Bispecific anti-lrrc15 and cd3e antibodies

Abstract

Disclosed are multispecific binding compounds that bind to LRRC15 and CD3; and methods of making such binding compounds; compositions, including pharmaceutical compositions, comprising such binding compounds; and uses of the multispecific binding compounds and the compositions for treating disorders characterized by expression of LRRC15.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 880,347, filed July 30, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 21, 2020, named QLS-0001-WO_SL.txt, and is 535,066 bytes in size. Technical Field

[0005] This invention relates to multispecific binding compounds that bind to LRRC15 and CD3. The invention also relates to methods for preparing such binding compounds, compositions comprising such binding compounds (including pharmaceutical compositions), and their use in treating conditions characterized by LRRC15 expression. Background Technology

[0006] LRRC15

[0007] Leucine-rich repeats (LRRs) are sequence motifs of 20 to 29 residues found in many proteins with diverse functions, such as hormone receptor interaction, enzyme inhibition, cell adhesion, and cell transport. The primary function of these motifs appears to provide a general structural framework for the formation of protein-protein interactions. One protein containing an LRR sequence motif is LRRC15, a 581-amino acid leucine-rich transmembrane protein.

[0008] LRRC15 (protein 15 containing leucine-rich repeats, UniProt Q8TF66), also known as HLib and LIB, has been identified as highly expressed in a variety of solid tumor indications, with limited expression in normal tissues. Purcell et al., Cancer Res; 78(14); 4059–72. LRRC15 is a type I membrane protein with no obvious intracellular signal transduction domain. Ibid. This protein has been found to be highly expressed on the cell surface of stromal fibroblasts in many solid tumors. Ibid. Due to the limited expression of LRRC15 in normal tissues, it is an attractive target for the treatment of malignancies characterized by LRRC15 expression. Monoclonal antibodies specific to LRRC15 have been described in the literature, for example, in US Patent Publication No. US2017 / 0151343, the disclosure of which is incorporated herein by reference in its entirety.

[0009] RNA expression analysis showed that LRRC15 was highly expressed in subgroups of invasive breast cancer, colonic adenocarcinoma, diffuse large B-cell lymphoma with lymphoid vegetations, esophageal cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, and rectal adenocarcinoma (http: / / gepia.cancer-pku.cn / detail.php?gene=LRRC15). LRRC15 expression was increased in smaller subgroups of bladder epithelial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, sarcoma, cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumors, and uterine carcinosarcoma. (Ibid.)

[0010] LRRC15 is not overexpressed in adrenocortical carcinoma, kidney chromophobe, clear cell renal carcinoma, papillary renal carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, pheochromocytoma and paraganglioma of the liver, prostate adenocarcinoma, thyroid cancer, thymoma, and endometrial cancer of the uterine body. (Ibid.)

[0011] Purcell et al. reported a histological analysis of LRRC15 expression. Notably, tumors from osteosarcoma, undifferentiated sarcoma, glioblastoma, and melanoma directly expressed LRRC15, as well as in the surrounding stroma. Interestingly, other tumors showed expression in the tumor-associated stroma but not in the tumor cells themselves, such as pancreatic cancer, breast cancer, squamous cell lung cancer, ovarian cancer, testicular cancer, gastric cancer, head and neck cancer, and colorectal cancer. LRRC15 expression was described in several cell lines, including U87 MG and U-118 MG (glioblastoma), RPMI-7951 and SKMEL2 (melanoma), and SAOS-2 (sarcoma).

[0012] In light of the foregoing, the therapeutic development of multispecific binding compounds (e.g., bispecific antibodies) may be effective in treating various cancer patients expressing LRRC15, in matrix-surrounded cancers in which both tumor cells and the matrix express LRRC15, and potentially in cancers in which LRRC15 is expressed only in the matrix surrounding the tumor. Summary of the Invention

[0013] Aspects of the present invention include multispecific binding compounds comprising a first binding unit having binding affinity for LRRC15 and a second binding unit having binding affinity for CD3ε, wherein the first binding unit comprises a heavy chain variable region having at least 95% sequence identity with any of the sequences of SEQ ID NO:24-26 and / or a light chain variable region having at least 95% sequence identity with the sequence of SEQ ID NO:27.

[0014] In some embodiments, the first binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO:24-26 and / or a light chain variable region sequence of SEQ ID NO:27. In some embodiments, the first binding unit comprises a heavy chain variable region sequence of SEQ ID NO:25 and a light chain variable region sequence of SEQ ID NO:27. In some embodiments, the second binding unit comprises a heavy chain variable region having at least 95% sequence identity with any of the sequences of SEQ ID NO:28-80 and / or a light chain variable region having at least 95% sequence identity with any of the sequences of SEQ ID NO:81-132. In some embodiments, the second binding unit comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO:28-80 and / or a light chain variable region sequence selected from the group consisting of SEQ ID NO:81-132. In some embodiments, the second binding unit comprises: (a) the heavy chain variable region sequence of SEQ ID NO:55 and the light chain variable region sequence of SEQ ID NO:107; or (b) the heavy chain variable region sequence of SEQ ID NO:28 and the light chain variable region sequence of SEQ ID NO:81; or (c) the heavy chain variable region sequence of SEQ ID NO:29 and the light chain variable region sequence of SEQ ID NO:82. In some embodiments, the second binding unit comprises a single-chain Fv (scFv) containing a first variable region sequence, a second variable region sequence, and a connector sequence linking the first variable region sequence and the second variable region sequence. In some embodiments, the connector sequence comprises the sequence of SEQ ID NO:229.

[0015] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), and a second heavy chain polypeptide (H2), wherein: L1 comprises a variable region sequence (L1V). L ) and constant region sequence (L1C) L H1 contains the variable region sequence (H1V); H C H 1. Constant region sequence (H1C) H 1) C H 2. Constant region sequence (H1C)H 2) and C H 3. Constant region sequence (H1C) H 3); and H2 contains: a sequence containing the first variable region (H2scFv) H ), second variable region sequence (H2scFv) L ), and H2scFv H Sequence and H2scFv L The single-stranded Fv(H2scFv) of the connector sequence for sequence ligation; C H 2. Constant region sequence (H2C) H 2); and C H 3. Constant region sequence (H2C) H 3); where: L1V L and H1V H The sequences together form a binding unit with binding affinity for LRRC15; H2scFv has binding affinity for CD3ε; L1C L and H1C H The sequence 1 is optionally linked by disulfide bonds; the H1 and H2 polypeptide chains optionally include hinge regions, wherein the H1 and H2 polypeptide chains are optionally linked by at least one disulfide bond; and H1C H 3 and H2C H The 3 sequence contains an asymmetric interface that facilitates proper pairing between the H1 and H2 polypeptide chains.

[0016] In some implementations, L1V L The sequence comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:27. In some embodiments, L1V L The sequence contains the sequence of SEQ ID NO:27. In some embodiments, H1V H The sequence comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:24-26. In some embodiments, H1V H The sequence is selected from the group consisting of SEQ ID NO:24-26. In some embodiments, H2scFv contains a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:133-185. In some embodiments, H2scFv contains a sequence selected from the group consisting of SEQ ID NO:133-185.

[0017] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises a variable region sequence (L1V). L ) and constant region sequence (L1C)L H1 contains the variable region sequence (H1V); H C H 1. Constant region sequence (H1C) H 1) C H 2. Constant region sequence (H1C) H 2) C H 3. Constant region sequence (H1C) H 3) and sequences containing the first variable region (H1scFv) H ), second variable region sequence (H1scFv) L ) and H1scFv H Sequence and H1scFv L The single-stranded Fv (H1scFv) of the connector sequence is linked by sequence ligation; and H2 contains the variable region sequence (H2V). H C H 1. Constant region sequence (H2C) H 1) C H 2. Constant region sequence (H2C) H 2) C H 3. Constant region sequence (H2C) H 3) and sequences containing the first variable region (H2scFv) H ), second variable region sequence (H2scFv) L ), and H2scFv H Sequence and H2scFv L The single-stranded Fv (H2scFv) of the connector sequence is used for sequence ligation; and L2 contains the variable region sequence (L2V). L ) and constant region sequences (L2C) L ); where: the L1V L and H1V H The sequences together form a binding unit with binding affinity for LRRC15; the L2V L and H2V H The sequences together form a binding unit with binding affinity for LRRC15; the H1scFv and H2scFv have binding affinity for CD3ε; the L1C L and H1C H The L2C sequence is optionally linked by disulfide bonds; L and H2C H The H1 and H2 polypeptide chains are optionally linked by disulfide bonds; the H1 and H2 polypeptide chains optionally include hinge regions, wherein the H1 and H2 polypeptide chains are optionally linked by at least one disulfide bond.

[0018] In some embodiments, L1 and L2 contain the same sequence. In some embodiments, H1 and H2 contain the same sequence. In some embodiments, L1 and L2 contain different sequences. In some embodiments, H1 and H2 contain different sequences. In some embodiments, L1V L The sequence comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:27. In some embodiments, L1V L The sequence contains the sequence of SEQ ID NO:27. In some embodiments, H1V H The sequence comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:24-26. In some embodiments, H1V H The sequence is selected from the group consisting of SEQ ID NO:24-26. In some embodiments, H1scFv contains a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:133-185. In some embodiments, H1scFv contains a sequence selected from the group consisting of SEQ ID NO:133-185. In some embodiments, L2V L The sequence comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:27. In some embodiments, L2V L The sequence contains the sequence of SEQ ID NO:27. In some embodiments, H2V H The sequence comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:24-26. In some embodiments, H2V H The sequence is selected from the group consisting of SEQ ID NO:24-26. In some embodiments, H2scFv comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:133-185. In some embodiments, H2scFv comprises a sequence selected from the group consisting of SEQ ID NO:133-185. In some embodiments, H1 comprises the following sequence order from the N-terminus to the C-terminus: H1V H H1C H 1. H1C H 2. H1C H 3. H1scFv. In some implementations, H2 comprises the following sequence from the N-end to the C-end: H2V H H2C H 1. H2C H 2. H2C H 3. H2scFv. In some implementations, H1 comprises the following sequence from the N-end to the C-end: H1V HH1C H 1. H1scFv, H1C H 2. H1C H 3. In some implementations, H2 comprises the following sequence from the N-terminus to the C-terminus: H2V H H2C H 1. H2scFv, H2C H 2. H2C H 3.

