FGFR3 binding molecules and methods of use thereof

Abstract

The present disclosure relates to molecules capable of binding to FGFR3 and methods of their use.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 479,861, filed January 13, 2023, U.S. Provisional Application No. 63 / 587,699, filed October 3, 2023, and U.S. Provisional Application No. 63 / 590,140, ​​filed October 13, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically and is incorporated herein by reference in its entirety. The copy created on January 11, 2024 is named RGN-008WO_SL.xml and is 140,165 bytes in size. [Background technology]

[0003] 3. Background technology Fibroblast growth factor receptors (FGFRs) are highly conserved transmembrane tyrosine kinase receptors expressed throughout the body. In humans, there are four FGFRs (FGFR1-4), which are key players in embryonic development, tissue homeostasis, and cell metabolism and survival (Eswarakumar et al., 2005, Cytokine Growth Factor Rev. 16:139-149; Turner & Grose, 2010, Nat Rev Cancer. 10(2):116-129).

[0004] FGFRs consist of an extracellular ligand-binding region, two or three immunoglobulin-like domains (Ig-I, Ig-II, and Ig-III), a transmembrane region consisting of a single α-helix, and a cytoplasmic tyrosine kinase domain. Signaling through FGFRs is initiated by the binding of fibroblast growth factors (FGFs) and heparin, leading to FGFR dimerization. This receptor dimerization activates the kinase domains by bringing them into close proximity, allowing for the phosphorylation of cytoplasmic substrates (Sarabipour & Hristova, 2016, Nat Commun. 7, 10262).

[0005] Constitutively active FGFRs are involved in various disease states, including cancer (Acevedo et al., 2009, Cell Cycle 8(4):580-588). For example, overexpression of constitutively active FGFR3 is sufficient to induce oncogenic transformation in hematopoietic cells and fibroblasts. Furthermore, gain-of-function mutations in FGFR3 are associated with 60-70% of papillary carcinomas and 16-20% of muscle-invasive bladder cancers (Qing et al., 2009, J Clin Invest. 119(5):1216-1229), and thus aberrant FGFR3 signaling is also involved in bladder cancer.

[0006] In the United States, bladder cancer is the fourth most common cancer among men. Current treatment options for bladder cancer patients are limited. Generally, these treatments are associated with severe side effects and / or low-to-moderate response rates. For example, cisplatin-based chemotherapy is considered the current standard of care for locally advanced or metastatic disease, but half of patients cannot receive this treatment due to other conditions, such as renal dysfunction or heart failure (Wong & Rosenberg, 2021, Expert Opin Biol Ther. 21(7):863-873). Meanwhile, patients receiving cisplatin have a median overall survival of approximately 14 months and suffer from various side effects due to off-target effects (Wong & Rosenberg, 2021, Expert Opin Biol Ther. 21(7):863-873).

[0007] Given the implications of aberrant FGFR3 signaling in bladder cancer, targeting this signaling cascade has become an attractive therapeutic option. However, early-phase clinical trials evaluating targeted therapies targeting FGF / FGFR signaling have produced mixed results, highlighting the complexity of FGFR signaling in cancer (Kommalapati et al., 2021, Cancers. 13:2968). For example, small molecule drugs that inhibit tyrosine kinase activity can inhibit aberrant FGFR3 signaling but are also associated with various side effects, including hyperphosphatemia or ocular toxicity due to inhibition of FGFR1 or FGFR2, respectively (Kommalapati et al., 2021, Cancers. 13:2968).

[0008] Thus, there is a clear need for specific molecules that bind to and inhibit FGFR3, including wild-type and constitutively active FGFR3 mutant variants, with high inhibitory activity and an acceptable side effect profile. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Cytokine Growth Factor Reviews, 2005, 16: 139-149 [Non-patent document 2] Nature Reviews Cancer, 2010, 10(2): 116-129 [Non-patent document 3] Nature Communications, 2016, 7: 10262 [Non-patent document 4] Cell Cycle, 2009, 8(4): 580-588 [Non-Patent Document 5] Journal of Clinical Investigation, 2009, 119(5): 1216-1229 [Non-patent document 6] Expert Opinion on Biological Therapy, 2021, 21(7): 863-873 [Non-Patent Document 7] Cancers, 2021, 13: 2968 Summary of the Invention

[0010] 4. Summary of the Invention The present disclosure provides multispecific binding molecules ("MBMs") that contain at least two antigen binding domains ("ABDs"), a first of which binds to a first epitope of FGFR3 and a second of which binds to a second, different epitope of FGFR3. Without being bound by theory, it is believed that the inclusion of two antigen binding domains, each with specificity for a different epitope of FGFR3, results in an MBM with greater inhibition of FGFR3 dimerization and / or signaling.

