.

[0004] There is therefore a need for enhanced multispecific antibodies that bind specifically to distinct cell populations, including tumor cell populations, with improvements including increased affinity or avidity, reduced non-targeted binding and / or reduced unintended immune activation.

[0005] Several tumors can demonstrate cell surface expression of transmembrane receptor tyrosine kinase (ROR) antigens, as described in more detail in Gentile, et al. (Cancer Res; 71(8) April 15, 2011), Rebagay, et al. (Front. Oncol., April 18, 2012), Zhang, et al. (American Journal of Pathology, Vol. 181, No. 6, December 2012), Henry, et al. (Oncotarget, Vol. 6, No. 37 2015), Zhang, et al. (PLoS ONE 7(3): e31127), and Bainbridge, et al. (PLoS ONE 9(7): e102695), each incorporated herein by reference in its entirety. Furthermore, ROR expression may not be present, or may only demonstrate limited expression, in tissue Petition 870260031312, dated 02 / 04 / 2026, page 11 / 417 3 / 285 normal, i.e., non-cancerous, as described in Balakrishnan et al. (Clin Cancer Res. June 15, 2017; 23(12): 3061-3071), incorporated herein in its entirety. Thus, ROR antigens can be used as a tumor-specific marker in certain tumors. Examples of tumors and cancers with demonstrated ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, as described in Gohil et al. (Oncoimmunology. 2017; 6(7): e1326437), incorporated herein in their entirety. Other cancers include, but are not limited to, hematologic cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer. The use of multispecific antibodies for ROR, formatted on various antibody platforms, to target tumors, is described in Gohil, et al., in international application WO 2017 / 053469, in international application WO 2014 / 167022, in US Publication No. 2017 / 0198045, in international application WO 2016 / 094873, in international application WO 2017 / 127499 and in international application WO 2016 / 142768, each of which is incorporated herein by reference in its entirety.

[0006] Thus, ROR antigen-binding molecules have therapeutic potential in cancer treatment. Multispecific ROR-binding molecules that bind to antigens on the surface of T cells, in addition to an ROR antigen, have the potential to cause redirected T cell killing of cancer cells that express ROR. Petition 870260031312, dated 02 / 04 / 2026, page 12 / 417 4 / 285

[0007] There is therefore a need for ROR antigen-binding molecules, including multispecific ROR antigen-binding molecules. There is also a need for ROR antigen-binding molecules that have improvements, including higher affinity or avidity, reduced non-targeted binding and / or unintended immune activation. There is a particular need for a multispecific ROR antigen-binding molecule that has enhanced manufacturing capabilities and is easily purified. 4. SUMMARY

[0008] In one aspect, antigen-binding molecules are provided. In all embodiments, the antigen-binding molecule includes at least one antigen-binding site specific to an antigen of the ROR; thus, the antigen-binding molecules are called ROR antigen-binding molecules.

[0009] Transmembrane receptor tyrosine kinase (ROR) antigen-binding molecules are described in the present invention comprising: A) amino acid sequences of a CDR1, a CDR2 and a CDR3 of a variable light chain (VL) region of an ROR antigen-binding site, wherein the CDR1, CDR2 and CDR3 sequences of the VL are selected from Table 6; and B) comprising amino acid sequences of a CDR1, a CDR2 and a CDR3 of a variable heavy chain (VH) region of the ROR antigen-binding site, wherein the CDR1, CDR2 and CDR3 sequences of the VH are selected from Table 6, wherein the ROR antigen-binding site is a first antigen-binding site and is specific for (i) ROR1 and ROR2, (ii) ROR1 or (iii) ROR2. Petition 870260031312, dated 02 / 04 / 2026, p. 13 / 417 5 / 285 In certain respects, the ROR antigen-binding molecule additionally comprises a second antigen-binding site. In certain respects, the second antigen-binding site is the same as the first antigen-binding site. In certain respects, the second antigen-binding site is specific for a second antigen different from the ROR antigen of the first antigen-binding site. In certain respects, the second antigen is a CD3 antigen.

[0010] The present invention describes transmembrane receptor tyrosine kinase (ROR) antigen-binding molecules comprising: a first and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and wherein the A domain has a VL amino acid sequence, the B domain has a CH3 amino acid sequence, the D domain has a CH2 amino acid sequence, and the E domain has a constant region domain amino acid sequence; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a VH amino acid sequence and the G domain has a CH3 amino acid sequence;and (c) the first and second polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G to form the ROR antigen-binding molecule, wherein the interaction between domain A and domain F; Petition 870260031312, dated 02 / 04 / 2026, page 14 / 417 6 / 285 forms a first antigen-specific binding site for an antigen of the ROR.

[0011] In certain respects, the ROR antigen is ROR1. In certain respects, the ROR antigen is ROR2. In certain respects, the ROR antigen is both ROR1 and ROR2. In certain respects, the ROR antigen is a selected domain from the group consisting of: an ROR1 Frizzle domain, an ROR2 Frizzle domain, an Ig-type ROR1 domain, an Ig-type ROR2 domain, an ROR1 Kringle domain, and an ROR2 Kringle domain. In certain respects, the ROR antigen comprises a human ROR antigen.

[0012] In certain respects, domain A comprises amino acid sequences of a CDR1, a CDR2, and a CDR3 of a variable light chain (VL) region specific to an ROR antigen-binding site, wherein the CDR1, CDR2, and CDR3 sequences of the VL are selected from Table 6, and domain F comprises amino acid sequences of a CDR1, a CDR2, and a CDR3 of a variable heavy chain (VH) region specific to the ROR antigen-binding site, wherein the CDR1, CDR2, and CDR3 sequences of the VH are selected from Table 6. In certain respects, the specific ROR antigen-binding site is I2A-10, I2A-10 D54E Y55Q, or I2A-27 from Table 6.

[0013] In certain respects, domain A comprises a VL with one or two amino acid mutations compared to an antibody VL sequence in Table 6, where the one or two amino acid mutations are in one or more CDR regions in the VL. In certain respects, domain F comprises a VH with one or two amino acid mutations in Petition 870260031312, dated 02 / 04 / 2026, page 15 / 417 7 / 285 comparison with an antibody VH sequence in Table 6, where the one or two amino acid mutations are in one or more CDR regions in the VH. In certain respects, domain A comprises a VL with the I2A-10 VL sequence with one or more mutations in one or more CDR regions. In certain respects, domain A comprises a VL with the I2A-27 VL sequence with one or more mutations in one or more CDR regions. In certain respects, domain F comprises a VL with the I2A-10 VL sequence with one or more mutations in one or more CDR regions. In certain respects, domain F comprises a VL with the I2A-27 VL sequence with one or more mutations in one or more CDR regions.

[0014] In certain respects, the amino acid sequences of the B domain and the G domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0015] In certain respects, the amino acid sequences of the B domain and the G domain are different and comprise separately, and respectively, orthogonal modifications in an endogenous CH3 sequence, in which the B domain interacts with the G domain, and in which neither the B domain nor the G domain interact significantly with a CH3 domain that does not have the orthogonal modification.

[0016] In certain respects, the orthogonal modifications of the B domain and the G domain comprise mutations that generate modified disulfide bridges between the B domain and the G domain. In certain respects, the mutations of the B domain and the G domain that generate modified disulfide bridges are an S354C mutation in one of the B and G domains, and a 349C mutation in the other domain. Petition 870260031312, dated 02 / 04 / 2026, page 16 / 417 8 / 285

[0017] In certain respects, the orthogonal modifications of the B domain and the G domain comprise knob-in-hole mutations. In certain respects, the knob-in-hole mutations are a T366W mutation in one of the B and G domains, and a T366S, L368A, and aY407V mutation in the other domain.

[0018] In certain respects, orthogonal modifications of the B domain and the G domain comprise charge pair mutations. In certain respects, charge pair mutations are a T366K mutation in one of the B domain or the G domain, and an L351D mutation in the other domain.

[0019] In certain aspects, the E domain has a CH3 amino acid sequence.

[0020] In certain respects, the amino acid sequences of the E domain and the K domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0021] In certain respects, the amino acid sequences of the E domain and the K domain are different. In certain respects, the different sequences separately comprise, respectively, orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain interacts significantly with a CH3 domain that does not have the orthogonal modification.

[0022] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between the E domain and the K domain. In certain respects, the mutations that generate the modified disulfide bridges are an S354C mutation in one of the E and K domains, and a 349C mutation in the other domain. Petition 870260031312, dated 02 / 04 / 2026, page 17 / 417 9 / 285

[0023] In certain respects, orthogonal modifications in the E domain and the K domain comprise knob-in-hole mutations. In certain respects, knob-in-hole mutations are a T366W mutation in one of the E domains or the K domain and a T366S, L368A, and aY407V mutation in the other domain.

[0024] In certain respects, orthogonal modifications in the E domain and the K domain comprise charge-pair mutations. In certain respects, charge-pair mutations are a T366K mutation in one of the E domains or the K domain, and a corresponding L351D mutation in the other domain.

[0025] In certain respects, the amino acid sequences of the E domain and the K domain are endogenous sequences of two different antibody domains, which are selected to have a specific interaction that promotes the specific association between the first and third polypeptides. In certain respects, the two different amino acid sequences are a CH1 sequence and a CL sequence.

[0026] In certain respects, the sequence that forms the junction between domain A and domain B is IKRTPREP or IKRTVREP.

[0027] In certain respects, the sequence that forms the junction between domain F and domain G is SSASPREP.

[0028] In certain respects, at least one CH3 amino acid sequence has a tripeptide insertion at the C-terminus connecting the CH3 amino acid sequence to a hinge amino acid sequence, wherein the tripeptide insertion is selected from the group consisting of PGK, KSC, and GEC. Petition 870260031312, dated 02 / 04 / 2026, page 18 / 417 10 / 285

[0029] In certain respects, sequences are human sequences.

[0030] In certain respects, at least one CH3 amino acid sequence is an IgG sequence. In certain respects, IgG sequences are IgG1 sequences.

[0031] In certain respects, at least one CH3 amino acid sequence has one or more isoallotype mutations. In certain respects, the isoallotype mutations are D356E and L358M.

[0032] In certain respects, the CL amino acid sequence is a C-kappa sequence.

[0033] In certain aspects, the CH2 sequences have one or more modified mutations that reduce the effector Fc function. In certain aspects, one or more manipulated mutations are at positions L234, L235, and P329. In certain aspects, one or more manipulated mutations are L234A, L235A, and P329G. In certain aspects, one or more manipulated mutations are L234A, L235A, and P329K.

[0034] Also described in the present invention are transmembrane receptor tyrosine kinase (ROR) antigen-binding molecules comprising: a first, second, third and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has an amino acid sequence of VL, the B domain has an amino acid sequence of CH3, the D domain has an amino acid sequence of CH2, and the E domain has an amino acid sequence of Petition 870260031312, dated 02 / 04 / 2026, page 19 / 417 11 / 285 constant region domain; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a VH amino acid sequence and the G domain has a CH3 amino acid sequence; (c) the third polypeptide chain comprises an H domain, an I domain, a J domain, and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the H domain has a variable region domain amino acid sequence, the I domain has a constant region domain amino acid sequence, the J domain has a CH2 amino acid sequence, and the K domain has a constant region domain amino acid sequence;(d) the fourth polypeptide chain comprises an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a variable region domain amino acid sequence and the M domain has a constant region domain amino acid sequence; (e) the first and second polypeptides are associated through an interaction between the A and F domains, and an interaction between the B and G domains; (f) the third and fourth polypeptides are associated through an interaction between the H and L domains and an interaction between the I and M domains; and (g) the first and third polypeptides are associated through an interaction between the D and J domains and an interaction between the E and K domains to form the ROR antigen-binding molecule, wherein the interaction between the A domain and the F domain forms a first site of; Petition 870260031312, dated 02 / 04 / 2026, page 20 / 417 12 / 285 antigen binding, the interaction between the H domain and the L domain forms a second antigen-binding site, where the first antigen-binding site, the second antigen-binding site, or both the first and second antigen-binding sites are specific for an antigen of the ROR.

[0035] In certain respects, the first antigen-binding site is specific to the ROR antigen. In certain respects, the second antigen-binding site is specific to the ROR antigen. In certain respects, both the first and second antigen-binding sites are specific to the ROR antigen.

[0036] In certain respects, the ROR antigen is ROR1. In certain respects, the ROR antigen is ROR2. In certain respects, the ROR antigen is both ROR1 and ROR2. In certain respects, the ROR antigen is a selected domain from the group consisting of: an ROR1 Frizzle domain, an ROR2 Frizzle domain, an Ig-type ROR1 domain, an Ig-type ROR2 domain, an ROR1 Kringle domain, and an ROR2 Kringle domain. In certain respects, the ROR antigen comprises a human ROR antigen.

[0037] In certain respects, domain A comprises amino acid sequences of a CDR1, a CDR2, and a CDR3 of a variable region of the light chain (VL) specific to an antigen-binding site of the ROR, wherein the sequences of the CDR1, CDR2, and CDR3 of the VL are selected from Table 6, and domain F comprises amino acid sequences of a CDR1, a CDR2, and a CDR3 of a variable region of the heavy chain (VH) specific to the antigen-binding site of the ROR, in Petition 870260031312, dated 02 / 04 / 2026, page 21 / 417 13 / 285 that the CDR1, CDR2, and CDR3 sequences of HV are selected from Table 6. In certain respects, the antigen-binding site of the specific ROR is I2A-10, I2A-10 D54E Y55Q, or I2A-27 from Table 6.

[0038] In certain respects, domain A comprises a VL with one or two amino acid mutations compared to an antibody VL sequence in Table 6, wherein the one or two amino acid mutations are in one or more CDR regions in the VL. In certain respects, domain F comprises a VH with one or two amino acid mutations compared to an antibody VH sequence in Table 6, wherein the one or two amino acid mutations are in one or more CDR regions in the VH. In certain respects, domain A comprises a VL with the I2A-10 VL sequence with one or more mutations in one or more CDR regions. In certain respects, domain A comprises a VL with the I2A-27 VL sequence with one or more mutations in one or more CDR regions. In certain respects, domain F comprises a VL with the I2A-10 VL sequence with one or more mutations in one or more CDR regions.In certain aspects, the F domain comprises a VL with the I2A-27 VL sequence with one or more mutations in one or more CDR regions.

[0039] In certain respects, the second antigen-binding site comprises: A) in the third polypeptide chain, a specific amino acid sequence of the variable region of the light chain (VL) selected from the group consisting of: SEQ ID NO: 69 and SEQ ID NO: 73; and B) in the fourth polypeptide chain, a specific amino acid sequence of the variable region of the heavy chain (VH). Petition 870260031312, dated 02 / 04 / 2026, page 22 / 417 14 / 285 selected from the group consisting of: SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

[0040] In certain respects, the second antigen-binding site comprises: A) in the fourth polypeptide chain, a specific amino acid sequence of the variable region of the light chain (VL) selected from the group consisting of: SEQ ID NO: 69 and SEQ ID NO: 73; and B) in the third polypeptide chain, a specific amino acid sequence of the variable region of the heavy chain (VH) selected from the group consisting of: SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

[0041] In certain respects, the amino acid sequences of the B domain and the G domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0042] In certain respects, the amino acid sequences of the B domain and the G domain are different and comprise separately, and respectively, orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain interact significantly with a CH3 domain that does not have the orthogonal modification.

[0043] In certain respects, the orthogonal modifications of the B domain and the G domain comprise mutations that generate modified disulfide bridges between the B domain and the G domain. In certain respects, the mutations of the B domain and the G domain that generate modified disulfide bridges are an S354C mutation in one of the B and G domains, and a 349C mutation in the other domain. Petition 870260031312, dated 02 / 04 / 2026, page 23 / 417 15 / 285

[0044] In certain respects, the orthogonal modifications of the B domain and the G domain comprise knob-in-hole mutations. In certain respects, the knob-in-hole mutations are a T366W mutation in one of the B and G domains, and a T366S, L368A, and aY407V mutation in the other domain.

[0045] In certain respects, orthogonal modifications of the B domain and the G domain comprise charge pair mutations. In certain respects, charge pair mutations are a T366K mutation in one of the B domain or the G domain, and an L351D mutation in the other domain.

[0046] In certain aspects, the E domain has a CH3 amino acid sequence.

[0047] In certain respects, the amino acid sequences of the E domain and the K domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0048] In certain respects, the amino acid sequences of the E domain and the K domain are different. In certain respects, the different sequences separately comprise, respectively, orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain interacts significantly with a CH3 domain that does not have the orthogonal modification.

[0049] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between the E domain and the K domain. In certain respects, the mutations that generate the modified disulfide bridges are an S354C mutation in one of the E and K domains, and a 349C mutation in the other domain. Petition 870260031312, dated 02 / 04 / 2026, page 24 / 417 16 / 285

[0050] In certain respects, orthogonal modifications in the E domain and the K domain comprise knob-in-hole mutations. In certain respects, knob-in-hole mutations are a T366W mutation in one of the E domains or the K domain and a T366S, L368A, and aY407V mutation in the other domain.

[0051] In certain respects, orthogonal modifications in the E domain and the K domain comprise charge-pair mutations. In certain respects, charge-pair mutations are a T366K mutation in one of the E domains or the K domain, and a corresponding L351D mutation in the other domain.

[0052] In certain respects, the amino acid sequences of the E domain and the K domain are endogenous sequences of two different antibody domains, which are selected to have a specific interaction that promotes the specific association between the first and third polypeptides. In certain respects, the two different amino acid sequences are a CH1 sequence and a CL sequence.

[0053] In certain respects, domain I has a CL sequence and domain M has a CH1 sequence.

[0054] In certain respects, the H domain has a VL sequence and the L domain has a VH sequence.

[0055] In certain respects, the H domain has a VL amino acid sequence; the I domain has a CL amino acid sequence; the K domain has a CH3 amino acid sequence; the L domain has a VH amino acid sequence; and the M domain has a CH1 amino acid sequence.

[0056] In certain aspects, the ROR antigen-binding molecule also comprises: a fifth chain Petition 870260031312, dated 02 / 04 / 2026, page 25 / 417 17 / 285 polypeptide, wherein: (a) the first polypeptide chain further comprises an N domain and an O domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a NOABDE orientation, and wherein the N domain has a variable region domain amino acid sequence, the O domain has a constant region domain amino acid sequence; (b) the ROR antigen-binding molecule additionally comprises a fifth polypeptide chain, comprising: a P domain and a Q domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a PQ orientation, and wherein the P domain has a variable region domain amino acid sequence and the Q domain has a constant region domain amino acid sequence; and (c) the first and fifth polypeptides are associated through an interaction between the N and P domains and an interaction between the O and Q domains to form the ROR antigen-binding molecule.

[0057] In certain respects, (a) the amino acid sequences of the N domain and the A domain are identical, the amino acid sequence of the H domain is different from the sequence of the N domain and the A domain, the amino acid sequences of the O domain and the B domain are identical, the amino acid sequence of the I domain is different from the sequences of the O domain and the B domain, the amino acid sequences of the P domain and the F domain are identical, the amino acid sequence of the L domain is different from the sequences of the P domain and the F domain, the amino acid sequences of the Q domain and the G domain are identical, the amino acid sequence of the M domain is different from the Petition 870260031312, dated 02 / 04 / 2026, page 26 / 417 18 / 285 sequences of the Q domain and the G domain; and (b) wherein the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the interaction between the N domain and the P domain forms a third antigen-specific binding site for the first antigen. In certain respects, the first antigen is the ROR antigen. In certain respects, the second antigen is a CD3 antigen.

[0058] In certain respects, (a) the amino acid sequences of the N domain, the A domain, and the H domain are different, the amino acid sequences of the O domain, the B domain, and the I domain are different, the amino acid sequences of the P domain, the F domain, and the L domain are different, and the amino acid sequences of the Q domain, the G domain, and the M domain are different; (b) the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the N domain and the P domain form a third antigen-specific binding site for a third antigen, and (c) the first, second, or third antigen is the ROR antigen.

[0059] In certain respects, the ROR antigen-binding molecule additionally comprises: a sixth polypeptide chain, wherein: (a) the third polypeptide chain further comprises an R domain and an S domain, wherein the Petition 870260031312, dated 02 / 04 / 2026, page 27 / 417 (a) 19 / 285 domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation, wherein the R domain has a variable region domain amino acid sequence and the S domain has a constant domain amino acid sequence; (b) the ROR antigen-binding molecule further comprises a sixth polypeptide chain, comprising: a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation, wherein the T domain has a variable region domain amino acid sequence and the U domain has a constant domain amino acid sequence. And (c) the third and sixth polypeptides are associated through an interaction between the R and T domains and an interaction between the S and U domains to form the ROR antigen-binding molecule.

[0060] In certain respects, (a) the amino acid sequences of the R domain and the A domain are identical, the amino acid sequence of the H domain is different from the sequence of the R domain and the A domain, the amino acid sequences of the S domain and the B domain are identical, the amino acid sequence of the I domain is different from the sequences of the S domain and the B domain, the amino acid sequences of the T domain and the F domain are identical, the amino acid sequence of the L domain is different from the sequences of the T domain and the F domain, the amino acid sequences of the U domain and the G domain are identical, the amino acid sequence of the M domain is different from the sequences of the U domain and the G domain; and (b) the interaction between the A domain and the F domain forms a first site of Petition 870260031312, dated 02 / 04 / 2026, page 28 / 417 20 / 285 antigen-specific binding for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the interaction between the R domain and the T domain forms a third antigen-specific binding site for the first antigen. In certain respects, the first antigen is the ROR antigen. In certain respects, the second antigen is a CD3 antigen.

[0061] In certain respects, (a) the amino acid sequences of the R domain and the H domain are identical, the amino acid sequence of the A domain is different from the sequence of the R domain and the H domain, the amino acid sequences of the S domain and the I domain are identical, the amino acid sequence of the B domain is different from the sequences of the S domain and the I domain, the amino acid sequences of the T domain and the L domain are identical, the amino acid sequence of the F domain is different from the sequences of the T domain and the L domain, the amino acid sequences of the U domain and the M domain are identical, the amino acid sequence of the G domain is different from the sequences of the U domain and the M domain;(b) the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, the interaction between the H domain and the L domain forms a second antigen-binding site specific to a second antigen, and the interaction between the R domain and the T domain forms a third antigen-binding site specific to the second antigen. In certain respects, the second site of; Petition 870260031312, dated 02 / 04 / 2026, page 29 / 417 21 / 285 antigen binding is specific to the ROR antigen. In certain respects, the first antigen is a CD3 antigen.

[0062] In certain respects, (a) the amino acid sequences of the R domain, the A domain, and the H domain are different, the amino acid sequences of the S domain, the B domain, and the I domain are different, the amino acid sequences of the T domain, the F domain, and the L domain are different, and the amino acid sequences of the U domain, the G domain, and the M domain are different; (b) the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the interaction between the R domain and the T domain forms a third antigen-specific binding site for a third antigen, and (c) the first, second, or third antigen is the ROR antigen.

[0063] In certain respects, the ROR antigen-binding molecule further comprises: a fifth and a sixth polypeptide chain, wherein: (a) the first polypeptide chain further comprises an N domain and an O domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a NOABDE orientation; (b) the third polypeptide chain further comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation; (c) the ROR antigen-binding molecule further comprises a fifth and a sixth polypeptide chain, wherein: the fifth polypeptide chain comprises a P domain and a Q domain, Petition 870260031312, dated 02 / 04 / 2026, page 30 / 417 22 / 285 wherein the domains are arranged, from the N-terminus to the C-terminus, in a PQ orientation, and the sixth polypeptide chain comprises a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation; and (d) the first and fifth polypeptides are associated through an interaction between the N- and P domains and an interaction between the O- and Q domains, and the third and sixth polypeptides are associated through an interaction between the R- and T domains and an interaction between the S- and U domains to form the ROR antigen-binding molecule.

[0064] In certain respects, (a) the amino acid sequences of the N domain and the A domain are identical, the amino acid sequences of the H domain and the R domain are identical, the amino acid sequences of the O domain and the B domain are identical, the amino acid sequences of the I domain and the S domain are identical, the amino acid sequences of the P domain and the F domain are identical, the amino acid sequences of the L domain and the T domain are identical, the amino acid sequences of the Q domain and the G domain are identical, the amino acid sequences of the M domain and the U domain are identical;and (b) wherein the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the N domain and the P domain form a second antigen-specific binding site for the first antigen, the interaction between the H domain and the L domain forms a third antigen-specific binding site for a second antigen, the interaction between the H domain and the L domain forms a third; Petition 870260031312, dated 02 / 04 / 2026, page 31 / 417 23 / 285 antigen-binding site specific to a second antigen, and the interaction between the R domain and the T domain forms a fourth antigen-binding site specific to the second antigen.

[0065] In certain respects, (a) the amino acid sequences of the H domain and the A domain are identical, the amino acid sequences of the N domain and the R domain are identical, the amino acid sequences of the I domain and the B domain are identical, the amino acid sequences of the O domain and the S domain are identical, the amino acid sequences of the L domain and the F domain are identical, the amino acid sequences of the P domain and the T domain are identical, the amino acid sequences of the M domain and the G domain are identical, the amino acid sequences of the Q domain and the U domain are identical;and (b) wherein the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, the N domain and the P domain form a second antigen-binding site specific to a second antigen, the interaction between the H domain and the L domain forms a third antigen-binding site specific to a first antigen, and the interaction between the R domain and the T domain forms a fourth antigen-binding site specific to the second antigen.

[0066] In certain respects, the sequence that forms the junction between domain A and domain B is IKRTPREP or IKRTVREP.

[0067] In certain respects, the sequence that forms the junction between domain F and domain G is SSASPREP. Petition 870260031312, dated 02 / 04 / 2026, page 32 / 417 24 / 285

[0068] In certain respects, at least one CH3 amino acid sequence has a tripeptide insertion at the C-terminus connecting the CH3 amino acid sequence to a hinge amino acid sequence, wherein the tripeptide insertion is selected from the group consisting of PGK, KSC, and GEC.

[0069] In certain respects, sequences are human sequences.

[0070] In certain respects, at least one CH3 amino acid sequence is an IgG sequence. In certain respects, IgG sequences are IgG1 sequences.

[0071] In certain respects, at least one CH3 amino acid sequence has one or more isoallotype mutations. In certain respects, the isoallotype mutations are D356E and L358M.

[0072] In certain respects, the CL amino acid sequence is a C-kappa sequence.

[0073] In certain aspects, the CH2 sequences have one or more modified mutations that reduce the effector Fc function. In certain aspects, one or more manipulated mutations are at positions L234, L235, and P329. In certain aspects, one or more manipulated mutations are L234A, L235A, and P329G. In certain aspects, one or more manipulated mutations are L234A, L235A, and P329K.

[0074] Also described in the present invention are antigen-binding molecules of the transmembrane receptor of protein tyrosine kinase (ROR) comprising: a first antigen-binding site specific for an ROR antigen, wherein the first binding site to Petition 870260031312, dated 02 / 04 / 2026, p. 33 / 417 25 / 285 antigen comprises: A) amino acid sequences of a CDR1, a CDR2 and a CDR3 of a variable light chain (VL) region of a specific ROR antigen-binding site, wherein the CDR1, CDR2 and CDR3 sequences of the VL are selected from Table 6; and B) comprises amino acid sequences of a CDR1, a CDR2 and a CDR3 of a variable heavy chain (VH) region of the ROR antigen-binding site, wherein the CDR1, CDR2 and CDR3 sequences of the VH are selected from Table 6.

[0075] In certain respects, the first antigen-binding site comprises a VL with one or two amino acid mutations compared to an antibody VL sequence in Table 6, wherein the one or two amino acid mutations are in one or more CDR regions in the VL. In certain respects, the first antigen-binding site comprises a VH with one or two amino acid mutations compared to an antibody VH sequence in Table 6, wherein the one or two amino acid mutations are in one or more CDR regions in the VH.

[0076] In certain respects, the first antigen-binding site is specific for ROR1. In certain respects, the first antigen-binding site is specific for ROR2. In certain respects, the first antigen-binding site is specific for both ROR1 and ROR2. In certain respects, the ROR antigen is a selected domain from the group consisting of: an ROR1 Frizzle domain, an ROR2 Frizzle domain, an Ig-like ROR1 domain, an Ig-like ROR2 domain, an ROR1 Kringle domain, and an ROR2 Kringle domain. In Petition 870260031312, dated 02 / 04 / 2026, page 34 / 417 26 / 285 In certain aspects, the ROR antigen comprises a human ROR antigen.

[0077] In certain respects, the ROR antigen-binding molecule additionally comprises a second antigen-binding site. In certain respects, the second antigen-binding site is specific for the ROR antigen. In certain respects, the second antigen-binding site is specific for a second antigen different from the ROR antigen. In certain respects, the second antigen is a CD3 antigen. In certain respects, the antigen-binding site is specific for an epitope of the CD3 antigen. In certain respects, the second antigen-binding site comprises: A) a specific amino acid sequence of the variable region of the light chain (VL) selected from the group consisting of: SEQ ID NO: 69 and SEQ ID NO: 73; and B) a specific amino acid sequence of the variable region of the heavy chain (VH) selected from the group consisting of: SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.In certain respects, the second antigen-binding site comprises: A) a specific amino acid sequence of the variable region of the light chain (VL) selected from the group consisting of: SEQ ID NO: 69 and SEQ ID NO: 73; and B) a specific amino acid sequence of the variable region of the heavy chain (VH) selected from the group consisting of: SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

[0078] In certain respects, the ROR antigen-binding molecule comprises a selected antibody format from the group consisting of: complete antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, diabodies, sc Petition 870260031312, dated 02 / 04 / 2026, page 35 / 417 27 / 285 diabodies, DARTs, tandAbs and minibodies. In certain respects, the ROR antigen-binding molecule comprises: a first and a second polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, wherein the A domain has a variable region domain amino acid sequence, and wherein the B domain, D domain and E domain have a constant region domain amino acid sequence;(b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a variable region domain amino acid sequence and the G domain has a constant region domain amino acid sequence; (c) the first and second polypeptides are associated through an interaction between the A domain and the F domain and an interaction between the B domain and the G domain to form the ROR antigen-binding molecule, and wherein the interaction between the A domain and the F domain forms a first antigen-binding site.

[0079] In certain respects, the ROR antigen-binding molecule additionally comprises: a third and a fourth polypeptide chain, wherein (a) the third polypeptide chain comprises an H domain, an I domain, a J domain and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the H domain has a variable region domain amino acid sequence and the I, J and K domains have a Petition 870260031312, dated 02 / 04 / 2026, page 36 / 417 (a) The 28 / 285 constant region domain amino acid sequence; (b) The fourth polypeptide chain comprises an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a variable region domain amino acid sequence and the M domain has a constant region amino acid sequence; (c) The third and fourth polypeptides are associated through an interaction between the H and L domains and an interaction between the I and M domains; and (d) The first and third polypeptides are associated through an interaction between the D and J domains and an interaction between the E and K domains to form the ROR antigen-binding molecule, and wherein the interaction between the H and L domains forms a second antigen-binding site. In certain respects, the first antigen-binding site is specific for the ROR antigen.In certain aspects, the second antigen-binding site is specific for CD3.