[0019] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises a variable region sequence (L1V). L ) and constant region sequence (L1C) L H1 contains the variable region sequence (H1V); H C H 1. Constant region sequence (H1C) H 1) C H 2. Constant region sequence (H1C) H 2) and C H 3. Constant region sequence (H1C) H 3); H2 contains variable region sequences (H2V) H C H 1. Constant region sequence (H2C) H 1) C H 2. Constant region sequence (H2C) H 2) C H 3. Constant region sequence (H2C) H 3) and sequences containing the first variable region (H2scFv) H ), second variable region sequence (H2scFv) L ) and the H2scFv H The sequence and the H2scFv L The single-stranded Fv (H2scFv) of the connector sequence is used for sequence ligation; and L2 contains the variable region sequence (L2V). L ) and constant region sequences (L2C) L ); where: the L1V L Sequence and the H1V H The sequences together form a binding unit with binding affinity for LRRC15; the L2V L Sequence and the H2V H The sequences together form a binding unit with binding affinity for LRRC15; the H2scFv has binding affinity for CD3ε; the L1C L Sequence and the H1C HThe L2C sequence is optionally linked by disulfide bonds; L Sequence and the H2C H The H1 and H2 polypeptide chains are optionally linked by disulfide bonds; the H1 and H2 polypeptide chains optionally include hinge regions, wherein the H1 and H2 polypeptide chains are optionally linked by at least one disulfide bond; and the H1C H 3. Sequence and the H2C H The 3 sequence contains an asymmetric interface that facilitates proper pairing between the H1 and H2 polypeptide chains.

[0020] In some implementations, L1 and L2 contain the same sequence. In some implementations, L1 and L2 contain different sequences. In some implementations, L1V L The sequence comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:27. In some embodiments, L1V L The sequence contains the sequence of SEQ ID NO:27. In some embodiments, H1V H The sequence comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:24-26. In some embodiments, H1V H The sequence is selected from the group consisting of SEQ ID NO:24-26. In some embodiments, H2scFv comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:133-185. In some embodiments, H2scFv comprises a sequence selected from the group consisting of SEQ ID NO:133-185. In some embodiments, L2V L The sequence comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:27. In some embodiments, L2V L The sequence contains the sequence of SEQ ID NO:27. In some embodiments, H2V H The sequence comprises a sequence having at least 95% sequence identity with any of the sequences in SEQ ID NO:24-26. In some embodiments, H2V H The sequence is selected from the group consisting of SEQ ID NO:24-26. In some embodiments, H1 comprises the following sequence order from the N-terminus to the C-terminus: H1V H H1C H 1. H1C H 2. H1C H 3. In some implementations, H2 comprises the following sequence from the N-terminus to the C-terminus: H2V H H2C H 1. H2C H 2. H2CH 3. H2scFv. In some implementations, H2 comprises the following sequence from the N-terminus to the C-terminus: H2V H H2C H 1. H2scFv, H2C H 2. H2C H 3.

[0021] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), and a second heavy chain polypeptide (H2), wherein: L1 comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:194; H1 comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:201; and H2 comprises a sequence having at least 95% sequence identity with any one of the sequences of SEQ ID NO:224 to 228 or 232.

[0022] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), and a second heavy chain polypeptide (H2), wherein: L1 comprises the sequence of SEQ ID NO:194; H1 comprises the sequence of SEQ ID NO:201; and H2 comprises a sequence selected from the group consisting of SEQ ID NO:224 to 228 or 232.

[0023] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), and a second heavy chain polypeptide (H2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; and H2 comprises SEQ ID NO:225.

[0024] The present invention includes a multispecific binding compound comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:194; H1 comprises a sequence having at least 95% sequence identity with any one of the sequences of SEQ ID NO:195-223, 231, 233-240; H2 comprises a sequence having at least 95% sequence identity with any one of the sequences of SEQ ID NO:195-223, 231, 233-240; and L2 comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO:194.

[0025] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises the sequence of SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:195-223, 231, and 233-240; H2 comprises a sequence selected from the group consisting of SEQ ID NO:195-223, 231, and 233-240; and L2 comprises the sequence of SEQ ID NO:194.

[0026] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:195 and 198; H2 comprises a sequence selected from the group consisting of SEQ ID NO:195 and 198; and L2 comprises SEQ ID NO:194.

[0027] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:196 and 199; H2 comprises a sequence selected from the group consisting of SEQ ID NO:196 and 199; and L2 comprises SEQ ID NO:194.

[0028] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:197 and 200; H2 comprises a sequence selected from the group consisting of SEQ ID NO:197 and 200; and L2 comprises SEQ ID NO:194.

[0029] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:233 and 234; H2 comprises a sequence selected from the group consisting of SEQ ID NO:233 and 234; and L2 comprises SEQ ID NO:194.

[0030] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:202, 206, 209, and 212; and L2 comprises SEQ ID NO:194.

[0031] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:203, 207, 210, and 213; and L2 comprises SEQ ID NO:194.

[0032] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:204, 208, 211, and 214; and L2 comprises SEQ ID NO:194.

[0033] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises SEQ ID NO:205; and L2 comprises SEQ ID NO:194.

[0034] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:231; H2 comprises a sequence selected from the group consisting of SEQ ID NO:235, 236, and 237; and L2 comprises SEQ ID NO:194.

[0035] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:215, 219, 221, and 239; H2 comprises a sequence selected from the group consisting of SEQ ID NO:215, 219, 221, and 239; and L2 comprises SEQ ID NO:194.

[0036] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:216, 220, 222, and 240; H2 comprises a sequence selected from the group consisting of SEQ ID NO:216, 220, 222, and 240; and L2 comprises SEQ ID NO:194.

[0037] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:217 and 223; H2 comprises a sequence selected from the group consisting of SEQ ID NO:217 and 223; and L2 comprises SEQ ID NO:194.

[0038] Aspects of the present invention include multispecific binding compounds comprising a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:218; H2 comprises SEQ ID NO:218; and L2 comprises SEQ ID NO:194.

[0039] Aspects of the invention include pharmaceutical compositions comprising the multispecific binding compounds described herein. Aspects of the invention include treatment methods comprising administering an effective dose of the multispecific binding compound or pharmaceutical composition described herein to an individual in need. Aspects of the invention include methods for treating a condition characterized by LRRC15 expression, the methods comprising administering the multispecific binding compound or pharmaceutical composition described herein to a subject suffering from the condition.

[0040] Aspects of the present invention include the use of multispecific binding compounds as described herein in the preparation of medicaments for treating conditions characterized by LRRC15 expression. Aspects of the present invention include multispecific binding compounds as described herein for treating conditions characterized by LRRC15 expression.

[0041] In some implementations, the disease is selected from the group consisting of: sarcoma, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, and large B-cell lymphoma.

[0042] Aspects of the present invention include polynucleotides encoding multispecific binding compounds as described herein, vectors comprising polynucleotides as described herein, and cells comprising vectors as described herein.

[0043] Aspects of the invention include a method for generating a multispecific binding compound as described herein, the method comprising growing cells as described herein under conditions that allow expression of the multispecific binding compound, and isolating the multispecific binding compound.

[0044] These and other aspects will be further explained in the remainder of this disclosure, including the embodiments. Attached Figure Description

[0045] Figure 1 This is a table showing the percentage of aggregates, monomers, and melting points of multispecific binding compounds according to embodiments of the present invention.

[0046] Figure 2 This is a table illustrating the binding kinetics of multispecific binding compounds according to embodiments of the present invention.

[0047] Figure 3 Figure AF is a schematic illustration of various multispecific binding compounds according to embodiments of the present invention.

[0048] Figure 4 This is a graph showing the results of protein thermal displacement measurements performed on various multispecific binding compounds according to embodiments of the present invention.

[0049] Figure 5 Figures A and B show the changes in binding as concentration occurs when the anti-CD3 scFv-Fc clone binds to human Jurkat cells (Figure A) and to cynomolgus monkey H-SCF cells (Figure B).

[0050] Figure 6Figure AF illustrates the binding of various bispecific binding compound forms to CD3+ cells. Figure A shows the binding of the scFv-Fc compound to CD3+ cells. Figure B shows the binding of type 1 compound to CD3+ cells. Figure C shows the binding of type 2 compound to CD3+ cells. Figures D, E, and F compare the binding of type 4, type 5, and scFv-Fc compounds to CD3+, CD4+ T cells, and CD3+, CD8+ T cells.

[0051] Figure 7 Figures A and B show how the binding to LRRC15+U118MG and U87MG cells varies with the concentration of various binding compound forms, respectively.

[0052] Figure 8 Figure AE is a graph showing how T cell activation varies with the concentration of various bound compound forms.

[0053] Figure 9 Figure AE is a graph showing how T cell proliferation varies with the concentration of various bound compound forms.

[0054] Figure 10 Figure AC is a graph showing how cytokine release varies with the concentration of various bound compound forms.

[0055] Figure 11 The graph AG is a graph showing how the percentage of cytotoxicity varies with the concentration of various forms of the bound compounds.

[0056] Figure 12 This is a graph showing how tumor volume changes over time as various forms of the compound or controls are administered to animals. Detailed Implementation

[0057] Unless otherwise indicated, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. These techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by R.R. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M. Usubel et al., 1987, and updated regularly); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); Harlow, Lane, and Harlow, "Using Antibodies: A Laboratory Manual: Portable Protocol No. I," Cold Spring Harbor. Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0058] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of that range (unless the context clearly indicates otherwise, the intermediate value is one-tenth of the lower limit unit) and any other specified or intermediate value within that range are included in this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and also covered in this invention, and any extreme values ​​within the specified range may be specifically excluded. When the specified range includes one or both of the extreme values, the range excluding any one or both of those included extreme values ​​is also included in this invention.

[0059] Unless otherwise indicated, antibody residues in this article are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0060] In the following description, numerous specific details are set forth to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures familiar to those skilled in the art have not been described in order to avoid obscuring the invention.

[0061] All references cited in this disclosure (including patent applications and publications) are incorporated herein by reference in their entirety.