[0011] In certain aspects, the first epitope and the second epitope are in different regions of FGFR3. In certain embodiments, the first epitope comprises a sequence in the D2 domain and / or D3 domain of FGFR3 (e.g., a linear or conformational epitope spanning the D2 domain, the D3 domain, or the D2 domain and the D3 domain), and the second epitope comprises a sequence in the D1 domain of FGFR3 (e.g., a linear or conformational epitope).

[0012] Without being bound by theory, it is believed that the MBMs of the present disclosure, in some embodiments, can have a lower KD for binding to FGFR3, stronger EC50 values ​​in cell-based binding assays, and / or stronger EC50 values ​​in cell (e.g., cancer cell) toxicity or cell viability assays than the corresponding parent monospecific antibody (e.g., a monospecific antibody comprising the first ABD or the second ABD, but not both).

[0013] Exemplary FGFR3 binding molecules are disclosed in Section 6.2 and numbered embodiments 1 to 128. Exemplary FGFR3 ABDs are disclosed in Section 6.2.1.

[0014] The present disclosure further provides nucleic acids encoding the MBMs of the present disclosure (either in a single nucleic acid or multiple nucleic acids), as well as recombinant host cells and cell lines engineered to express the nucleic acids and MBMs of the present disclosure. Exemplary nucleic acids, host cells, and cell lines, and methods of their use to produce the MBMs of the present disclosure, are described in Section 6.5 and numbered embodiments 129-133. The present disclosure further provides pharmaceutical compositions comprising the MBMs. Exemplary pharmaceutical compositions are described in Section 6.6 below and numbered embodiment 134.

[0015] Further provided herein are methods of using the ABM proproteins and pharmaceutical compositions of the present disclosure to treat proliferative conditions (e.g., cancer) in which target molecules such as FGFR3 are expressed. Exemplary methods and indications are described in Sections 6.7 and 6.8, and in numbered embodiments 135-170. [Brief explanation of the drawings]