[0080] In certain aspects, the B domain and the G domain have a CH3 amino acid sequence.

[0081] In certain respects, the amino acid sequences of the B domain and the G domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0082] In certain respects, the amino acid sequences of the B domain and the G domain are different and comprise separately, and respectively, orthogonal modifications in an endogenous CH3 sequence, in which the B domain interacts with the G domain, and in which neither the B nor the G domain interacts. Petition 870260031312, dated 02 / 04 / 2026, page 37 / 417 29 / 285 the G domain interacts significantly with a CH3 domain that does not have the orthogonal modification.

[0083] In certain respects, the orthogonal modifications of the B domain and the G domain comprise mutations that generate modified disulfide bridges between the B domain and the G domain. In certain respects, the mutations of the B domain and the G domain that generate modified disulfide bridges are an S354C mutation in one of the B and G domains, and a 349C mutation in the other domain.

[0084] In certain respects, the orthogonal modifications of the B domain and the G domain comprise knob-in-hole mutations. In certain respects, the knob-in-hole mutations of the B domain and the G domain are a T366W mutation in one of the B and G domains, and a T366S, L368A, and aY407V mutation in the other domain.

[0085] In certain respects, orthogonal modifications of the B domain and the G domain comprise charge pair mutations. In certain respects, the charge pair mutations of the B domain and the G domain are a T366K mutation in one of the B or G domains, and an L351D mutation in the other domain.

[0086] In certain respects, the B domain and the G domain have an IgM CH2 amino acid sequence or an IgE CH2 amino acid sequence. In certain respects, the IgM CH2 amino acid sequence or the IgE CH2 amino acid sequence comprises orthogonal modifications.

[0087] In certain respects, domain I has a CL sequence and domain M has a CH1 sequence. In certain respects, domain I has a CH1 sequence and domain M has a CL sequence. In certain respects, each of the Petition 870260031312, dated 02 / 04 / 2026, page 38 / 417 30 / 285 CH1 sequence and CL sequence comprise one or more orthogonal modifications, wherein a domain that has the CH1 sequence does not interact significantly with a domain that has a CL sequence without the orthogonal modification.

[0088] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between at least one CH1 domain and one CL domain, with the selected mutations being from the group consisting of: a modified cysteine ​​at position 138 of the CH1 sequence and at position 116 of the CL sequence; a modified cysteine ​​at position 128 of the CH1 sequence and at position 119 of the CL sequence, and a modified cysteine ​​at position 129 of the CH1 sequence and at position 210 of the CL sequence.

[0089] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between at least one CH1 domain and a CL domain, wherein the mutations comprise modified cysteines at position 128 of the CH1 sequence and at position 118 of a kappa CL sequence.

[0090] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between at least one CH1 domain and one CL domain, the selected mutations from the group consisting of: an F118C mutation in the CL sequence with a corresponding A141C in the CH1 sequence; an F118C mutation in the CL sequence with a corresponding L128C in the CH1 sequence; and S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence. Petition 870260031312, dated 02 / 04 / 2026, page 39 / 417 31 / 285

[0091] In certain respects, orthogonal modifications comprise charge pair mutations between at least one CH1 domain and one CL domain, the charge pair mutations selected from the group consisting of: an F118S mutation in the CL sequence with a corresponding A141L in the CH1 sequence; an F118A mutation in the CL sequence with a corresponding A141L in the CH1 sequence; an F118V mutation in the CL sequence with a corresponding A141L in the CH1 sequence; and a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence.

[0092] In certain respects, orthogonal modifications comprise charge pair mutations between at least one CH1 domain and one CL domain, with the selected charge pair mutations from the group consisting of: an N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence, and an N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence.

[0093] In certain respects, domain A has a VL amino acid sequence and domain F has a VH amino acid sequence. In certain respects, domain A has a VH amino acid sequence and domain F has a VL amino acid sequence.

[0094] In certain respects, the H domain has a VL amino acid sequence and the L domain has a VH amino acid sequence. In certain respects, the H domain has a VH amino acid sequence and the L domain has a VL amino acid sequence.

[0095] In certain aspects, the D domain and the J domain have a CH2 amino acid sequence. Petition 870260031312, dated 02 / 04 / 2026, page 40 / 417 32 / 285

[0096] In certain aspects, the E domain has a CH3 amino acid sequence.

[0097] In certain respects, the amino acid sequences of the E domain and the K domain are identical, wherein the sequence is an endogenous CH3 sequence.

[0098] In certain respects, the amino acid sequences of the E domain and the K domain are different. In certain respects, the different sequences separately and respectively comprise orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain interacts significantly with a CH3 domain that does not have the orthogonal modification.

[0099] In certain respects, orthogonal modifications comprise mutations that generate modified disulfide bridges between the E domain and the K domain. In certain respects, the mutations that generate the modified disulfide bridges are an S354C mutation in one of the E and K domains, and a 349C mutation in the other domain. In certain respects, orthogonal modifications in the E and K domains comprise knob-in-hole mutations. In certain respects, the knob-in-hole mutations are a T366W mutation in one of the E or K domains and a T366S, L368A, and aY407V mutation in the other domain. In certain respects, orthogonal modifications in the E and K domains comprise charge-pair mutations. In certain respects, the charge-pair mutations are a T366K mutation in one of the E or K domains, and a corresponding L351D mutation in the other domain. Petition 870260031312, dated 02 / 04 / 2026, page 41 / 417 33 / 285

[0100] In certain respects, the amino acid sequences of the E domain and the K domain are endogenous sequences of two different antibody domains, which are selected to have a specific interaction that promotes the specific association between the first and third polypeptides. In certain respects, the two different amino acid sequences are a CH1 sequence and a CL sequence.

[0101] In certain respects, the ROR antigen-binding molecule additionally comprises a third antigen-binding site. In certain respects, the third antigen-binding site is specific for an ROR antigen. In certain respects, the first antigen-binding site and the third antigen-binding site are specific for the same ROR antigen. In certain respects, the first antigen-binding site and the third antigen-binding site are specific for different ROR antigens.

[0102] In certain respects, the ROR antigen-binding molecule comprises a fifth polypeptide chain, wherein: (a) the first polypeptide chain further comprises an N domain and an O domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a NO-ABDE orientation, and wherein the N domain has a variable region amino acid sequence, the O domain has a constant region amino acid sequence; (b) the fifth polypeptide chain comprises a P domain and a Q domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a PQ orientation, and wherein the P domain Petition 870260031312, dated 02 / 04 / 2026, page 42 / 417 34 / 285 has a variable region domain amino acid sequence and the Q domain has a constant region amino acid sequence; and (c) the first and fifth polypeptides are associated through an interaction between the N and P domains and an interaction between the O and Q domains to form the ROR antigen-binding molecule.

[0103] In certain respects, (a) the amino acid sequences of the N domain and the A domain are identical, the amino acid sequence of the H domain is different from the sequence of the N domain and the A domain, the amino acid sequences of the O domain and the B domain are identical, the amino acid sequence of the I domain is different from the sequences of the O domain and the B domain, the amino acid sequences of the P domain and the F domain are identical, the amino acid sequence of the L domain is different from the sequences of the P domain and the F domain, the amino acid sequences of the Q domain and the G domain are identical, the amino acid sequence of the M domain is different from the sequences of the Q domain and the G domain;and (b) wherein the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, the interaction between the H domain and the L domain forms a second antigen-binding site specific to a second antigen, and the interaction between the N domain and the P domain forms a third antigen-binding site specific to the first antigen. In certain respects, the first antigen is an ROR antigen. In certain respects, the second antigen is a CD3 antigen. Petition 870260031312, dated 02 / 04 / 2026, page 43 / 417 35 / 285

[0104] In certain respects, (a) the amino acid sequences of the N domain, the A domain, and the H domain are different, the amino acid sequences of the O domain, the B domain, and the I domain are different, the amino acid sequences of the P domain, the F domain, and the L domain are different, and the amino acid sequences of the Q domain, the G domain, and the M domain are different; and (b) the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the interaction between the N domain and the P domain forms a third antigen-specific binding site for a third antigen.

[0105] In certain respects, the ROR antigen-binding molecule comprises a sixth polypeptide chain, wherein: (a) the third polypeptide chain further comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RS-HIJK orientation, and wherein the R domain has a variable region amino acid sequence and the S domain has a constant domain amino acid sequence; (b) the sixth polypeptide chain comprises: a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation, and wherein the T domain has a variable region amino acid sequence and the U domain has a constant domain amino acid sequence; and (c) the third and sixth polypeptides are associated through an interaction between the R and T domains and a Petition 870260031312, dated 02 / 04 / 2026, page 44 / 417 36 / 285 interaction between the S and U domains to form the ROR antigen-binding molecule.

[0106] In certain respects, (a) the amino acid sequences of the R domain and the A domain are identical, the amino acid sequence of the H domain is different from the sequence of the R domain and the A domain, the amino acid sequences of the S domain and the B domain are identical, the amino acid sequence of the I domain is different from the sequences of the S domain and the B domain, the amino acid sequences of the T domain and the F domain are identical, the amino acid sequence of the L domain is different from the sequences of the T domain and the F domain, the amino acid sequences of the U domain and the G domain are identical, the amino acid sequence of the M domain is different from the sequences of the U domain and the G domain;(b) the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, the interaction between the H domain and the L domain forms a second antigen-binding site specific to a second antigen, and the interaction between the R domain and the T domain forms a third antigen-binding site specific to the first antigen. In certain respects, the first antigen is an ROR antigen. In certain respects, the second antigen is a CD3 antigen.

[0107] In certain respects, (a) the amino acid sequences of the R domain and the H domain are identical, the amino acid sequence of the A domain is different from the sequence of the R domain and the H domain, the amino acid sequences of the S domain and the I domain are identical, a Petition 870260031312, dated 02 / 04 / 2026, page 45 / 417 37 / 285 The amino acid sequence of domain B is different from the sequences of domain S and domain I; the amino acid sequences of domain T and domain L are identical; the amino acid sequence of domain F is different from the sequences of domain T and domain L; the amino acid sequences of domain U and domain M are identical; the amino acid sequence of domain G is different from the sequences of domain U and domain M; and (b) the interaction between domain A and domain F forms a first antigen-specific binding site for a first antigen; the interaction between domain H and domain L forms a second antigen-specific binding site for a second antigen; and the interaction between domain R and domain T forms a third antigen-specific binding site for the second antigen. In certain respects, the second antigen is an ROR antigen. In certain respects, the first antigen is a CD3 antigen.

[0108] In certain respects, (a) the amino acid sequences of the R domain, the A domain, and the H domain are different, the amino acid sequences of the S domain, the B domain, and the I domain are different, the amino acid sequences of the T domain, the F domain, and the L domain are different, and the amino acid sequences of the U domain, the G domain, and the M domain are different; and (b) the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the interaction between the R domain and the T domain forms Petition 870260031312, dated 02 / 04 / 2026, pp. 46 / 417 38 / 285 a third antigen-specific binding site for a third antigen.

[0109] Also described in the present invention are purified ROR antigen-binding molecules, wherein the purified ROR antigen-binding molecules comprise any of the ROR antigen-binding molecules described in the present invention. In certain respects, the purified ROR antigen-binding molecules are purified by a purification method comprising a CH1 affinity purification step. In certain respects, the purification method is a single-step purification method.

[0110] Also described in the present invention are pharmaceutical compositions comprising any of the ROR antigen-binding molecules described herein, and a pharmaceutically acceptable diluent.

[0111] Methods for treating an individual with cancer are also described in the present invention, wherein the methods comprise administering a therapeutically effective amount of any of the pharmaceutical compositions described in the present invention. In certain respects, the cancer is selected from the group consisting of: pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematologic cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer. 5. BRIEF DESCRIPTION OF THE DRAWINGS Petition 870260031312, dated 02 / 04 / 2026, page 47 / 417 39 / 285

[0112] Figure 1 shows an alignment of the IgG1 CH3-CH3 CH1-CL dimer pair. The quaternary structures align with an RMSD of ~1.6 Å2.

[0113] Figure 2 presents schematic architectures, with their respective nomenclature conventions, for various binding molecules (also called antibody constructs) described in the present invention.

[0114] Figure 3 presents a higher resolution scheme of the polypeptide chains and their domains, with the respective nomenclature conventions, for the 1x1 bivalent antibody constructs described in the present invention.

[0115] Figure 4 shows the architecture of an exemplary bivalent and monospecific building.

[0116] Figure 5 shows data from a biolayer interferometry (BLI) experiment, described in Example 1, in which a bivalent monospecific binding molecule having the architecture illustrated in Figure 4 [polypeptide 1: VLCH3(Knob)-CH2-CH3 / polypeptide 2: VH-CH3(Hole)] was evaluated. The antigen-binding site was specific for TNFα. The BLI response of the immobilized binding molecule and TNFα binding to the immobilized construct demonstrates robust, specific, and bivalent binding to the antigen. The data are consistent with a molecule with a high percentage of intended polypeptide 1 and polypeptide 2 pairing.

[0117] Figure 6 illustrates the characteristics of an exemplary bivalent 1x1 bispecific bonding molecule, BC1. Petition 870260031312, dated 02 / 04 / 2026, pp. 48 / 417 40 / 285

[0118] Figure 7A shows the size exclusion chromatography (SEC) analysis of BC1, demonstrating that a one-step CH1 affinity purification step (CaptureSelect™ CH1 affinity resin) produces a single, monodisperse peak via gel filtration, where >98% is composed of non-aggregated bivalent protein. Figure 7B shows comparative literature data from the SEC analysis of a CrossMab bivalent antibody construct [data from Schaefer et al. (Proc Natl Acad Sci USA. July 5, 2011; 108(27):11187-92)].

[0119] Figure 8A is a cation exchange chromatography elution profile of “BC1 after a one-step purification using the CH1 CaptureSelect™ affinity resin, showing a single compact peak. Figure 8B is a cation exchange chromatography elution profile of “BC1 after purification using standard protein A purification.

[0120] Figure 9 shows non-reducing SDS-PAGE gels of “BC1 at various purification stages.

[0121] Figures 10A and 10B compare the SDSPAGE gels of “BC1” after one-step affinity purification of CH1 under non-reducing and reducing conditions (Figure 10A) with SDS-PAGE gels of a bivalent bispecific antibody CrossMab under non-reducing and reducing conditions, as published in the referenced literature (Figure 10B).

[0122] Figures 11A and 11B show the mass spectrometry analysis of “BC1, demonstrating two distinct heavy chains (figure 11A) and two distinct light chains (figure 11B), under reducing conditions. Petition 870260031312, dated 02 / 04 / 2026, pp. 49 / 417 41 / 285

[0123] Figure 12 presents a mass spectrometry analysis of purified BC1 under non-reducing conditions, confirming the absence of incomplete pairing after purification.

[0124] Figure 13 presents the accelerated stability test data, demonstrating the stability of BC1” for 8 weeks at 40 °C, compared with two IgG control antibodies.

[0125] Figure 14 illustrates features of an exemplary bivalent 1x1 bonding molecule, BC6, further described in Example 3.

[0126] Figure 15A shows the size exclusion chromatography (SEC) analysis of BC6” after one-step purification using the CaptureSelect™ CH1 affinity resin, demonstrating that one-step CH1 affinity purification produces a single monodisperse peak and the absence of non-covalent aggregates. Figure 15B shows an SDS-PAGE gel of BC6” under non-reducing conditions.

[0127] Figure 16 illustrates features of an exemplary bivalent bispecific bonding molecule, BC28”, further described in Example 4.

[0128] Figure 17 shows the SDS-PAGE analysis under non-reducing conditions after affinity purification of CH1 in a step of BC28”, BC29”, BC30”, BC31” and BC32”.

[0129] Figure 18 shows the SEC analysis of BC28” and BC30”, each after one-step purification using the CaptureSelect™ CH1 affinity resin. Petition 870260031312, dated 02 / 04 / 2026, page 50 / 417 42 / 285

[0130] Figure 19 illustrates features of an exemplary bivalent bispecific bonding molecule, BC44, further described in Example 5.

[0131] Figures 20A and 20B show size exclusion chromatography (SEC) data of two bivalent bonding molecules, BC15” and BC16”, respectively, under accelerated stability test conditions. BC15” and BC16” have different variable junctions of the CH3 region.

[0132] Figure 21 presents a scheme of five polypeptide chains and their domains, with the respective nomenclature conventions, for the 2x1 trivalent antibody constructs described herein, in which, according to the nomenclature convention, chain 5 is called “polypeptide chain 5” in the schematic representation.

[0133] Figure 22 illustrates the characteristics of an exemplary trivalent 2x1 bispecific bonding molecule, BC1-2x1”, further described in example 7.

[0134] Figure 23 shows the non-reducing SDS-PAGE of BC1” and the BC1-2x1” protein expressed using the ThermoFisher Expi293 transient transfection system, at various purification stages.

[0135] Figure 24 compares the avidity of the bivalent 1x1 BC1” construct with the avidity of the trivalent 2x1 BC1-2x1” construct using an Octet biolayer interferometry analysis (Pall ForteBio).

[0136] Figure 25 illustrates important features of a 2x1 trivalent construction, TB111”.

[0137] Figure 26 presents a diagram of five polypeptide chains and their domains, with their respective Petition 870260031312, dated 02 / 04 / 2026, page 51 / 417 43 / 285 nomenclature conventions, for the 1x2 trivalent antibody constructs described herein, wherein, according to the nomenclature convention, chain 5 is called the 6th polypeptide chain in the schematic representation.

[0138] Figure 27 illustrates features of an exemplary trivalent construction 1x2 CTLA4-4 x Nivo x CTLA44, further described in example 10.

[0139] Figure 28 is an SDS-PAGE gel in which the lines showing the trivalent 1x2 CTLA4-4 x Nivo x CTLA4-4 construction under non-reducing (-DTT) and reducing (+DTT) conditions are contained within the rectangle.

[0140] Figure 29 shows a comparison of antigen binding between two antibodies: the bivalent 1x1 CTLA4-4 x OX40-8 construct and the trivalent 1x2 CTLA4-4 x Nivo x CTLA4-4 construct. CTLA4-4 x OX40-8 binds to CTLA4 monovalently, while CTLA4-4 x Nivo x CTLA4-4 binds to CTLA4 bivalently.

[0141] Figure 30 illustrates features of an exemplary tri-specific 1x2 trivalent construction, BC281x1x1a, further described in example 11.

[0142] Figure 31 shows the size exclusion chromatography of BC28-1x1x1a after transient expression and purification of the CH1 affinity resin in a single step, showing a single well-defined peak.

[0143] Figure 32 shows the SDS-PAGE results with bivalent and trivalent constructs, each after transient expression and one-step purification using the CH1 CAPTURESelect™ affinity resin, under non-reducing and reducing conditions, as described in Example 12. Petition 870260031312, dated 02 / 04 / 2026, p. 52 / 417 44 / 285

[0144] Figures 33A to 33C show Octet binding analyses for 3 antigens: PD1, antigen A, and CTLA4. As further described in Example 13, Figure 33A shows the binding of BC1 to PD1 and antigen A; Figure 33B shows the binding of a bivalent bispecific construct CTLA4-4 x OX40-8 to CTLA4, antigen A, and PD1; Figure 33C shows the binding of the trivalent trispecific BC28-1x1x1a to PD1, antigen A, and CTLA4.

[0145] Figure 34 presents a schematic representation of six polypeptide chains and their domains, with respective nomenclature conventions, for certain 2x2 tetravalent constructions described in the present invention.

[0146] Figure 35 illustrates certain relevant features of the exemplary tetravalent 2x2 construction, BC222x2, further described in example 14.

[0147] Figure 36 is a non-reducing SDS-PAGE gel comparing the tetravalent 2x2 BC22-2x2 construction with a trivalent 1x2 BC12-1x2 construction and a trivalent 2x1 BC21-2x1 construction at different purification stages.

[0148] Figure 37 provides the architecture for an exemplary 2x2 tetravalent construction.

[0149] Figure 38 presents a schematic representation of six polypeptide chains and their domains, with the respective nomenclature conventions, for certain tetravalent constructions described in the present invention, wherein, according to the nomenclature convention, chain 5 Petition 870260031312, dated 02 / 04 / 2026, page 53 / 417 In the schematic representation, 45 / 285 is called the 7th polypeptide chain and chain 6 is called the 8th polypeptide chain.

[0150] Figure 39 provides an exemplary architecture of a two-specific tetravalent construction.

[0151] Figure 40 provides an exemplary architecture for a tetravalent trispecific construction using a common light chain strategy.

[0152] Figure 41 shows the bispecific antigen binding by the tetravalent construct schematized in Figure 39, demonstrating that this construct was capable of simultaneous binding. The biolayer interferometry (BLI) responses of B-body immobilization and TNFα binding to the immobilized construct are consistent with a molecule with a high percentage of intended chain pairing.

[0153] Figure 42 provides a flow cytometry analysis of B-body binding to cell surface antigen. The crosshatch signal indicates antigen-free cells; the dotted signal indicates cells transiently transfected with the surface antigen.

[0154] Figure 43 provides an exemplary architecture of a trivalent construction.

[0155] Figure 44 provides an exemplary architecture of a trivalent construction.

[0156] Figure 45 shows the SDS-PAGE results with bivalent and trivalent constructs, each after transient expression and one-step purification using the CH1 CaptureSelect™ affinity resin, under non-reducing and reducing conditions, as described in Example 17. Petition 870260031312, dated 02 / 04 / 2026, page 54 / 417 46 / 285

[0157] Figure 46 shows the differences in thermal transitions for BC24jv, BC26jv and BC28jv measured to assess the pairing stability of the junctional variants.

[0158] Figure 47 shows the Octet biolayer interferometry analysis (Pall ForteBio) of a two-fold serial dilution (200 to 12.5 nM) used to determine the CD3 binding affinity for a non-mutated monovalent SP34-89 B-body.

[0159] Figures 48A to 48B demonstrate the Octet biolayer interferometry analysis (Pall ForteBio) of a two-fold serial dilution (200 to 12.5 nM) used to determine the ROR1 binding affinity for two ROR antigen binding site candidates (Figure 48A, clone I2-A10; Figure 48B, clone I2-A27).

[0160] Figure 49 shows that the bispecific 1x1 and 1x2 B-bodies for RORxCD3 resulted in the activation of reporter T cells when mixed with tumor cell lines expressing ROR1 (HOP-92), but no activation when mixed with tumor cell lines that did not express ROR1 (B16).

[0161] Figure 50 shows that the bispecific rorxCD3 1x2 I2A-10 B-body resulted in cytotoxic T cell-mediated killing when mixed with ROR1-expressing tumor cell lines (MDA-MD-231), but did not result in cytotoxicity when a bispecific CD3 B-body with an ABS-irrelevant tumor antigen (e.g., a tumor antigen not expressed in MDA-MD-231) was added to the mixture. Petition 870260031312, dated 02 / 04 / 2026, page 55 / 417 47 / 285

[0162] Figures 51A to 51E show that the bispecific 1x1 and 1x2 B-bodies for RORxCD3 resulted in the activation of reporter T cells when mixed with tumor cell lines expressing ROR1 HOP-92 (Figure 51A), A549 (Figure 51B), MDA-MD-231 (Figure 51C), JeKo-1 (Figure 51D), and RPMI8226 (Figure 51E), but not in activation when mixed with tumor cell lines that do not express ROR1 (B16).

[0163] Figure 52 shows that the bispecific 1x2 B bodies of RORxCD3 I2A-1, I2A-3, I2A-10, I2A-14, I2A22 and I2A-27 resulted in cytotoxic T cell-mediated killing when mixed with ROR1-expressing tumor cell lines (MDA-MD-231), but the 1x2 B bodies I2A-16 and I2A-22 did not result in potent cytotoxicity.

[0164] Figure 53A illustrates the published ROR1 expression data for the MDA-MD-231 and RPMI8226 tumor cell lines. Figures 53B and 53C demonstrate that cytotoxicity efficacy correlates with ROR1 in the MDA-MD-231 and RPMI-8226 tumor cell lines.

[0165] Figures 54A to 54F show that B-bodies I2A-10 and I2A-27 activated CD8+ T cells in a PBMC population, as determined by the quantification of CD25 (Figure 54A), CD69 (Figure 54C), and both CD25 and CD69 (Figure 54E), as well as activating CD4+ T cells in a PBMC population, as determined by the quantification of CD25 (Figure 54A), CD69 (Figure 54D), and both CD25 and CD69 (Figure 54F).

[0166] Figure 55 shows that 26% and 36% of candidates I2A-10 (top panel) and I2A-27 (panel Petition 870260031312, dated 02 / 04 / 2026, page 56 / 417 48 / 285 lower) were internalized after a 2-hour incubation period with MDA-MB-231 cells, respectively.

[0167] Figure 56 shows the size exclusion chromatography (SEC) analysis, demonstrating that a single affinity purification step of CH1 produces single, monodisperse peaks by gel filtration, where > 98% is non-aggregated protein for the Bbody 1x2 candidates I2A-10 (top panel) and I2A-27 (bottom panel).

[0168] Figure 57A shows non-reducing SDS-PAGE gels of the 1x2 B-body candidates I2A-10 (left panel) and I2A-27 (right panel), demonstrating a large band of fully assembled constructs (high migration 250 kDa band).

[0169] Figure 57B shows the analysis with the bioanalyzer (Agilent) of unreduced samples for the ts a B-body 1x2 I2A-10 and I2A-27, indicating a large band of fully assembled constructs.

[0170] Figure 58 shows the SDS-PAGE analysis of bispecific antibodies comprising standard knob-hole orthogonal mutations introduced into the CH3 domains found in their native positions within the Fc portion of the bispecific bivalent antibody that were purified using a one-step CH1 affinity purification step (CaptureSelect™ CH1 affinity resin).

[0171] Figures 59A and 59B show biolayer interferometry analysis on Octet (Pall ForteBio) demonstrating the binding of FcyRIa to trastuzumab (Figure 59A, “WT IgG1), but not to sFc10 (Figure 59B). Petition 870260031312, dated 02 / 04 / 2026, page 57 / 417 49 / 285

[0172] Figure 60 shows death by trastuzumab (herceptin, “wild-type IgG1”), but not by sFc7 or sFc10 in an ADCC assay.

[0173] Figure 61 shows the binding of C1q by trastuzumab (herceptin, “wild-type IgG1”) but not by sFc1, sFc7 or sFc10 in a C1q ELISA.

[0174] Figures 62A to 62C show the monitored tumor volume for mice grafted with tumor cells, humanized with PBMCs (left solid arrow), and then treated IV (right dashed arrow) with PBS (figure 62A), B-body candidate 1x2 I2-A10 (figure 62B), or B-body candidate 1x2 I2-A27 (figure 62C).

[0175] Figure 63 shows tumor volume at the conclusion of the study for each of the mice, with mean and standard deviation for each group shown. The open square for group I2-A27 was removed from the analysis due to probable non-humanization by PMBCs.

[0176] Figure 64A shows the binding of SP34-89 to cynologous and Jurkat T cells. Figure 64B shows the binding of a bispecific antibody B-body™ 1x2 I2-A27 to the delta and epsilon heterodimer of cynologous CD3.

[0177] Figure 65A shows the binding of an IgG I2-A27 antibody to ROR1 in a monovalent binding assay. Figure 65B shows the minimum binding of an IgG I2A27 antibody to ROR2. Figure 65C shows the binding of a B-body™ 1x2 I2-A27 to ROR1 in a monovalent binding assay. Figure 65D shows the minimum binding of a B-body™ 1x2 I2-A27 to ROR2. Figure 65E shows the binding of a B-body™ 1x2 I2-A27 to ROR1 in a bivalent binding assay. Petition 870260031312, dated 02 / 04 / 2026, page 58 / 417 50 / 285

[0178] Figure 66A shows the SEC analysis of a 1x2 I2-A27 B-body™. Figure 66B shows the SMAC analysis of a 1x2 I2-A27 B-body™. Figure 66C shows the CIH analysis of a 1x2 I2-A27 B-body™.

[0179] Figure 67A shows the activity of a B-body™ 1x2 I2-A27 in a Jurkat assay with MDA-MB-231 cells (expressing ROR1). Figure 67B shows the activity of a B-body™ 1x2 I2-A27 in a Jurkat assay with RPMI8226 cells (expressing ROR1 and ROR2). Figure 67C shows the inactivity of a B-body™ 1x2 I2-A27 in a Jurkat assay with K562 cells (expressing ROR2). Figure 67D shows the inactivity of a B-body™ 1x2 I2-A27 in a Jurkat assay in the absence of a target cell line.

[0180] Figure 68A shows the expression of CD69 with various concentrations of a B-body™ 1x2 I2-A27 and MDAMB-231 cells expressing ROR1. Figure 68B shows the expression of CD69 with various concentrations of a B-body™ 1x2 I2-A27 and RPMI-8226 cells expressing ROR1. Figure 68C shows the expression of CD25 with various concentrations of a B-body™ 1x2 I2-A27 and MDA-MB-231 cells expressing ROR1. Figure 68D shows the expression of CD25 with various concentrations of a B-body™ 1x2 I2-A27 and RPMI-8226 cells expressing ROR1.

[0181] Figure 69A shows LDH release with various concentrations of a 1x2 I2-A27 B-body™ and MDAMB-231 cells expressing ROR1. Figure 69B shows LDH release with various concentrations of a 1x2 I2-A27 B-body™ and RPMI-8226 cells expressing ROR1.

[0182] Figure 70A shows granzyme B with various concentrations of a B-body™ 1x2 I2-A27 and MDA-MB-231 cells. Petition 870260031312, dated 02 / 04 / 2026, page 59 / 417 Figure 70B shows granzyme B with various concentrations of a B-body™ 1x2 I2-A27 and RPMI-8226 cells expressing ROR1. Figure 70C shows TNFα secretion with various concentrations of a B-body™ 1x2 I2-A27 and MDAMB-231 cells expressing ROR1. Figure 70D shows TNFα secretion with various concentrations of a B-body™ 1x2 I2-A27 and RPMI-8226 cells expressing ROR1. Figure 70E shows IFNγ release with various concentrations of a B-body™ 1x2 I2-A27 and MDA-MB-231 cells expressing ROR1. Figure 70F shows the release of IFNγ with various concentrations of a Bbody™ 1x2 I2-A27 and RPMI-8226 cells expressing ROR1.

[0183] Figure 71A shows the activity for B-body™ 1x2 I2-A27 samples stored in human serum at 4°C or 37°C for 1 week. Figure 71B shows the inactivity of the samples in a Jurkat assay in the absence of ROR1-expressing cells.

[0184] Figure 72 shows stability tests on B-body™ 1x2 I2-A27 samples under accelerated conditions.

[0185] Figure 73 shows stability tests on B-body™ 1x2 I2-A27 samples under real-time conditions.

[0186] Figure 74 shows acid stability tests of a B-body™ 1x2 I2-A27.

[0187] Figure 75 shows the attachment of a B-body™ 1x2 I2-A10 D54E Y55Q to the cynological delta and epsilon CD3 heterodimer.