[0062] I. definition

[0063] "Comprising" means that the stated elements are required in the composition / method / kit, but may include other elements to form a composition / method / kit, etc., within the scope of the claims.

[0064] "consisting essentially of..." means to limit the scope of the described composition or method to specified materials or steps that do not substantially affect the essential and novel features of the invention.

[0065] "Composed of" means excluding any element, step or component not specified in the claims from the composition, method or kit.

[0066] The antibody residues in this article are numbered according to the Kabat numbering system and the EU numbering system. When referring to residues in the variable domain (approximately residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., Kabat et al., EU index as reported above). “EU index as in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise specified herein, references to the numbering of residues in the variable domain of an antibody mean the residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to the numbering of residues in the constant domain of an antibody mean the residue numbering according to the EU numbering system.

[0067] Antibodies, also known as immunoglobulins, typically comprise at least one heavy chain and one light chain, wherein the amino-terminal domains of the heavy and light chains are sequence-variable and are therefore commonly referred to as variable region domains or variable heavy (VH) or variable light (VH) domains. These two domains typically associate to form a specific binding region; however, as will be discussed here, specific binding can also be obtained using only the variable heavy chain sequence, and various non-natural antibody conformations are known and used in the art.

[0068] "Functional" or "bioactive" antibodies or conjugates are antibodies or conjugates capable of exerting one or more activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other conjugate may have the ability to specifically bind to an antigen, and this binding may initiate or alter cellular or molecular events such as signal transduction or enzyme activity. Functional antibodies or other conjugates may also block ligand activation of receptors or act as agonists or antagonists. The ability of an antibody or other conjugate to exert one or more activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0069] In this article, the term "antibody" is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, polymers, multispecific antibodies (e.g., bispecific antibodies), triple-chain antibodies, single-chain Fvs (scFvs), nanobodies, and also antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0070] The term antibody may refer to full-length heavy chain, full-length light chain, or intact immunoglobulin molecules; or the immunoactive portion of any of these polypeptides, i.e., a polypeptide or portion thereof containing an antigen-binding site that specifically binds to an antigen of a target, including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. The immunoglobulins may be derived from any species.

[0071] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, unlike conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The monoclonal antibodies according to the invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be prepared, for example, via recombinant protein production methods (see, for example, U.S. Patent No. 4,816,567).

[0072] The term "variability" used in conjunction with antibodies refers to the fact that certain portions of the antibody's variable domain vary significantly in sequence across multiple antibodies, contributing to the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. In the light and heavy chain variable domains, it is concentrated in three segments called hypervariable regions. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which are mostly beta-sheet oriented and linked by three hypervariable regions, forming loops that connect the beta-sheet structure and sometimes constitute part of the beta-sheet structure. The hypervariable regions in each chain are held together very closely by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains do not directly participate in the binding of antibodies to antigens, but exhibit various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity (ADCC).

[0073] The term "hypervariant region," as used herein, refers to the amino acid residues in an antibody responsible for antigen binding. Hypervariant regions generally comprise amino acid residues from the complementarity-determining region (CDR) or CDR (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the hypervariant loop (HLO) residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Frame region" or "FR" residues are variable domain residues other than those defined in the hypervariant region herein.

[0074] Exemplary CDR names are shown in this document; however, those skilled in the art will understand that various CDR definitions are commonly used, including the Kabat definition (see “Zhao et al., A germline knowledge-based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010; 47:694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al., “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877–883).Alternative definitions of target CDRs include, but are not limited to, those published by: Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001; 309:657–670; Ofran et al., “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008; 181:6230–6235; Almagro, “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004; 17:132–143; and Padlan et al., “Identification of specificity-determining residues in antibodies.” Faseb J. 1995; 9:133–139, each of which is specifically incorporated herein by reference.

[0075] As used herein, the term "multispecific binding compound" refers to a binding compound containing two or more antigen-binding sites. A multispecific binding compound according to embodiments of the invention may be an antibody-like molecule comprising, substantially comprising, or composed of two, three, or four polypeptide subunits, wherein any one polypeptide subunit may contain one or more variable region domains having binding affinity for a target antigen (e.g., LRRC15). In some embodiments, the multispecific binding compound comprises a pair of variable region domains (e.g., a heavy chain variable region domain and a light chain variable region domain) that together form a binding unit. In some embodiments, the multispecific binding compound comprises a pair of variable region domains in the form of a single-chain Fv (scFv), wherein the first and second variable region domains are linked by a linker and together form a binding unit. The subject multispecific binding compound may have any suitable combination or configuration of binding units, including but not limited to the specific configurations described herein.

[0076] The multispecific binding compounds described herein can belong to any immunoglobulin subclass, including IgG, IgM, IgA, IgD, and IgE subclasses. In specific embodiments, the multispecific binding compound is an IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. This document further describes modifications to the CH domain that alter effector function.

[0077] As used herein, a “complete antibody chain” is a chain containing a full-length variable region and a full-length constant region (Fc). A complete “conventional” antibody contains a complete light chain and a complete heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domain structures CH1, hinge, CH2, and CH3 (for secreted IgG). Other isotypes (e.g., IgM or IgA) may have different CH domains. The constant region can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. A complete antibody may have one or more “effective functions,” which refer to those biological activities attributable to the antibody’s Fc constant region (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter effector characteristics, binding to Fc receptors, etc.

[0078] Antibodies and various antigen-binding proteins are classified into different classes based on the amino acid sequence of their heavy chain Fc (constant domain). There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the Fc corresponding to different antibody classes are respectively designated as α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known. Ig forms include hinge-modified or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). Based on the amino acid sequence of its constant domains, the light chain of antibodies from any vertebrate species can be designated as one of two types (called κ and λ).

[0079] A “functional Fc region” possesses the “effective function” of the native Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the interaction of the Fc region with receptors such as FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors and low-affinity FcRn receptors; and can be assessed using a variety of analyses known in the art. A “dead” or “silent” Fc is an Fc that has been mutated to retain, for example, activity related to prolonged serum half-life, but does not activate high-affinity Fc receptors, or has a reduced affinity for Fc receptors.

[0080] The “natural sequence Fc region” contains the same amino acid sequence as the Fc region that exists in nature. Natural sequence human Fc regions include, for example, the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0081] The “variant Fc region” comprises an amino acid sequence that distinguishes it from the native Fc region due to at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions in the native Fc region or the Fc region of the parent polypeptide, and preferably about one to about five amino acid substitutions. The variant Fc region as used herein will preferably have at least about 80% homology with the native Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology.

[0082] The amino acid sequences of human IgG1 are provided by UniProtKB No. P01857 (in whole or in part, incorporated herein by reference). The amino acid sequences of human IgG2 are provided by UniProtKB No. P01859 (in whole or in part, incorporated herein by reference). The amino acid sequences of human IgG3 are provided by UniProtKB No. P01860 (in whole or in part, incorporated herein by reference). The amino acid sequences of human IgG4 are provided by UniProtKB No. P01861 (in whole or in part, incorporated herein by reference).

[0083] The variant Fc sequence may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563; Hezareh et al., (2001) J. Virology 75:12161; U.S. Patent No. 5,624,821, the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the variant Fc sequence may include the following amino acid substitutions: L234A; L235A; and G237A. When these three amino acid substitutions are present in the IgG1 Fc sequence, they may be referred to as G1AAA or LALAGA.

[0084] Two amino acid substitutions at complement C1q binding sites at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at human IgG1 or IgG2 residues at positions 233–236 and at IgG4 residues at positions 327, 330, and 331 significantly reduce ADCC and CDC (see, for example, Armour KL et al., 1999 Eur J Immunol. 29(8):2613–24; and Shields RL et al., 2001 J Biol Chem. 276(9):6591–604).

[0085] Other Fc variants are also possible, including, but not limited to, variants that lack a region capable of forming a disulfide bond, or variants that eliminate certain amino acid residues at the N-terminus of the native Fc, or variants that add methionine residues thereto. Thus, in some embodiments, one or more Fc moieties of the binding compound may contain one or more mutations in the hinge region to eliminate the disulfide bond. In yet another embodiment, the hinge region of the Fc may be completely removed. In still another embodiment, the binding compound may contain an Fc variant.

[0086] Furthermore, Fc variants can be constructed to remove or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to achieve complement binding or Fc receptor binding. For example, but not limited to, deletions can occur at complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0087] The term "antibody containing an Fc region" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Therefore, antibodies containing an Fc region according to the present invention may include antibodies with or without K447.

[0088] Various aspects of the present invention include binding compounds having multispecific configurations (including, but not limited to, bispecific, trispecific, etc.). Various methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0089] Various methods for generating multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on the peptide are linked by a peptide linker. A non-limiting example of such a peptide linker is the GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue (SEQ ID NO:192), wherein said sequence is repeated n times, where n is an integer in the range of 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO:192) (n=1) and GGGGSGGGGS (SEQ ID NO:193) (n=2). Other suitable linkers may also be used, and said linkers are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0090] Antibodies and multispecific binding compounds as described herein can be in the form of dimers, wherein two heavy chains are bonded by disulfide bonds or otherwise covalently or non-covalently attached to each other, and may optionally include an asymmetric interface between two or more CH domains to facilitate proper pairing between polypeptide chains (commonly referred to as a "knob-into-hole" interface). The engineering of knob-into-hole antibodies with heterodimerization of heavy chains is discussed in Ridgway et al., Protein Eng. July 1996; 9(7):17-21 and U.S. Patent No. 8,216,805 (the disclosure of which is incorporated herein by reference in its entirety). The Fc region containing the asymmetric interface may be referred to herein by the abbreviation "KiH," meaning knob-into-hole. For example, aspects of the invention include variant Fc region sequences (such as the G1AAA sequence) containing the asymmetric interface and referred to herein as "G1AAA KiH."

[0091] The terms “LRRC15” and “protein 15 containing leucine-rich repeat sequences” refer to the LRRC15 protein of any human or non-human animal species, and specifically include human LRRC15 and LRRC15 of non-human mammals.

[0092] As used herein, the term “human LRRC15” includes any variant, isotype, and species homologue of human LRRC15 (UniProt Q8TF66), regardless of its origin or preparation method. Thus, “human LRRC15” includes human LRRC15 naturally expressed in cells as well as LRRC15 expressed in cells transfected with the human LRRC15 gene.