[0016] 5. Brief description of the drawings [Figure 1A] 1 shows the domain structure and oncogenic mutations of the FGFR3 protein. Figure 1A is a diagram of the human FGFR3 protein with its domains presented in the N- to C-terminal direction. [Figure 1B] The domain structure and oncogenic mutations of the FGFR3 protein are shown. Figure 1B is a cartoon representation of the FGFR3 signaling pathway. Under normal circumstances, FGFR3 is a monomer in the absence of ligands such as FGF and dimerizes upon ligand binding. This dimerization is essential for the intracellular tyrosine kinase domain of FGFR3 to activate downstream signaling cascades, such as the MAPK pathway. [Figure 1C] Figure 1C shows the domain structure and oncogenic mutations of the FGFR3 protein. Figure 1C shows examples of oncogenic FGFR3 dimerization mutations (S249C) and fusion mutations (FGFR3-TACC3) associated with constitutively active FGFR signaling. [Figure 1D] The domain structure and oncogenic mutations of the FGFR3 protein are shown in Figure 1D. Figure 1D is a ribbon structure from PDB #1RY7 showing the Ig-II and Ig-III domains of human FGFR3c, with the ligand-binding site between Ig-II and Ig-III (black) shown in complex with the ligand FGF1. [Figure 2A] Figure 2A shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. Figure 2A shows the VH and VL components used to construct different Fab and / or scFv portions, whereby the VL components of the Fabs or scFvs can be derived from the same or different mAbs. [Figure 2B] 2B shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. Figure 2B shows an antibody in Davis body format with its Fab domain comprising two different heavy chains. [Figure 2C] Figure 2C shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. Figure 2C shows a 2+1 N-Fab AF antibody with asymmetric Fab arms, i.e., two consecutive Fab portions on one side and one Fab portion on the other side, linked to the N-terminus of the Fc portion. [Figure 2D]Figure 2D shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. Figure 2D shows a 2+1 N-Fab AF antibody with asymmetric Fab arms, i.e., two consecutive Fab portions on one side and one Fab portion on the other side, linked to the N-terminus of the Fc portion. [Figure 2E] Figure 2E shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. Figure 2E shows a 2+1 N-Fab AF antibody with asymmetric Fab arms, i.e., two consecutive Fab portions on one side and one Fab portion on the other side, linked to the N-terminus of the Fc portion. [Figure 2F] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2F shows a 2+1 N-scFv AF antibody with a Fab portion contiguous with an scFv portion on one side and a Fab portion on the other side linked to the N-terminus of the Fc portion. [Figure 2G] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2G shows a 2+1 N-scFv AF antibody with a Fab portion contiguous with an scFv portion on one side and a Fab portion on the other side linked to the N-terminus of the Fc portion. [Figure 2H] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2H shows a 2+2 N-Fab AF antibody with two Fab arms, each with two consecutive Fab portions, linked to the N-terminus of the Fc portion. [Figure 2I] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2I shows a 2+2 N-Fab AF antibody with two Fab arms, each with two consecutive Fab portions, linked to the N-terminus of the Fc portion. [Figure 2J]Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2J shows a 2+2 N-Fab AF antibody with two Fab arms, each with two consecutive Fab portions, linked to the N-terminus of the Fc portion. [Figure 2K] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2K shows a 2+2 N-Fab AF antibody with two Fab arms, each with two consecutive Fab portions, linked to the N-terminus of the Fc portion. [Figure 2L] Figure 2L shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fab or scFv. Figure 2L shows an antibody in a 2+2 N-scFv format with consecutive Fab portions, each with an scFv portion connected to the N-terminus of the Fc portion. [Figure 2M] Figure 2M shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs. The Fab portion is linked to the N-terminus, and the scFv portion is linked to the C-terminus of both Fc portions in a 2+2 C-scFv format. [Figure 2N] Figure 2N shows an exemplary AF antibody structure with a combination of distinct anti-FGFR3 Fabs or scFvs: a 2+2 C-Fab antibody with a Fab portion linked to the N-terminus and another Fab portion linked to the C-terminus of both Fc portions. [Figure 2O] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2O shows a 2+2 C-Fab antibody with a Fab portion linked to the N-terminus and another Fab portion linked to the C-terminus of each Fc portion. [Figure 2P] Exemplary AF antibody structures with distinct anti-FGFR3 Fab or scFv combinations: Figure 2P shows a 2+2 C-Fab antibody with a Fab portion linked to the N-terminus and another Fab portion linked to the C-terminus of each Fc portion. [Figure 3A]Figure 3 shows the results of AF antibody screening in a bladder cancer cell spheroid proliferation assay. Figure 3A shows a cartoon representation of FGFR3 activating mutations expressed in bladder cancer cell lines. The UMUC14 cell line endogenously expresses the S249C mutation, which leads to receptor dimerization via disulfide bond formation. The RT4 cell line endogenously expresses an in-frame C-terminal fusion of the coiled-coil domain of the TACC3 protein (FGFR3-TACC3). [Figure 3B] Figure 3B shows the results of AF antibody screening in a bladder cancer cell spheroid proliferation assay. Figure 3B shows the