[0188] Figure 76A shows the binding of an IgG I2-A10 D54E Y55Q antibody to ROR1 in a monovalent binding assay. Figure 76B shows the binding of an IgG I2-A10 D54E Y55Q antibody to ROR2 in a monovalent binding assay. A Petition 870260031312, dated 02 / 04 / 2026, page 60 / 417 Figure 76C shows the bonding of a B-body™ 1x2 I2-A10 D54E Y55Q to ROR1 in a monovalent bonding test. Figure 76D shows the bonding of a B-body™ 1x2 I2-A10 D54E Y55Q to ROR2 in a monovalent bonding test. Figure 76E shows the bonding of a B-body™ 1x2 I2-A10 D54E Y55Q to ROR1 in a bivalent bonding test. Figure 76F shows the bonding of a B-body™ 1x2 I2-A10 D54E Y55Q to ROR2 in a bivalent bonding test.

[0189] Figure 77 shows the binding of a B-body™ 1x2 I2-A10 D54E Y55Q to the Ig-like domain of ROR1.

[0190] Figure 78A shows the SEC analysis of a 1x2 I2-A10 D54E Y55Q B-body™. Figure 78B shows the SMAC analysis of a 1x2 I2-A10 D54E Y55Q B-body™. Figure 78C shows the CIH analysis of a 1x2 I2-A10 D54E Y55Q B-body™.

[0191] Figure 79A shows the activity of a B-body™ 1x2 I2-A10 D54E Y55Q in a Jurkat assay with MDAMB-231 cells (expressing ROR1). Figure 79B shows the activity of a B-body™ 1x2 I2-A10 D54E Y55Q in a Jurkat assay with RPMI-8226 cells (expressing ROR1 and ROR2). Figure 79C shows the activity of a B-body™ 1x2 I2-A10 D54E Y55Q with K562 cells (expressing ROR2). Figure 79D shows the inactivity of a B-body™ 1x2 I2-A10 in a Jurkat assay in the absence of a target cell line.

[0192] Figure 80A shows the expression of CD69 at various concentrations in a B-body™ 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1. Figure 80B shows the expression of CD69 at various concentrations in a B-body™ 1x2 I2-A10 D54E Y55Q and RPMI-8226 cells expressing ROR1 and ROR2. Figure 80C shows the expression of CD25 at various Petition 870260031312, dated 02 / 04 / 2026, page 61 / 417 Figure 80D shows the expression of CD25 at various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1.

[0193] Figure 81A shows LDH release at various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1. Figure 81B shows LDH release at various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and RPMI-8226 cells expressing ROR1 and ROR2.

[0194] Figure 82A shows granzyme B with various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1. Figure 82B shows granzyme B with various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and RPMI-8226 cells expressing ROR1 and ROR2. Figure 82C shows TNFα secretion with various concentrations of a B-body 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1. Figure 82D shows TNFα secretion with various concentrations of a B-body™ 1x2 I2-A10 D54E Y55Q and RPMI-8226 cells expressing ROR1 and ROR2. Figure 82E shows IFNr secretion at various concentrations from a B-body™ 1x2 I2-A10 D54E Y55Q and MDA-MB-231 cells expressing ROR1. Figure 82F shows IFNr secretion at various concentrations from a B-body™ 1x2 I2-A10 D54E Y55Q and RPMI-8226 cells expressing ROR1 and ROR2.

[0195] Figure 83A shows the activity of B-body™ 1x2 I2-A10 D54E Y55Q samples stored in human serum at 4°C or 37°C for 1 week. Figure 83B shows the inactivity of the samples in a Jurkat assay in the absence of ROR1-expressing cells. Petition 870260031312, dated 02 / 04 / 2026, page 62 / 417 54 / 285

[0196] Figure 84 shows stability tests of B-body™ 1x2 I2-A10 D54E Y55Q samples under accelerated conditions.

[0197] Figure 85 shows stability tests of B-body™ 1x2 I2-A10 D54E Y55Q samples under real-time conditions.

[0198] Figure 86 shows acid stability tests of a B-body™ 1x2 I2-A10 D54E Y55Q.

[0199] Figure 87 shows the in vivo efficacy of a B-body™ 1x2 I2-27 and a B-body™ 1x2 I2-A10 D54E Y55Q in reducing tumor volume (mm3) in a mouse model for cancer study.

[0200] Figure 88 shows the in vivo efficacy of multiple doses of a B-body™ 1x2 I2-27 and a B-body™ 1x2 I2-A10 D54E Y55Q in reducing tumor size (mm3) in a mouse model for cancer study.

[0201] The figures represent various embodiments of the present invention for illustrative purposes only. A person skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein can be employed without departing from the principles of the invention described herein. 6. Detailed description 6.1. Definitions

[0202] Unless defined otherwise, all technical and scientific terms used in the present invention have the meaning commonly understood by a person skilled in the art, to whom this invention pertains. Petition 870260031312, dated 02 / 04 / 2026, page 63 / 417 55 / 285 As used in this document, the following terms have the meanings assigned to them below.

[0203] “Antigen-binding site” means a region of a ROR-binding molecule that specifically recognizes or binds to a particular antigen or epitope.

[0204] “B-Body”, as used in the present invention and with reference to Figure 3, refers to linking molecules comprising the attributes of a first and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an AB-DE orientation, and wherein the A domain has a VL amino acid sequence, the B domain has a CH3 amino acid sequence, the D domain has a CH2 amino acid sequence and the E domain has a constant region domain amino acid sequence; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a VH amino acid sequence and the G domain has a CH3 amino acid sequence;(c) the first and second polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G to form the linker molecule. The B-bodies are described in more detail in International Patent Application No. PCT / US2017 / 057268, which is incorporated herein by reference in its entirety. Petition 870260031312, dated 02 / 04 / 2026, page 64 / 417 56 / 285

[0205] As used in the present invention, the terms treat or treatment refer to both therapeutic treatment and prophylactic or preventive measures, where the objective is to prevent or mitigate (reduce) an undesirable physiological alteration or disorder, such as the progression of multiple sclerosis, arthritis, or cancer. Desired beneficial or clinical outcomes include, but are not limited to, symptom relief, reduction in disease extent, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), whether detectable or undetectable. Treatment may also mean prolonging survival compared to the expected survival if no treatment is received.People who need treatment include those who already have the condition or disorder, as well as those who are prone to having the condition or disorder, or those in whom the condition or disorder should be avoided.

[0206] By subject, individual, animal, patient or mammal, is meant any individual, particularly a mammal, for whom diagnosis, prognosis or therapy is desired. Mammalian individuals include humans, domestic animals, farm animals and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows and so forth.

[0207] The term sufficient quantity means a quantity sufficient to produce a desired effect, by Petition 870260031312, dated 02 / 04 / 2026, page 65 / 417 57 / 285 for example, a sufficient quantity to modulate protein aggregation in a cell.

[0208] The term “therapeutically effective amount” is an amount that is effective in improving a symptom of a disease. A therapeutically effective amount may be a “prophylactically effective amount,” since prophylaxis may be considered therapy. 6.2. Other conventions of interpretation

[0209] Unless otherwise specified, all references to sequences in the present invention are to amino acid sequences.

[0210] Unless otherwise specified, the numbering of antibody constant region residues conforms to the index I, as described in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html #refs (accessed August 22, 2017) and in Edelman et al., Proc. Natl. Acad. USA, 63:78-85 (1969), which are incorporated by reference in their entirety, and identifies the residue according to its location in an endogenous constant region sequence, regardless of the physical location of the residue within a chain of ROR-binding molecules described in the present invention. By “endogenous sequence” or “native sequence” is meant any sequence, including nucleic acid and amino acid sequences, that originates from an organism, tissue, or cell and has not been artificially modified or mutated.

[0211] The numbers of the polypeptide chain (e.g., a “first” polypeptide chain, a “second” polypeptide chain, etc., or “chain 1”, “chain 2” Petition 870260031312, dated 02 / 04 / 2026, page 66 / 417 58 / 285 polypeptide etc.) are used in the present invention as a unique identifier for specific polypeptide chains that form a linking molecule and are not intended to denote the order or quantity of the different polypeptide chains in the linking molecule.

[0212] In this disclosure, comprehends, including, containing, having, includes, and linguistic variants thereof have the meaning ascribed to them in U.S. patent law, allowing for the presence of additional components beyond those explicitly cited.

[0213] It is understood that the ranges given here are abbreviations for all values ​​within the range, including for the extreme points of the range mentioned. For example, a range from 1 to 50 is understood to include any number, combination of numbers, or sub-ranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0214] Unless specifically stated or evident from the context, the term "or," as used herein, is interpreted as inclusive. Unless specifically stated or evident from the context, as used in the present invention, the terms "a," "an," and "the" are interpreted as being singular or plural.

[0215] Unless specifically stated or evident from the context, as used in Petition 870260031312, dated 02 / 04 / 2026, page 67 / 417 59 / 285 of the present invention, the term “about” is understood to mean being within a normal tolerance range in the art, such as, for example, within 2 standard deviations from the mean. About can be understood to mean being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless clear from the context, all numerical values ​​given herein are modified by the term about. 6.3. Antigen-binding molecules of the ROR

[0216] In one aspect, antigen-binding molecules are provided. In all embodiments, the antigen-binding molecule includes at least one primary antigen-binding site specific to an ROR antigen; thus, the binding molecules are called ROR antigen-binding molecules.

[0217] The ROR-binding molecules described in the present invention bind specifically to ROR antigens.

[0218] As used in the present invention, “ROR antigens” refers to members of the transmembrane receptor tyrosine kinase (ROR) family, including ROR1 and ROR2 members. In certain embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically only to ROR1. In other embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically only to ROR2. In still other embodiments, the ROR-binding molecule has antigen-binding sites that are Petition 870260031312, dated 02 / 04 / 2026, p. 68 / 417 60 / 285 cross-reactive and bind specifically to both ROR1 and ROR2.

[0219] ROR1 and ROR2 proteins typically consist of at least four protein domains: three extracellular domains—Ig-like, FZ, and kringle domains—as well as the intracellular protein kinase domain. In some embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically to the extracellular portion of the ROR antigen. In certain embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically only to the Ig-like domain. In other embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically to the FZ domain. In still other embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically to the kringle domain. In particular embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically to at least one portion of a single ROR domain.In particular embodiments, the ROR-binding molecule possesses antigen-binding sites that bind specifically to at least one portion of more than one ROR domain, such as the junction between a first and a second ROR domain. ROR domains may refer to ROR1 domains or ROR2 domains.

[0220] In specific embodiments, the ROR antigen is human. Uniprot accession number Q01973 describes a canonical human ROR1 protein, including its sequences and domain specificities, and is incorporated by reference. Petition 870260031312, dated 02 / 04 / 2026, page 69 / 417 61 / 285 in its entirety. SEQ ID NO: 94 provides the complete protein sequence of ROR1. With reference to the complete sequence from the N-terminus to the C-terminus, the Ig-type domain is defined as amino acids 42 to 147, the FZ domain as amino acids 165 to 299, and the kringle domain as amino acids 312 to 391. Uniprot accession number Q01974 describes a canonical human ROR2 protein, including its sequences and domain specificities, and is incorporated by reference in its entirety. SEQ ID NO: 95 provides the complete protein sequence of ROR2. With reference to the complete sequence from the N-terminus to the C-terminus, the Ig-type domain is defined as amino acids 55 to 145, the FZ domain as amino acids 169 to 303, and the kringle domain as amino acids 316 to 394.

[0221] Several tumors can demonstrate cell surface expression of ROR antigens, as described in more detail in Gentile, et al. (Cancer Res; 71(8) April 15, 2011), Rebagay, et al. (Front. Oncol., April 18, 2012), Zhang, et al. (American Journal of Pathology, Vol. 181, No. 6, December 2012), Henry, et al. (Oncotarget, Vol. 6, No. 37 2015), Zhang, et al. (PLoS ONE 7(3): e31127.), and Bainbridge, et al. (PLoS ONE 9(7): e102695.), each incorporated herein by reference in its entirety. Furthermore, ROR expression may not be achieved, or may only show limited expression, in normal, i.e., non-cancerous tissue, as described in Balakrishnan et al. (Clin Cancer Res. June 15, 2017; 23(12): 3061-3071), which is incorporated herein in its entirety. Thus, ROR antigens can be used as a tumor-specific marker in Petition 870260031312, dated 02 / 04 / 2026, page 70 / 417 62 / 285 certain tumors. Examples of tumors and cancers with demonstrated ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, as described in Gohil et al. (Oncoimmunology. 2017; 6(7): e1326437.), herein incorporated in their entirety. Other cancers include, but are not limited to, hematologic cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer.

[0222] In several embodiments, the ROR-binding molecule binds specifically to at least one antigen in addition to an ROR antigen.

[0223] In one specific embodiment, the ROR-binding molecule is a bispecific bivalent molecule. In another embodiment, the ROR-binding molecule is a bispecific trivalent molecule. In particular embodiments, the ROR-binding molecule has antigen-binding sites that bind specifically to the ROR antigen and to a molecule expressed on the surface of T cells. In one specific embodiment, the ROR-binding molecule has antigen-binding sites that bind specifically to the ROR antigen and to a CD3 protein expressed on the surface of T cells. Without wishing to delve into the theory, the ROR-binding molecule that binds specifically to the ROR antigen and to the molecule expressed on the surface of T cells (i.e., CD3) can direct T cell-mediated death (cytotoxicity) of cells expressing the ROR antigen by redirecting T cells. Petition 870260031312, dated 02 / 04 / 2026, p. 71 / 417 63 / 285 for cells expressing ROR (i.e., target cells). T cell-mediated killing using bispecific anti-CD3 molecules is described in detail in North American Publication No. 2006 / 0193852, which is incorporated herein by reference in its entirety. In some embodiments, the molecule expressed on the surface of T cells is selected from any molecule capable of redirecting T cells to a target cell.

[0224] With reference to figure 3, in a number of embodiments, ROR-binding molecules comprise a first and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, wherein the A domain has a variable region domain amino acid sequence, and wherein the B domain, the D domain and the E domain have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a variable region domain amino acid sequence and the G domain has a constant region domain amino acid sequence;(c) the third polypeptide chain comprises an H domain, an I domain, a J domain and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the H domain has a variable region domain amino acid sequence, the I domain has an amino acid sequence; Petition 870260031312, dated 02 / 04 / 2026, page 72 / 417 64 / 285 (d) the fourth polypeptide chain comprises an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a variable region domain amino acid sequence, and wherein the fourth polypeptide chain comprises the CH1 domains and the M domain is the CH1 domain, or a portion thereof; (e) the first and second polypeptides are associated through an interaction between the A and F domains and an interaction between the B and G domains; (f) the third and fourth polypeptides are associated through an interaction between the H and L domains and an interaction between the I and M domains; (g) the first and third polypeptides are associated through an interaction between the D and J domains and an interaction between the E and K domains to form the ROR-binding molecule.

[0225] In a number of embodiments, (a) the first polypeptide chain comprises an A domain, a B domain, a D domain, and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, wherein the A domain has a variable region domain amino acid sequence, and wherein the B domain, the D domain, and the E domain have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a variable region domain amino acid sequence and the Petition 870260031312, dated 02 / 04 / 2026, page 73 / 417 65 / 285 domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises an H domain, an I domain, a J domain and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the third polypeptide chain comprises the CH1 domain and the I domain is in the CH1 domain, or a portion thereof, the H domain has a variable region domain amino acid sequence, and the J and K domains have a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a variable region domain amino acid sequence, and wherein the M domain has a CL amino acid sequence;(e) the first and second polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G; (f) the third and fourth polypeptides are associated through an interaction between domains H and L and an interaction between domains I and M; (g) the first and third polypeptides are associated through an interaction between domains D and J and an interaction between domains E and K to form the ROR-binding molecule. 6.3.1. Domain A (variable region)

[0226] In ROR-binding molecules, domain A has a variable region domain amino acid sequence. The variable region domain amino acid sequences, as described in the present invention, are Petition 870260031312, dated 02 / 04 / 2026, page 74 / 417 66 / 285 amino acid sequences of the variable region domain of an antibody, including the VL and VH antibody domain sequences. The VL and VH sequences are described in greater detail below, in sections 0 and 0, respectively. In a preferred embodiment, the A domain has a VL antibody domain sequence and the F domain has a VH antibody domain sequence. 6.3.1.1. VL Regions

[0227] The VL amino acid sequences useful in the ROR-binding molecules described in the present invention are variable domain sequences of the antibody light chain. In a typical arrangement, in natural antibodies and in the antibody constructs described in the present invention, a specific VL amino acid sequence associates with a VH amino acid sequence to form an antigen-binding site. In various embodiments, the VL amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or combinations of human, non-human mammalian, mammalian, and / or synthesized sequences, as described in more detail below in sections 0 and 0.

[0228] In several embodiments, the amino acid sequences of the VL are mutant sequences of sequences that occur in nature. In certain embodiments, the amino acid sequences of the VL are sequences of the variable domain of the lambda (λ) light chain. In certain embodiments, the amino acid sequences of the VL are sequences of the variable domain of the kappa (κ) light chain. In a preferred embodiment, the Petition 870260031312, dated 02 / 04 / 2026, page 75 / 417 67 / 285 amino acid sequences of the VL are sequences of the variable domain of the kappa (κ) light chain.

[0229] In the ROR-binding molecules described in the present invention, the C-terminus of domain A is linked to the N-terminus of domain B. In certain embodiments, domain A has a VL amino acid sequence that is modified by mutation at its C-terminus at the junction between domain A and domain B, as described in more detail below in Section 0 and Example 6. 6.3.1.2. Regions determining complementarity

[0230] The amino acid sequences of the VL comprise highly variable sequences called “complementarity-determining regions” (CDRs), typically three CDRs (CDR1, CD2, and CDR3). In several embodiments, the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CDRs are human sequences. In several embodiments, the CDRs are naturally occurring sequences. In several embodiments, the CDRs are naturally occurring sequences that have been mutated to alter the antigen-binding site affinity for a particular antigen or epitope. In certain embodiments, naturally occurring CDRs have been mutated in a host in vivo through affinity maturation and somatic hypermutation.In certain modalities, CDRs have been modified by in vitro mutation through methods including, but not limited to, PCR mutagenesis and chemical mutagenesis. In several... Petition 870260031312, dated 02 / 04 / 2026, p. 76 / 417 68 / 285 modes, CDRs are synthesized sequences, including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. 6.3.1.3. Regions of structure and grafting with CDR

[0231] The amino acid sequences of the VL comprise “framework region” (FR) sequences. FRs are generally conserved sequence regions that act as a framework for interspersed CDRs (see section 0.), typically in an FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from the N-terminus to the C-terminus). In various embodiments, FRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, FRs are human sequences. In various embodiments, FRs are naturally occurring sequences. In various embodiments, FRs are synthesized sequences, including, but not limited to, rationally designed sequences.

[0232] In several modalities, FRs and CDRs are both of the same naturally occurring variable domain sequence. In several modalities, FRs and CDRs are of different variable domain sequences, where CDRs are grafted onto the FR structure with the CDRs providing specificity for a specific antigen. In certain modalities, the grafted CDRs are all derived from the same naturally occurring variable domain sequence. In certain modalities, the grafted CDRs are derived from different variable domain sequences. In Petition 870260031312, dated 02 / 04 / 2026, page 77 / 417 69 / 285 In certain embodiments, grafted CDRs are synthesized sequences, including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries. In certain embodiments, Frs and grafted CDRs are of the same species. In certain embodiments, the Frs and grafted CDRs are of different species. In a preferred grafted CDR embodiment, an antibody is humanized, where the grafted CDRs are non-human mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, and the FRs are human sequences. Humanized antibodies are discussed in more detail in U.S. Patent No. 6,407,213, the entirety of which is incorporated herein by reference for all that it teaches.In several embodiments, specific portions or sequences of FRs from one species are used to substitute specific portions or sequences of FRs from another species. 6.3.1.4. Regions of the VH

[0233] The VH amino acid sequences in the ROR-binding molecules described in the present invention are variable domain sequences of the antibody heavy chain. In a typical arrangement of both natural antibodies and ROR-binding molecules described in the present invention, a specific VH amino acid sequence associates with a specific VL amino acid sequence to form an antigen-binding site. In various embodiments, the VH amino acid sequences are mammalian sequences, including human sequences, Petition 870260031312, dated 02 / 04 / 2026, p. 78 / 417 70 / 285 synthesized sequences, or combinations of non-human mammalian, mammalian, and / or synthesized sequences, as described in more detail above in sections 0 and 0. In several embodiments, VH amino acid sequences are mutant sequences of sequences that occur in nature. 6.3.2. Domain B (constant region)

[0234] In ROR-binding molecules, domain B has a constant region domain sequence. The constant region domain amino acid sequences, as described in the present invention, are sequences of a constant region domain of an antibody.

[0235] In several embodiments, the constant region sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the constant region sequences are human sequences. In certain embodiments, the constant region sequences are of an antibody light chain. In particular embodiments, the constant region sequences are of a lambda or kappa light chain. In certain embodiments, the constant region sequences are of an antibody heavy chain. In particular embodiments, the constant region sequences are an antibody heavy chain sequence that is an isotype of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the constant region sequences are of an IgG isotype. In a preferred embodiment, the sequences Petition 870260031312, dated 02 / 04 / 2026, p. 79 / 417 71 / 285 of the constant region are of an IgG1 isotype. In specific preferred modalities, the sequence of the constant region is a CH3 sequence. CH3 sequences are described in more detail below in section 0. In other preferred modalities, the sequence of the constant region is an orthologous CH2 sequence. Orthologous CH2 sequences are described in more detail below in section 0.

[0236] In particular embodiments, the constant region sequence has been modified to include one or more orthogonal mutations. In a preferred embodiment, domain B has a constant region sequence that is a CH3 sequence comprising knob-hole orthogonal mutations (synonymously, knob-in-hole, KIH), as described in more detail below in Section 0, and an S354C or Y349C mutation that forms a modified disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in more detail below in Section 0. In some preferred embodiments, the knob-hole orthogonal mutation is a T366W mutation. 6.3.2.1. CH3 Regions

[0237] The CH3 amino acid sequences, as described in the present invention, are sequences of the C-terminal domain of an antibody heavy chain.

[0238] In several embodiments, the CH3 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH3 sequences are human sequences. In certain embodiments, the CH3 sequences are of an IgA1 isotype, Petition 870260031312, dated 02 / 04 / 2026, p. 80 / 417 72 / 285 IgA2, IgD, IgE, IgM, IgG1, IgG2, IgG3, IgG4, or CH4 sequences of an IgE or IgM isotype. In a specific embodiment, the CH3 sequences are of an IgG isotype. In a preferred embodiment, the CH3 sequences are of an IgG1 isotype.

[0239] In certain embodiments, CH3 sequences are endogenous sequences. In particular embodiments, the CH3 sequence is amino acids 244-330 of Uniprot accession number P01857. In several embodiments, a CH3 sequence is a segment of an endogenous CH3 sequence. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the N-terminal amino acids G224 and Q225. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks the C-terminal amino acids P328, G329, and K330. In particular embodiments, a CH3 sequence has an endogenous CH3 sequence that lacks both the N-terminal amino acids G224 and Q225 and the C-terminal amino acids P328, G329, and K330.In preferred embodiments, a ROR-binding molecule has multiple domains that have CH3 sequences, where a CH3 sequence can refer to either a complete endogenous CH3 sequence or a CH3 sequence that lacks N-terminal amino acids, C-terminal amino acids, or both.

[0240] In certain embodiments, CH3 sequences are endogenous sequences that have one or more mutations. In particular embodiments, the mutations are one or more orthogonal mutations that are introduced into an endogenous CH3 sequence to direct specific pairing of Petition 870260031312, dated 02 / 04 / 2026, p. 81 / 417 73 / 285 specific CH3 sequences, as described in more detail below in Sections 0-0.

[0241] In certain embodiments, CH3 sequences are designed to reduce antibody immunogenicity by replacing specific amino acids of one allotype with those of another allotype, and referred to in the present invention as isoallotype mutations, as described in more detail in Stickler et al. (Genes Immun. April 2011; 12(3):213-221), which is incorporated herein by reference for all that it teaches. In particular embodiments, specific amino acids of the G1m1 allotype are substituted. In a preferred embodiment, D356E and L358M isoallotype mutations are made in the CH3 sequence.

[0242] In a preferred embodiment, domain B has a human IgG1 CH3 amino acid sequence with the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K. In other preferred embodiments, domain B has a human IgG1 CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C. In still other preferred embodiments, domain B has a human IgG1 CH3 sequence with the following mutational changes: Y349C and a tripeptide insertion, 445P, 446G, 447K.

[0243] In certain embodiments, domain B has a human IgG1 CH3 sequence with a 447C mutation incorporated into an endogenous CH3 sequence.

[0244] In the ROR-binding molecules described in the present invention, the N-terminus of the B domain is linked to Petition 870260031312, dated 02 / 04 / 2026, p. 82 / 417 74 / 285 C-terminus of domain A. In certain embodiments, domain B has a CH3 amino acid sequence that is mutated at its N-terminus at the junction between domain A and domain B, as described in more detail below in section 0 and example 6.

[0245] In ROR-binding molecules, the C-terminus of domain B is linked to the N-terminus of domain D. In certain embodiments, domain B has a CH3 amino acid sequence that is extended at the C-terminus at the junction between domain B and domain D, as described in more detail below in section 0. 6.3.2.2. Orthologous CH2 Regions

[0246] The CH2 amino acid sequences, as described in the present invention, are sequences of the third domain of an antibody heavy chain, with reference to the N-terminus to the C-terminus. The CH2 amino acid sequences, in general, are discussed in more detail below in section 0. In a number of embodiments, an ROR-binding molecule has more than one paired set of CH2 domains that have CH2 sequences, where a first set has CH2 amino acid sequences from a first isotype and one or more orthologous sets of CH2 amino acid sequences from another isotype. The orthologous CH2 amino acid sequences, as described herein, are capable of interacting with CH2 amino acid sequences from a shared isotype, but not of interacting significantly with CH2 amino acid sequences from another isotype present in the ROR-binding molecule. In particular embodiments, all sets of CH2 amino acid sequences are of the same species.In preferred modes, all sets of. Petition 870260031312, dated 02 / 04 / 2026, p. 83 / 417 75 / 285 CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the sets of CH2 amino acid sequences are from different species. In particular embodiments, the first set of CH2 amino acid sequences is of the same isotype as the other non-CH2 domains in the ROR-linking molecule. In one specific embodiment, the first set has CH2 amino acid sequences of an IgG isotype, and one or more sets of orthologs have CH2 amino acid sequences of an IgM or IgE isotype. In certain embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences. In other embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences that have one or more mutations. In particular embodiments, one or more mutations are orthogonal knob-in-hole mutations, orthogonal charge-pair mutations, or orthogonal hydrophobic mutations.The orthologous CH2 amino acid sequences useful for ROR-binding molecules are described in more detail in international PCT applications WO2017 / 011342 and WO2017 / 106462, which are incorporated herein by reference in their entirety. 6.3.3. Domain D (constant region)

[0247] In the ROR-binding molecules described in the present invention, the D domain has a constant region amino acid sequence. The constant region amino acid sequences are described in more detail in section 0.

[0248] In a preferred series of embodiments, the D domain has an amino acid sequence of CH2. The sequences Petition 870260031312, dated 02 / 04 / 2026, page 84 / 417 76 / 285 of CH2 amino acids, as described in the present invention, are CH2 amino acid sequences of the third domain of a native antibody heavy chain, referenced from the N-terminus to the C-terminus. In various embodiments, the CH2 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH2 sequences are human sequences. In certain embodiments, the CH2 sequences are of an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH2 sequences are of an IgG1 isotype.

[0249] In certain embodiments, the CH2 sequences are endogenous sequences. In particular embodiments, the sequence is formed by amino acids 111-223 of the Uniprot registry number P01857. In a preferred embodiment, the CH2 sequences possess an N-terminal hinge region peptide that connects the N-terminal constant domain variable domain segment to the CH2 domain, as discussed in more detail below in section 0.

[0250] In ROR-binding molecules, the N-terminus of the D domain is linked to the C-terminus of the B domain. In certain embodiments, the B domain has a CH3 amino acid sequence that is extended at the C-terminus at the junction between the D and B domains, as described in more detail below in section 0. 6.3.4. Domain E (constant region)

[0251] In ROR-binding molecules, the E domain has a region domain amino acid sequence Petition 870260031312, dated 02 / 04 / 2026, page 85 / 417 77 / 285 constant. The amino acid sequences of the constant region are described in more detail in section 0.

[0252] In certain embodiments, the constant region sequence is a CH3 sequence. CH3 sequences are described in more detail above in section 0. In particular embodiments, the constant region sequence has been modified to include one or more orthogonal mutations. In a preferred embodiment, the E domain has a constant region sequence that is a CH3 sequence comprising knob-hole orthogonal mutations (synonymously, “knob-hole,” “KIH”), as described in more detail below in section 0, and an S354C or Y349C mutation that forms a modified disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in more detail below in section 0. In some preferred embodiments, the knob-hole orthogonal mutation is a T366W mutation.

[0253] In certain embodiments, the constant region domain sequence is a CH1 sequence. In particular embodiments, the CH1 amino acid sequence of the E domain is the only CH1 amino acid sequence in the ROR-binding molecule. In certain embodiments, the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in more detail below in 0. In certain embodiments, the constant region sequence is a CL sequence. In certain embodiments, the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in more detail below in 0. The CH1 and CL sequences are described in more detail in section 0. 6.3.5. F-domain (variable region) Petition 870260031312, dated 02 / 04 / 2026, page 86 / 417 78 / 285

[0254] In ROR-binding molecules, the F domain has a variable region domain amino acid sequence. The variable region domain amino acid sequences, as discussed in more detail in section

[0225] , are antibody variable region domain amino acid sequences, including VL and VH antibody domain sequences. The VL and VH sequences are described in more detail below, in sections 0 and 0, respectively. In a preferred embodiment, the F domain has a VH antibody domain sequence. 6.3.6. Domain G (constant region)

[0255] In ROR-binding molecules, the G domain has a constant region amino acid sequence. The constant region amino acid sequences are described in more detail in section 0.

[0256] In specific preferred embodiments, the sequence of the constant region is a CH3 sequence. CH3 sequences are described in more detail below in section 0. In other preferred embodiments, the sequence of the constant region is an orthologous CH2 sequence. Orthologous CH2 sequences are described in more detail below in section 0.