[0093] The terms “anti-LRRC15 antibody,” “LRRC15 antibody,” “anti-LRRC15 binding compound,” and “LRRC15 binding compound” are used interchangeably herein to refer to an antibody or binding compound as defined herein that specifically binds to LRRC15 (including human LRRC15) as defined herein.

[0094] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the amino acid sequence identity % value.

[0095] "Isolated" antibodies or binding compounds are antibodies or binding compounds that have been identified and isolated and / or recovered from components of their native environment. The contaminating components of their native environment are materials that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody will be purified (1) to more than 95% by weight of antibody, as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a turn-cup sequencer, or (3) to a degree to which homogeneity is found by SDS-PAGE using Coomassie blue or preferably silver staining under reducing or non-reducing conditions. Isolated antibodies include in situ antibodies within recombinant cells, since at least one component of the antibody's native environment will be absent. However, typically, isolated antibodies will be prepared by at least one purification step.

[0096] The binding compounds of the present invention include multispecific binding compounds. Multispecific binding compounds have more than one binding specificity. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinity, such as higher-order multi-epitope specificity, and tetravalent antibodies and antibody fragments. The terms "multispecific antibody" and "multispecific binding compound" are used herein in the broadest sense and cover all antibodies and antibody-like molecules having more than one binding specificity. The multispecific anti-LRRC5 binding compounds of the present invention specifically include binding compounds that bind immunospecifically to epitopes on LRRC15 proteins (such as human LRRC15) and epitopes on different proteins (e.g., CD3 proteins).

[0097] An epitope is a site on the surface of an antigen molecule where a single antibody molecule binds. Generally, an antigen has several or more different epitopes and reacts with a variety of different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0098] Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed from a continuous amino acid sequence in a protein. Conformational epitopes are formed from discontinuous amino acids in a protein sequence, but are aggregated together when the protein folds into its three-dimensional structure.

[0099] As used in this article, the term "valence" refers to a specified number of binding sites in an antibody molecule or binding compound.

[0100] A "monovalent" bound compound has a single binding site. Therefore, a monovalent bound compound is also monospecific.

[0101] "Multivalent" binding compounds have two or more binding sites. Therefore, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two, three, and four binding sites, respectively. Thus, the bispecific binding compounds according to the invention are at least bivalent and can be trivalent, tetravalent, or otherwise multivalent. Bivalent binding compounds according to embodiments of the invention can have two binding sites targeting the same epitope (i.e., bivalent, monocomplementary) or two different epitopes (i.e., bivalent, bicomplementary).

[0102] Various methods and protein configurations are known and used to prepare bispecific monoclonal antibodies (BsMABs) and binding compounds, trispecific antibodies and binding compounds, etc.

[0103] The term "human antibody" is used herein to refer to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies as used herein may include amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo. Specifically, the term "human antibody" includes antibodies and binding compounds having sequences of the human heavy chain variable region.

[0104] As used herein, the term "chimeric antibody" refers to an antibody having a variable sequence derived from a non-human immunoglobulin (such as a rat or mouse antibody) and a constant region of a human immunoglobulin (typically selected from a human immunoglobulin template). Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, all of which are incorporated herein by reference in their entirety. The term "chimeric antibody" specifically includes antibodies and binding compounds having a variable region sequence derived from a non-human immunoglobulin and a constant region sequence of a human immunoglobulin.

[0105] As used herein, the term "humanized antibody" refers to an antibody or binding compound containing a minimal sequence derived from a non-human immunoglobulin. Typically, a humanized antibody will contain at least one and usually substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of the non-human immunoglobulin and all or substantially all of the frame (FR) regions are those FR regions of the human immunoglobulin sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the common sequence of human immunoglobulins. Methods of antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, and 5,693,762; and U.S. Patent No. 6,180,370 to Queen et al.; EP239400; PCT Publication WO U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332, all of which are incorporated herein by reference in their entirety.

[0106] As used herein, the term "effective cell" refers to immune cells that participate in the effector phase of an immune response, in contrast to the cognitive and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells (such as natural killer cells) are capable of inducing antibody-dependent cytotoxicity (ADCC). For example, monocytes and macrophages expressing Fc receptors participate in the specific killing of target cells and presenting antigens to other components of the immune system, or binding to antigen-presenting cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0107] "Human effector cells" are leukocytes that express receptors (such as T-cell receptors or FcRs) and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among which NK cells are preferred. As described herein, effector cells can be isolated from their natural sources, such as blood or PBMCs.

[0108] The term “immune cells” is used in the broadest sense herein, including but not limited to cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0109] Antibody "effective function" refers to those biological activities attributable to the Fc region of the antibody (either the native Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR).

[0110] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells and subsequently cause the lysis of the target cells. Primary cells (NK cells) used to mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0111] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve its target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., an antibody) that is complexed with a homologous antigen. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0112] As used interchangeably in this article, "directed T cell-mediated cytotoxicity" and "redirected T cell-mediated cytotoxicity" refer to a cell-mediated response in which cross-linked molecules (e.g., bispecific antibodies) cross-link surface antigens (e.g., CD3) on T cells and antigens on target cells (e.g., surface antigens on cancer cells). The cross-linking between T cells and target cells promotes the killing of target cells by T cells through their cytotoxic activity. For example, redirected T cell-mediated cytotoxicity is described in Velasquez et al., Blood 2018 131:30-38.

[0113] "Binding affinity" refers to the strength of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed as an equilibrium dissociation constant (KD). Affinity can be measured using common methods known in the art. Low-affinity antibodies generally bind antigens slowly and tend to dissociate rapidly, while high-affinity antibodies generally bind antigens more quickly and tend to maintain the binding.

[0114] As used herein, “KD” or “KD value” refers to the dissociation constant determined in kinetic mode using an Octet Red96 instrument (Fortebio Inc., Menlo Park, CA) via biolayer interferometry. For example, an anti-mouse Fc sensor was loaded with a mouse-Fc fusion antigen and then immersed in wells containing antibodies to measure the concentration-dependent association rate (k-association). In a final step, the antibody dissociation rate (k-dissociation) was measured, in which the sensor was immersed in wells containing only buffer. KD is the ratio of k-dissociation to k-association. (For more details, see Concepcion, J et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0115] The terms “treatment”, “treating”, etc., are generally used herein to mean achieving the desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals and includes: (a) preventing the occurrence of the disease in a subject who is susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; or (c) alleviating the disease, i.e. causing its remission. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease that stabilizes or reduces unwanted clinical symptoms in the patient is of particular interest. It is desirable to perform such treatment before complete loss of function of the affected tissue. The therapies of the present invention may be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0116] "Therapeutic effective amount" refers to the amount of active agent necessary for a subject to impart a therapeutic benefit. For example, "therapeutic effective amount" is the amount that induces, improves, or otherwise causes pathological symptoms, disease progression, or improvement in disease-related physiological conditions or resistance to disease.

[0117] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. More specific examples of this type of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., epithelial bladder cancer), testicular (germ cell tumor) cancer, hepatocellular carcinoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma, or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Other examples of cancer include, but are not limited to, retinoblastoma, theca cell tumor, ovarian androma, hepatocellular carcinoma, hematologic malignancies (including non-Hodgkin's lymphoma (NHL), multiple myeloma, and acute hematologic malignancies), endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal carcinoma, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, Schwann cell tumor, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and urinary tract cancer.

[0118] The term "metastatic cancer" refers to a cancerous condition in which cancer cells from the primary tissue spread from the primary site to one or more other sites in the body via blood vessels or lymphatic vessels, forming one or more secondary tumors in one or more organs outside the primary tissue. A prominent example is metastatic breast cancer.

[0119] The term "characterized by LRRC15 expression" refers to any disease or condition in which LRRC15 expression is associated with or involved in one or more pathological processes characteristic of the disease or condition. Specifically, but not limitingly, diseases or conditions characterized by LRRC15 expression include, for example, cancers in which tumor cells express LRRC15 and / or tumor-associated stroma exhibits LRRC15 expression. Such conditions include, but are not limited to: aggressive breast cancer, colonic adenocarcinoma, lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, rectal adenocarcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and cervical endogenous adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme, sarcoma (e.g., undifferentiated sarcoma), cutaneous melanoma, gastric adenocarcinoma, testicular germ cell tumors, uterine carcinosarcoma, osteosarcoma, glioblastoma, melanoma, ovarian, gastric, and colorectal cancers.

[0120] The terms "cellular proliferative disorder" and "proliferative disorder" refer to a condition associated with some degree of abnormal cell proliferation. In one implementation, a cellular proliferative disorder is cancer.

[0121] As used in this article, “tumor” refers to all proliferative cell growth and proliferation (whether malignant or benign) as well as all precancerous and cancerous cells and tissues.

[0122] As used herein, the terms “treat,” “treatment,” or “treating” refer to therapeutic treatments and prophylactic or preventative measures aimed at preventing or alleviating the symptoms or condition of a targeted pathology. Subjects requiring treatment include those who already have a specific symptom or condition, those who are susceptible to the condition, or those who wish to prevent the condition.

[0123] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to a mammal that is being evaluated for and / or undergoing treatment. In one embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” cover, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly those that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0124] The term "pharmaceutical formulation" refers to a preparation in a form that allows for the effective bioactivity of the active ingredient and does not contain any other components that would have unacceptable toxicity to a subject to whom the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (carriers, additives) are those that can be reasonably administered to a test mammal to provide an effective dose of the active ingredient used.

[0125] "Sterile" preparations are sterile or contain no or substantially no live microorganisms and their spores. "Frozen" preparations are preparations kept at temperatures below 0°C.

[0126] A “stable” formulation is one in which the protein substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, during storage. The storage period is generally selected based on the expected shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and *Jones. A. Adv. Drug Delivery Rev. 10:29-90* (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and / or quantitatively in a variety of ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary band electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced antibodies with intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing antibody bioactivity or antigen-binding function; etc. Instability may involve one or more of the following: aggregates, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), trimming / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine ​​residues, N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0127] II. Detailed Explanation

[0128] Anti-LRRC15 binding compounds

[0129] Aspects of the present invention include multispecific binding compounds having binding affinity for LRRC15 and CD3ε. The multispecific binding compounds may comprise various configurations, and each binding unit may comprise a set of CDR sequences. CDR sequences are provided in Tables 1 and 2. Anti-LRRC15 heavy chain CDR sequences include SEQ ID NO: 1, 3, and 13, and anti-LRRC15 light chain CDR sequences include SEQ ID NO: 15, 19, and 22. Anti-CD3ε heavy chain CDR sequences include SEQ ID NO: 2, 4-12, and 14, and anti-CD3ε light chain CDR sequences include SEQ ID NO: 16-18, 20-21, and 23. In some embodiments, the multispecific binding compound comprises a CDR sequence having two or fewer amino acid substitutions in any of SEQ ID NO: 1-23.

[0130] The multispecific binding compound according to embodiments of the present invention may comprise any suitable combination of heavy and light chain variable region sequences, as listed in Tables 3, 4, 5, and 6. The anti-LRRC15 heavy chain variable region sequence comprises SEQ ID NO: 24-26. The anti-LRRC15 light chain variable region sequence comprises SEQ ID NO: 27. The anti-CD3ε heavy chain variable region sequence comprises SEQ ID NO: 28-80. The anti-CD3ε light chain variable region sequence comprises SEQ ID NO: 81-132. In some embodiments, the multispecific binding compound comprises a variable region sequence having at least about 80% identity with any of the variable region sequences in SEQ ID NO: 24-132, such as about 85%, about 90%, about 95%, about 99%, or about 99.9% identity.

[0131] The multispecific binding compound according to embodiments of the present invention may comprise one or more anti-CD3εscFv sequences, as listed in Table 7. The anti-CD3εscFv sequences include SEQ ID NO:133-185. In some embodiments, the multispecific binding compound comprises an scFv sequence having at least about 80% identity with any of SEQ ID NO:133-185, such as about 85%, about 90%, about 95%, about 99%, or about 99.9% identity.

[0132] The multispecific binding compounds described herein offer numerous benefits for use as clinical therapeutic agents. Multispecific binding compounds include members with multiple binding unit configurations, allowing for the selection of specific molecules that exhibit therapeutic benefits.

[0133] Suitable binding compounds may be selected from the binding compounds provided herein for development and therapeutic or other uses (including, but not limited to, use as bispecific binding compounds) (e.g., such as...). Figure 3(As shown in Figure AF). Figure 3 Schematic illustrations of non-limiting examples of multispecific binding compounds according to embodiments of the present invention are provided. In some embodiments, a pestle-and-mortar technique is used to pair the two heavy chains.

[0134] Turning Figure 3 The binding compound is depicted in Figure A, which shows a scFv-Fc form molecule containing two heavy chains, each of which contains a scFv, a hinge region, and an Fc region that have binding affinity for CD3ε.

[0135] Figure 3 Figure B depicts an asymmetric bispecific binding compound comprising a first light chain, a first heavy chain, and a second heavy chain. The first light chain contains a variable region (L1V). L ) and constant region (L1C) L The first heavy chain contains the variable region H1V. H and containing hinge area and C H 1. C H 2 and C H 3. Constant regions of the structural domains. Combined, L1V L and H1V H The region is used for binding units with binding affinity to LRRC15. The second heavy chain contains scFv with binding affinity to CD3ε, the hinge region, and C. H 2 and C H 3. Structural Domains. C H 2 and C H The three structural domains constitute the Fc region. In some embodiments, the binding compound is contained in the C16 of the first and second heavy chains. H 2. Structural domains and / or C H The asymmetric interface between the three structural domains ensures proper pairing between the first and second heavy chains. Figure 3 The bound compounds depicted in Figure B are referred to in this paper as type I bound compounds.

[0136] Figure 3 Figure C depicts a symmetrical, bispecific binding compound comprising a first light chain, a first heavy chain, a second heavy chain, and a second light chain. The first light chain contains a variable region (L1V). L ) and constant region (L1C) L The first heavy chain contains the variable region H1V. H Contains hinge area and C H 1. C H 2 and C H The constant region of the 3-structural domain, and the scFv with binding affinity for CD3ε. Combined, L1V L and H1V HThe region forms binding units with binding affinity for LRRC15. The second heavy chain contains variable H2V regions. H Contains hinge area and C H 1. C H 2 and C H 3. The constant region of the structural domain, and the scFv with binding affinity for CD3ε. C H 2 and C H The three structural domains constitute the Fc region. The second light chain contains the variable region (L2V). L ) and constant region (L2C) L Combined, L2V L and H2V H The region forms binding units that have binding affinity for LRRC15. Figure 3 The binding compound depicted in Figure C is referred to in this paper as a type 2 binding compound.

[0137] Figure 3 Figure D depicts an asymmetric bispecific binding compound comprising a first light chain, a first heavy chain, a second heavy chain, and a second light chain. The first light chain contains a variable region (L1V). L ) and constant region (L1C) L The first heavy chain contains the variable region H1V. H and containing hinge area and C H 1. C H 2 and C H 3. Constant regions of the structural domains. Combined, L1V L and H1V H The region forms binding units with binding affinity for LRRC15. The second heavy chain contains variable H2V regions. H Hinge area, C H 1. C H 2 and C H 3. A structural domain, and scFv with binding affinity for CD3ε. C H 2 and C H The three structural domains constitute the Fc region. In some embodiments, the binding compound is contained in the C16 of the first and second heavy chains. H 2. Structural domains and / or C H The asymmetric interface between the three structural domains ensures proper pairing between the first and second heavy chains. The second light chain contains a variable region (L2V). L ) and constant region (L2C) L Combined, L2V L and H2V H The region forms binding units that have binding affinity for LRRC15. Figure 3 The binding compound depicted in Figure D is referred to in this paper as a type 3 binding compound.

[0138] Figure 3 Figure E depicts an asymmetric bispecific binding compound comprising a first light chain, a first heavy chain, a second heavy chain, and a second light chain. The first light chain contains a variable region (L1V). L ) and constant region (L1C) L The first heavy chain contains the variable region H1V. H and containing hinge area and C H 1. C H 2 and C H 3. Constant regions of the structural domains. Combined, L1V L and H1V H The region forms binding units with binding affinity for LRRC15. The second heavy chain contains variable H2V regions. H C H 1. Structural domain, first hinge region and / or first joint region, scFv with binding affinity to CD3ε, second hinge region and C H 2 and C H 3. Structural Domains. C H 2 and C H The three structural domains constitute the Fc region. In some embodiments, the binding compound is contained in the C16 of the first and second heavy chains. H 2. Structural domains and / or C H The asymmetric interface between the three structural domains ensures proper pairing between the first and second heavy chains. The second light chain contains a variable region (L2V). L ) and constant region (L2C) L Combined, L2V L and H2V H The region forms binding units that have binding affinity for LRRC15. Figure 3 The binding compounds depicted in Figure E are referred to in this paper as type 4 binding compounds.

[0139] Figure 3 Figure F depicts a symmetrical, bispecific binding compound comprising a first light chain, a first heavy chain, a second heavy chain, and a second light chain. The first light chain contains a variable region (L1V). L ) and constant region (L1C) L The first heavy chain contains the variable region H1V. H C H 1. Structural domain, first hinge region and / or first joint region, scFv with binding affinity to CD3ε, second hinge region and C H 2 and C H 3 structural domains. Taken together, L1V L and H1V H The region forms binding units with binding affinity for LRRC15. The second heavy chain contains variable H2V regions. HC H 1. Structural domain, first hinge region and / or first joint region, scFv with binding affinity to CD3ε, second hinge region and C H 2 and C H 3. Structural Domains. C H 2 and C H The three structural domains constitute the Fc region. The second light chain contains the variable region (L2V). L ) and constant region (L2C) L Combined, L2V L and H2V H The region forms binding units that have binding affinity for LRRC15. Figure 3 The binding compounds depicted in Figure F are referred to in this paper as type 5 binding compounds.

[0140] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:224.

[0141] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:225.

[0142] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:226.

[0143] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:227.

[0144] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:228.

[0145] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide containing the sequence SEQ ID NO:194, a first heavy chain polypeptide containing the sequence SEQ ID NO:201, and a second heavy chain polypeptide containing the sequence SEQ ID NO:232.

[0146] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:195 and 198; H2 comprises a sequence selected from the group consisting of SEQ ID NO:195 and 198; and L2 comprises SEQ ID NO:194.

[0147] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:196 and 199; H2 comprises a sequence selected from the group consisting of SEQ ID NO:196 and 199; and L2 comprises SEQ ID NO:194.

[0148] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:197 and 200; H2 comprises a sequence selected from the group consisting of SEQ ID NO:197 and 200; and L2 comprises SEQ ID NO:194.

[0149] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:233 and 234; H2 comprises a sequence selected from the group consisting of SEQ ID NO:233 and 234; and L2 comprises SEQ ID NO:194.

[0150] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein: L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:202, 206, 209, and 212; and L2 comprises SEQ ID NO:194.

[0151] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:203, 207, 210, and 213; and L2 comprises SEQ ID NO:194.

[0152] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises a sequence selected from the group consisting of SEQ ID NO:204, 208, 211, and 214; and L2 comprises SEQ ID NO:194.

[0153] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:201; H2 comprises SEQ ID NO:205; and L2 comprises SEQ ID NO:194.

[0154] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:231; H2 comprises a sequence selected from the group consisting of SEQ ID NO:235, 236, and 237; and L2 comprises SEQ ID NO:194.

[0155] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:215, 219, 221, and 239; H2 comprises a sequence selected from the group consisting of SEQ ID NO:215, 219, 221, and 239; and L2 comprises SEQ ID NO:194.

[0156] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:216, 220, 222, and 240; H2 comprises a sequence selected from the group consisting of SEQ ID NO:216, 220, 222, and 240; and L2 comprises SEQ ID NO:194.

[0157] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises a sequence selected from the group consisting of SEQ ID NO:217 and 223; H2 comprises a sequence selected from the group consisting of SEQ ID NO:217 and 223; and L2 comprises SEQ ID NO:194.

[0158] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε, and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:218; H2 comprises SEQ ID NO:218; and L2 comprises SEQ ID NO:194.