[0257] In certain preferred embodiments, the G domain has a human IgG1 CH3 sequence with the following mutational alterations: S354C; and a tripeptide insertion, 445P, 446G, 447K. In some preferred embodiments, the G domain has a human IgG1 CH3 sequence with the following mutational alterations: S354C; and a tripeptide insertion, 445P, 446G, 447K. In some preferred embodiments, the G domain has a CH3 sequence Petition 870260031312, dated 02 / 04 / 2026, page 87 / 417 79 / 285 of human IgG1 with the following alterations: L351D; and a tripeptide insertion of 445G, 446E, 447C. 6.3.7. Domain H (variable region)

[0258] In ROR-binding molecules, the L domain has a variable region domain amino acid sequence. The variable region domain amino acid sequences, as discussed in more detail in section

[0225] , are antibody variable region domain amino acid sequences, including VL and VH antibody domain sequences. The VL and VH sequences are described in more detail below, in sections 0 and 0, respectively. In a preferred embodiment, the H domain has a VL antibody domain sequence. 6.3.8. Domain I (constant region)

[0259] In ROR-binding molecules, domain I has a constant region domain amino acid sequence. The constant region domain amino acid sequences are described in more detail above in section 0. In a number of preferred embodiments of ROR-binding molecules, domain I has a CL amino acid sequence. In another series of embodiments, domain I has a CH1 amino acid sequence. The CH1 and CL amino acid sequences are described in more detail in section 0. 6.3.8.1. Regions CH1 and CL

[0260] The CH1 amino acid sequences, as described in the present invention, are sequences of the second domain of an antibody heavy chain, with reference to the N-terminus to the C-terminus. In certain embodiments, the CH1 sequences are endogenous sequences. In several Petition 870260031312, dated 02 / 04 / 2026, page 88 / 417 In 80 / 285 embodiments, the CH1 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH1 sequences are human sequences. In certain embodiments, the CH1 sequences are of an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH1 sequences are of an IgG1 isotype. In preferred embodiments, the CH1 sequence is formed by amino acids 1-98 of the Uniprot registry number P01857.

[0261] The CL amino acid sequences useful in the ROR-binding molecules described in the present invention are constant domain sequences of the antibody light chain. In certain embodiments, the CL sequences are endogenous sequences. In several embodiments, the CL sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CL sequences are human sequences.

[0262] In certain embodiments, the CL amino acid sequences are constant domain sequences of the lambda (λ) light chain. In particular embodiments, the CL amino acid sequences are constant domain sequences of the human lambda light chain. In preferred embodiments, the lambda (λ) light chain sequence is the Uniprot registration number P0CG04.

[0263] In certain embodiments, the CL amino acid sequences are sequences of the constant kappa (κ) light chain domain. In a preferred embodiment, the sequences Petition 870260031312, dated 02 / 04 / 2026, p. 89 / 417 81 / 285 of the amino acid CL are sequences of the constant domain of the human kappa (κ) light chain. In a preferred embodiment, the kappa light chain sequence is the Uniprot registration number P01834.

[0264] In certain embodiments, the CH1 sequence and the CL sequences are both endogenous sequences. In certain embodiments, the CH1 sequence and the CL sequences comprise, separately and respectively, orthogonal modifications to the endogenous CH1 and CL sequences, as discussed in more detail below in section 0. It should be understood that orthogonal mutations in the CH1 sequence do not eliminate the specific binding interaction between the CH1-binding reagent and the CH1 domain. However, in some embodiments, orthogonal mutations may reduce, though not eliminate, the specific binding interaction. The CH1 and CL sequences may also be portions thereof, either of an endogenous or modified sequence, such that a domain with the CH1 sequence, or a portion thereof, may associate with a domain with the CH1 sequence, or a portion thereof. Furthermore, the ROR-binding molecule with a portion of the CH1 sequences described above may be bound by the CH1-binding reagent.

[0265] Without wishing to dwell on theory, the CH1 domain is also unique in that its folding is typically the rate-limiting step in IgG secretion (Feige et al. Mol Cell. June 12, 2009; 34(5): 569-79; incorporated here by reference in its entirety). Thus, purification of ROR-binding molecules based on the rate-limiting component of CH1 comprising polypeptide chains may provide a means to purify Petition 870260031312, dated 02 / 04 / 2026, p. 90 / 417 82 / 285 complete complexes of incomplete chains, how to purify complexes with a limiting CH1 domain from complexes that have only one or more chains comprising non-CH1.

[0266] Although limiting CH1 expression may be beneficial in some respects, as discussed, there is the potential for CH1 to limit the overall expression of complete ROR-binding molecules. Thus, in certain embodiments, the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted to improve the efficiency of ROR-binding molecules forming complete complexes. In an illustrative example, the ratio of a plasmid vector constructed to express the polypeptide chain comprising the CH1 sequence(s) may be increased relative to plasmid vectors constructed to express the other polypeptide chains. In another illustrative example, the polypeptide chain comprising the CH1 sequence(s), compared to the polypeptide chain comprising the CL sequence(s), may be the shorter of the two polypeptide chains.In another specific embodiment, the expression of the polypeptide chain comprising the CH1 sequence(s) can be adjusted by controlling which polypeptide chain has the CH1 sequence(s). For example, the ROR-binding molecule can be manipulated so that the CH1 domain is present in a two-domain polypeptide chain (e.g., the 4th polypeptide chain described in the present invention), instead of the native position of the CH1 sequence in a four-domain polypeptide chain (e.g., the 3rd polypeptide chain described in the present invention). Petition 870260031312, dated 02 / 04 / 2026, page 91 / 417 83 / 285 invention) to control the expression of the polypeptide chain comprising the CH1 sequence(s). However, in other respects, a relative expression level of CH1-containing chains that is too high compared to the other chains may result in incomplete complexes that possess the CH1 chain but not each of the other chains. Thus, in certain embodiments, the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted both to reduce the formation of incomplete complexes lacking the CH1-containing chain and to reduce the formation of incomplete complexes with the CH1-containing chain but lacking the other chains present in a complete complex. 6.3.8.2. Orthogonal modifications in CH1 and CL

[0267] In certain embodiments, the CH1 sequence and the CL sequences comprise, separately and respectively, orthogonal modifications in the endogenous CH1 and CL sequences. Orthogonal mutations, in general, are described in more detail below in sections 0 to 0.

[0268] In particular embodiments, the orthogonal modifications in endogenous CH1 and CL sequences are a modified disulfide bridge selected from modified cysteines at position 138 of the CH1 sequence and position 116 of the CL sequence, at position 128 of the CH1 sequence and position 119 of the CL sequence, or at position 129 of the CH1 sequence and position 210 of the CL sequence, as numbered and discussed in more detail in U.S. Patent No. 8,053,562 and U.S. Patent No. 9,527,927, each of which is incorporated herein by reference in its entirety. In a preferred embodiment, the cysteines Petition 870260031312, dated 02 / 04 / 2026, page 92 / 417 The modified 84 / 285 are located at position 128 of the CH1 sequence and at position 118 of the CL kappa sequence, as numbered by the index Eu.

[0269] In a number of preferred embodiments, the mutations that provide non-endogenous cysteine ​​amino acids are an F118C mutation in the CL sequence with a corresponding A141C in the CH1 sequence, or an F118C mutation in the CL sequence with a corresponding L128C in the CH1 sequence, or S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence, as numbered by the index I.

[0270] In several embodiments, orthogonal mutations in the CL sequence and the CH1 sequence are charge pair mutations. In specific embodiments, charge pair mutations are F118S, F118A, or F118V mutations in the CL sequence with a corresponding A141L in the CH1 sequence, or a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence, as numbered by the index I and described in more detail in Bonisch et al. (Protein Engineering, Design & Selection, 2017, pages 1-12), here incorporated by reference for all that it teaches. In a number of preferred embodiments, charge pair mutations are an N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence, or an N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence, numbered by the index I. 6.3.9. J Domain (CH2)

[0271] In ROR-binding molecules, the J domain has a CH2 amino acid sequence. The sequences of Petition 870260031312, dated 02 / 04 / 2026, page 93 / 417 The 85 / 285 CH2 amino acid sequence is described in more detail above in section 0. In a preferred embodiment, the CH2 amino acid sequence has a hinge region at the N-terminus that connects the J domain to the I domain, as described in more detail below in section 0.

[0272] In ROR-binding molecules, the C-terminus of the J-domain is linked to the N-terminus of the K-domain. In particular embodiments, the J-domain is connected to the N-terminus of the K-domain which has a CH1 amino acid sequence or a CL amino acid sequence, as described in more detail below in section 0. 6.3.10. Domain K (constant region)

[0273] In ROR-binding molecules, the K domain has a constant region domain amino acid sequence. The constant region domain amino acid sequences are described in more detail above in section 0. In a preferred embodiment, the K domain has a constant region sequence that is a CH3 sequence comprising orthogonal knob-hole mutations, as described in more detail below in section 0; isoallotype mutations, as described in more detail above in 0; and an S354C or Y349C mutation that forms a modified disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in more detail below in section 0. In some preferred embodiments, the orthogonal knob-hole mutations combined with the isoallotype mutations are the following mutational changes: D356E, L358M, T366S, L368A, and Y407V.

[0274] In certain embodiments, the constant region domain sequence is a CH1 sequence.In particular embodiments, the amino acid sequence CH1 of the K domain is... Petition 870260031312, dated 02 / 04 / 2026, page 94 / 417 86 / 285 is the only CH1 amino acid sequence in the ROR-binding molecule. In certain embodiments, the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in more detail below in section 0. In certain embodiments, the constant region sequence is a CL sequence. In certain embodiments, the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in more detail below in section 0. The CH1 and CL sequences are described in more detail in section 0. 6.3.11. L domain (variable region)

[0275] In ROR-binding molecules, the L domain has a variable region domain amino acid sequence. The variable region domain amino acid sequences, as discussed in more detail in section

[0225] , are antibody variable region domain amino acid sequences, including VL and VH antibody domain sequences. The VL and VH sequences are described in more detail above, in sections 0 and 0, respectively. In a preferred embodiment, the L domain has a VH antibody domain sequence. 6.3.12. Domain M (constant region)

[0276] In ROR-binding molecules, the M domain has a constant region domain amino acid sequence. The constant region domain amino acid sequences are described in more detail above in section 0. In a series of preferred embodiments of ROR-binding molecules, the I domain has a CH1 amino acid sequence. In another series of preferred embodiments, the I domain has a CL amino acid sequence. The sequences of Petition 870260031312, dated 02 / 04 / 2026, page 95 / 417 The 87 / 285 amino acids CH1 and CL are described in more detail in the section. 0. 6.3.13. Matching of domains A and F

[0277] In ROR-binding molecules, an amino acid sequence from the VL or VH of the A domain and an amino acid sequence from the VL or VH of the F domain are associated and form an antigen-binding site (ABS). The A:F antigen-binding site (ABS) is capable of specifically binding to an epitope of an antigen. Antigen binding by an ABS is described in more detail below in section 0.

[0278] In a variety of multivalent embodiments, the ABS formed by the A and F domains (A:F) has the same sequence as one or more ABS in the ROR-binding molecule and therefore has the same recognition specificity as one or more other ABS with identical sequence within the ROR-binding molecule.

[0279] In several multivalent embodiments, ABS A:F does not have the same sequence as one or more other ABS within the ROR-binding molecule. In certain embodiments, ABS A:F has a recognition specificity different from that of one or more other non-identical sequence ABS in the ROR-binding molecule. In particular embodiments, ABS A:F recognizes a different antigen from that recognized by at least one other non-identical sequence ABS in the ROR-binding molecule. In particular embodiments, ABS A:F recognizes a different epitope of an antigen that Petition 870260031312, dated 02 / 04 / 2026, p. 96 / 417 88 / 285 is also recognized by at least one other non-identical sequence ABS in the ROR-binding molecule. In these embodiments, the ABS formed by domains A and F recognizes an antigen epitope, where one or more other ABS in the ROR-binding molecule recognize the same antigen, but not the same epitope. 6.3.13.1. Antigen binding by ABS

[0280] We say that an ABS, and the ROR-binding molecule comprising such ABS, recognizes the epitope (or more generally, the antigen) to which the ABS specifically binds, and we say that the epitope (or more generally, the antigen) is the recognition specification or the binding specificity of the ABS.

[0281] ABS is said to bind to its specific antigen or epitope with a particular affinity. As described in the present invention, affinity refers to the intensity of the interaction of non-covalent intermolecular forces between one molecule and another. Affinity, i.e., the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), where a lower kD value refers to a stronger interaction between the molecules. The KD values ​​of antibody constructs are measured by methods well known in the art, including, but not limited to, biolayer interferometry (e.g., on the Octet / FORTEBIO® platform), surface plasmon resonance (SPR) technology (e.g., Biacore®), and cell binding assays. For purposes of the present invention, affinities are the constants of Petition 870260031312, dated 02 / 04 / 2026, page 97 / 417 89 / 285 dissociation equilibrium measured by biolayer interferometry using the Octet / FORTEBIO® platform.

[0282] “Specific binding”, as used in the present invention, refers to an affinity between an ABS and its cognate antigen or epitope wherein the KD value is below 10-6 M, 10-7 M, 10-8 M, 10-9 M or 10-10 M.

[0283] The number of ABS in an ROR-binding molecule, as described in the present invention, defines the “valence” of the ROR-binding molecule, as schematized in Figure 2. An ROR-binding molecule with a single ABS is “monovalent”. An ROR-binding molecule with multiple ABS is considered “multivalent”. A multivalent ROR-binding molecule with two ABS is “bivalent”. A multivalent ROR-binding molecule with three ABS is “trivalent”. A multivalent ROR-binding molecule with four ABS is “tetravalent”.

[0284] In several multivalent embodiments, all the plurality of ABS have the same recognition specificity. As illustrated in Figure 2, such an ROR-binding molecule is a “monospecific” “multivalent” binding construct. In other multivalent embodiments, at least two of the plurality of ABS have different recognition specificities. Such ROR-binding molecules are multivalent and “multispecific”. In multivalent embodiments in which the ABS collectively have two recognition specificities, the ROR-binding molecule is “bispecific”. In multivalent embodiments in which ABS Petition 870260031312, dated 02 / 04 / 2026, p. 98 / 417 90 / 285 collectively have three recognition specificities; the ROR binding molecule is trispecific.

[0285] In multivalent modalities where ABS collectively have a plurality of recognition specificities for different epitopes present on the same antigen, the ROR-binding molecule is multiparatopic. Multivalent modalities where ABS collectively recognize two epitopes on the same antigen are biparatopic.

[0286] In several multivalent embodiments, the multivalence of the ROR-binding molecule enhances the avidity of the ROR-binding molecule for a specific target. As described in the present invention, avidity refers to the overall strength of the interaction between two or more molecules, for example, a multivalent ROR-binding molecule for a specific target, where avidity is the cumulative strength of the interaction provided by the affinities of multiple ABS. Avidity can be measured by the same methods used to determine affinity, as described above. In certain embodiments, the avidity of an ROR-binding molecule for a specific target is such that the interaction is a specific binding interaction, in which the avidity between two molecules has a KDa value below 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M.In certain modalities, the avidity of a ROR-binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, whereas one or more individual ABS affinities do not have a KD value that qualifies as specific binding of their respective antigens or epitopes on their own. In certain... Petition 870260031312, dated 02 / 04 / 2026, page 99 / 417 In 91 / 285 modalities, avidity is the cumulative interaction strength provided by the affinities of multiple ABS for separate antigens in a specific shared target or complex, such as separate antigens found in an individual cell. In certain modalities, avidity is the cumulative interaction strength provided by the affinities of multiple ABS for separate epitopes in a shared individual antigen. 6.3.14. B & G Domain Pairing

[0287] In the ROR-binding molecules described in the present invention, an amino acid sequence from the constant region of the B domain and an amino acid sequence from the constant region of the G domain are associated. The amino acid sequences of the constant region domain are described in more detail above in section 0.

[0288] In a number of preferred embodiments, the B domain and the G domain have CH3 amino acid sequences. The CH3 sequences are described in more detail above in section 0. In several embodiments, the amino acid sequences of the B and G domains are identical. In some of these embodiments, the sequence is an endogenous CH3 sequence.

[0289] In a variety of embodiments, the amino acid sequences of the B and G domains are different, and comprise separately, and respectively, orthogonal modifications in an endogenous CH3 sequence, in which the B domain interacts with the G domain, and in which neither the B domain nor the G domain interact significantly with a CH3 domain that does not have the orthogonal modification. Petition 870260031312, dated 02 / 04 / 2026, page 100 / 417 92 / 285

[0290] “Orthogonal modifications” or synonymously, “orthogonal mutations,” as described in the present invention, are one or more mutations by manipulation in an amino acid sequence of an antibody domain that increase the binding affinity of a first domain having an orthogonal modification for a second domain having a complementary orthogonal modification. In certain embodiments, orthogonal modifications decrease the affinity of a domain having the orthogonal modifications for a domain that does not have the complementary orthogonal modifications. In certain embodiments, orthogonal modifications are mutations in an endogenous antibody domain sequence. In a variety of embodiments, orthogonal modifications are N-terminus or C-terminus modifications of an endogenous antibody domain sequence, including, but not limited to, amino acid additions or deletions.In particular embodiments, orthogonal modifications include, but are not limited to, modified disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in more detail below in sections 0-0. In particular embodiments, orthogonal modifications include a combination of selected, but not limited to, modified disulfide bridges, knob-in-hole mutations, and charge-pair mutations. In particular embodiments, orthogonal modifications may be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations, as described in more detail above in section 0. 6.3.14.1. Orthogonal modified disulfide bridges Petition 870260031312, dated 02 / 04 / 2026, page 101 / 417 93 / 285

[0291] In a variety of embodiments, orthogonal modifications comprise mutations that generate modified disulfide bridges between a first and a second domain. As described in the present invention, “modified disulfide bridges” are mutations that provide non-endogenous cysteine ​​amino acids in two or more domains, such that a non-native disulfide bond is formed when the two or more domains associate. Modified disulfide bridges are described in more detail in Merchant et al. (Nature Biotech(1998) 16:677681), the entirety of which is incorporated by reference in relation to all that it teaches. In certain embodiments, the modified disulfide bridges enhance the orthogonal association between specific domains. In one particular embodiment, the mutations that generate modified disulfide bridges are a K392C mutation in one of a first or second CH3 domain, and a D399C mutation in the other CH3 domain.In a preferred embodiment, the mutations that generate modified disulfide bridges are an S354C mutation in one of the first or second CH3 domains, and a Y349C mutation in the other CH3 domain. In another preferred embodiment, the mutations that generate modified disulfide bridges are a 447C mutation in the first and second CH3 domains that are provided by the C-terminal extension of a CH3 domain incorporating a KSC tripeptide sequence. 6.3.14.2. Orthogonal knob-in-hole mutations

[0292] In various embodiments, orthogonal modifications comprise knob-in-hole mutations (synonymously, knob-in-hole). As described in the present invention, the Petition 870260031312, dated 02 / 04 / 2026, page 102 / 417 94 / 285 Knob-hole mutations are mutations that alter the steric surface characteristics of a first domain such that the first domain will preferentially associate with a second domain with complementary steric mutations relative to the association with domains without complementary steric mutations. Knob-hole mutations are described in more detail in U.S. Patent No. 5,821,333 and U.S. Patent No. 8,216,805, each of which is incorporated herein in its entirety. In several embodiments, knob-hole mutations are combined with modified disulfide bridges, as described in more detail in Merchant et al. (Nature Biotech (1998) 16:677-681), incorporated in the present invention by reference in its entirety. In several embodiments, knobhole mutations, isoallotype mutations, and manipulation disulfide mutations are combined.

[0293] In certain embodiments, knob-in-hole mutations are a T366Y mutation in a first domain, and a Y407T mutation in a second domain. In certain embodiments, knob-in-hole mutations are an F405A mutation in a first domain and a T394W mutation in a second domain. In certain embodiments, knob-in-hole mutations are a T366Y mutation and an F405A mutation in a first domain, and a T394W mutation and a Y407T mutation in a second domain. In certain embodiments, knob-in-hole mutations are a T366W mutation in a first domain and a Y407A mutation in a second domain. In certain embodiments, combined knob-in-hole mutations and manipulation disulfide mutations are S354C and T366W mutations in a first domain, and a Y349C, T366S, L368A, and AY407V mutation in a second domain. In a Petition 870260031312, dated 02 / 04 / 2026, page 103 / 417 In the 95 / 285 preferred modality, the knob-in-hole mutations, isoallotype mutations, and disulfide mutations combined by manipulation are S354C and T366W mutations in a first domain, and Y349C, D356E, L358M, T366S, L368A, and AY407V mutations in a second domain. 6.3.14.3. Orthogonal charge pair mutations

[0294] In several embodiments, orthogonal modifications are charge-pair mutations. As used in the present invention, charge-pair mutations are mutations that affect the charge of an amino acid on the surface of a domain, such that the domain will preferentially associate with a second domain with complementary charge-pair mutations relative to association with domains without complementary charge-pair mutations. In certain embodiments, charge-pair mutations improve orthogonal association between specific domains. Charge-pair mutations are described in more detail in U.S. Patent No. 8,592,562, U.S. Patent No. 9,248,182, and U.S. Patent No. 9,358,286, each of which is incorporated by reference into the present invention for all that it teaches. In certain embodiments, charge-pair mutations improve stability between specific domains.In a preferred embodiment, the charge pair mutations are a T366K mutation in one domain and an L351D mutation in the other domain. 6.3.15. E and K domain pairing

[0295] In several embodiments, the E domain has a CH3 amino acid sequence. Petition 870260031312, dated 02 / 04 / 2026, p. 104 / 417 96 / 285

[0296] In several embodiments, the K domain has a CH3 amino acid sequence.

[0297] In several embodiments, the amino acid sequences of the E and K domains are identical, where the sequence is an endogenous CH3 sequence.

[0298] In several embodiments, the sequences of the E and K domains are different. In several embodiments, the different sequences separately comprise, respectively, orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain interacts significantly with a CH3 domain that does not have the orthogonal modification. In certain embodiments, the orthogonal embodiments include, but are not limited to, modified disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in more detail below in sections 00. In particular embodiments, the orthogonal modifications include a combination of selected, but not limited to, orthogonal modifications, modified disulfide bridges, knob-in-hole mutations, and charge-pair mutations.In particular modalities, orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isotype mutations. 6.3.16. Pairing of Domains I and M and of Domains H and L

[0299] In several modalities, the I domain has a CL sequence and the M domain has a CH1 sequence. In several modalities, the H domain has a VL sequence and the L domain Petition 870260031312, dated 02 / 04 / 2026, p. 105 / 417 97 / 285 has a VH sequence. In a preferred embodiment, the H domain has a VL amino acid sequence, the I domain has a CL amino acid sequence, the L domain has a VH amino acid sequence, and the M domain has a CH1 amino acid sequence. In another preferred embodiment, the H domain has a VL amino acid sequence, the I domain has a CL amino acid sequence, the L domain has a VH amino acid sequence, the M domain has a CH1 amino acid sequence, and the K domain has a CH3 amino acid sequence.

[0300] In a variety of embodiments, the amino acid sequences of domains I and M separately comprise, respectively, orthogonal modifications in an endogenous sequence, wherein domain I interacts with domain M, and wherein neither domain I nor domain M interact significantly with a domain that does not have the orthogonal modification. In a number of embodiments, orthogonal mutations in domain I are in a CL sequence and orthogonal mutations in domain M are in the CH1 sequence. Orthogonal mutations in the CH1 and CL sequences are described in more detail above in section 0.

[0301] In a variety of embodiments, the amino acid sequences of the H and L domains separately comprise, respectively, orthogonal modifications in an endogenous sequence, wherein the H domain interacts with the L domain, and wherein neither the H domain nor the L domain interacts significantly with a domain that does not have the orthogonal modification. In a number of embodiments, the orthogonal mutations in the H domain are in a VL sequence. Petition 870260031312, dated 02 / 04 / 2026, page 106 / 417 98 / 285 and the orthogonal mutations in the L domain are in the VH sequence. In specific embodiments, the orthogonal mutations are charge-pair mutations at the VH / VL interface. In preferred embodiments, the charge-pair mutations at the VH / VL interface are a Q39E in VH with a corresponding Q38K in VL, or a Q39K in VH with a corresponding Q38E in VL, as described in more detail in Igawa et al. (Protein Eng. Des. Sel., 2010, vol. 23, 667-677), which is incorporated here by reference for all that it teaches.

[0302] In certain embodiments, the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, and the interaction between the H domain and the L domain forms a second antigen-binding site specific to a second antigen. In certain embodiments, the interaction between the A domain and the F domain forms a first antigen-binding site specific to a first antigen, and the interaction between the H domain and the L domain forms a second antigen-binding site specific to the first antigen. 6.3.17. Trivalent ROR-binding molecules

[0303] In another set of embodiments, ROR-binding molecules have three antigen-binding sites and are therefore termed trivalent.

[0304] With reference to figure 21, in several trivalent embodiments, the ROR-binding molecules further comprise a fifth polypeptide chain, wherein (a) the first polypeptide chain further comprises an N domain and an O domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a NOABDE orientation, Petition 870260031312, dated 02 / 04 / 2026, p. 107 / 417 99 / 285 and in which the N domain has a VL amino acid sequence, the O domain has a constant region amino acid sequence; (b) the ROR-binding molecule further comprises a fifth polypeptide chain comprising: a P domain and a Q domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a PQ orientation, and wherein the P domain has a VH amino acid sequence and the Q domain has a constant amino acid sequence; and (c) the first and fifth polypeptides are associated through an interaction between the N and P domains and an interaction between the O and Q domains to form the ROR-binding molecule. As illustrated in Figure 2, these trivalent embodiments are termed 2x1 trivalent constructs.

[0305] With reference to Figure 26, in a further series of trivalent embodiments, the ROR-binding molecules additionally comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation, and wherein the R domain has a VL amino acid sequence and the S domain has a constant-domain amino acid sequence; (b) the ROR-binding molecule additionally comprises a sixth polypeptide chain, comprising: a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation, and wherein the T domain has a VH amino acid sequence and the U domain has a constant-domain amino acid sequence; and (c) the third and the Petition 870260031312, dated 02 / 04 / 2026, page 108 / 417 100 / 285 sixth polypeptides are associated through an interaction between the R and T domains and an interaction between the S and U domains to form the ROR-binding molecule. As illustrated in Figure 2, these trivalent arrangements are termed 1x2 trivalent constructs.

[0306] In a variety of embodiments, the O domain is connected to the A domain via a peptide linker. In a variety of embodiments, the S domain is connected to the H domain via a peptide linker. In a preferred embodiment, the peptide linker that connects the O domain to the A domain, or connects the S domain to the H domain, is a 6-amino acid GSGSGS peptide sequence, as described in more detail in section 0. 6.3.17.1. Trivalent bispecific constructions 2x1 [2(AA)x1(B)]

[0307] With reference to Figure 21, in a variety of embodiments, the amino acid sequences of the N domain and the A domain are identical, the amino acid sequence of the H domain is different from the N and A domains, the amino acid sequences of the O and B domains are identical, the amino acid sequence of the I domain is different from the O and B domains, the amino acid sequences of the P and F domains are identical, the amino acid sequence of the L domain is different from the P and F domains, the amino acid sequences of the Q and G domains are identical, the amino acid sequence of the M domain is different from the Q and G domains; and the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H and Petition 870260031312, dated 02 / 04 / 2026, page 109 / 417 101 / 285 the L domain forms a second antigen-binding site specific to a second antigen, and the N domain and the P domain form a third antigen-binding site specific to the first antigen. 6.3.17.2. Trivalent bispecific constructions 2x1 [2(AB)x1(A)]

[0308] With reference to figure 21, in a variety of embodiments, the amino acid sequences of the N domain and the H domain are identical, the amino acid sequence of the A domain is different from the N and H domains, the amino acid sequences of the O domain and the I domain are identical, the amino acid sequence of the B domain is different from the O and I domains, the amino acid sequences of the P domain and the L domain are identical, the amino acid sequence of the F domain is different from the P and L domains, the amino acid sequences of the Q domain and the M domain are identical, the amino acid sequence of the G domain is different from the Q and M domains;The interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen; the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen; and the N domain and the P domain form a third antigen-specific binding site for the second antigen. 6.3.17.2. Trivalent three-specific constructions 2x1 [2(AB)x1(C)]

[0309] With reference to figure 21, in a variety of embodiments, the amino acid sequences of the N domain, the A domain, and the H domain are different, the sequences of Petition 870260031312, dated 02 / 04 / 2026, page 110 / 417 102 / 285 amino acids of the O domain, the B domain, and the I domain are different; the amino acid sequences of the P domain, the F domain, and the L domain are different; and the amino acid sequences of the Q domain, the G domain, and the M domain are different; and the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the N domain and the P domain form a third antigen-specific binding site for a third antigen.

[0310] In certain embodiments, the O domain has a constant region sequence that is a CL of a kappa light chain and the Q domain has a constant region sequence that is a CH1 of an IgG1 isotype, as discussed in more detail in section 0. In a preferred embodiment, the O domain and the Q domain have CH3 sequences, so that they specifically associate with each other, as discussed in more detail above in section 0. 6.3.17.4. Trivalent bispecific constructions 1x2 [1(A)x2(BA)]

[0311] With reference to Figure 26, in a variety of embodiments, the amino acid sequences of the R domain and the A domain are identical, the amino acid sequence of the H domain is different from the R and A domains, the amino acid sequences of the S domain and the B domain are identical, the amino acid sequence of the I domain is different from the S and B domains, the amino acid sequences of the T domain and the F domain are identical, the amino acid sequence of Petition 870260031312, dated 02 / 04 / 2026, page 111 / 417 103 / 285 The L domain is different from the T and F domains, the amino acid sequences of the U and G domains are identical, the amino acid sequence of the M domain is different from the U and G domains, and the interaction between the A and F domains forms a first antigen-specific binding site for a first antigen, the interaction between the H and L domains forms a second antigen-specific binding site for a second antigen, and the R and T domains form a third antigen-specific binding site for the first antigen. 6.3.17.5. Trivalent bispecific constructions 1x2 [1(A)x2(BB)]

[0312] In a variety of embodiments, the ROR-binding molecule further comprises a second CH1 domain, or part thereof. With reference to Figure 26, in specific embodiments, the amino acid sequences of the R domain and the H domain are identical, the amino acid sequence of the A domain is different from the R and H domains, the amino acid sequences of the S and I domains are identical, the amino acid sequence of the B domain is different from the S and I domains, the amino acid sequences of the T and L domains are identical, the amino acid sequence of the F domain is different from the T and L domains, the amino acid sequences of the U and M domains are identical, the amino acid sequence of the G domain is different from the U and M domains, and the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-binding site Petition 870260031312, dated 02 / 04 / 2026, page 112 / 417 104 / 285 specific for a second antigen, and the R domain and the T domain form a third antigen-binding site specific for the second antigen.

[0313] In particular embodiments, the amino acid sequences of the S domain and I domain are CH1 sequences. In particular embodiments, the amino acid sequences of the U domain and M domain are CH1 sequences. 6.3.17.6. Trivalent three-specific constructions 1x2 [1(A)x2(BC)]

[0314] With reference to figure 26, in a variety of embodiments, the amino acid sequences of the R domain, the A domain, and the H domain are different, the amino acid sequences of the S domain, the B domain, and the I domain are different, the amino acid sequences of the T domain, the F domain, and the L domain are different, and the amino acid sequences of the U domain, the G domain, and the M domain are different; and the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the R domain and the T domain form a third antigen-specific binding site for a third antigen.

[0315] In particular embodiments, the S domain has a constant region sequence that is a CL of a kappa light chain and the U domain has a constant region sequence that is a CH1 of an IgG1 isotype, as discussed in more detail in section 0. In a preferred embodiment, the S domain and the U domain have CH3 sequences, so that Petition 870260031312, dated 02 / 04 / 2026, page 113 / 417 105 / 285 are specifically associated with each other, as discussed in more detail above in section 0.

[0316] In certain embodiments, the ROR-binding molecule also comprises a second CH1 domain, or part thereof. In particular embodiments, the amino acid sequences of the S domain and I domain are CH1 sequences. In particular embodiments, the amino acid sequences of the U domain and M domain are CH1 sequences. 6.3.18. Tetravalent 2x2 bonding molecules

[0317] In a variety of embodiments, ROR-binding molecules have 4 antigen-binding sites and are therefore termed tetravalent.

[0318] With reference to figure 34, in a further series of embodiments, the ROR-binding molecules additionally comprise a fifth and a sixth polypeptide chain, wherein: (a) the first polypeptide chain additionally comprises an N domain and an O domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a NOABDE orientation; (b) the third polypeptide chain further comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation; (c) the ROR antigen-binding molecule further comprises a fifth and a sixth polypeptide chain, wherein the fifth polypeptide chain comprises a P domain and a Q domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a PQ orientation, and the sixth polypeptide chain comprises a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation; and (d) the Petition 870260031312, dated 02 / 04 / 2026, page 114 / 417 106 / 285 The first and fifth polypeptides are associated through an interaction between the N and P domains and an interaction between the O and Q domains, and the third and sixth polypeptides are associated through an interaction between the R and T domains and an interaction between the S and U domains to form the ROR-binding molecule.

[0319] In several embodiments, the O domain is connected to the A domain via a peptide linker and the S domain is connected to the H domain via a peptide linker. In a preferred embodiment, the peptide linker that connects the O domain to the A domain, and connects the S domain to the H domain is a 6-amino acid GSGSGS peptide sequence, as described in more detail in section 0. 6.3.18.1. Tetravalent 2x2 bispecific constructions

[0320] With reference to Figure 34, in a series of tetravalent 2x2 bispecific ROR-binding molecules, the amino acid sequences of the N domain and the A domain are identical, the amino acid sequences of the H domain and the R domain are identical, the amino acid sequences of the O domain and the B domain are identical, the amino acid sequences of the I domain and the S domain are identical, the amino acid sequences of the P domain and the F domain are identical, the amino acid sequences of the L domain and the T domain are identical, the amino acid sequences of the Q domain and the G domain are identical, the amino acid sequences of the M domain and the U domain are identical; and wherein the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first Petition 870260031312, dated 02 / 04 / 2026, page 115 / 417 107 / 285 antigen, the N domain and the P domain form a second antigen-binding site specific to the first antigen, the interaction between the H domain and the L domain forms a third antigen-binding site specific to a second antigen, the interaction between the H domain and the L domain forms a third antigen-binding site specific to a second antigen, and the interaction between the R domain and the T domain forms a fourth antigen-binding site specific to the second antigen.

[0321] With reference to figure 34 in another series of tetravalent 2x2 bispecific ROR-binding molecules, the amino acid sequences of the H domain and the A domain are identical, the amino acid sequences of the N domain and the R domain are identical, the amino acid sequences of the I domain and the B domain are identical, the amino acid sequences of the O domain and the S domain are identical, the amino acid sequences of the L domain and the F domain are identical, the amino acid sequences of the P domain and the T domain are identical, the amino acid sequences of the M domain and the G domain are identical, the amino acid sequences of the Q domain and the U domain are identical, and wherein the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, the N domain and the P domain form a second antigen-specific binding site for a second antigen,The interaction between the H domain and the L domain forms a third antigen-binding site specific to the first antigen, and the interaction between the R domain and... Petition 870260031312, dated 02 / 04 / 2026, page 116 / 417 108 / 285 the T domain forms a fourth antigen-specific binding site for the second antigen. 6.3.19. Domain Joins 6.3.19.1. Joins connecting the VL and CH3 domains

[0322] In a variety of embodiments, the amino acid sequence that forms a junction between the C-terminus of a VL domain and the N-terminus of a CH3 domain is a modified sequence. In certain embodiments, one or more amino acids are deleted or added at the C-terminus of the VL domain. In certain embodiments, the junction connecting the C-terminus of a VL domain and the N-terminus of a CH3 domain is one of the sequences described in Table 2 below, in Section 0. In particular embodiments, A111 is deleted at the C-terminus of the VL domain. In certain embodiments, one or more amino acids are deleted or added at the N-terminus of the CH3 domain. In particular embodiments, p343 is deleted at the N-terminus of the CH3 domain. In particular embodiments, P343 and R344 are deleted at the N-terminus of the CH3 domain. In certain embodiments, one or more amino acids are deleted or added to both the C-terminus of the VL domain and the N-terminus of the CH3 domain.In particular embodiments, A111 is deleted at the C terminal of the VL domain and P343 is deleted at the N terminal in the CH3 domain. In a preferred embodiment, A111 and V110 are deleted at the C terminal of the VL domain. In another preferred embodiment, A111 and V110 are deleted at the C terminal of the VL domain and the N terminal of the CH3 domain has a P343V mutation. 6.3.19.2. Junctions connecting the VH and CH3 domains Petition 870260031312, dated 02 / 04 / 2026, page 117 / 417 109 / 285

[0323] In a variety of embodiments, the amino acid sequence that forms a junction between the C-terminus of a VH domain and the N-terminus of a CH3 domain is a modified sequence. In certain embodiments, one or more amino acids are deleted or added at the C-terminus of the VH domain. In certain embodiments, the junction connecting the C-terminus of a VH domain and the N-terminus of a CH3 domain is one of the sequences described in Table 3 below, in Section 0. In particular embodiments, K117 and G118 are deleted at the C-terminus of the VH domain. In certain embodiments, one or more amino acids are deleted or added at the N-terminus of the CH3 domain. In particular embodiments, P343 is deleted at the N-terminus of the CH3 domain. In particular embodiments, P343 and R344 are deleted at the N-terminus of the CH3 domain. In particular embodiments, P343, R344, and E345 are deleted at the N-terminus of the CH3 domain.In certain embodiments, one or more amino acids are deleted or added to either the C-terminus of the VH domain or the N-terminus of the CH3 domain. In a preferred embodiment, T116, K117, and G118 are deleted at the C-terminus of the VH domain. 6.3.19.3. Junctions connecting terminal C CH3 to terminal N CH2 (hinge)

[0324] In the ROR-binding molecules described in the present invention, the N-terminus of the CH2 domain has an amino acid sequence from the hinge region. As used in the present invention, hinge regions are sequences of an antibody heavy chain that link the variable-constant domain segment of the N-terminus of an antibody and a CH2 domain of an antibody. Furthermore Petition 870260031312, dated 02 / 04 / 2026, page 118 / 417 110 / 285 In addition, the hinge region typically provides both flexibility between the variable-constant domain segment of the N-terminal and the CH2 domain, as well as portions of the amino acid sequence that form disulfide bridges between heavy chains (e.g., the first and third polypeptide chains). As used in the present invention, the amino acid sequence of the hinge region is SEQ ID NO: 56.

[0325] In a variety of embodiments, a CH3 amino acid sequence is extended at the C-terminus at the junction between the C-terminus of the CH3 domain and the N-terminus of a CH2 domain. In certain embodiments, a CH3 amino acid sequence is extended at the C-terminus at the junction between the C-terminus of the CH3 domain and a hinge region, which in turn is connected to the N-terminus of a CH2 domain. In a preferred embodiment, the CH3 amino acid sequence is extended by inserting a PGK tripeptide sequence, followed by the DKTHT portion of an IgG1 hinge region.

[0326] In a particular embodiment, the C-terminal extension of the CH3 domain incorporates amino acid sequences that can form a disulfide bond with the orthogonal C-terminal extension of another CH3 domain. In a preferred embodiment, the C-terminal extension of the CH3 domain incorporates a KSC tripeptide sequence that is followed by the DKTHT portion of an IgG1 hinge region that forms a disulfide bond with the orthogonal C-terminal extension of another CH3 domain that incorporates a GEC portion of a kappa light chain. Petition 870260031312, dated 02 / 04 / 2026, page 119 / 417 111 / 285 6.3.19.4. Junctions connecting terminal C CL and terminal N CH2 (hinge)

[0327] In a variety of embodiments, a CL amino acid sequence is connected via its C-terminus to a hinge region which, in turn, is connected to the N-terminus of a CH2 domain. The hinge region sequences are described in more detail above in section 0. In a preferred embodiment, the hinge region amino acid sequence is SEQ ID NO: 56. 6.3.19.5. Junctions connecting the C CH2 terminal to the constant region domain.

[0328] In several embodiments, a CH2 amino acid sequence is connected through its C-terminus to the N-terminus of a constant region domain. Constant regions are described in more detail above in section 0. In a preferred embodiment, the CH2 sequence is connected to a CH3 sequence through its endogenous sequence. In other embodiments, the CH2 sequence is connected to a CH1 or CL sequence. Examples discussing the connection of a CH2 sequence to a CH1 or CL sequence are described in more detail in U.S. Patent No. 8,242,247, which is incorporated herein in its entirety. 6.3.19.6. Junctions connecting the O domain to the A domain or the S domain to the H domain in trivalent and tetravalent molecules.

[0329] In several embodiments, the heavy chains of antibodies (e.g., the first and third polypeptide chains) are extended at their N-terminus to include additional domains that provide additional ABS. With Petition 870260031312, dated 02 / 04 / 2026, p. 120 / 417 112 / 285 Referring to Figure 21, Figure 26, and Figure 34, in certain embodiments, the C-terminus of the constant region domain amino acid sequence of an O domain and / or an S domain is connected to the N-terminus of the variable region domain amino acid sequence of an A domain and / or an H domain, respectively. In some preferred embodiments, the constant region domain is a CH3 amino acid sequence and the variable region domain is a VL amino acid sequence. In some preferred embodiments, the constant region domain is a CL amino acid sequence and the variable region domain is a VL amino acid sequence. In certain embodiments, the constant region domain is connected to the variable region domain via a peptide linker. In a preferred embodiment, the peptide linker is a 6-amino acid GSGSGS peptide sequence.

[0330] In a variety of embodiments, the light chains of antibodies (e.g., the second and fourth polypeptide chains) are extended at their N-terminus to include additional variable-constant domain segments of an antibody. In certain embodiments, the constant region domain is a CH1 amino acid sequence and the variable region domain is a VH amino acid sequence. 6.4. Specific bivalent ROR binding molecules

[0331] In a further aspect, bivalent ROR binding molecules are provided.

[0332] With reference to figure 3, in a number of embodiments, the ROR-binding molecules comprise a Petition 870260031312, dated 02 / 04 / 2026, page 121 / 417 113 / 285 first, second, third and fourth polypeptide chains, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has a VL amino acid sequence, the B domain has a CH3 amino acid sequence, the D domain has a CH2 amino acid sequence, and the E domain has a constant region domain amino acid sequence; (b) the second polypeptide chain comprises an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a VH amino acid sequence and the G domain has a CH3 amino acid sequence;(c) the third polypeptide chain comprises an H domain, an I domain, a J domain, and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the H domain has a variable region domain amino acid sequence, the I domain has a constant region domain amino acid sequence, the J domain has a CH2 amino acid sequence, and the K domain has a constant region domain amino acid sequence; (d) the fourth polypeptide chain comprises an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a variable region domain amino acid sequence and the M domain has a constant region domain amino acid sequence; (e) the first and second polypeptides are associated through an interaction between the A and F domains; Petition 870260031312, dated 02 / 04 / 2026, page 122 / 417 114 / 285 and an interaction between domains B and G; (f) the third and fourth polypeptides are associated through an interaction between domains H and L and an interaction between domains I and M; and (g) the first and third polypeptides are associated through an interaction between domains D and J and an interaction between domains E and K to form the ROR-binding molecule.

[0333] In a preferred embodiment, the E domain has a CH3 amino acid sequence, the H domain has a VL amino acid sequence, the I domain has a CL amino acid sequence, the K domain has a CH3 amino acid sequence, the L domain has a VH amino acid sequence, and the M domain has a CH1 amino acid sequence.

[0334] In certain embodiments, the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, and the interaction between the H domain and the L domain forms a second antigen-specific binding site for a second antigen, and the ROR-binding molecule is a bivalent bispecific ROR-binding molecule. In certain embodiments, the interaction between the A domain and the F domain forms a first antigen-specific binding site for a first antigen, and the interaction between the H domain and the L domain forms a second antigen-specific binding site for the first antigen, and the ROR-binding molecule is a monovalent bivalent ROR-binding molecule. 6.4.1. Bivalent bispecific B-body BC1 Petition 870260031312, dated 02 / 04 / 2026, page 123 / 417 115 / 285

[0335] With reference to Figure 3 and Figure 6, in a series of embodiments, the ROR-binding molecule has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain, and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has a first amino acid sequence VL, the B domain has a CH3 amino acid sequence of human IgG1 with a T366K mutation and a C-terminus extension incorporating a KSC tripeptide sequence that is followed by the DKTHT portion of an IgG1 hinge region, the D domain has a CH2 amino acid sequence of IgG1, and the E domain has CH3 amino acids of human IgG1 with an S354C and T366W mutation;(b) the second polypeptide chain has an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a first amino acid sequence VH and the G domain has an amino acid sequence CH3 of IgG1, with an L351D mutation and a C-terminus extension incorporating a GEC amino acid disulfide moiety; (c) the third polypeptide chain has an H domain, an I domain, a J domain and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HIJK orientation, and wherein the H domain has a second amino acid sequence VL, the I domain has a human kappa Cl amino acid sequence, the J domain has a human IgG1 CH2 amino acid sequence and the K domain has a human IgG1 CH3 amino acid sequence with a Y349C, a D356E mutation, an L358M, a T366S mutation; Petition 870260031312, dated 02 / 04 / 2026, page 124 / 417 116 / 285 one L368A and one Y407V; (d) the fourth polypeptide chain has an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a second amino acid sequence VH and the M domain has an amino acid sequence CH1 of IgG1; (e) the first and second polypeptides are associated through an interaction between the A and F domains and an interaction between the B and G domains; (f) the third and fourth polypeptides are associated through an interaction between the H and L domains and an interaction between the I and M domains; (g) the first and third polypeptides are associated through an interaction between the D and J domains and an interaction between the E and K domains to form the ROR-binding molecule; (h) the A domain and the F domain form a first antigen-specific binding site for a first antigen;and (i) the H domain and the L domain form a second antigen-binding site specific for a second antigen.;

[0336] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 8, the second polypeptide chain has the sequence SEQ ID NO: 9, the third polypeptide chain has the sequence SEQ ID NO: 10, and the fourth polypeptide chain has the sequence SEQ ID NO: 11. 6.4.2. BC6 bivalent bispecific B-body

[0337] With reference to Figure 3 and Figure 14, in a number of embodiments, the ROR-binding molecule has a first, second, third and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain and a Petition 870260031312, dated 02 / 04 / 2026, page 125 / 417 (a) the first polypeptide chain has an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has a first amino acid sequence VL, the B domain has a CH3 amino acid sequence of human IgG1 with a C-terminal extension incorporating a KSC tripeptide sequence that is followed by the DKTHT portion of an IgG1 hinge region, the D domain has a CH2 amino acid sequence of IgG1, and the E domain has CH3 amino acids of human IgG1 with an S354C and T366W mutation; (b) the second polypeptide chain has an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a first amino acid sequence VH and the G domain has a CH3 amino acid sequence of IgG1, with a C-terminal extension incorporating a GEC amino acid disulfide portion;(c) the third polypeptide chain has an H domain, an I domain, a J domain, and a K domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a HI-JK orientation, and wherein the H domain has a second VL amino acid sequence, the I domain has a human kappa CL amino acid sequence, the J domain has a human IgG1 CH2 amino acid sequence, and the K domain has a human IgG1 CH3 amino acid sequence with a Y349C, a D356E, an L358M, a T366S, an L368A, and a Y407V mutation; (d) the fourth polypeptide chain has an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a second VH amino acid sequence and the M domain has a human IgG1 amino acid sequence; (e) the first and the second; Petition 870260031312, dated 02 / 04 / 2026, page 126 / 417 118 / 285 polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G; (f) the third and fourth polypeptides are associated through an interaction between domains H and L and an interaction between domains I and M; (g) the first and third polypeptides are associated through an interaction between domains D and J and an interaction between domains E and K to form the ROR-binding molecule; (h) domain A and domain F form a first antigen-specific binding site for a first antigen; and (i) domain H and domain L form a second antigen-specific binding site for a second antigen. 6.4.3. Bivalent bispecific B-body BC28

[0338] With reference to Figure 3 and Figure 16, in a series of embodiments, the ROR-binding molecule has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain, and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has a first amino acid sequence VL, the B domain has a CH3 amino acid sequence of human IgG1 with a Y349C mutation and a C-terminus extension incorporating a PGK tripeptide sequence that is followed by the DKTHT portion of an IgG1 hinge region, the D domain has a CH2 amino acid sequence of IgG1, and the E domain has CH3 amino acids of human IgG1 with an S354C and T366W mutation; (b) the second polypeptide chain has an F domain and a G domain, wherein the domains are arranged, from the N-terminus Petition 870260031312, dated 02 / 04 / 2026, page 127 / 417 (a) the first polypeptide chain has a 119 / 285 C-terminal, in an FG orientation, wherein the F domain has a first amino acid sequence VH and the G domain has a CH3 amino acid sequence of IgG1, with an S354C mutation and a C-terminal extension incorporating a PGK tripeptide sequence; (b) the third polypeptide chain has an H domain, an I domain, a J domain, and a K domain, wherein the domains are arranged, from the N-terminal to the C-terminal, in a HIJK orientation, and wherein the H domain has a second amino acid sequence VL, the I domain has a human kappa CL amino acid sequence, the J domain has a human IgG1 CH2 amino acid sequence, and the K domain has a human IgG1 CH3 amino acid sequence with a Y349C, a D356E, an L358M, a T366S, an L368A, and a Y407V mutation;(d) the fourth polypeptide chain has an L domain and an M domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an LM orientation, and wherein the L domain has a second amino acid sequence VH and the M domain has an amino acid sequence CH1 of IgG1; (e) the first and second polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G; (f) the third and fourth polypeptides are associated through an interaction between domains H and L and an interaction between domains I and M; (g) the first and third polypeptides are associated through an interaction between domains D and J and an interaction between domains E and K to form the ROR-binding molecule; (h) the A domain and the F domain form a first antigen-specific binding site for a first antigen; and (i) the; Petition 870260031312, dated 02 / 04 / 2026, page 128 / 417 The 120 / 285 H domain and the L domain form a second antigen-binding site specific for a second antigen.

[0339] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 24, the second polypeptide chain has the sequence SEQ ID NO: 25, the third polypeptide chain has the sequence SEQ ID NO: 10 and the fourth polypeptide chain has the sequence SEQ ID NO: 11. 6.4.4. Bivalent bispecific B-body BC44

[0340] With reference to Figure 3 and Figure 19, in a series of embodiments, the ROR-binding molecule has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises an A domain, a B domain, a D domain, and an E domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an ABDE orientation, and the A domain has a first amino acid sequence VL, the B domain has a CH3 amino acid sequence of human IgG1 with a Y349C mutation, a P343V mutation, and a C-terminus extension incorporating a PGK tripeptide sequence that is followed by the DKTHT portion of an IgG1 hinge region, the D domain has a CH2 amino acid sequence of human IgG1, and the E domain has CH3 amino acids of human IgG1 with an S354C mutation and a T366W mutation;(b) the second polypeptide chain has an F domain and a G domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an FG orientation, and wherein the F domain has a first amino acid sequence VH and the G domain has a CH3 amino acid sequence of human IgG1, with an S354C mutation; Petition 870260031312, dated 02 / 04 / 2026, page 129 / 417 121 / 285 an extension of the C-terminal incorporating a PGK tripeptide sequence; (c) the third polypeptide chain has an H domain, an I domain, a J domain and a K domain, wherein the domains are arranged, from the N-terminal to the C-terminal, in a HIJK orientation, and wherein the H domain has a second VL amino acid sequence, the I domain has a human kappa CL amino acid sequence, the J domain has a human IgG1 CH2 amino acid sequence and the K domain has a human IgG1 CH3 amino acid sequence with a Y349C, T366S, L368A and aY407V; (d) the fourth polypeptide chain has an L domain and an M domain, wherein the domains are arranged, from the N-terminal to the C-terminal, in an LM orientation, and wherein the L domain has a second VH amino acid sequence and the M domain has a human IgG1 amino acid sequence; (e) the first and second polypeptides are associated through an interaction between domains A and F and an interaction between domains B and G;(f) the third and fourth polypeptides are associated through an interaction between the H and L domains and an interaction between the I and M domains; and (g) the first and third polypeptides are associated through an interaction between the D and J domains and an interaction between the E and K domains to form the ROR-binding molecule; (h) the A domain and the F domain form a first antigen-specific binding site for a first antigen; and (i) the H domain and the L domain form a second antigen-specific binding site for a second antigen.

[0341] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 32, the second Petition 870260031312, dated 02 / 04 / 2026, page 130 / 417 The 122 / 285 polypeptide chain has the sequence SEQ ID NO: 25, the third polypeptide chain has the sequence SEQ ID NO: 10, and the fourth polypeptide chain has the sequence SEQ ID NO: 11. 6.5. Specific trivalent ROR binding molecules 6.5.1. Trivalent bispecific B-body 1x2 BC28-1x2

[0342] With reference to section 0 and figure 26, in a number of embodiments, the ROR-binding molecules additionally comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain additionally comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation, and wherein the R domain has the first amino acid sequence VL and the S domain has an amino acid sequence CH3 of IgG1 with a Y349C mutation and an extension at the C-terminus incorporating a tripeptide sequence PGK which is followed by the GSGSGS linking peptide connecting the S domain to the H domain;(b) the ROR-binding molecule further comprises a sixth polypeptide chain comprising: a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation, and wherein the T domain has the first amino acid sequence VH and the U domain has a CH3 amino acid sequence of human IgG1 with an S354C mutation and an extension at the C-terminus incorporating a PGK tripeptide sequence; (c) the third and sixth polypeptides are associated through an interaction between the R and T domains and an interaction between the S and U domains to form the; Petition 870260031312, dated 02 / 04 / 2026, page 131 / 417 123 / 285 ROR binding molecule, and (d) the R domain and the T domain form a third antigen-specific binding site for the first antigen.

[0343] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 24, the second polypeptide chain has the sequence SEQ ID NO: 25, the third polypeptide chain has the sequence SEQ ID NO: 37, the fourth polypeptide chain has the sequence SEQ ID NO: 11 and the sixth polypeptide chain has the sequence SEQ ID NO: 25. 6.5.2. Trivalent trispecific B-body 1x2 “BC281x1x1a

[0344] With reference to section 0 and figures 26 and 30, in a number of embodiments, the ROR-binding molecules additionally comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain additionally comprises an R domain and an S domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in an RSHIJK orientation, and wherein the R domain has a third amino acid sequence VL and the S domain has a CH3 amino acid sequence of human IgG1 with a T366K mutation and a C-terminus extension incorporating a KSC tripeptide sequence that is followed by the GSGSGS linker peptide connecting the S domain to the H domain; (b) the ROR-binding molecule further comprises a sixth polypeptide chain comprising: a T domain and a U domain, wherein the domains are arranged, from the N-terminus to the C-terminus, in a TU orientation, and wherein the T domain has a third amino acid sequence VH and the domain Petition 870260031312, dated 02 / 04 / 2026, p. 132 / 417 124 / 285 U has a human IgG1 CH3 amino acid sequence with an L351D mutation and a C-terminus extension incorporating a GEC amino acid disulfide moiety; and (c) the third and sixth polypeptides are associated through an interaction between the R and T domains and an interaction between the S and U domains to form the ROR-binding molecule, and (d) the R domain and the T domain form a third antigen-specific binding site for a third antigen.

[0345] In preferred embodiments, the first polypeptide chain has the sequence SEQ ID NO: 24, the second polypeptide chain has the sequence SEQ ID NO: 25, the third polypeptide chain has the sequence SEQ ID NO: 45, the fourth polypeptide chain has the sequence SEQ ID NO: 11, and the sixth polypeptide chain has the sequence SEQ ID NO: 53. 6.6. Other ROR binding molecule platforms

[0346] The various antibody platforms described above are not limiting. The antigen-binding sites described in the present invention, including specific CDR subsets, can be formatted into any binding molecule platform, including, but not limited to, complete antibodies, fab fragments, Fvs, scFvs, tandem scFvs, diabodies, scdiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibodies and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), which is incorporated herein by reference for all that is taught. Petition 870260031312, dated 02 / 04 / 2026, page 133 / 417 125 / 285 6.7. Antigen Specificities

[0347] Other antigens to which a ROR-binding molecule, as described in the present invention, can specifically bind, in addition to an ROR antigen, can be selected from a wide variety of molecular targets. For example, an antigen-binding site or sites may bind specifically to ECad, CLDN7, FGFR2b, N-Cad, Cad-11, FGFR2c, ERBB2, ERBB3, FGFR1, FOLR1, IGF-Ira, GLP1R, PDGFRa, PDGFRb, EPHB6, ABCG2, CXCR4, CXCR7, integrin-avb3, SPARC, VCAM, ICAM, annexin, ROR1, ROR2, TNFa, CD137, angiopoietin 2, angiopoietin 3, BAFF, beta-amyloid, C5, CA-125, CD147, CD152, CD19, CD20, CD22, CD23, CD24, CD25, CD274, CD28, CD3, CD30, CD33, CD37, CD4, CD40, CD44, CD44v4, CD44v6, CD44v7, CD50, CD51, CD52, CEA, CSF1R, CTLA-2, DLL4, EGFR, EPCAM, HER3, ganglioside GD2, GDF-8, Her2 / neu, CD2221, IL-17A, IL-12, IL-23, IL-13, IL-6, IL-23, an integrin, CD11a, MUC1, Notch,TAG-72, TGF3, TRAIL-R2, VEGF-A, VEGFR-1, VEGFR2, VEGFc, hematopoietins (four-helix bundles) (such as EPO (erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF), IL-4 (BCGF-1, BSF-1), IL-5 (BCGF2), IL-6, IL-4 (IFN-p2, BSF-2, BCDF), IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growth factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), OSM (OM, oncostatin M) and LIF (leukemia inhibitory factor)); interferons (such as IFN-γ, IFN-α and IFN-β); superfamily of immunoglobulins (such as B7.1 (CD80) and B7.2 (B70, CD86)); family of TNF (such as TNF-α, Petition 870260031312, dated 02 / 04 / 2026, page 134 / 417 126 / 285 (caquectin), TNF-β (lymphotoxin, LT, LT-α), LT-β, ​​Fas, CD27, CD30 and 4-1BBL); and those not assigned to a particular family (such as TGF-β, IL 1α, IL-1β, IL-1RA, IL-10 (inhibitor of cytokine F synthesis), IL-12 (NK cell stimulating factor), MIF, IL-16, IL-17 (mCTLA-8) and / or IL-18 (IGIF, interferon γ-inducible factor)); in modalities related to bispecific antibodies, the antibody may bind, for example, to two of these targets. Furthermore, the Fc portion of an antibody's heavy chain can be used to target cells that express the Fc receptor, such as using the Fc portion of an IgE antibody to target mast cells and basophils.

[0348] Other antigens to which a ROR-binding molecule, as described in the present invention, can specifically bind, in addition to an ROR antigen, may be selected, which specifically bind to the TNF family of receptors including, but not limited to, TNFR1 (also known as CD120a and TNFRSF1A), TNFR2 (also known as CD120b and TNFRSF1B), TNFRSF3 (also known as LTeR), TNFRSF4 (also known as OX40 and CD134), TNFRSF5 (also known as CD40), TNFRSF6 (also known as FAS and CD95), TNFRSF6B (also known as DCR3), TNFRSF7 (also known as CD27), TNFRSF8 (also known as CD30), TNFRSF9 (also known as 4-1BB), TNFRSF10A (also known as TRAILR1, DR4 and CD26), TNFRSF10B (also known as TRAILR2, DR5 and CD262), TNFRSF10C (also known as TRAILR3, DCR1, CD263), TNFRSF10D (also known as TRAILR4, DCR2 and CD264), TNFRSF11A (also known as RANK and CD265),TNFRSF11B (also known as OPG), TNFRSF12A (also known as FN14, Petition 870260031312, dated 02 / 04 / 2026, page 135 / 417 127 / 285 TWEAKR and CD266), TNFRSF13B (also known as TACI and CD267), TNFRSF13C (also known as BAFFR, BR3 and CD268), TNFRSF14 (also known as HVEM and CD270), TNFRSF16 (also known as NGFR, p75NTR and CD271) or TNFRSF17 (also known as BCMA and CD269), TNFRSF18 (also known as GITR and CD357), TNFRSF19 (also known as TROY, TAJ and TRADE), TNFRSF21 (also known as CD358), TNFRSF25 (also known as Apo-3, TRAMP, LARD or WS-1) and EDA2R (also known as XEDAR).

[0349] Other antigens to which a ROR-binding molecule, as described in the present invention, may specifically bind, in addition to an ROR antigen, may be selected from immuno-oncological targets including, but not limited to, checkpoint inhibitor targets such as PD1, PDL1, CTLA-4, PDL2, B7-H3, B7-H4, BTLA, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, BY55 and CGEN-15049. 6.8. Other modifications

[0350] In a further series of embodiments, the ROR-binding molecule has additional modifications. 6.8.1. Antibody-drug conjugates

[0351] In several embodiments, the ROR-binding molecule is conjugated with a therapeutic agent (e.g., drug) to form an ROR-binding molecule-drug conjugate. Therapeutic agents include, but are not limited to, chemotherapeutic agents, imaging agents (e.g., radioisotopes), immunomodulators (e.g., cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g., cytotoxic agents). In certain embodiments, the therapeutic agents Petition 870260031312, dated 02 / 04 / 2026, p. 136 / 417 128 / 285 are linked to the ROR-binding molecule via a peptide linker, as discussed in more detail below in section 0.