[0159] In a preferred embodiment, the multispecific binding compound has binding affinity for LRRC15 and CD3ε and comprises a first light chain polypeptide (L1), a first heavy chain polypeptide (H1), a second heavy chain polypeptide (H2), and a second light chain polypeptide (L2), wherein L1 comprises SEQ ID NO:194; H1 comprises SEQ ID NO:238; H2 comprises SEQ ID NO:238; and L2 comprises SEQ ID NO:194.

[0160] Affinity determinations for candidate proteins can be performed using methods known in the art, such as the Biacore measurement. Multispecific binding compounds as described herein exhibit affinity for LRRC15 or CD3ε with a Kd of approximately 10. -6 Up to approximately 10 -11 Including but not limited to: approximately 10 -6 Up to approximately 10 -10 Approximately 10 -6 Up to approximately 10 -9 Approximately 10 -6 Up to approximately 10 -8 Approximately 10 -8 Up to approximately 10 -11 Approximately 10 -8 Up to approximately 10 -10 Approximately 10 -8 Up to approximately 10 -9 Approximately 10 -9 Up to approximately 10 -11 Approximately 10 -9 Up to approximately 10 -10Or any value within these ranges. Affinity selection can be confirmed through biological assessments (including in vitro assays, preclinical models, and clinical trials) used to modulate the biological activity of LRRC15 or CD3ε, as well as assessments of potential toxicity.

[0161] Various forms of multispecific binding compounds are within the scope of this invention, including but not limited to two-chain, three-chain, and four-chain peptides as described herein. Specifically, multispecific binding compounds include bispecific binding compounds with binding affinity for both LRRC15 and CD3ε (e.g., anti-LRRC15 x anti-CD3ε binding compounds). Such bispecific binding compounds induce effective T cell-mediated killing of cells expressing LRRC15 and / or tumor cell-associated matrix expressing LRRC15. Sequence information is provided in Tables 1-14.

[0162] Table 1: Heavy Chain CDR Sequences

[0163]

[0164]

[0165]

[0166]

[0167] Table 2: Light Chain CDR Sequences

[0168]

[0169]

[0170]

[0171] Table 3: Variable region sequence of the anti-LRRC15 heavy chain

[0172]

[0173] Table 4: Variable region sequence of the anti-LRRC15 light chain

[0174]

[0175] Table 5: Anti-CD3 VH sequences

[0176]

[0177]

[0178]

[0179]

[0180] Table 6: Anti-CD3 VL sequences

[0181]

[0182]

[0183]

[0184]

[0185] Table 7: Anti-CD3 scFv sequence (VH-connector-VL)

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] Table 8: LRRC15 sequences

[0194]

[0195]

[0196] Table 9: Mixed Sequences

[0197]

[0198] Table 10: Full-length light chain sequence

[0199]

[0200]

[0201] Table 11: Full-length heavy chain sequences, type 2 form

[0202]

[0203]

[0204]

[0205] Table 12: Full-length heavy chain sequences, type 4

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] Table 13: Full-length heavy chain sequences, 5 types

[0216]

[0217]

[0218]

[0219]

[0220]

[0221] Table 14: Full-length heavy chain sequences, type 1 form

[0222]

[0223]

[0224]

[0225] Preparation of compound

[0226] The multispecific binding compounds of the present invention can be prepared by methods known in the art. For example, the binding compounds and their antigen-binding fragments can also be generated by recombinant DNA technology, by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), Escherichia coli, or yeast.

[0227] Pharmaceutical compositions, uses and treatment methods

[0228] Another aspect of the invention provides pharmaceutical compositions comprising a mixture of one or more multispecific binding compounds of the invention with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are, for example, but not limited to, adjuvants, solid carriers, water, buffer solutions, or other carriers or combinations thereof used in the art to support therapeutic components.

[0229] In one embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds to LRRC15. In another embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds specifically to two or more non-overlapping epitopes on the LRRC15 protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific binding compound that binds specifically to LRRC15 and also binds specifically to binding targets on effector cells (e.g., binding targets on T cells, such as CD3 protein on T cells).

[0230] Pharmaceutical compositions of the conjugated compounds used according to the invention are prepared for storage (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)) by mixing proteins of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers, such as in lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (small) Polypeptides (approximately 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium ions; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0231] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practices (GMP) conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., doses for a single administration). The formulation depends on the chosen route of administration. The conjugated compounds described herein may be administered intravenously or by infusion or subcutaneously. For injection administration, the conjugated compounds described herein may be formulated in an aqueous solution (preferably in a physiologically compatible buffer) to reduce discomfort at the injection site. The solution may contain the carriers, excipients, or stabilizers discussed above. Alternatively, the conjugated compounds may be in lyophilized form for reconstitution with a suitable medium (e.g., sterile, pyrogen-free water) prior to use.

[0232] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used with the binding compounds of the present invention. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.

[0233] How to use

[0234] The multispecific binding compounds and pharmaceutical compositions described herein may be used to treat diseases and symptoms characterized by LRRC15 expression, including but not limited to the symptoms and diseases described above.

[0235] LRRC15 has been identified as highly expressed in a variety of solid tumor indications (including in some tumor-associated stromas) and expressed in limited amounts in normal tissues. Purcell et al., Cancer Res; 78(14); 4059–72. Due to the limited expression of LRRC15 in normal tissues, it is an attractive target for the treatment of malignancies characterized by LRRC15 expression.

[0236] In one aspect, the multispecific binding compounds and pharmaceutical compositions described herein can be used to treat cancers characterized by LRRC15 expression. As used herein, cancers "characterized by LRRC15 expression" include, but are not limited to, cancers in which one or more tumor cells express LRRC15 and / or in which the tumor-associated stroma exhibits LRRC15 expression. Such cancers include, but are not limited to, hematologic malignancies characterized by LRRC15 expression, including, but not limited to, large B-cell lymphomas. In another aspect, the multispecific binding compounds and pharmaceutical compositions described herein can be used to treat solid tumors characterized by LRRC15 expression, including, but not limited to, breast cancer, lung cancer, pancreatic cancer, and ovarian cancer. In yet another aspect, the multispecific binding compounds and pharmaceutical compositions described herein can be used to treat sarcomas characterized by LRRC15 expression.

[0237] The effective dosage of the compositions of the present invention for treating diseases varies depending on a variety of factors, including the route of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human, but non-human mammals such as companion animals (e.g., dogs, cats, horses), laboratory mammals (e.g., rabbits, mice, rats), etc., can also be treated. The therapeutic dose can be measured using titration to optimize safety and efficacy.

[0238] Dosage levels can be readily determined by a competent clinician and can be modified as needed, such as to modify the subject's response to the therapy. The amount of active ingredient that can be combined with a carrier to produce a single dosage form varies depending on the host being treated and the specific administration method. Dosage units typically contain between about 1 mg and about 500 mg of the active ingredient.

[0239] In some embodiments, the therapeutic dose of the agent can be in the range of about 0.0001 to 100 mg / kg, and more typically 0.01 to 5 mg / kg of host body weight. For example, the dose can be 1 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once every two weeks, once a month, or once every 3 to 6 months. The therapeutic entities of the present invention are typically administered in a variety of situations. The time interval between individual doses can be weekly, monthly, or yearly. The time interval can also be irregular, as indicated by measuring the blood level of the therapeutic entity in the patient. Alternatively, the therapeutic entities of the present invention can be administered as a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the peptide in the patient.

[0240] Typically, compositions are prepared in injectable form (liquid solutions or suspensions); solid forms are also prepared for dissolution or suspension in a liquid medium prior to injection. The pharmaceutical compositions described herein can be administered directly or after reconstitution of a solid (e.g., lyophilized) composition for intravenous or subcutaneous administration. The formulations can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolic acid, or copolymers to enhance the effect of the adjuvant, as discussed above. (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The agents of the present invention can be administered in the form of reservoir injections or implantable formulations, which can be formulated in such a manner as to allow for sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration.

[0241] The toxicity of the antibodies and antibody structures described herein can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50 (the dose that is lethal to 50% of the population) or LD100 (the dose that is lethal to 100% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dose range that is non-toxic to humans. The dosage of the antibodies described herein is preferably within a circulating concentration range that includes effective doses with little or no toxicity. The dosage may vary within this range depending on the dosage form and route of administration. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition.

[0242] Compositions intended for administration typically contain antibodies or other agents (e.g., another ablative agent) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as buffered physiological saline. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized using conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients required to mimic physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the specific administration modality chosen and the patient's needs (e.g., Remington's Pharmaceutical Science (15th edition, 1980) and Goodman and Gillman, The Pharmacological Basis of Therapeutics (edited by Hardman et al., 1996)).

[0243] Kits containing the active agents and formulations thereof, as well as instructions for use, are also within the scope of this invention. The kits may also contain at least one additional reagent, such as a chemotherapeutic agent. Kits typically include a label indicating the intended use of the kit contents. As used herein, the term "label" includes any written or recorded material on, supplied with, or otherwise accompanying the kit.

[0244] The present invention has been fully described, and it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0245] Example

[0246] Example 1: Library construction and phage display

[0247] Humanized / optimized libraries were constructed based on the following principles: Mouse CDRs were left unchanged to maintain binding with human and cynomolgus monkey CD3ε. Homology between human and mouse frames was analyzed. The diversity of frame differences was based on the amino acid composition of mouse and human frames. Furthermore, assuming that amino acids retained throughout frame evolution could lead to improved stability, antibodies from 890 frame families were analyzed to preserve the amino acid diversity of covariates. The combinatorial diversity of VH was 1.26E7, and that of VL was 2.56E2.

[0248] This diversity is encoded in the Ultramer (IDT) containing designed amino acids at each position. In splicing PCR, overlapping conserved regions facilitate the construction of the V region. Full-length VL and VH segments were rescued using high-fidelity PCR via terminal primers. The ScFv library insert set was completed by PCR of the VH and VL sets with a (G4S)3 adapter (SEQ ID NO:229) in the form of VH-linker-VL-6his tag-myc tag-amber terminator_g3p. The complete library fragment set was digested with the SfiI restriction endonuclease and cloned using Electroligase (New England Biolabs) into the Bgl1-digested vector pADL23c (Antibody Design Labs). Finally, NEB5F'Iq electron-competent cells were transformed with the cloned phage particles, and the cultures were grown to the logarithmic growth phase. Phages expressing the scFv clone were packaged with helper M13K07 and cultured overnight. The purified phage library was heated to 65°C for 10 minutes to reduce poorly folded clones in the library. Phages expressing intact scFv clones were captured by preparative Nanolink streptavidin magnetic beads coated with biotinylated goat anti-Myc antibody and propagated in NEB F'Iq phage cultures.