[0352] Methods for preparing antibody-drug conjugates (ADCs) that can be adapted to conjugate drugs with the ROR-binding molecules disclosed in the present invention are described, for example, in U.S. Patent No. 8,624,003 (container method), U.S. Patent No. 8,163,888 (one-step method), U.S. Patent No. 5,208,020 (two-step method), U.S. Patent No. 8,337,856, U.S. Patent No. 5,773,001, U.S. Patent No. 7,829,531, U.S. Patent No. 5,208,020, U.S. Patent No.7,745, 394, WO 2017 / 136623, WO 2017 / 015502, WO 2017 / 015496, WO 2017 / 015495, WO 2004 / 010957, WO 2005 / 070, WO 2005 / 02020, WO 2006 / 065533, WO 2007 / 030642, WO 2007 / 103288, WO 2013 / 173337, WO 2015 / 057699, WO 2015 / 095755, WO 2015 / 1, WO 23679 2015 / 157286, WO 2017 / 165851, WO 2009 / 073445, WO 2010 / 068759, WO 2010 / 138719, WO 2012 / 171020, WO 2014 / 00035, WO 2014 / 0394, WO 2010 / 068759 WO 2014 / 093640, WO 2014 / 160360, WO 2015 / 054659, WO 2015 / 195925, WO 2017 / 160754, Storz (MAbs. 2015, Nov-Dec; 7(6): 99-99), Lambert et al. (Adv Ther, 2017 34: 1015), Diamantis et al. (British Journal of Cancer, 2016, 114, 362367), Carrico et al. (Nat Chem Biol, 2007. 3: 321-2), We et al. (Proc Natl Acad Sci USA, 2009. 106: 3000-5), Rabuka et al. (Curr Opin Chem Biol., 2011 14: 790-6), Hudak et al. (Angew Chem Int Ed Engl., 2012: 4161-5), Rabuka et al. (Nat Protoc., 2012 7:1052-67), Agarwal et al. (Proc Natl Acad Sci. Petition 870260031312, of 02 / 04 / 2026, p. 137 / 417 129 / 285 USA., 2013, 110: 46-51), Agarwal et al. (Bioconjugate Chem., 2013, 24: 846-851), Barfield et al. (Drug Dev. e D., 2014, 14:34-41), Drake et al. (Bioconjugate Chem., 2014, 25:133-141), Liang et al. (J Am Chem Soc., 2014, 136:10850-3), Drake et al. (Curr Opin Chem Biol., 2015, 28:174-80) and York et al. (BMC Biotechnology, 2016, 16(1):23), each of them incorporated by reference in their entirety, for all that they teach. 6.8.2. Additional linking portions

[0353] In several embodiments, the ROR-binding molecule has modifications comprising one or more additional binding moieties. In certain embodiments, the binding moieties are antibody fragments or antibody formats including, but not limited to, complete antibodies, fab fragments, Fvs, scFvs, tandem scFvs, diabodies, scdiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art. Exemplary antibodies and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), which is incorporated here by reference for all that it teaches.

[0354] In particular embodiments, one or more additional linking portions are attached to the C-terminus of the first or third polypeptide chain. In particular embodiments, one or more additional linking portions are attached to the C-terminus of the first and third polypeptide chains. In particular embodiments, one or more additional linking portions are attached to the C-terminus of both Petition 870260031312, dated 02 / 04 / 2026, p. 138 / 417 130 / 285 of the first and third polypeptide chains. In certain embodiments, individual portions of one or more additional linking portions are separately attached to the C-terminus of the first and third polypeptide chains, so that the portions form a functional linking portion.

[0355] In particular embodiments, one or more additional linking portions are attached to the N-terminus of any of the polypeptide chains (for example, the first, second, third, fourth, fifth, or sixth polypeptide chains). In certain embodiments, individual portions of the additional linking portions are separately attached to the N-terminus of different polypeptide chains, so that the portions form the functional linking portion.

[0356] In certain embodiments, one or more additional binding portions are specific for a different antigen or epitope of the ABS within the ROR-binding molecule. In certain embodiments, one or more additional binding portions are specific for the same antigen or epitope of the ABS within the ROR-binding molecule. In certain embodiments, where the modification is two or more additional binding portions, the additional binding portions are specific for the same antigen or epitope. In certain embodiments, where the modification is two or more additional binding portions, the additional binding portions are specific for different antigens or epitopes.

[0357] In certain embodiments, one or more additional binding moieties are attached to the ROR-binding molecule using in vitro methods including, but not limited to, reactive chemistry and affinity labeling systems, such as Petition 870260031312, dated 02 / 04 / 2026, p. 139 / 417 131 / 285 discussed in more detail below in section 0. In certain embodiments, one or more additional binding moieties are attached to the ROR-binding molecule via Fc-mediated binding (e.g., A / G protein). In certain embodiments, one or more additional binding moieties are attached to the ROR-binding molecule using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the ROR-binding molecule and the additional binding moieties in the same expression vector (e.g., plasmid). 6.8.3. Functional / Reactive Groups

[0358] In several embodiments, the ROR-binding molecule has modifications comprising functional groups or chemically reactive groups, which can be used in downstream processes such as binding to additional moieties (e.g., drug conjugates and additional binding moieties, as discussed in more detail above in sections 0 and 0) and in downstream purification processes.

[0359] In certain embodiments, the modifications are chemically reactive groups, including, but not limited to, reactive thiols (e.g., maleimide-based reactive groups), reactive amines (e.g., N-hydroxysuccinimide-based reactive groups), “click chemistry” (e.g., alkyne-reactive groups), and formylglycine-containing aldehydes (FGly). In certain embodiments, the modifications are functional groups including, but not limited to, affinity peptide sequences (e.g., HA, HIS, FLAG, GST, MBP, and Strep systems, etc.). In certain embodiments, the functional groups or chemically reactive groups have a Petition 870260031312, dated 02 / 04 / 2026, page 140 / 417 132 / 285 cleavable peptide sequence. In particular embodiments, the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reduction conditions, and pH conditions. In particular embodiments, protease cleavage is performed by intracellular proteases. In particular embodiments, protease cleavage is performed by extracellular or membrane-associated proteases. ADC therapies employing protease cleavage are described in more detail in Choi et al. (Theranostics, 2012; 2(2): 156-178), the entirety of which is incorporated herein by reference for all that it teaches. 6.8.4. Reduced effector function

[0360] In certain embodiments, the ROR-binding molecule has one or more manipulation mutations in the amino acid sequence of an antibody domain that reduces the effector functions naturally associated with antibody binding. Effector functions include, but are not limited to, cellular functions that result from Fc receptor binding to an Fc portion of an antibody, such as antibody-dependent cellular cytotoxicity (ADCC, also referred to as antibody-dependent cell-mediated cytotoxicity), complement fixation (e.g., C1q binding), antibody-dependent cell-mediated phagocytosis (ADCP), and opsonization. Manipulation mutations that reduce effector functions are described in more detail in North American Publication No. 2017 / 0137530, in Armour, et al. (Eur. J. Immunol. 29(8) (1999) 2613-2624), Shields, et al. (J. Biol. Chem. 276(9) (2001) 6591-6604), and Petition 870260031312, dated 02 / 04 / 2026, pp. 141 / 417 133 / 285 in Oganesyan, et al. (Acta Cristallographica D64 (2008) 700704), each incorporated herein by reference in its entirety.

[0361] In specific embodiments, the ROR-binding molecule has one or more manipulation mutations in an amino acid sequence of an antibody domain that reduce the binding of an Fc portion of the ROR-binding molecule by FcR receptors. In some embodiments, the FcR receptors are FcRy receptors. In particular embodiments, the FcR receptors are FcyRIIa and / or FcyRIIIA receptors.

[0362] In specific embodiments, one or more manipulation mutations that reduce effector function are mutations in a CH2 domain of an antibody. In several embodiments, one or more manipulation mutations are at positions L234 and L235 of the CH2 domain. In particular embodiments, one or more manipulation mutations are L234A and L235A of the CH2 domain. In other embodiments, one or more manipulation mutations are at positions L234, L235, and P329 of the CH2 domain. In particular embodiments, one or more manipulation mutations are L234A, L235A, and P329G of the CH2 domain. In particular embodiments, one or more manipulation mutations are L234A, L235A, and P329K of the CH2 domain. 6.9. Purification methods

[0363] The present invention provides a method for purifying a ROR-binding molecule comprising a B-Body platform. Petition 870260031312, dated 02 / 04 / 2026, page 142 / 417 134 / 285

[0364] In a number of embodiments, the method comprises the steps of: i) contacting a sample comprising the ROR-binding molecule with a CH1-binding reagent, wherein the ROR-binding molecule comprises at least one first, second, third and fourth polypeptide chains associated in a complex, wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one less than the valence of the complex, and wherein the contact is made under conditions sufficient for the CH1-binding reagent to bind to the CH1 domain, or portion thereof; and ii) purifying the complex from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, second, third and fourth polypeptide chains.

[0365] In a typical antibody occurring in nature, two heavy chains are associated, each having a CH1 domain as a second domain, numbered from the N-terminus to the C-terminus. Thus, a typical antibody has two CH1 domains. The CH1 domains are described in more detail in section 0. In several of the ROR-binding molecules described in the present invention, the CH1 domain typically found in the protein has been replaced by another domain, such that the number of CH1 domains in the protein is effectively reduced. In a non-limiting illustrative example, the CH1 domain of a typical antibody can be replaced by a CH3 domain, generating an antigen-binding protein with only a single CH1 domain. Petition 870260031312, dated 02 / 04 / 2026, page 143 / 417 135 / 285

[0366] ROR-binding molecules may also refer to molecules based on antibody architectures that have been modified in such a way that they no longer possess a typical antibody architecture. For example, an antibody may be extended at its N- or C-terminus to increase the valence (described in more detail in section 0) of the antigen-binding protein, and in certain cases, the number of CH1 domains is also increased beyond the typical two CH1 domains. Such molecules may also have one or more of their CH1 domains substituted, such that the number of CH1 domains in the protein is at least one less than the valence of the antigen-binding protein. In some embodiments, the number of CH1 domains that are substituted by other domains generates an ROR-binding molecule with only a single CH1 domain.In other embodiments, the number of CH1 domains substituted by another domain generates an ROR-binding molecule with two or more CH1 domains, but at least one less than the valence of the antigen-binding protein. In particular embodiments, where an ROR-binding molecule has two or more CH1 domains, the multiple CH1 domains may all be on the same polypeptide chain. In other particular embodiments, where an ROR-binding molecule has two or more CH1 domains, the multiple CH1 domains may be a single CH1 domain in multiple copies of the same polypeptide chain present in the complete complex. 6.9.1. CH1 bonding reagents

[0367] In exemplary non-limiting purification methods of ROR-binding molecules, a sample that Petition 870260031312, dated 02 / 04 / 2026, p. 144 / 417 136 / 285 comprises the ROR-binding molecules that come into contact with the CH1-binding reagents. The CH1-binding reagents, as described in this invention, can be any molecule that specifically binds to a CH1 epitope. The various CH1 sequences that provide the CH1 epitope are described in more detail in section 0, and the specific binding is described in more detail in section 0.

[0368] In some embodiments, the CH1 binding reagents are derived from immunoglobulin proteins and have an antigen-binding site (ABS) that specifically binds to the CH1 epitope. In particular embodiments, the CH1 binding reagent is an antibody, also referred to as an “anti-CH1 antibody.” The anti-CH1 antibody can be derived from various species. In particular embodiments, the anti-CH1 antibody is a mammalian antibody, including, but not limited to, antibodies from mice, rats, hamsters, rabbits, camels, donkeys, goats, and humans. In specific embodiments, the anti-CH1 antibody is a single-domain antibody. Single-domain antibodies, as described in the present invention, have a single variable domain that forms an ABS and specifically binds to the CH1 epitope.Exemplary single-domain antibodies include, but are not limited to, camel- and shark-derived heavy-chain antibodies, as described in more detail in international application WO 2009 / 011572, incorporated herein by reference for all that it teaches. In a preferred embodiment, the anti-CH1 antibody is a camel-derived antibody (also referred to as a “camellid antibody”). Exemplary camelid antibodies include, but are not limited to. Petition 870260031312, dated 02 / 04 / 2026, p. 145 / 417 137 / 285 limit the antibodies to human IgG-CH1 CaptureSelect™ (ThermoFisher, No. 194320010) and human IgA-CH1 (ThermoFisher, No. 194311010). In some embodiments, the anti-CH1 antibody is a monoclonal antibody. Monoclonal antibodies are typically produced from cultured antibody-producing cell lines. In other embodiments, the anti-CH1 antibody is a polyclonal antibody, that is, a collection of different anti-CH1 antibodies, where each recognizes the CH1 epitope. Polyclonal antibodies are typically produced by collecting antibody-containing serum from an animal immunized with the antigen of interest, or a fragment thereof, in this case CH1.

[0369] In some embodiments, the CH1-binding reagents are molecules not derived from immunoglobulin proteins. Examples of such molecules include, but are not limited to, aptamers, peptoids, and aphibodies, as described in more detail in Perret and Boschetti (Biochimie, February 2018, vol 145: 98-112). 6.9.2. Solid supports

[0370] In exemplary, non-limiting methods for purifying ROR-binding molecules, the CH1-binding reagent can be attached to a solid support in various embodiments of the invention. Solid supports, as described in the present invention, refer to a material to which other entities can be attached or immobilized, such as, for example, the CH1-binding reagent. Solid supports, also called carriers, are described in more detail in international application WO 2009 / 011572. Petition 870260031312, dated 02 / 04 / 2026, p. 146 / 417 138 / 285

[0371] In specific embodiments, the solid support comprises a sphere or nanoparticle. Examples of spheres and nanoparticles include, but are not limited to, agarose spheres, polystyrene spheres, magnetic nanoparticles (e.g., Dynabeads™Λ from ThermoFisher), polymers (e.g., dextran), synthetic polymers (e.g., Sepharose™) or any other material suitable for binding the binding reagent to CH1. In particular embodiments, the solid support is modified to allow the binding reagent to be attached to CH1. Examples of solid support modifications include, but are not limited to, chemical modifications that form covalent bonds with proteins (e.g., activated aldehyde groups) and modifications that specifically pair with a cognate modification of a CH1-binding reagent (e.g., biotin-streptavidin pairs, disulfide linkages, polyhistidine-nickel, or "click chemistry" modifications such as azidoalkynyl pairs).

[0372] In certain embodiments, the CH1-binding reagent is attached to the solid support before the CH1-binding reagent comes into contact with the ROR-binding molecules, herein also referred to as an “anti-CH1 resin”. In some embodiments, the anti-CH1 resins are dispersed in a solution. In other embodiments, the anti-CH1 resins are “packed” in a column. The anti-CH1 resin is then brought into contact with the ROR-binding molecules and the CH1-binding reagents bind specifically to the ROR-binding molecules. Petition 870260031312, dated 02 / 04 / 2026, p. 147 / 417 139 / 285

[0373] In other embodiments, the CH1-binding reagent is fixed to the solid support after the CH1-binding reagent comes into contact with the ROR-binding molecules. As a non-limiting illustration, a CH1-binding reagent with a biotin modification can come into contact with the ROR-binding molecules, and subsequently, the CH1-binding reagent / ROR-binding molecule mixture can be brought into contact with the streptavidin-modified solid support to fix the CH1-binding reagent to the solid support, including CH1-binding reagents specifically bound to ROR-binding molecules.

[0374] In methods where CH1-binding reagents are attached to solid supports, in a variety of embodiments, the bound ROR-binding molecules are released, or eluted, from the solid support forming an eluate with the ROR-binding molecules. In some embodiments, the bound ROR-binding molecules are released by inverting paired modifications (e.g., disulfide bond reduction), adding a reagent to compete with ROR-binding molecules (e.g., adding imidazole competing with a polyhistidine to bind to nickel), cleaving ROR-binding molecules (e.g., a cleavable moiety may be included in the modification), or otherwise interfering with the specific binding of the CH1-binding reagent to the ROR-binding molecule. Methods that interfere with specific binding include, but are not limited to, contacting the bound ROR-binding molecules with Petition 870260031312, dated 02 / 04 / 2026, pp. 148 / 417 140 / 285 CH1 binding reagents with a low pH solution. In the preferred embodiment, the low pH solution comprises 0.1 M acetic acid at pH 4.0. In other embodiments, the bound ROR-binding molecules can be brought into contact with a range of low pH solutions, i.e., with a gradient. 6.9.3. Additional purification

[0375] In some embodiments of the exemplary non-limiting methods, a single iteration of the method using the steps of contacting the ROR-binding molecules with the CH1-binding reagents, followed by elution of the ROR-binding molecules, is used to purify the ROR-binding molecules from one or more incomplete complexes. In particular embodiments, no further purification steps are performed. In other embodiments, one or more additional purification steps are performed to further purify the ROR-binding molecules from one or more incomplete complexes. The one or more additional purification steps include, but are not limited to, the purification of the ROR-binding molecules based on other protein characteristics such as size (e.g., size exclusion chromatography), charge (e.g., ion exchange chromatography), or hydrophobicity (e.g., hydrophobic interaction chromatography).In a preferred embodiment, an additional cation exchange chromatography analysis is performed. Furthermore, the ROR-binding molecules can be further purified by repeating the contact of the ROR-binding molecules. Petition 870260031312, dated 02 / 04 / 2026, page 149 / 417 141 / 285 ROR with CH1-binding reagents, as described above, as well as modifying the CH1 purification method between iterations, for example, using a stepwise elution for the first iteration and a gradient elution for a subsequent elution. 6.9.4. Assembly and purity of the complexes

[0376] In embodiments of the present invention, at least four distinct polypeptide chains associate to form a complete complex, for example, the ROR-binding molecule. However, incomplete complexes may also form that do not contain at least four distinct polypeptide chains. For example, incomplete complexes may form that have only one, two, or three polypeptide chains. In other examples, an incomplete complex may contain more than three polypeptide chains but not contain at least four distinct polypeptide chains, for example, the incomplete complex inappropriately associates with more than one copy of a distinct polypeptide chain. The method of the invention purifies the complex, for example, the fully assembled ROR-binding molecule, from incomplete complexes.

[0377] The methods for evaluating the effectiveness and efficiency of purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion-exchange chromatography, size-exclusion chromatography, and mass spectrometry. Purity can also be evaluated according to a Petition 870260031312, dated 02 / 04 / 2026, page 150 / 417 142 / 285 variety of criteria. Examples of criteria include, but are not limited to: 1) evaluating the percentage of total protein in an eluate that is provided by the fully assembled ROR-binding molecule, 2) evaluating the enrichment in times or the percentage increase of the purification method of the desired products, for example, by comparing the total protein provided by the fully assembled ROR-binding molecule in the eluate with that of an initial sample, 3) evaluating the percentage of total protein or the percentage decrease of undesirable products, for example, the incomplete complexes described above, including determining the percentage or percentage decrease of specific undesirable products (e.g., unassociated single polypeptide chains, dimers of any combination of polypeptide chains or trimers of any combination of polypeptide chains). Purity can be evaluated after any combination of the methods described in the present invention.For example, purity can be evaluated after a single iteration of the use of the CH1 anti-binding reagent, as described in the present invention, or after additional purification steps, as described in more detail in section 0. The effectiveness and efficiency of the purification steps can also be used to compare the methods described using the CH1 anti-binding reagent with other purification methods known to those skilled in the art, such as the purification of protein A. 6.10. Manufacturing methods

[0378] The ROR-binding molecules described in the present invention can be readily manufactured by expression using free translation approaches. Petition 870260031312, dated 02 / 04 / 2026, page 151 / 417 143 / 285 standard cells, transient transfection, and stable transfection are currently used for antibody production. In specific embodiments, Expi293 (ThermoFisher) cells can be used for the production of ROR-binding molecules using ThermoFisher protocols and reagents, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethyleneimine, as described in detail in Fang et al. (Biological Procedures Online, 2017, 19:11), which is incorporated here by reference for all that it teaches.

[0379] As further described in the examples below, expressed proteins can be readily separated from unwanted proteins and protein complexes using a CH1 affinity resin, such as CaptureSelect CH1 resin and the ThermoFisher protocol provided. Other purification strategies include, but are not limited to, the use of protein A, protein G, or protein A / G reagents. Further purification can be performed using ion-exchange chromatography, as is routinely used in the technique. 6.11. Pharmaceutical compositions

[0380] In another aspect, pharmaceutical compositions are provided comprising the ROR-binding molecule, as described in the present invention, and a pharmaceutically acceptable vehicle or diluent. In typical embodiments, the pharmaceutical composition is sterile.

[0381] In various embodiments, the pharmaceutical composition comprises the ROR-binding molecule at a concentration of 0.1 mg / mL to 100 mg / mL. In embodiments Petition 870260031312, dated 02 / 04 / 2026, page 152 / 417 144 / 285 specific, the pharmaceutical composition comprises the ROR-binding molecule at a concentration of 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, or 10 mg / mL. In some embodiments, the pharmaceutical composition comprises the ROR-binding molecule at a concentration of more than 10 mg / mL. In certain embodiments, the ROR-binding molecule is present at a concentration of 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or even 50 mg / mL or more. In particular embodiments, the ROR-binding molecule is present at a concentration of more than 50 mg / mL.

[0382] In various embodiments, the pharmaceutical compositions are described in more detail in U.S. Patent No. 8,961,964, U.S. Patent No. 8,945,865, U.S. Patent No. 8,420,081, U.S. Patent No. 6,685,940, U.S. Patent No. 6,171,586, U.S. Patent No. 8,821,865, U.S. Patent No. 9,216,219, U.S. Application No. 10 / 813,483, WO 2014 / 066468, WO 2011 / 104381 ​​and WO 2016 / 180941, each of which is incorporated herein in its entirety. 6.12. Treatment methods

[0383] In another aspect, treatment methods are provided, comprising administering an ROR-binding molecule (e.g., antibody), as described in the present invention, to an individual in an amount effective to treat the individual. Such ROR antigen-binding molecules are useful in the treatment of ROR-expressing cancers, including cancers expressing a Petition 870260031312, dated 02 / 04 / 2026, page 153 / 417 145 / 285 antigen to ROR1, cancers that express an antigen to ROR2 and / or cancers that express an antigen to ROR1 and an antigen to ROR2.

[0384] In some embodiments, an antibody of the present disclosure can be used to treat a variety of cancers. Cancer can be bladder cancer, blood cancer (acute and chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), bone cancer, bone marrow cancer, brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), breast cancer, colon cancer, esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastrointestinal cancer, gum cancer, head cancer, kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma), liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, prostate cancer (adenocarcinoma, sarcoma, castration-resistant prostate cancer), skin cancer, stomach cancer (carcinoma, lymphoma, leiomyosarcoma), testicular (seminoma, teratoma,Embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma), of the tongue or uterus. In some forms, the cancer may be a malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; Petition 870260031312, dated 02 / 04 / 2026, page 154 / 417 146 / 285 pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis of the colon; solid carcinoma; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma;Serous papillary cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; Petition 870260031312, dated 02 / 04 / 2026, page 155 / 417 147 / 285 glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid melanocytoma; malignant blue nevus; sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma); fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Müllerian tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian stromal tumor; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;Mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillar astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma (sarcoma of; Petition 870260031312, dated 02 / 04 / 2026, page 156 / 417 148 / 285 reticulum cell); Hodgkin's disease; Hodgkin's paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other unspecified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative disease of the small intestine; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; myxoma; rhabdomyoma; fibroma; squamous cell carcinoma of the head and neck; laryngeal and hypopharyngeal cancer; Nasal cavity or paranasal sinus cancer; nasopharyngeal cancer; salivary gland cancer; oral cancer; oropharyngeal cancer;Bronchogenic carcinoma (squamous cell carcinoma, small undifferentiated cell carcinoma, large undifferentiated cell carcinoma, adenocarcinoma, non-small cell lung cancer); alveolar (bronchiolar) carcinoma; bronchial adenoma; chondromatous hamartoma; colorectal cancer; gastrointestinal stromal tumors; carcinoids; Turcot syndrome; gastric cancer; adenocarcinoma of the gastroesophageal junction; pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, virgoma); small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large intestine cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); metastatic breast cancer; carcinoma; Petition 870260031312, dated 02 / 04 / 2026, page 157 / 417 149 / 285 ductal carcinoma in situ; invasive ductal carcinoma; tubular carcinoma; mucinous carcinoma; lobular carcinoma in situ; triple-negative breast cancer; bladder and urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma); clear cell carcinoma; hepatoma (hepatocellular carcinoma); angiosarcoma; hepatocellular adenoma; hemangioma; osteogenic sarcoma (osteosarcoma); malignant fibrous histiocytoma; giant cell malignant tumor chordoma; osteochondroma (osteocartilaginous exostoses); benign chondroma; chondromyxofibroma; osteoid osteoma; giant cell tumors; medullary thyroid cancer; differentiated thyroid cancer; papillary thyroid cancer; follicular thyroid cancer; Hurthle cell carcinoma; anaplastic thyroid cancer; skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans); meninges (meningioma, meningiosarcoma, gliomatosis);of the spinal cord (neurofibroma, meningioma, glioma, sarcoma); of the uterus (clear); of the cervix (cervical carcinoma, cervical predysplasia); of the ovary (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma); of the vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma); of the vagina (clear cell carcinoma, squamous cell carcinoma); botryoid sarcoma (embryonal rhabdomyosarcoma); of the fallopian tubes (carcinoma); non-Hodgkin lymphoma [malignant lymphoma]; Karposi's sarcoma; soft dysplastic nevi; angioma; Petition 870260031312, dated 02 / 04 / 2026, page 158 / 417 150 / 285 dermatofibroma; keloids; psoriasis; neuroblastoma; adrenocortical carcinoma; pheochromocytomas; paragangliomas; Merkel cell carcinoma; neuroendocrine tumors; carcinoid tumors; pancreatic cancers; gastroesophageal cancers; clear cell renal cell carcinoma; and primary peritoneal cancer.

[0385] An antibody of the present disclosure may be administered to an individual by itself or in the form of a pharmaceutical composition for the treatment of, for example, cancer, autoimmunity, transplant rejection, post-traumatic immune responses, graft-versus-host disease, ischemia, stroke and infectious diseases (for example, by targeting viral antigens such as HIV gp120).

[0386] In another aspect, a ROR-binding molecule (e.g., antibody), as described in the present invention, can be used in a method of treating an individual with cancer in combination with one or more additional therapies. The additional therapies that can be used in combination with a ROR antigen-binding molecule (e.g., antibody) described in the present invention include, but are not limited to: (i) surgery; (ii) radiotherapy; (iii) endocrine therapy; (iv) immunotherapy (including adjuvant therapy and cell therapy, such as CAR T-cell therapy); and (v) chemotherapy, including cytotoxic agents and chemotherapeutic agents.

[0387] Any therapy that has activity against a cancer can be used in conjunction with a ROR antigen-binding molecule (e.g., antibody), Petition 870260031312, dated 02 / 04 / 2026, page 159 / 417 151 / 285 provided in the present invention. Examples of such agents for the treatment of cancer can be found, for example, at https: / / www.cancer.gov / about-cancer / treatment / drugs (last accessed January 22, 2019) and in publicly available sources such as Cancer Principles and Practice of Oncology, by V.T. Devita and S. Hellman (editors), 11th edition (2018), Lippincott Williams & Wilkins Publishers. A person skilled in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the type of cancer involved.

[0388] In certain modalities, additional therapy is radiotherapy including, for example, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals. The radiation source may be external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Exemplary radioactive elements include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

[0389] In certain modalities, the additional therapy is immunotherapy. Immunotherapy (also called biological response modifying therapy, biological therapy, biotherapy, immunotherapy, or biological therapy) is treatment that uses parts of the immune system to Petition 870260031312, dated 02 / 04 / 2026, page 160 / 417 152 / 285 to fight diseases. Immunotherapy can help the immune system recognize cancer cells, or improve a response against cancer cells. Immunotherapies include active and passive immunotherapies. Active immunotherapies, including immunotherapeutic agents, stimulate the body's own immune system (e.g., vaccines), while passive immunotherapies, including immunotherapeutic agents, generally use components of the immune system created outside the body (e.g., antibodies), antibodies conjugated with drugs, toxins, or radionuclides, and targeted therapies.

[0390] Exemplary immunotherapeutic agents include immune checkpoint inhibitors. In some modalities, the immune checkpoint inhibitor used in treatment methods may reduce, inhibit, interfere with, or modulate, wholly or partially, one or more checkpoint proteins that regulate T cell activation or function. Several checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0391] In certain embodiments, the checkpoint inhibitor is an OX40 (CD134) agonist. In some embodiments, the checkpoint inhibitor is an anti-OX40 antibody. In some embodiments, the anti-OX40 antibody is anti-OX-40. In some embodiments, the anti-OX40 antibody is MEDI6469. Petition 870260031312, dated 02 / 04 / 2026, p. 161 / 417 153 / 285

[0392] In certain embodiments, the checkpoint inhibitor is an OX40 agonist. In some embodiments, the checkpoint inhibitor is an anti-OX40 antibody. In some embodiments, the anti-CD40 antibody is CF-870.893.

[0393] In certain embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in U.S. Patents Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238. In some embodiments, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the trade name Yervoy™.

[0394] In certain embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include, but are not limited to, those described in U.S. Patents Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and in PCT Patent Application Publications Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400 and WO2011161699.

[0395] In certain embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is BGB-A317, Petition 870260031312, dated 02 / 04 / 2026, p. 162 / 417 154 / 285 nivolumab (also known as ONO-4538, BMS-936558, or MDX1106) or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is marketed under the trade name Opdivo™. In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized IgG4 monoclonal antibody and is marketed under the trade name Keytruda™. In some embodiments, the anti-PD-1 antibody is CT-011, a humanized antibody. CT-011, administered alone, has not shown a response in the treatment of relapsed acute myeloid leukemia (AML). In some embodiments, the anti-PD-1 antibody is AMP-224, a fusion protein. In some embodiments, the PD-1 antibody is BGB-A317.BGB-A317 is a monoclonal antibody in which the ability to bind to the Fc gamma I receptor is specifically modified by manipulation, and which has a unique binding signature with PD-1, with high affinity and specificity for the superior target.

[0396] In certain embodiments, the checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In some embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In some embodiments, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriq®).

[0397] In certain embodiments, the checkpoint inhibitor is a PD-L2 inhibitor. In some Petition 870260031312, dated 02 / 04 / 2026, p. 163 / 417 In embodiments 155 / 285, the PD-L2 inhibitor is an anti-PDL2 antibody. In some embodiments, the anti-PD-L2 antibody is rHIgM12B7A.

[0398] In certain embodiments, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. In some embodiments, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brigrone et al., J. Immunol., 2007, 179, 4202-4211). In some embodiments, the LAG-3 inhibitor is BMS-986016.

[0399] In certain embodiments, the checkpoint inhibitor is a B7 inhibitor. In some embodiments, the B7 inhibitor is a B7-H3 inhibitor or a B7H4 inhibitor. In some embodiments, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).

[0400] In certain embodiments, the checkpoint inhibitor is a TIM3 inhibitor (T-cell immunoglobulin domain and mucin domain 3) (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).

[0401] In certain embodiments, the checkpoint inhibitor is a GITR agonist. In some embodiments, the checkpoint inhibitor is an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518.

[0402] In certain embodiments, the checkpoint inhibitor is a CD137 agonist. In some embodiments, the checkpoint inhibitor is an anti-CD137 antibody. In some embodiments, the anti-CD137 antibody is urelumab. Petition 870260031312, dated 02 / 04 / 2026, page 164 / 417 156 / 285 In some forms, the anti-CD137 antibody is PF05082566.

[0403] In certain embodiments, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL15).

[0404] In certain embodiments, the checkpoint inhibitor is an IDO inhibitor. In some embodiments, the IDO inhibitor is INCB024360. In some embodiments, the IDO inhibitor is indoximode.

[0405] Other exemplary immunotherapies include adjuvant therapies, including immunotherapeutic agents such as cytokines, chemokines, interferons, interleukins, or lymphokines. Examples include cytokines such as granulocyte-macrophage colony-stimulating factor (GMCSF), granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (alum); Bacillus Calmette-Guerin (BCG); California limpet hemocyanin (KLH); Incomplete Freund's adjuvant (IFA); QS-21; DETOX; levamisole; and dinitrophenyl (DNP), and combinations thereof, such as combinations of interleukins, for example, IL-2, with other cytokines, such as IFN-alpha.