[0249] The resulting libraries were stored and used for plate-based and bead-based phage display of cynomolgus monkey CD3ε1-27aa-Fc. Furthermore, cynomolgus monkey CD3ε1-27aa-Fc was panned and subsequently bound to human CD3ε1-27aa-Fc in multiple rounds to maintain cross-reactive clones. In some panning experiments, binding at room temperature was followed by incubation at 65°C and rigorous washing to enrich stable clones. Target-bound phages were eluted and neutralized with acidic glycine buffer, then plated, titrated, and sequenced. HB2151 *E. coli* was infected with the eluted, target-enriched phages to express soluble scFv protein, and evaluated using an enzyme-linked immunosorbent assay (ELISA).

[0250] Example 2: Vector Construction

[0251] The vector pcDNA3.4TOPO (Invitrogen) was ligated to a short multi-linker containing EcoRI, XhoI, and NotI. The resulting plasmid was digested with EcoRI and NotI restriction endonucleases and purified by gel electrophoresis. For heavy chain clones, the prepared vector was assembled with the appropriate DNA fragment encoding the VH region and the human IgG1 AAA fragment (CH1 to CH3 domains) using the Gibson method. Variable light chain regions were constructed using a similar method, assembling the Vκ region with a gblock fragment encoding constant κ (Ck) using gblock. The plasmid expressing ScFv-Fc used a gblock fragment (IDT) to encode scFv and a PCR fragment to encode the antibody hinge to the CH3 domain of IgG1. Symmetrical forms of scFv-Fc, type 2, and type 5 ( Figure 3 Figures A, C, and F) contain the IGHG1 Fc sequence with three mutations that disrupt Fcg receptor interaction and complement binding: L234A, L235A, and G237A, which produce the sequence (from the hinge region) EPKSCDKTHTCPPCPAPEAAGA (SEQ ID NO: 230). In a preferred embodiment, the C220S mutation is included in the upper hinge region in the form of scFv attached to the hinge Fc (SEQ ID NO: 241 and 242) (see, for example, US 2010 / 0233173A1; WO2018 / 071919A1; US2016 / 0009824A1; WO2014 / 144357; and EP3511342A1, the disclosures of which are incorporated herein by reference in their entirety). Asymmetric forms (such as type 1 and type 4) Figure 3Figures B and E) share the same Fc sequence with the addition of a "groove" or "mortar" mutation (see, for example, Ridgway et al., Protein Eng. July 1996; 9(7):17-21; U.S. Patent No. 8,216,805). In a preferred embodiment, the mutations S354C (groove Fc) and Y349C (mortar Fc) (Merchant et al., Nature Biotech. July 1998; 16:677-681) create disulfide bonds between the CH3 domains to aid in the generation of bispecific antibodies. In another preferred embodiment, the protein A non-binding mutations H435R and Y436F (Jendeberg et al., J. Immuno. Methods 1997; 201:25-34) are added to the grove Fc to promote the purification of the asymmetric form. By removing the terminal lysine from the Fc domain and cloning the scFv and linkers containing G and S amino acid residues (Table 9 shows the various linkers evaluated), a type 2 form of anti-CD3εscFv was constructed at the C-terminus of the Fc domain. Type 4 and type 5 extended heavy chain fragments were cloned with the following structures: [anti-LRRC15 VH]-[CH1]-[linker 1]-[anti-CD3 scFv]-[hinge-CH2-CH3], where linker 1 contains the sequences EPKSCDKTHT (SEQ ID NO: 189) or EPKSSDKTHT (SEQ ID NO: 241), EPKSCDGGSGGSGGSG (SEQ ID NO: 190), or EPKSCDGGGGSGGGGS (SEQ ID NO: 191). All assemblies were performed using the Gibson method (NEB).

[0252] Example 3: Protein Expression

[0253] Plasmids were prepared and transfected into Expi293 or ExpiCHO cells using a transient expression system (Thermo Fisher). Briefly, plasmids were transfected into 3e6 cells / mL of culture at a total plasmid DNA concentration of 1 μg. Heavy and light chain plasmids were mixed at a 1:1 ratio. Bisspecific binding compounds were used to transfect the culture with an increased light chain plasmid compared to the two isolated heavy chain plasmids. The cultures were incubated at 37°C with shaking. After 16 hours, transfection enhancers 1 and 2 were added to the cultures, and incubation continued for 6 days. The supernatant was filtered, and protein titers were determined using the Octet Red96 (Pall) assay according to the IgG quantification protocol. IgG was purified using the Mab Select Sure Protein A column purification method on the ACTA PURE system and dialyzed overnight in PBS. Asymmetric bispecific antibodies (types 1 and 4, ...) Figure 3(Figures B and E) typically require additional purification, which usually involves preparative size exclusion chromatography (pSEC).

[0254] Example 4: Humanized anti-LRRC15 x Protein thermal displacement of CD3 dual-specific binding compounds

[0255] Ten (10) μg / mL of the anti-LRRC15xCD3 bispecific binding compound was mixed with 2 μL of 50X protein thermal shift dye and PBS, resulting in a final volume of 100 μL. The sample was quadrupled into PCR 96-tube plates (25 μL / well). Protein thermal shift reaction was measured using an Applied Biosystems StepOne real-time PCR instrument with a continuous temperature gradient of 1°C every 5 seconds from 22-95°C. Tm was analyzed using the derivative method. The results showed that when the three clones (i.e., 160C9, 4G2, and 1B4) were presented in different forms with different adapters, the scFv Tm ranged from 60-66°C. Figure 4 The Tm of anti-CD3 clone 160C9 scFv-Fc is approximately 64 °C (see [link]). Figure 1 and 4 Furthermore, when scFv is presented at the C-terminus in the T2a form (the type 2 form with a GS connector), it decreases to approximately 62°C. A decrease in Tm was also observed in two other clones (see [link to clone]). Figure 4 ), usually, with Figure 3 Compared to the scFv-Fc form depicted in Figure A, the types 4 and 5 show similar or slightly improved Tm.

[0256] Example 5: Flow cytometry analysis of the binding of anti-CD3ε antibody to T cells, Jurkat cells, and H-SCF cells

[0257] Peripheral blood mononuclear cells (PBMCs), Jurkat cells, or H-SCF (cynomolgus monkey T cells) were prepared using standard methods, washed with FACS buffer, and distributed at 200,000 cells / well in 96-well V-bottom polypropylene plates. scFv-Fc protein or a positive control antibody (BD Biosciences 556610) was serially diluted from 40 μg / mL and used for staining cells on ice for 20 min. Cells were washed in FACS buffer, stained on ice for 25 min with 1:500 secondary antibody (goat anti-human IgG Fc-AF647, with goat anti-mouse IgG Fc-AF647 used as a control), washed again, and resuspended in buffer containing 7-AAD before analysis by flow cytometry. In addition to the bispecific antibody detected by goat anti-human IgG Fc-AF647 as described above, PBMCs were also stained against CD3-FITC, CD4-APC-H7, and CD8-PE expression. The results showed that several scFv-Fc clones were compatible with human Jukat cells ( Figure 5 A) Cynomolgus monkey T cell line H-SCF ( Figure 5 B) and CD3+ cells in prepared human PBMCs ( Figure 6 A) Stable bonding. In type 1 ( Figure 6 B) and Type 2 ( Figure 6 In C), binding to CD3+ PBMCs was reduced. Similarly, compared to scFv-Fc, type 5 bispecific binding to CD4+ T cells was reduced, and type 4 binding was further reduced. Figure 6 D). Figure 6 E and Figure 6 F showed similar binding patterns with CD8+ and pan-T cells, with the binding signal weakened in type 5 and further weakened in type 4.

[0258] Example 6: Flow cytometry combined with analysis of U118MG cells

[0259] U118MG or U87MG cells were harvested via trypsin, washed with FACS buffer, resuspended at 5E6 cells / mL, and aliquoted into 96-well plates at 1E5 cells / well. Cells were stained on ice for 45 min with serially diluted anti-LRRC15 binding compound, bispecific binding compound, positive control antibody "C1-IgG1", or isotype IgG1 (starting concentration 50 nM), followed by washing and secondary staining with 1:500 diluted goat anti-human IgG-AF647. Cells were analyzed by flow cytometry after final washing and addition of 7-AAD. Results showed… Figure 7 In, and demonstrated with LRRC15 positive U118MG ( Figure 7 A) and U87MG( Figure 7 B) Stable cell binding.

[0260] Example 7: Assay for Tumor-Dependent T Cell Activation

[0261] RPMI7951, U118MG, U87MG, or A431 (LRRC15 negative) tumor cells were prepared at 2E5 cells / mL in medium containing RPMI 10% human serum and distributed at 1E4 cells / well in flat-bottomed 96-well plates and incubated overnight. The next day, fresh PBMCs were prepared at 4E6 cells / mL in RPMI + 10% human serum using standard techniques. These cells and diluted bispecific binding compounds or control binding compounds were then added to wells containing tumor cells at a 10:1 effector cell:target cell ratio, and the plates were incubated for 48 hours before flow cytometry analysis. Control wells lacked tumor cells to test the direct activation of T cells by the anti-CD3 bispecific binding compound. The supernatant was frozen for cytokine detection. Cells were analyzed by flow cytometry using the following first-reagent antibodies diluted in FACS buffer: CD3-FITC, CD4-APC-H7, CD8-PE, CD25-BV421, CD69-APC, 7-AAD, allotype-APC, allotype-BV421, and allotype-APC. After sample collection, data were analyzed using FlowJo by gating single-cell pairs (P1) FSC / SSC, (P2) FSC-AxFSC-H, followed by (P3) CD3x7AAD live / dead cell gating, and then CD4(P4)xCD8(P5) gating. Activation was assessed using CD25 and CD69 markers for CD3+ / CD8+ T cells and CD3+ / CD4+ T cells. Results showed that the LRRC15xCD3 type 1 clone activated CD8+ T cells in the presence of U118MG tumor cells, but did not activate them in the absence of tumor cells. Figure 8 A). Similarly, LRRC15xCD3 type 2 clone ( Figure 8 B), type 4 and type 5 clones ( Figure 8 C and Figure 8 E) Activate CD8+ T cells in a U118MG tumor cell-dependent manner.