[0406] Other exemplary immunotherapies include cell therapies, for example, a population of immune cells, such as leukocytes (nucleated white blood cells), comprising (for example, expressing) a receptor that Petition 870260031312, dated 02 / 04 / 2026, page 165 / 417 157 / 285 binds to an antigen of interest. A leukocyte in this disclosure may be, for example, a neutrophil, eosinophil, basophil, lymphocyte, or monocyte. In some embodiments, a leukocyte is a lymphocyte. Examples of lymphocytes include T cells, B cells, natural killer (NK) cells, or NKT cells. In some embodiments, a T cell is a CD4+ Th cell (T helper), a CD8+ cytotoxic T cell, a γδT cell, or a regulatory (suppressor) T cell. In some embodiments, an immune cell is a dendritic cell. In some embodiments, cell therapies are CAR T cell therapies. In some embodiments, a bispecific CAR is composed of two distinct antigen recognition domains present in tandem in a single transgenic receptor (referred to as a TanCAR; see, for example, Grada Z et al. Molecular Therapy Nucleic Acids 2013; 2:e105, incorporated herein by reference in its entirety).Thus, the methods, in some modalities, involve providing a tumor with a combination comprising a ROR antigen-binding molecule (e.g., antibody) and an immunotherapeutic agent, wherein the immunotherapeutic agent is a modified nucleic acid encoding an antigen, or delivering to a tumor a modified nucleic acid that induces the expression of an autoantigen, and delivering to the tumor an immune cell expressing a bispecific CAR that binds to two antigens, one of which is encoded by the modified nucleic acid.

[0407] Other exemplary immunotherapies include immunotherapeutic agents, such as cancer vaccines, which can be used to elicit an immune response in a Petition 870260031312, dated 02 / 04 / 2026, page 166 / 417 158 / 285 individual against a cancer antigen. An exemplary method involves administering to the individual an RNA vaccine comprising at least one RNA polynucleotide with an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, inducing in the individual a specific immune response to the antigenic polypeptide or a fragment thereof, in combination with the administration of an ROR antigen-binding molecule (e.g., antibody) in the same composition or in a separate composition, administered at the same time, or dosed sequentially, wherein the anti-antigenic polypeptide antibody titer in the individual is increased after vaccination relative to the anti-antigenic polypeptide antibody titer in an individual vaccinated with a prophylactically effective dose of a traditional cancer vaccine.

[0408] In certain modalities, additional therapies include chemotherapy, such as one or more cytotoxic agents or one or more chemotherapeutic agents. A cytotoxic agent can inhibit or prevent a cellular function and / or cause cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 Pb212e radioactive isotopes of Lu); chemotherapeutic agents; growth-inhibiting agents; enzymes and fragments thereof, such as nucleotide enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, origin. Petition 870260031312, dated 02 / 04 / 2026, page 167 / 417 159 / 285 plant or animal, including fragments and / or variants thereof.

[0409] In certain modalities, additional therapy includes one or more chemotherapeutic agents. Chemotherapeutic agents include chemical compounds useful in the treatment of cancer. Chemotherapeutic agents include (i) anti-hormonal agents that act to regulate or inhibit hormonal action in tumors such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the aromatase enzyme, which regulates estrogen production in the adrenal glands; (iii) antiandrogens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, including those that inhibit gene expression in signaling pathways involved in the proliferation of aberrant cells; (viii) vaccines such as gene therapy vaccines. Chemotherapeutic agents may also include antibodies.

[0410] Exemplary kinase inhibitors include erlotinibe (Tarceva®), gefitinibe (Iressa®), dasatinibe (Sprycel®), nilotinibe (Tasigna®), crizotinibe (Xalkori®), ruxolitinibe (Jakafi®), vemurafenib (Zelboraf®), vandetanib (Caprelsa®), pazopanibe (Votrient®), afatinibe, alisertibe, amuvatinibe, axitinibe, baricitinibe, bosutinibe, brivanibe, canertinibe, cabozantinibe (Cabometyx®), cediranib, ceritinibe, crenolanib, dabrafenib, dacomitinibe, danusertibe, dovitinibe, foretinibe, ganetespibe, ibrutinibe, Idelalisibe, Imatinibe, Iniparibe, Lapatinibe, Lenvatinibe, Linifanibe, Linsitinibe, Petition 870260031312, 02 / 04 / 2026, p. 168 / 4 160 / 285 masitinib, momelotinib, motesanib, neratinib, nintedanib, niraparib, oprozomib, olaparib, palbociclib, pictilisib, pirfenidone, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, saracatinib, saridegib, sorafenib, sunitinib, tandutinib, tasocitinib, telatinib, tivantinib, tivozanib, tofacitinib, trametinib, veliparib, vismodegib, volasertib, cobimetinib (Cotellic®), XL-147, XL-765, XL-499, XL-880 and others. In some embodiments, a ROR antigen-binding molecule (e.g., antibody) can be used in combination with an HSP90 inhibitor (e.g., XL888), liver X receptor (LXR) modulators, retinoid-related orphan gamma receptor (RORy) modulators, a CK1 inhibitor, a CK1-a inhibitor, a WNT pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor (e.g., esaxerenone or XL-550) for cancer treatment.

[0411] Kinase inhibitors can be tyrosine kinase inhibitors, such as EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors, such as mubritonib (TAK165, Takeda); CP-724,714 (Axon Medchem BV, an oral selective inhibitor of ErbB2 receptor tyrosine kinase); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; lapatinib (GSK572016; available from GlaxoSmithKline), an oral inhibitor of HER2 and EGFR tyrosine kinase; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors, such as Petition 870260031312, dated 02 / 04 / 2026, page 169 / 417 161 / 285 the antisense agent ISIS-5132 available from ISIS Pharmaceuticals, which inhibit Raf-1 signaling; non-HER-targeted TK inhibitors, such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib (SUTENT®, available from Pfizer); VEGF tyrosine kinase receptor inhibitors, such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); extracellular MAPK-regulated kinase 1 inhibitor CI-1040 (available near Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; Pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7Hpyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis-(4-fluoroanilino)phthalimide); nitrothiophene moieties containing tripostines; antisense molecules (e.g., those that bind to nucleic acid encoding HER);quinoxalines (U.S. Patent No. 5,804,396); trifostines (U.S. Patent No. 5,804,396); Affinitac (ISIS 3521; Isis / Lilly); PKI166 (Novartis); Semaxinib (Pfizer); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO 1999 / 06396 (Warner Lambert); WO 1996 / 30347 (Pfizer, Inc); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca) and WO 1996 / 33980 (Zeneca).; Petition 870260031312, dated 02 / 04 / 2026, page 170 / 417 162 / 285

[0412] Combined treatment with the ROR antigen-binding molecule (e.g., antibody) provided in the present invention and additional therapy, such as a therapeutic agent, may be simultaneous, separate, or sequential, in any order. For combinations of therapeutic agents, such as a molecule that provides ROR antigen binding and another therapeutic agent, such as an immunotherapeutic agent or a chemotherapeutic agent, simultaneous administration of the therapeutic agents may occur as a single composition or as separate compositions, as appropriate. 6.13. Examples

[0413] The following examples are provided for illustrative purposes only and not as a limitation. 6.13.1. Methods

[0414] Illustrative, non-limiting methods for the purification of various antigen-binding proteins and their use in various assays are described in more detail below. 6.13.1.1. Expression of Expi293

[0415] The various antigen-binding proteins tested were expressed using the Expi293 transient transfection system according to the manufacturer's instructions. Briefly, four plasmids encoding four individual chains were mixed in a mass ratio of 1:1:1:1, unless otherwise indicated, and transfected with the ExpiFectamine 293 transfection kit for Expi 293 cells. The cells were cultured at 37 °C with 8% CO2, 100% humidity and by shaking at 125 rpm. Petition 870260031312, dated 02 / 04 / 2026, page 171 / 417 163 / 285 The transfected cells were fed once 16 to 18 hours after transfection. Cells were collected on day 5 by centrifugation at 2,000 g for 10 minutes. The supernatant was collected for purification by affinity chromatography. 6.13.1.2. Purification of Protein A and anti-CH1

[0416] Clean supernatants containing the various antigen-binding proteins were separated using either a protein A (ProtA) resin or an anti-CH1 resin on an AKTA purification FPLC. In examples where a peer-to-peer comparison was performed, the supernatants containing the various antigen-binding proteins were split into two equal samples. For protA purification, a 1 mL protein A column (GE Healthcare) was equilibrated with PBS (5 mM sodium potassium phosphate, pH 7.4, 150 mM sodium chloride). The sample was loaded onto the column at 5 mL / min. The sample was eluted using 0.1 M acetic acid at pH 4.0. Elution was monitored by absorbance at 280 nm, and the elution peaks were pooled for analysis. For anti-CH1 purification, a 1 mL CaptureSelect™ XL column (ThermoFisher) was equilibrated with PBS. The sample was loaded into the column at 5 mL / min. The sample was eluted using 0.1 M acetic acid at pH 4.0.Elution was monitored by absorbance at 280 nm, and the elution peaks were pooled for analysis. 6.13.1.3. SDS-PAGE Analysis

[0417] Samples containing the various separated antigen-binding proteins were analyzed by reductive and non-reductive SDS-PAGE for the presence of complete product, Petition 870260031312, dated 02 / 04 / 2026, page 172 / 417 164 / 285 incomplete product and overall purity. 2 μg of each sample were added to 15 μL of SDS loading buffer. Reducing samples were incubated in the presence of 10 mM reducing agent at 75 °C for 10 minutes. Non-reducing samples were incubated at 95 °C for 5 minutes without reducing agent. Reducing and non-reducing samples were loaded onto a TGX gel with a 4-15% gradient (BioRad) with analysis buffer, and analyzed for 30 minutes at 250 volts. After completing the analysis, the gel was washed with DI water and stained using GelCode Blue Safe protein dye (ThermoFisher). The gels were decolorized with DI water before analysis. Densitometry analysis of the digitized images of the decolorized gels was performed using standard image analysis software to calculate the relative abundance of bands in each sample. 6.13.1.4. Chromatography IEX

[0418] Samples containing the various separated antigen-binding proteins were analyzed by cation-exchange chromatography for the ratio of complete product to incomplete product, and impurities. Clean supernatants were analyzed with a 5 mL MonoS column (GE Lifesciences) on an AKTA purifying FPLC. The MonoS column was equilibrated with 10 mM MES buffer A at pH 6.0. Samples were loaded onto the column at 2 mL / min. The sample was eluted using a 0 to 30% gradient with buffer B (10 mM MES, at pH 6.0, and 1 M sodium chloride) at 6 CV. Elution was monitored by absorbance at 280 nm, and sample purity was calculated by peak integration to identify the abundance of monomer peaks and peaks. Petition 870260031312, dated 02 / 04 / 2026, page 173 / 417 165 / 285 of contaminants. The monomer peak and the contaminant peaks were separated for analysis by SDS-PAGE, as described above. 6.13.1.5. Analytical SEC Chromatography

[0419] Samples containing the various separated antigen-binding proteins were analyzed by analytical size exclusion chromatography for the monomer-to-high molecular weight product-to-impurity ratio. Clean supernatants were analyzed using an industry-standard TSK G3000SWxl column (Tosoh Bioscience) on an Agilent HPLC 1100. The TSK column was equilibrated with PBS. 25 μL of each sample at 1 mg / mL were loaded onto the column at 1 mL / min. The sample was eluted using an isocratic flow of PBS to 1.5 CV. Elution was monitored by absorbance at 280 nm, and elution peaks were analyzed by peak integration. 6.13.1.6. Mass spectrometry

[0420] Samples containing the various separated antigen-binding proteins were analyzed by mass spectrometry to confirm the correct species by molecular weight. All analyses were performed by a third-party research organization. Briefly, the samples were treated with an enzyme cocktail to remove glycosylation. Both samples were tested in reduced form to specifically identify each chain by molecular weight. All samples were tested under non-reducing conditions to identify the molecular weights of all complexes in the samples. The analysis Petition 870260031312, dated 02 / 04 / 2026, page 174 / 417 Mass spectrometry (166 / 285) was used to identify the number of unique products based on molecular weight. 6.13.1.7. Antibody discovery by phage display

[0421] Phage display of human Fab libraries was performed using standard protocols. Biotinylated extracellular domains of human ROR1 and ROR2 proteins were acquired from Acro Biosystems and biotinylated with NHS EZ-Link biotin (Thermo Scientific catalog No. 20217). Phage clones were examined for the ability to ligate the extracellular domains of ROR1 (Acro catalog No. RO1-H522y) and ROR2 (ACro catalog No. ACroRO2H52E5) by phage-ELISA using standard protocols. Briefly, Fab-format phage libraries were constructed using expression vectors capable of replicating and expressing in the phage (also referred to as a phagemid). Both the light chain and the heavy chain were encoded in the same expression vector, where the heavy chain was fused to a truncated variant of the pIII phage coat protein.The light chain and the pIII heavy chain fusion are expressed as separate polypeptides and assembled in the bacterial periplasm, where the redox potential allows the formation of disulfide bonds, to form the phage-displaying antibody containing the ABS candidate.

[0422] The library was created using sequences derived from a specific human heavy chain variable domain (VH3-23) and a specific human light chain variable domain (Vk-1). The light chain variable domains within the tested library generated with diversity were introduced into the CDR3 of VL (L3) and where the CDR1 (L1) and Petition 870260031312, dated 02 / 04 / 2026, page 175 / 417 167 / 285 The CDR2 (L2) light chain remained the human germline sequence. For the tested library, all three CDRs of the VH domain were diversified to match the positional amino acid frequency by CDR length found in the human antibody repertoire. The heavy chain (SEQ ID NO: 74) and light chain (SEQ ID NO: 75) phage display structures used in the library are listed below, where a lowercase “x” represents CDR amino acids that varied to create the library, and those written in italics and bold represent CDR sequences that were constant.

[0423] Diversity was created through Kunkel mutagenesis using primers to introduce diversity in VL CDR3 and VH CDR1 (H1), CDR2 (H2), and CDR3 (H3) to mimic the diversity found in the natural antibody repertoire, as described in more detail in Kunkel, TA (PNAS, January 1, 1985. 82 (2) 488-492), here incorporated by reference in its entirety. In summary, single-stranded DNAs were prepared from isolated phage using standard procedures, and Kunkel mutagenesis was performed. The chemically synthesized DNA was then electroporated into TG1 cells, followed by recovery. The recovered cells were subcultured and infected with the helper phage M13K07 to produce the phage library.

[0424] Phage immobilization was performed using standard procedures. Briefly, the first round of phage immobilization was performed with the target immobilized in streptavidin magnetic beads, which Petition 870260031312, dated 02 / 04 / 2026, p. 176 / 417 168 / 285 were subjected to ~5x10¹² phages from the prepared library in a 1 mL volume in 2% PBST-BSA. After one hour of incubation, the bead-bound phages were separated from the supernatant using a magnetic support. The beads were washed three times to remove non-specifically bound phages and then added to ER2738 cells (5 mL) at OD600 ~0.6. After 20 minutes, the infected cells were subcultured in 25 mL 2xYT + ampicillin and with helper phage M13K07, and allowed to multiply overnight at 37 °C with vigorous shaking. The following day, the phages were prepared using standard procedures by precipitation with PEG. Pre-clearance of the SAV-coated bead-specific phages was performed before immobilization. The second immobilization was performed using the KingFisher magnetic bead manipulator with antigen immobilized on beads at 100 nM, using standard procedures.In total, 3 to 4 rounds of phage immobilization were performed to enrich Fabs displaying phages specific to the target antigen. Target-specific enrichment was confirmed using polyclonal and monoclonal phage-ELISA. DNA sequencing was used to determine isolated Fab clones containing an ABS candidate.

[0425] To measure binding affinity in ROR ligand discovery campaigns, the VL and VH domains identified in the phage analysis described above were formatted into a complete human bivalent, native, monospecific IgG1 architecture and immobilized on a biosensor in an Octet biolayer interferometer (Pall Petition 870260031312, dated 02 / 04 / 2026, page 177 / 417 169 / 285 (ForteBio). Soluble ROR antigens, including the extracellular domains of ROR1 (Acro catalog No. RO1H522y) and ROR2 (Acro catalog No. RO2-H52E5), as well as the individual ROR1 Frizzled (Acro catalog No. RO1H5222), Ig-type (Acro catalog No. RO2-H52E5), Ig-type (Acro catalog No. RO1-H5221), and Kringle (Acro catalog No. RO1-H5223) domains, were then added to the system, and binding was measured.

[0426] For experiments performed using the B-Body format, the variable regions of the VL of the individual clones were formatted in the A and / or H domain, and the VH region in the F and / or L domain of a 1x1 bivalent B-Body structure “BC1” shown below and with reference to figure 3.

[0427] BC1 Structure: 1 polypeptide chain (SEQ ID NO: 78) Domain A = A / H structure of the B-Body domain of antigen 1 (SEQ ID NO: 76) Domain B = CH3 (T366K; tripeptide insertion 445K, 446S, 447C) Domain D = CH2 Domain E = CH3 (T366W, S354C) 2nd polypeptide chain (SEQ ID NO: 79): F domain = F / L structure of the B-Body domain of antigen 1 (SEQ ID NO: 77) Domain G = CH3 (L351D; tripeptide insertion 445G, 446E, 447C) 3rd polypeptide chain (SEQ ID NO: 80): Domain H = A / H structure of the B-Body domain of antigen 2 (SEQ ID NO: 76) Petition 870260031312, dated 02 / 04 / 2026, page 178 / 417 170 / 285 Domain I = CL (kappa) J domain (CH2) Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) 4polypeptide chain (SEQ ID NO: 81): L domain = F / L structure of the B-Body domain of antigen 2 (SEQ ID NO: 77) Domain M = CH1 *

[0428] For ROR ABS candidates formatted in a 1x1 bivalent bispecific format with SP34-89 anti-CD3, the H domain has the amino acid sequence of SEQ ID NO: 69 and the L domain has the amino acid sequence of SEQ ID NO: 68, while the A domain has the candidate sequence of the ROR VL and the F domain had the candidate sequence of the ROR VH.

[0429] For the BC1 1x2 formats, the variable domains were formatted in the 1(A)x2(BA) format described in section 0, which refers to figure 26. Figure 26 presents a scheme of five polypeptide chains and their domains, with the respective nomenclature conventions, for the 1x2 trivalent antibody constructs described herein, wherein, according to the nomenclature convention, chain 5 is called the “6th polypeptide chain” in the schematic representation. Unless otherwise specified, the antigen-binding site (ABS) ROR is the bivalent ligand (specificity “A”) and CD3 is the monovalent ligand (specificity “B”). The structure of the 3 SP34-89 1x2 chain has the sequence SEQ ID NO: 82, where the junction between the S domain and the H domain is a 10-amino acid linker with the sequence TASSGGSSSG (SEQ ID NO: 83), unless it is Petition 870260031312, dated 02 / 04 / 2026, p. 179 / 417 171 / 285 indicated otherwise. Polypeptide chains 2 and 5 are identical in the format 1(A)x2(BA) (see, for example, Figure 2 6, where, according to the nomenclature convention, chain 5 is called “polypeptide chain 6” in the diagram). 6.13.1.8. NFkB GFP T cell stimulation assay Jurkat

[0430] The NFuB / Jurkat / GFP transcriptional reporter cell line was purchased from System Biosciences (Catalog No. TR850-1). The anti-CD28 antibody used for co-stimulation was purchased from BD Pharmingen (Catalog No. 555725). The background signal suppression dye solution C was purchased from Life Technologies (K1037). Briefly, Jurkat cells (effector cells, E) were mixed with tumor cells (T) in an E:T ratio of 2:1 to 4:1 in the presence of a series of dilutions of B-body™ antibodies and an anti-CD28 antibody at 1 ug / mL in a 96-well clear-bottom, black-walled plate. The plate was incubated at 37°C / 5% CO2 for 6 hours, after which a 6X solution of the background signal suppressor solution C was added to the plate, and the GFP fluorescence was read on a plate reader. The EC50 values, referring to the antibody concentration that produces half the maximum response, were determined from serial dilution. 6.13.1.9. Primary T-cell cytotoxicity assay

[0431] Cells expressing the target tumor antigen (T) and effector cells (E) were mixed in a Petition 870260031312, dated 02 / 04 / 2026, pp. 180 / 417 The E:T ratio was 172 / 285, ranging from 3:1 to 10:1. Effector cells used included PBMCs or isolated cytotoxic CD8+ T cells. The T cell redirection candidate antibody was added in a series of dilutions to the cells. Controls included medium-only controls, tumor cell-only controls, and untreated E:T cell controls. Mixed cells under control conditions were incubated at 37°C / 5% CO2 for 40 to 50 hours. The Cytotoxicity Detection Kit Plus (LDH) was purchased from Sigma (Catalog No. 4744934001) and the manufacturer's instructions were followed. Briefly, the lysis solution added to the tumor cells served as the 100% cytotoxicity control, and the untreated E:T cells served as the 0% cytotoxicity control. The lactate dehydrogenase (LDH) level in each sample was determined by absorbance at 490 nm and normalized to the 100% and 0% controls.The EC50 values, referring to the antibody concentration that produces half the maximum response, were determined from serial dilution. 6.13.2. Example 1: Monospecific bivalent construction and bispecific bivalent construction

[0432] A bivalent monospecific B-Body that recognizes TNFα was constructed with the following architecture (VL(certolizumab)-CH3(Knob)-CH2-CH3 / VH(certolizumab)CH3(Hole)) using standard molecular biology procedures. In this construction, we have: 1 polypeptide chain (SEQ ID NO: 1) Domain A = VL (certolizumab) Domain B = CH3 (IgG1) (knob: S354C+T366W) Petition 870260031312, dated 02 / 04 / 2026, p. 181 / 417 173 / 285 Domain D = CH2 (IgG1) Domain E = CH3 (IgG1) 2polypeptide chain (SEQ ID NO: 2) F domain = VH (certolizumab) Domain G = CH3 (IgG1) (hole: Y349C, T366S, L368A, Y407V) 3rd polypeptide chain: identical to the first polypeptide chain The fourth polypeptide chain is identical to the second polypeptide chain.

[0433] The domain and polypeptide chain references are in accordance with Figure 3. The overall construction architecture is illustrated in Figure 4. The sequence of the first polypeptide chain, with domain A abbreviated as (VL), is given in SEQ ID NO: 1. The sequence of the second polypeptide chain, with domain F abbreviated as (VH), is given in SEQ ID NO: 2.

[0434] The complete construct was expressed in an E. coli cell-free protein synthesis expression system for approximately 18 hours at 26 °C with gentle agitation. After expression, the cell-free extract was centrifuged to pelletize the insoluble material, and the supernatant was diluted 2x with 10x kinetic buffer (Forte Bio) and used as an analyte in biolayer interferometry.

[0435] Biotinylated TNFα was immobilized on a streptavidin sensor to produce a wave response shift of approximately 1.5 nm. After establishing a baseline with 10x kinetic buffer, the sensor was immersed Petition 870260031312, dated 02 / 04 / 2026, page 182 / 417 174 / 285 in the antibody construct analyte solution. The construct produced a response of approximately 3 nm, comparable to the shape of traditional certolizumab IgG, demonstrating the ability of the bivalent monospecific construct to be assembled into a functional complete antibody. The results are shown in Figure 5.

[0436] We also constructed a bispecific bivalent antibody with the following domain architecture: 1 polypeptide chain: VL-CH3-CH2-CH2 (knob) 2 polypeptide chain: VH-CH3 3rd polypeptide chain: VL-CL-CH2-CH3 (hole) 4th polypeptide chain: VH-CH1.

[0437] The sequences (except for the variable region sequences) are provided respectively in SEQ ID NO: 3 (1st polypeptide chain), SEQ ID NO: 4 (2nd polypeptide chain), SEQ ID NO: 5 (3rd polypeptide chain) and SEQ ID NO: 6 (4th polypeptide chain). 6.13.3. Example 2: Bivalent bispecific B-body BC1

[0438] We constructed a bivalent bispecific construct, called “BC1”, specific for PD1 and a second antigen, the “A antigen”). The relevant features of the “BC1” architecture are illustrated in Figure 6.

[0439] In more detail, with the domain and polypeptide chain references according to figure 3, and the native sequence modifications indicated in parentheses, the architecture was as follows: 1st polypeptide chain (SEQ ID NO: 8) Domain A = VL (“antigen A”) Petition 870260031312, dated 02 / 04 / 2026, page 183 / 417 175 / 285 Domain B = CH3 (T366K; tripeptide insertion 445K, 446S, 447C) Domain D = CH2 Domain E = CH3 (T366W, S354C) 2polypeptide chain (SEQ ID NO: 9): F domain = VH (“A antigen”) Domain G = CH3 (L351D; tripeptide insertion 445G, 446E, 447C) 3rd polypeptide chain (SEQ ID NO: 10): Domain H = VL (“Nivo”) Domain I = CL (kappa) J domain (CH2) Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) 4th polypeptide chain (SEQ ID NO: 11): Domain L = VH (“Nivo”) Domain M = CH1 *

[0440] Domain A (SEQ ID NO: 12) and domain F (SEQ ID NO: 16) form an antigen-binding site (A:F) specific for “antigen A”. Domain H has the VH sequence of nivolumab and domain L has the VL sequence of nivolumab; H and L associate to form an antigen-binding site (H:L) specific for human PD1.

[0441] Domain B (SEQ ID NO: 13) has the human IgG1 CH3 sequence with several mutations: T366K, 445K, 446S, and 447C insertions. The T366K mutation is a cognate charge pair of residue L351D in domain G. The “447C” residue in domain B is derived from the insertion of the KSC tripeptide at the C-terminus. Petition 870260031312, dated 02 / 04 / 2026, page 184 / 417 176 / 285

[0442] Domain D (SEQ ID NO: 14) has the CH2 sequence of human IgG1

[0443] Domain E (SEQ ID NO: 15) has the CH3 sequence of human IgG1 with the T366W and S354C mutations. 366W is the knob mutation. 354C introduces a cysteine ​​that is capable of forming a disulfide bond with the cognate 349C mutation in domain K.

[0444] The G domain (SEQ ID NO: 17) has the human IgG1 CH3 sequence with the following mutations: L351D, and the insertion of tripeptides 445G, 446E, 447C. The L351D mutation introduces a cognate charge pair to the T366K mutation of the B domain. Residue 447C in the G domain is derived from the insertion of the GEC tripeptide at the C-terminus.

[0445] Domain I (SEQ ID NO: 19) has the human kappa C light chain sequence (Ck).

[0446] The J domain [SEQ ID NO: 20] has the sequence of the CH2 domain of human IgG1, and is identical to the sequence of the D domain.

[0447] The K domain [SEQ ID NO: 21] has the human IgG1 CH3 sequence with the following alterations: Y349C, D356E, L358M, T366S, L368A, Y407V. The 349C mutation introduces a cysteine ​​that is capable of forming a disulfide bond with the cognate 354C mutation in the E domain. 356E and L358M introduce isoalloy amino acids that reduce immunogenicity. The 366S, 368A, and 407V mutations are hole-type mutations.

[0448] The M domain [SEQ ID NO: 23] has the sequence of the CH1 region of human IgG1. Petition 870260031312, dated 02 / 04 / 2026, page 185 / 417 177 / 285

[0449] “BC1 could be easily expressed at high levels using mammalian expression at concentrations greater than 100 μg / mL.

[0450] We discovered that the bivalent bispecific protein “BC1” could be easily purified in a single step using a CaptureSelect™ affinity resin specific for CH1, from ThermoFisher.

[0451] As illustrated in Figure 7A, SEC analysis demonstrates that a single-step CH1 affinity purification process produces a single monodisperse peak via gel filtration, where more than 98% corresponds to monomer. Figure 7B shows comparative literature data from SEC analysis of a CrossMab bivalent antibody construct.

[0452] Figure 8A is a cation-exchange chromatography elution profile of “BC1 after one-step purification using the CH1 CAPTURESelect™ affinity resin, showing a single compact peak. Figure 8B is a cation-exchange chromatography elution profile of “BC1 after purification using standard protein A purification, showing additional elution peaks consistent with co-purification of incomplete assembly products.

[0453] Figure 9 shows SDS-PAGE gels under non-reducing conditions. As seen in lane 3, single-step purification of “BC1” with CH1 affinity resin provides a nearly homogeneous single band, with lane 4 showing minimal additional purification with a subsequent cation exchange purification step. Lane 7, in comparison, Petition 870260031312, dated 02 / 04 / 2026, page 186 / 417 178 / 285 shows less substantial purification using standard protein A purification, with lanes 8 to 10 demonstrating greater purification of protein A material purified using cation exchange chromatography.

[0454] Figure 10 compares SDS-PAGE gels of “BC1 after single-step CH1 affinity purification under non-reducing and reducing conditions (panel A) with SDS-PAGE gels of a bivalent bispecific antibody CrossMab under non-reducing and reducing conditions, as published in the reference literature (panel B).

[0455] Figure 11 shows the mass spectrometry analysis of BC1, demonstrating two distinct heavy chains (Figure 11A) and two distinct light chains (Figure 11B) under reducing conditions. The mass spectrometry data in Figure 12 confirm the absence of incomplete pairing after purification.

[0456] Accelerated stability testing was performed to evaluate the long-term stability of the B-Body BC1 design. Purified B-Body was concentrated to 8.6 mg / ml in PBS buffer and incubated at 40 °C. Structural integrity was measured weekly using analytical size exclusion chromatography (SEC) with a Shodex KW-803 column. Structural integrity was determined by measuring the percentage of intact monomer (% monomer) relative to aggregate formation. Data are shown in Figure 13. IgG control 1 is a positive control with good stability properties. IgG control 2 is a negative control known to aggregate under incubation conditions. B-Body BC1 was incubated for 8 Petition 870260031312, dated 02 / 04 / 2026, page 187 / 417 179 / 285 weeks without any loss of structural integrity, as determined by SEC analytics.

[0457] We also determined that BC1 exhibits high thermostability, with a TM of the bivalent construction of approximately 72 °C.

[0458] Table 1 compares BC1 with CrossMab in relation to key development capability characteristics: Table 1 Parameter Unit Roche CrossMab* BC1” Purification yield after protein A / SEC mg / L 58.5 300 Homogeneity after purification % of area in SEC 50-85 98 Denaturation temperature (Tm) degrees C 69.2 72 *Data from Schaefer et al. (Proc Natl Acad Sci USA. July 5, 2011; 108 (27): 11187-92)]. 6.13.4. Example 3: Bivalent bispecific B-body BC6

[0459] We constructed a bivalent bispecific B-Body, called BC6”, which is identical to BC1 except that it retains wild-type residues in the B domain at residue 366 and in the G domain at residue 351. Thus, BC6” does not have the pair-charge cognates T366K and L351D that were designed to facilitate the correct pairing of the B domain and the G domain in BC1. The relevant features of the BC6” architecture are illustrated in Figure 14. Petition 870260031312, dated 02 / 04 / 2026, pp. 188 / 417 180 / 285

[0460] Notwithstanding the absence of the even-charge residues present in BC1”, we found that a single purification step of BC6” using the CH1 affinity resin resulted in a highly homogeneous sample. Figure 15A shows the SEC analysis of BC6” after one-step purification using the CaptureSelect™ CH1 affinity resin. The data demonstrate that single-step CH1 affinity purification produces a single monodisperse peak, similar to that observed with BC1”, demonstrating that the disulfide bridges between polypeptide chains 1 and 2 and between polypeptide chains 3 and 4 are intact. The chromatogram also shows the absence of non-covalent aggregates.

[0461] Figure 15B shows an SDS-PAGE gel under non-reducing conditions, with lane 1 loaded with a first batch of BC6” after a single-step affinity purification of CH1, and lane 2 loaded with a second batch of BC6” after a single-step affinity purification of CH1. Lanes 3 and 4 demonstrate that further purification can be achieved by ion-exchange chromatography after affinity purification of CH1. 6.13.5. Example 4: Bivalent bispecific B-bodies BC28, BC29, BC30 and BC31

[0462] We constructed bivalent 1x1 bispecific B-bodies BC28”, BC29”, BC30” and BC31” having a modified disulfide within the CH3 interface in the B and G domains as an alternative SS bond to the C-terminal disulfide present in BC1” and BC6”. The literature indicates that the disulfide bond at the CH3 interface is insufficient to reinforce orthogonality in the context of the CH3 domains. Petition 870260031312, dated 02 / 04 / 2026, page 189 / 417 181 / 285 of the FC. The general architecture of these B-Body constructions is schematized in Figure 16, with the relevant characteristics of BC28 summarized below: Polypeptide chain 1: chain 1 of BC28” (SEQ ID NO: 24) Domain A = VL (antigen A) Domain B = CH3 (Y349C; insertion of 445P, 446G, 447K) Domain D = CH2 Domain E = CH3 (S354C, T366W) Polypeptide chain 2: chain 2 of BC28” (SEQ ID NO: 25) F domain = VH (antigen A) Domain G = CH3 (S354C; insertion 445P, 446G, 447K) Polypeptide chain 3: chain 3 of BC1” (SEQ ID NO: 10) Domain H = VL (Nivo”) Domain I = CL (kappa) Domain J = (CH2) Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) Polypeptide chain 4: chain 4 of BC1” (SEQ ID NO: 11) Domain L = VH (Nivo”) Domain M = CH1.

[0463] The BC28” A:F antigen binding site is specific for the A” antigen. The BC28” H:L antigen binding site is specific for PD1 (nivolumab sequences). The BC28” B domain has the following Petition 870260031312, dated 02 / 04 / 2026, pp. 190 / 417 182 / 285 changes compared to wild-type CH3: Y349C; insertion 445P, 446G, 447K. BC28's E domain has the following changes compared to wild-type CH3: S354C, T366W. BC28's G domain has the following changes compared to wild-type: S354C; insertion 445P, 446G, 447K.

[0464] Thus, BC28” has a modified cysteine ​​at residue 349C of domain B and a modified cysteine ​​at residue 354C of domain G (349C-354C”).

[0465] BC29” has modified cysteines at residue 351C of domain B and 351C of domain G (351C-351C”). BC30” has a modified cysteine ​​at residue 354C of domain B and 349C of domain G (354C-349C”). BC31 has a modified cysteine ​​at residue 394C and a modified cysteine ​​at 394C of domain G (394C-394C”). BC32 has modified cysteines at residue 407C of domain B and 407C of domain G (407C-407C”).

[0466] Figure 17 shows SDS-PAGE analysis under non-reducing conditions after one-step purification using CaptureSelect™ CH1 affinity resin. Lanes 1 and 3 show high expression levels and substantial homogeneity of intact BC28” (lane 1) and BC30” (lane 3). Lane 2 shows oligomerization of BC29. Lanes 4 and 5 show poor expression of BC31 and BC32, respectively, and insufficient binding at BC32. Another construct, BC9, which had cysteines introduced at residue 392 in the B domain and 399 in the G domain (392C-399C”), a disulfide pairing reported by Genentech, demonstrated oligomerization on SDS-PAGE (data not shown). Petition 870260031312, dated 02 / 04 / 2026, pp. 191 / 417 183 / 285

[0467] Figure 18 shows the SEC analysis of BC28 and BC30” after one-step purification using the CaptureSelect™ CH1 affinity resin. We also demonstrate that BC28” can be easily purified using a single purification step using protein A resin (results not shown). 6.13.6. Example 5: Bivalent bispecific B-body BC44

[0468] Figure 19 shows the overall architecture of the bispecific 1x1 bivalent BC44” BBody, our currently preferred bispecific 1x1 bivalent construction. first polypeptide chain (chain 1 of BC44”) (SEQ ID NO: 32) Domain A = VL (antigen A) Domain B = CH3 (P343V; Y349C; insertion 445P, 446G, 447K) Domain E = CH2 Domain E = CH3 (S354C, T366W) second polypeptide chain: (= polypeptide chain 2 of BC28”) (SEQ NO: 25) F domain = VH (antigen A) Domain G = CH3 (S354C; insertion 445P, 446G, 447K) third polypeptide chain (= polypeptide chain 3 of BC1”) (SEQ ID NO: 10) Domain H = VL (Nivo”) Domain I = CL (kappa) Domain J = CH2 Petition 870260031312, dated 02 / 04 / 2026, page 192 / 417 184 / 285 Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) fourth polypeptide chain (= polypeptide chain 4 of BC1) (SEQ ID NO: 11) Domain L = VH (Nivo) Domain M = CH1. 6.13.7. Example 6: Modification of the variable-CH3 junction

[0469] We produced a series of variants in which we mutated the VL-CH3 junction between domains A and B and the VH-CH3 junction between domains F and G to evaluate the expression level, assembly, and stability of the 1x1 bivalent constructs of the B-Body. Although many solutions likely exist, to reduce the introduction of T-cell epitopes, we opted to use only residues found naturally in the VL, VH, and CH3 domains. Structural evaluation of the domain architecture further limits the desirable sequence combinations. Table 2 and Table 3 below show junctions for various junctional variants based on “BC1” and other bivalent constructs. Table 2 - Variants of variable domain / constant domain junctions for the 1st polypeptide chain VL CH3 Sequence Variant 106 107 108 109 110 111 343 344 345 346 BC1 IKRTPREP IKRTPREP (SEQ ID NO: 57) BC13 IKRTPREP IKRTPREP (SEQ ID NO: 57) BC14 IKRTPREP IKRTPREP (SEQ ID NO: 57) BC15 IKRTVREP IKRTVREP (SEQ ID NO: 58) BC16 IKRTREP IKRTREP (SEQ ID NO: 59) Petition 870260031312, dated 02 / 04 / 2026, page 193 / 417 185 / 285 Table 2 - variants of the variable domain / constant domain junctions for 1st polypeptide chain VL CH3 Sequence BC17 IKRTVPREP IKRTVPREP (SEQ ID NO: 60) BC24 IKRTPREP IKRTPREP (SEQ ID NO: 57) BC25 IKRTPREP IKRTPREP (SEQ ID NO: 57) BC2 6 INCREASED INCREASED (SEQ ID NO: 61) BC27 INCREASED INCREASED (SEQ ID NO: 62) BC44 INCREASED INCREASED (SEQ ID NO:58) BC45 INCREASED INSTEAD (SEQ ID NO: 57) BC5 INSTEAD ICRTPREP (SEQ ID NO: 57) BC6 ICRTPREP ICRTPREP (SEQ ID NO: 57) BC28 ICRTPREP ICRTPREP (SEQ ID NO: 57) BC30 ICRTPREP ICRTPREP (SEQ ID NO: 57) . Table 3 - Variants of variable domain / constant domain junctions for the 2nd polypeptide chain VH CH3 Sequence Variant 112 113 114 115 116 117 118 343 344 345 346 BC1 SSASPREP SSASPREP (SEQ ID NO: 63) BC13 SSASTREP SSASTREP (SEQ ID NO: 64) BC14 SSASTPREP SSASTPREP (SEQ ID NO: 65) BC15 SSASPREP SSASPREP (SEQ ID NO: 63) BC16 SSASPREP SSASPREP (SEQ ID NO: 63) Petition 870260031312, dated 02 / 04 / 2026, pp. 194 / 417 186 / 285 Table 3 - Variants of variable domain / constant domain junctions for the 2nd polypeptide chain VH CH3 Sequence BC17 SSASPREP SSASPREP (SEQ ID NO: 63) BC24 SSASTKGEP SSASTKGEP (SEQ ID NO: 66) BC25 SSASTKGREP SSASTKGREP (SEQ ID NO: 67) BC26 SSASPREP SSASPREP (SEQ ID NO: 63) BC27 SSASPREP SSASPREP (SEQ ID NO: 63) BC44 SSASPREP SSASPREP (SEQ ID NO: 63) BC45 SSASPREP SSASPREP (SEQ ID NO: 63) BC5 SSASPREP SSASPREP (SEQ ID NO: 63) BC6 SSASPREP SSASPREP (SEQ ID NO: 63) BC28 SSASPREP SSASPREP (SEQ ID NO: 63) BC30 SSASPREP SSASPREP (SEQ ID NO: 63)

[0470] Figure 20 shows the size exclusion chromatography of samples BC15 and BC16 at the indicated week of an accelerated stability test protocol at 40°C. BC15” remained stable; BC16” was unstable over time. 6.13.8. Example 7: Trivalent 2x1 bispecific construction of the B-Body (“BC1-2x1”)

[0471] We constructed a trivalent bispecific B-Body 2x1 BC1-2x1” based on BC1”. The relevant architectural features are illustrated in figure 22. Petition 870260031312, dated 02 / 04 / 2026, pp. 195 / 417 187 / 285

[0472] In more detail, using the domain and polypeptide chain references summarized in Figure 21, 1polypeptide chain Domain N = VL (“antigen A”) Domain O = CH3 (T366K, 447C) Domain A = VL (“Antigen A”) Domain B = CH3 (T366K, 447C) Domain D = CH2 Domain E = CH3 (knob, 354C) 5th polypeptide chain (= chain 2 of “BC1”) P domain = VH (“Antigen A”) Q domain = CH3 (L351D, 447C) 2nd polypeptide chain (= chain 2 of “BC1”) F domain = VH (“A antigen”) Domain G = CH3 (L351D, 447C) 3rd polypeptide chain (= chain 3 of “BC1”) Domain H = VL (“Nivo”) Domain I = CL (kappa) Domain J = CH2 Domain K = CH3 (hole, 349C) 4th polypeptide chain (= chain 4 of “BC1”) Domain L = VH (“Nivo”) Domain M = CH1.

[0473] Figure 23 shows the non-reducing SDS-PAGE of the protein expressed using the ThermoFisher Expi293 transient transfection system.

[0474] Lane 1 shows the eluate of the 2x1 trivalent protein “BC1-2X1” after one-step purification using CaptureSelect™ CH1 affinity resin. Lane Petition 870260031312, dated 02 / 04 / 2026, pp. 196 / 417 Lanes 188 / 285 show the lower molecular weight, faster-migrating bivalent BC1 protein after one-step purification using CaptureSelect™ CH1 affinity resin. Lanes 3 to 5 demonstrate the purification of BC1-2x1 using protein A. Lanes 6 and 7 show the purification of BC1-2x1 using CH1 affinity resin.

[0475] Figure 24 compares the avidity of the bivalent BC1” construct with the avidity of the trivalent 2x1 BC1-2x1” construct using an Octet analysis (Pall ForteBio). The biotinylated A” antigen is immobilized on the surface, and antibody constructs are passed over the surface for binding analysis. 6.13.9. Example 8: Trivalent 2x1 trispecific construction of the B-Body (TB111)

[0476] We designed a trivalent trispecific molecule 2x1 TB111”, having the architecture schematized in figure 25. With reference to the domain nomenclature conventions defined in figure 21, TB111 has the following architecture (Ada” indicates a V region of adalimumab): polypeptide chain 1 Domain N: VH (Ada”) Domain O:CH3 (T366K, 394C) Domain A:VL (Antigen A) Domain B:CH3 (T366K, 349C) Domain D:CH2 E:CH3 domain (knob, 354C) polypeptide chain 5 P:VL domain (Ada”) Q:CH3 domain (L351D, 394C) Petition 870260031312, dated 02 / 04 / 2026, page 197 / 417 189 / 285 polypeptide chain 2 F:VH domain (“Antigen A”) G:CH3 domain (L351D, 351C) polypeptide chain 3 Domain H:VL (“Nivo”) Domain I: CL (kappa) Domain J: CH2 K:CH3 domain (hole, 349C) polypeptide chain 4 (= chain 4 of “BC1”) Domain L: VH (“Nivo”) Domain M: CH1 This construction did not express itself. 6.13.10. Example 9: Trivalent bispecific construction 1x2 (“BC28-1x2”)

[0477] We construct a trivalent 1x2 bispecific B-body with the following domain and chain structure with reference to the domain and chain nomenclature defined in Figure 26: 1 polypeptide chain (= chain 1 “BC28”) (SEQ ID NO: 24) Domain A = VL (“Antigen A”) Domain B = CH3 (Y349C; insertion of 445P, 446G, 447K) Domain D = CH2 Domain E = CH3 (S354C, T366W) 2nd polypeptide chain (= chain 2 of “BC28”) (SEQ ID NO: 25) F domain = VH (antigen “A”) Domain G = CH3 (S354C; insertion 445P, 446G, 447K) Petition 870260031312, dated 02 / 04 / 2026, pp. 198 / 417 190 / 285 3-chain polypeptide (SEQ ID NO: 37) R domain = VL (antigen A) Domain S = CH3 (Y349C; insertion of 445P, 446G, 447K) Binder = GSGSGS Domain H = VL (Nivo) Domain I = CL Domain J = CH2 Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) 4th polypeptide chain: (= chain 4 of BC1) (SEQ ID NO: 11): Domain L = VH (Nivo) Domain M = CH1. 6th polypeptide chain (= chain 2 of BC28) (SEQ ID NO: 25) Domain T = VH (antigen A) Domain U = CH3 (S354C; insertion 445P, 446G, 447K)

[0478] The A:F antigen binding site is specific for Antigen A, as is the H:L antigen binding site. The R:T antigen binding site is specific for PD. Therefore, the specificity of this construct is antigen A x (PDl-antigen A). 6.13.11. Example 10: trivalent bispecific construction 1x2 (“CTLA4-4 x Nivo x CTLA4-4”)

[0479] We constructed a trivalent 1x2 bispecific molecule with the general structure outlined in Figure 27 (CTLA4-4 x Nivo x CTLA4-4). The domain nomenclature is defined in Figure 26. Petition 870260031312, dated 02 / 04 / 2026, p. 199 / 417 191 / 285

[0480] Figure 28 is an SDS-PAGE gel in which the lines showing the CTLA4-4 x Nivo x CTLA4-4 construction under non-reducing and reducing conditions are contained within the rectangle.

[0481] Figure 29 compares the antigen binding of two antibodies: CTLA4-4 x OX40-8” and CTLA4-4 x Nivo x CTLA4-4”. CTLA4-4 x OX40-8” binds to CTLA4 monovalently, while CTLA4-4 x Nivo x CTLA4-4” binds to CTLA4 bivalently. 6.13.12. Example 11: Trivalent three-specific construction 1x2 BC28-1x1x1a

[0482] We constructed a trivalent 1x2 trispecific molecule with the general structure shown in Figure 30. With regard to the domain and chain nomenclature established in Figure 26, 1 polypeptide chain (= chain 1 of BC28) [SEQ ID NO: 24] Domain A = VL (Antigen A) Domain B = CH3 (Y349C; insertion of 445P, 446G, 447K) Domain D = CH2 Domain E = CH3 (S354C, T366W) 2nd polypeptide chain (= chain 2 of BC28”) (SEQ ID NO: 25) F domain = VH (antigen A) Domain G = CH3 (S354C; insertion 445P, 446G, 447K) 3rd polypeptide chain (SEQ ID NO: 45) Domain R = VL (CTLA4-4) Petition 870260031312, dated 02 / 04 / 2026, pp. 200 / 417 192 / 285 Domain S = CH3 (T366K; insertion of 445K, 446S, 447C) Binder = GSGSGS Domain H = VL (Nivo) Domain I = CL Domain J = CH2 Domain K = CH3 (Y349C, D356E, L358M, T366S, L368A, Y407V) 4th polypeptide chain: (= chain 4 of BC1) (SEQ ID NO: 11) Domain L = VH (Nivo) Domain M = CH1 6th polypeptide chain (=chain 2 of hCTLA4-4) (SEQ ID NO: 53) Domain T = VH (CTLA4) Domain U = CH3 (L351D; insertion of 445G, 446E, 447C).

[0483] The antigen-binding sites of this trispecific construct were: The A:F antigen binding site was specific for the A antigen. The H:L antigen binding site was specific for PD1 (nivolumab sequence). The R:T antigen binding site was specific for CTLA4.

[0484] Figure 31 shows size exclusion chromatography with BC28-1x1x1a after transient expression and one-step purification using resin. Petition 870260031312, dated 02 / 04 / 2026, page 201 / 417 193 / 285 affinity of CH1 CaptureSelect™, demonstrating a single well-defined peak. 6.13.13. Example 12: SDS-PAGE analysis of bivalent and trivalent constructions

[0485] Figure 32 shows an SDS-PAGE gel with multiple constructs, each after transient expression and one-step purification using CaptureSelect™ CH1 affinity resin, under non-reducing and reducing conditions.

[0486] Lanes 1 (non-reducing conditions) and 2 (reducing conditions + DTT) are the bivalent bispecific construction 1x1 BC1”. Lanes 3 (non-reducing) and 4 (reducing) are the trivalent bispecific construction 2x1 BC1-2x1” (see example 7). Lanes 5 (non-reducing) and 6 (reducing) are the trivalent bispecific construction 1x2 CTLA4-4 x Nivo x CTLA4-4” (see example 10). Lanes 7 (non-reducing) and 8 (reducing) are the trivalent trispecific construction 1x2 BC28-1x1x1a described in example 11.

[0487] The SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct. 6.13.14. Example 13: Linkage analysis

[0488] Figure 33 shows the Octet binding analyses for 3 antigens: PD1, antigen A” and CTLA-4. In each case, the antigen is immobilized and B-Body is the analyte. For reference, bispecific BC1” and CTLA4-4 x OX40-8” 1x1 were also compared to demonstrate that 1x1 B-Bodies Petition 870260031312, dated 02 / 04 / 2026, p. 202 / 417 194 / 285 bind specifically only to antigens for which antigen-binding sites have been selected.

[0489] Figure 33A shows the binding of BC1 to PD1 and antigen A, but not to CTLA4. Figure 33B shows the binding of a bivalent 1x1 CTLA44 x OX40-8 bispecific construct to CTLA4, but not to antigen A or PD1. Figure 33C shows the binding of the trivalent 1x2 trispecific construct, BC28-1x1x1a, to PD1, antigen A, and CTLA4. 6.13.15. Example 14: Tetravalent constructions

[0490] Figure 35 shows the general architecture of a tetravalent 2x2 bispecific construction BC22 - 2x2. The tetravalent 2x2 bispecific construction was built using the BC1 structure by duplicating each segment of the variable domain-constant domain. The domain nomenclature is defined in Figure 34.

[0491] Figure 36 is an SDS-PAGE gel. Lanes 7 to 9 show the tetravalent BC22-2x2 construct, respectively, after one-step purification using CapturSelect™ CH1 affinity resin (CH1 eluate), and after further ion-exchange chromatography purification (lane 8, pk 1 after IEX; lane 9, pk 2 after IEX). Lanes 1 to 3 are the trivalent 2x1 BC21-2x1 construct after CH1 affinity purification (lane 1) and, in lanes 2 and 3, the subsequent ion-exchange chromatography. Lanes 4 to 6 are the trivalent 1x2 BC12-1x2 construct.

[0492] Figure 37 shows the general architecture of a 2x2 tetravalent construction. Petition 870260031312, dated 02 / 04 / 2026, page 203 / 417 195 / 285

[0493] Figures 39 and 40 schematically represent tetravalent constructions with alternative architectures. The domain nomenclature is presented in Figure 38. 6.13.16. Example 15: Binding of the bispecific antigen by the B-Body.

[0494] A tetravalent bispecific B-Body 2x2 BBody-IgG 2x2” was constructed. In more detail, using the domain and polypeptide chain nomenclature references summarized in Figure 38, 1 polypeptide chain Domain A = VL (certolizumab) Domain B = CH3 (IgG1, knob) Domain D = CH2 (IgG1) Domain E = CH3 (IgG1) Domain W = VH (antigen A) X domain = CH1 (IgG1) 3rd polypeptide chain (identical to the first polypeptide chain) Domain H = VL (certolizumab) Domain I = CH3 (IgG1, knob) Domain J = CH2 (IgG1) Domain K = CH3 (IgG1) Domain WW = VH (antigen A) Domain XX = CH1 (IgG1) 2nd polypeptide chain F domain = VH (certolizumab) Domain G = CH3 (IgG1, hole) Petition 870260031312, dated 02 / 04 / 2026, page 204 / 417 196 / 285 4th polypeptide chain (identical to the third polypeptide chain) F domain = VH (certolizumab) Domain G = CH3 (IgG1, hole) 7th polypeptide chain Domain Y = VH (“Antigen A”) Domain Z = CL kappa 8th polypeptide chain (identical to the seventh polypeptide chain) YY domain = VH (“Antigen A”) Domain ZZ = CL kappa.

[0495] This was cloned and expressed as described in Example 1. Here, the BLI experiment consisted of immobilizing the biotinized antigen “A” on a streptavidin sensor, followed by establishing a baseline with 10x kinetic buffer. The sensor was then immersed in cell-free “B-Body-IgG 2x2”, followed by establishing a new baseline. Finally, the sensor was immersed in 100 nM TNFα where a second binding event was observed, confirming the bispecific binding of both antigens by a single “B-Body-IgG 2x2” construct. The results are shown in Figure 41. 6.13.17. Example 16: Cell-antigen specific binding of “BB-IgG 2x2”.

[0496] Expi-293 cells were sham transfected or transiently transfected with antigen “B” using the Expi-293 transfection kit (Life Technologies). Forty-eight hours after transfection, Expi-293 cells were collected and fixed in paraformaldehyde. Petition 870260031312, dated 02 / 04 / 2026, page 205 / 417 197 / 285 4% for 15 minutes at room temperature. The cells were washed twice with PBS. 200,000 Expi-293 cells transfected with B antigen or sham-transfected were placed in a 96-well V-bottom plate in 100 μL of PBS. The cells were incubated with B-Body-IgG 2x2” at a concentration of 3 μg / mL for 1.5 hours at room temperature. The cells were centrifuged at 300 xg for 7 minutes, washed in PBS, and incubated with 100 μL of FITC-labeled goat anti-human secondary antibody at a concentration of 8 μg / mL for 1 hour at room temperature. The cells were centrifuged at 300 xg for 7 minutes and washed in PBS, and cell ligation was confirmed by flow cytometry using a Guava easyCyte. The results are shown in Figure 42. 6.13.18. Example 17: SDS-PAGE analysis of bivalent and trivalent constructions

[0497] Figure 45 shows an SDS-PAGE gel with multiple constructs, each after transient expression and one-step purification using CaptureSelect™ CH1 affinity resin, under non-reducing and reducing conditions.

[0498] Lanes 1 (non-reducing conditions) and 2 (reducing conditions + DTT) are the bispecific 1x1 bivalent construction “BC1”. Lanes 3 (non-reducing) and 4 (reducing) are the bispecific 1x1 bivalent construction BC28 (see example 4). Lanes 5 (non-reducing) and 6 (reducing) are the bispecific 1x1 bivalent construction BC44” (see example 5). Lanes 7 (non-reducing) and 8 (reducing) are the trispecific 1x2 bivalent construction BC28-1x2” (see example 9). Lane 9 (non-reducing) Petition 870260031312, dated 02 / 04 / 2026, pp. 206 / 417 198 / 285 and 10 (reducing gears) are the trivalent three-specific construction 1x2 BC28-1x1x1a described in example 11.

[0499] The SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct. 6.13.19. Example 18: Stability analysis of the variable-CH3 junction modification

[0500] The pairing stability between various combinations of junctional variants was evaluated. Differential scanning fluorimetry was performed to determine the melting temperature of various junctional variant pairings between the VL-CH3 polypeptides of chain 1 (domains A and B) and the VH-CH3 polypeptides of chain 2 (domains F and G). The junctional variants BC6jv”, BC28jv”, BC30jv”, BC44jv” and BC45jv”, each with the corresponding junctional sequences BC6”, BC28”, BC30”, BC44” and BC45” found in Table 2 and Table 3 above, demonstrate greater pairing stability with Tms in the range of 76 to 77 degrees (see Table 4). Figure 46 shows differences in thermal transitions for BC24jv”, BC26jv” and BC28jv”, with BC28jv” demonstrating the greatest stability of the three. The x-axis in the figure is the temperature and the y-axis is the change in fluorescence divided by the change in temperature (-dFluor / dTemp).The experiments were performed as described in Niesen et al. (Nature Protocols, (2007) 2, 2212 - 2221), which is incorporated into the present invention by reference, for all that it teaches. Petition 870260031312, dated 02 / 04 / 2026, pp. 207 / 417 199 / 285 TABLE 4 - MELTING TEMPERATURES OF JUNCTIONAL VARIANT PAIRS JUNCTIONAL VARIANT PAIR MELTING TEMPERATURE No. 1 (°C) MELTING TEMPERATURE No. 2 (°C) BC1jv 69.7 55.6 BC5jv 71.6 BC6jv 77 BC15jv 68.2 54 BC16jv 65.9 BC17jv 68 BC24jv 69.7 BC26jv 70.3 BC28jv 76.7 BC30jv 76.8 BC44jv 76.2 BC45jv 76 6.13.20. Example 19: Candidate RORxCD3 binding molecules

[0501] Several RORxCD3 antibodies were constructed and tested as described below. 6.13.20.1. Connection arm to CD3

[0502] A series of CD3 binding arm variants based on a humanized version of the anti-CD3 antibody SP34 (SP34-89, SEQ ID Nos: 68 and 69) were engineered with point mutations in both VH or VL amino acid sequences (SEQ ID Nos: 70 to 73). The various VH and VL sequences were paired as described in Table 5. Table 5: SP34 anti-CD3 binding arm variants VL / VH variants VL-WT SP34-89 (SEQ ID NO: 69) VL-W57G SP34-89 (SEQ ID NO: 73) VH-WT SP34-89 SEQ ID NO: 68 VL-WT SP34-89 / VH-WT SP34-89 VL-W57G SP34-89 / VH-WT SP34-89 VH-N30S SP34-89 SEQ ID NO: 70 VL-WT SP34-89 / VH-WT SP34-89 VL-W57G SP34-89 / VH-N30S SP34-89 VH-G65D SP34-89 SEQ ID NO: 71 VL-WT SP34-89 / VHG65D SP34-89 VL-W57G SP34-89 / VH-G65D SP34-89 VH-S68T SP34-89 SEQ ID NO: 72 VL-WT SP34-89 / VHS68T SP34-89 VL-W57G SP34-89 / VH-S68T SP34-89 Petition 870260031312, dated 02 / 04 / 2026, pp. 208 / 417 200 / 285

[0503] The VL and VH variants were cloned into one arm of a 1x1 BC1 B-Body construct, while the other arm contained an irrelevant antigen-binding site. Figure 47 demonstrates the binding affinity of the unmutated monovalent SP3489 B-Body, as determined by Octet biolayer interferometry (Pall ForteBio). A two-fold serial dilution (200 to 12.5 nM) of the construct was used to determine a binding affinity of 23 nM for SP34-89 (kon = 3 x 10⁵ M⁻¹s⁻¹, koff = 7.1 x 10⁻³s⁻¹), matching the affinity with other SP34 variants in the literature. The kinetic affinity also matched the equilibrium binding affinity. 6.13.20.2. Connecting arm to the ROR

[0504] A chemically synthetic Fab phage library with diversity introduced into Fab CDRs was tested against ROR antigens using a monoclonal phage ELISA format, where plate-immobilized ROR variants were evaluated for phage binding, as described above. Phage clones expressing the antigen-recognized Fabs were sequenced. A first screening campaign for ROR1 binding identified antigen-binding site (ABS) clones designated I2A in Table 6, and a second screening campaign for ROR2 binding identified ABS clones designated “I2C” in Table 6. Table 6: Cand...

Claims

1. Transmembrane receptor binding molecule for protein tyrosine kinase (ROR) characterized in that the ROR antigen-binding molecule comprises: an antigen-binding site specific for an ROR antigen, wherein the antigen-binding site comprises a variable region of the heavy chain (VH) of an antibody and a variable region of the light chain (VL) of an antibody, wherein the antigen-binding site comprises: a VH CDR1 comprising SEQ ID NO:314, a VH CDR2 comprising SEQ ID NO:315, a VH CDR3 comprising SEQ ID NO:316, a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, a VL CDR3 comprising SEQ ID NO:319, wherein the antigen-binding site is specific for ROR1.

2. ROR antigen-binding molecule according to claim 1, characterized in that the VH region comprises a VH region as defined by the amino acid sequence of SEQ ID NO: 104 and the VL region comprises a VL region as defined by the amino acid sequence of SEQ ID NO:

100.

3. ROR antigen-binding molecule, according to claim 1 or 2, characterized in that the ROR antigen is a Kringle ROR1 domain.

4. ROR antigen-binding molecule, according to any one of claims 1 to 3, characterized in that the ROR antigen comprises a human ROR antigen. Petition 870260031312, dated 02 / 04 / 2026, pp. 295 / 417 2 / 3 5. ROR antigen-binding molecule, according to any one of claims 1 to 4, characterized in that the ROR antigen-binding molecule additionally comprises one or more additional antigen-binding sites.

6. ROR antigen-binding molecule, according to any one of claims 1 to 5, characterized in that the ROR antigen-binding molecule comprises an antibody format selected from the group consisting of: complete antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, diabodies, sc-diabodies, DARTs, tandAbs and minibodies.

7. ROR antigen-binding molecule, according to any one of claims 1 to 6, characterized in that the molecule comprises a sequence of one or more constant regions.

8. ROR antigen-binding molecule according to claim 7, characterized in that the constant region is a CH1, CH2, CH3 and / or CL constant region.

9. Pharmaceutical composition characterized by comprising the ROR antigen-binding molecule as defined in any one of claims 1 to 8, and a pharmaceutically acceptable diluent.

10. Use of a pharmaceutical composition, according to claim 9, characterized by being in the manufacture of a medicament for the treatment of cancer in an individual, wherein the cancer is a cancer that expresses ROR antigen. Petition 870260031312, dated 02 / 04 / 2026, pp. 296 / 417 3 / 3 11. Use, according to claim 10, characterized in that the cancer expresses an ROR1 antigen, or an ROR1 antigen and an ROR2 antigen.

12. Use, according to claim 10 or 11, characterized in that the cancer is selected from the group consisting of: pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematologic cancer, prostate cancer, colon cancer, renal cancer, thyroid cancer, liver cancer, urothelial carcinoma, melanoma, endometrial cancer, clear cell renal cell carcinoma, clear cell carcinoma and uterine cancer.

13. Use, according to any one of claims 10 to 12, characterized in that the pharmaceutical composition is administered to the individual in combination with additional therapy.

14. Use according to claim 13, characterized in that the additional therapy is surgery, radiotherapy, endocrine therapy, immunotherapy or chemotherapy.

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