[0262] Example 8: T cell proliferation assay

[0263] RPMI7951, U118MG, U87MG, or A431 (LRRC15 negative) tumor cells were prepared at 2E5 cells / mL in medium containing 10% human serum from RPMI, distributed at 1E4 cells / well in flat-bottomed 96-well plates, and incubated overnight. The next day, T cells were prepared from fresh PBMCs and labeled with 5 μM CellTrace Violet in PBS in the dark for 15 minutes. After washing three times in medium, 5E4 labeled T cells were added to the wells (effective cell:target cell ratio of 5:1). A diluted bispecific binding compound or control compound was then added to the wells containing tumor cells, and the plates were incubated for 5 days prior to flow cytometry analysis. Cells were analyzed by flow cytometry using the following first-response antibodies: CD3-FITC, CD8-PE, 7AAD-PerCP, CellTrace Violet-PB, CD56-APC, CD4-APC-H7, and appropriate isotype control reagents. The results showed that in the presence of LRRC15-positive RPMI7951 cells, the LRRC15xCD3 bispecific antibody enhanced the proliferation of CD4+ and CD8+ T cells, but had no effect in the presence of A431 tumor cells that did not express LRRC15. Figure 9 A and 9B). Cloning 160C9_T4h showed improved potency compared to 160C9_T1 ( Figure 9 Similar results were obtained when U118MG tumor cells were used to prepare proliferation assays. See also Figure 9 C Figure 9 D and Figure 9 E.

[0264] Example 9: Cytokine Assay

[0265] In the previously described activation or proliferation assay, the plate was incubated for 2–5 days. The release of IFNγ and IL-2 in the supernatant was analyzed by ELISA according to the manufacturer's protocol (R&D Systems). Results were shown... Figure 10 In A-10C, dose-dependent production of IFNγ and IL-2 was demonstrated in the presence of tumor cells. PBMCs incubated with the highest concentration of the compound did not show IFNγ and IL-2 secretion.

[0266] Example 10: Cytotoxicity Assay

[0267] RPMI7951, U118MG, U87MG, or A431 (LRRC15-negative) tumor cells were prepared at 2E5 cells / mL in medium containing 10% human serum from RPMI, distributed at 1E4 cells / well in flat-bottomed 96-well white plates, and incubated overnight. The next day, CD8+ T cells were purified from PBMCs using the Miltenyi CD8 Isolation Kit. Then, serial dilutions of the bispecific binding compound, the control binding compound, and 5E4 freshly prepared T cells, starting at 1 nM (final concentration), were added to the wells. Cell concentrations represent a 1:5 target cell:effect cell ratio. Plates of RPMI7951 cells were incubated for 2 days, and plates of U118MG and A431 cells were incubated for 3 days. Plates were treated with the CytoTox-Glo kit according to the manufacturer's instructions, and luminescence was analyzed. The results showed that the LRRC15xCD3 bispecific antibody enhanced T cell killing against LRRC15-positive tumor cells. Figure 11 ). Figure 11 A-11C illustrates examples of T-cell-directed cytotoxicity of compounds type 1, 2, 4, and 5 on RPMI7951 cells. Similarly, Figure 11 D-11F demonstrates examples of T-cell-directed cytotoxicity of compounds type 2, 4, and 5 on U118MG cells. Finally, Figure 11 G illustrates an example of T cell-guided cytotoxicity in U87MG cells.

[0268] Example 11: In vivo efficacy study

[0269] 1E6 U118MG tumor cells were subcutaneously implanted into each immunodeficient NSG mouse (6-9 week old females from The Jackson Laboratory, #005557), and the mice were randomly assigned to different groups when the tumors grew to approximately 60 mm³. Fresh PBMCs from a single donor were intraperitoneally implanted into each mouse with 1E7 cells. Starting 3 days after PBMC implantation, animals (n=8 per group) received four doses of either a 1 mg / kg bispecific binding compound, OKT3 antibody, or PBS every two weeks. Tumors were measured every two weeks. Results were displayed... Figure 12 The study compared tumor volume with that of the four compounds relative to a PBS control. Dosing of all LRRC15xCD3 bispecific antibodies appeared to control and / or reduce tumor size.

[0270] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various modifications, variations, and substitutions will now be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. This means that the following claims define the scope of the invention and thus cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A multispecific binding compound comprising a first light chain polypeptide LI, a first heavy chain polypeptide HI, a second heavy chain polypeptide H2, and a second light chain polypeptide L2, wherein: L1 comprises variable region sequence L1V L and constant region sequence L1C L wherein said L1V L contains a light chain complementarity determining region 1 (CDRL1) of SEQ ID NO: 15, a light chain complementarity determining region 2 (CDRL2) of SEQ ID NO: 19, and a light chain complementarity determining region 3 (CDRL3) of SEQ ID NO: 22; H1 comprises variable region sequence H1V H , C H 1 constant region sequence H1C H 1, C H 2 constant region sequence H1C H 2 and C H 3 constant region sequence H1C H 3, wherein the H1V H comprises a heavy chain complementarity determining region 1 (CDRH1) set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDRH2) set forth in SEQ ID NO: 3, and a heavy chain complementarity determining region 3 (CDRH3) set forth in SEQ ID NO: 13; H2 comprises variable region sequence H2V H , C H 1 constant region sequence H2C H 1, C H 2 constant region sequence H2C H 2, C H 3 constant region sequence H2C H 3, and a single chain Fv (H2scFv) comprising a first variable region sequence H2scFv H , a second variable region sequence H2scFv L , and a linker sequence linking said H2scFv H sequence to said H2scFv L sequence; wherein said H2V H comprises CDRH1 as set forth in SEQ ID NO: 1, CDRH2 as set forth in SEQ ID NO: 3, and CDRH3 as set forth in SEQ ID NO: 13, said H2scFv H comprises CDRH1 as set forth in SEQ ID NO: 2, CDRH2 as set forth in SEQ ID NO: 4, and CDRH3 as set forth in SEQ ID NO: 14, said H2scFv L comprises CDRL1 as set forth in SEQ ID NO: 16, CDRL2 as set forth in SEQ ID NO: 20, and CDRL3 as set forth in SEQ ID NO: 23; and L2 comprises a variable region sequence L2V L and a constant region sequence L2C L wherein the L2V L contains a CDRL1 of SEQ ID NO: 15, a CDRL2 of SEQ ID NO: 19, and a CDRL3 of SEQ ID NO: 22; wherein: said L1V L sequence and said H1V H together form a binding unit having binding affinity for LRRC15; said L2V L sequence and said H2V H together form a binding unit having binding affinity for LRRC15; the H2 scFv has a binding affinity for CD3 epsilon; and said H1C H 3 sequence and said H2C H 3 sequence comprises an asymmetric interface that promotes correct pairing between said H1 polypeptide chain and said H2 polypeptide chain.

2. The multispecific binding compound of claim 1, wherein: the L1V L having the amino acid sequence of SEQ ID NO: 27; the H1V H having the amino acid sequence of SEQ ID NO: 25; The H2V H having the amino acid sequence of SEQ ID NO: 25; the H2scFv H having the amino acid sequence of SEQ ID NO: 28; the H2scFv L having the amino acid sequence of SEQ ID NO: 81; and / or The L2V L has the amino acid sequence of SEQ ID NO:

27.

3. The multi-specific binding compound of claim 1, wherein the L1C L sequences and the H1C H 1 sequences are linked by disulfide bonds.

4. The multi-specific binding compound of claim 1, wherein the L2C L sequences and the H2C H 1 sequences are linked by disulfide bonds.

5. The multispecific binding compound of claim 1, wherein the HI polypeptide chain and the H2 polypeptide chain comprise a hinge region.

6. The multispecific binding compound of claim 1, wherein the HI polypeptide chain and the H2 polypeptide chain are linked by at least one disulfide bond.

7. The multispecific binding compound of claim 1, wherein the H2 scFv comprises CDRH1 set forth in SEQ ID NO: 2, CDRH2 set forth in SEQ ID NO: 4, and CDRH3 set forth in SEQ ID NO: 14, and CDRL1 set forth in SEQ ID NO: 16, CDRL2 set forth in SEQ ID NO: 20, and CDRL3 set forth in SEQ ID NO: 23, and has at least 95% sequence identity to SEQ ID NO: 133 throughout its sequence.

8. The multispecific binding compound of claim 7, wherein the H2 scFv comprises the sequence of SEQ ID NO:

133.

9. The multi-specific binding compound of claim 1, wherein H1 comprises the following sequence order from N-terminus to C-terminus: H1V H , H1C H 1, H1C H 2, H1C H 3.

10. The multi-specific binding compound of claim 1, wherein H2 comprises the following sequence order from N-terminus to C-terminus: H2V H , H2C H 1, H2scFv, H2C H 2, H2C H 3.

11. A multispecific binding compound comprising a first light chain polypeptide LI, a first heavy chain polypeptide HI, a second heavy chain polypeptide H2, and a second light chain polypeptide L2, wherein: LI comprises SEQ ID NO: 194; HI comprises SEQ ID NO: 231; H2 comprises SEQ ID NO: 236; and L2 comprises SEQ ID NO:

194.

12. A pharmaceutical composition comprising the multispecific binding compound of any one of claims 1-11 for use in the treatment of a disorder characterized by LRRC15 expression.

13. Use of the multispecific binding compound of any one of claims 1-11 for the manufacture of a medicament for the treatment of a disorder characterized by LRRC15 expression, wherein the disorder is selected from the group consisting of: sarcoma, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, and large B-cell lymphoma.

14. A polynucleotide encoding the multispecific binding compound of any one of claims 1-11.

15. A vector comprising the polynucleotide of claim 14.

16. A cell comprising the vector of claim 15.

17. A method of producing the multispecific binding compound of any one of claims 1-11, comprising growing the cell of claim 16 under conditions that allow for expression of the multispecific binding compound, and isolating the multispecific binding compound.

Images