anti-proliferative effect of AF antibodies in a UMUC14 spheroid proliferation assay. [Figure 3C] Figure 3C shows the results of AF antibody screening in a bladder cancer cell spheroid proliferation assay. Figure 3C shows the anti-proliferative effect of AF antibodies in an RT4 spheroid proliferation assay. [Figure 4A] 4A and 4B are graphs illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4A shows the dose-dependent effect of a subset of AF antibodies in inhibiting UMUC14 cell growth. [Figure 4B] Figure 4B is a graph illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4B shows the dose-dependent effect of a subset of AF antibodies in inhibiting RT4 cell growth. [Figure 4C] Figure 4C is a graph illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4C shows the anti-proliferative activity of BiP063N2 compared to its parent antibodies, mAb063 and mAb117, in UMUC14 cells. [Figure 4D] Figure 4D is a graph illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4D shows the anti-proliferative activity of BiP063N2 compared to its parent antibodies, mAb063 and mAb117, in RT4 cells. [Figure 4E]Figure 4E is a graph illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4E shows the inhibitory activity of parental antibodies mAb063 and mAb117, assessed alone or in combination via a proliferation assay in UMUC14 cells. [Figure 4F] Figure 4F is a graph illustrating the dose-dependent inhibitory effect of AF antibodies on bladder cancer cell spheroid growth. Figure 4F shows the inhibitory activity of parental antibodies mAb063 and mAb117, assessed alone or in combination via a proliferation assay in RT4 cells. [Figure 5A] Figure 5 illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5A shows tumor growth inhibition in a UMUC14 xenograft model. SCID mice bearing established tumors (approximately 200 mm) were treated biweekly with IP injections of the antibody at the indicated doses. [Figure 5B] Figure 5B illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5B shows the dose-dependent effect of antibody treatment on tumor growth inhibition. SCID mice bearing established UMUC14 tumors (approximately 200 mm 3 ) were treated biweekly with IP injections of the antibody at the indicated doses. Antibody dosing was discontinued on day 32, and tumor growth inhibition was maintained until day 46. [Figure 5C] Figure 5C illustrates the in vivo characterization of 2+2 AF antibodies in tumor growth inhibition assays. Figure 5C shows tumor growth inhibition in SCID mice bearing RT112 tumors. Mice bearing established tumors (approximately 200 mm 3 ) were treated with biweekly IP injections of 15 mg / kg of isotype control, mAb063, mAb108, REGN6331, or equimolar concentrations of BiP063N2 at 20 mg / kg. [Figure 5D] Figure 5D illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5D shows tumor growth inhibition in a UMUC14 xenograft model in which mice bearing established tumors were treated biweekly with 2 mg / kg of parental antibodies alone or in combination, or with 2.66 mg / kg of BiP063N2. [Figure 5E]Figure 5E illustrates the in vivo characterization of the 2+2 AF antibody in a tumor growth inhibition assay. Figure 5E is a Western blot image showing the effect of BiP063N2 on MAPK phosphorylation after 72 hours of treatment. [Figure 5F] Figure 5F illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5F shows phospho-MAPK (pMAPK) levels relative to MAPK levels in tumor lysates 48 hours after treatment with parental antibodies mAb063, BiP063N2, or AZD4547. [Figure 5G] Figure 5G illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5G shows pMAPK levels relative to MAPK levels in tumor lysates 72 hours after treatment with parental antibodies mAb063, BiP063N2, or AZD4547. [Figure 5H] Figure 5H illustrates the in vivo characterization of 2+2 AF antibodies in a tumor growth inhibition assay. Figure 5H is a graph showing tumor growth inhibition in the LU-0813 PDX model expressing the FGFR3 S249C mutation. Mice bearing established tumors were treated biweekly with isotype control antibody or parental antibody mAb063 at 10 mg / kg, or with BiP063N2 at 13.3 mg / kg. [Figure 6A] Figure 6 illustrates the effects of FGFR3 AF antibodies on receptor dimerization, degradation, and downstream signaling. Figure 6A illustrates non-reducing and reducing SDS-PAGE analysis of FGFR3 S249C dimerization in UMUC14 cells treated with 33 nM of the indicated antibodies for 3 hours. Monomers and dimers were separated via non-reducing SDS-PAGE (top). The mobilities of FGFR3 dimers and monomers are shown. Total FGFR3 protein (middle) and actin (bottom) levels were separated by reducing SDS-PAGE and analyzed by Western blot. [Figure 6B]Figure 6B illustrates the effect of FGFR3 AF antibody on receptor dimerization, degradation, and downstream signal transduction. Figure 6B is a Western blot image showing the effect of AF antibody treatment on FGFR3 receptor degradation. UMUC14 cells were treated with the indicated antibodies for 16 hours, and the levels of total FGFR3 protein (top) and actin (bottom) were analyzed. [Figure 6C] Figure 6 illustrates the effect of FGFR3 AF antibody on receptor dimerization, degradation, and downstream signaling. Figure 6C shows a Western blot image showing the effect of AF antibody treatment on inhibition of MAPK phosphorylation. UMUC14 cells were treated with the indicated antibodies for 24 hours, and the levels of phosphorylated MAPK (pMAPK, top) and total MAPK (bottom) were analyzed. [Figure 7] 1 is a table demonstrating the affinity of BiP063N2 and its parent antibody for the FGFR3 isoforms, FGFR3b and FGFR3c. [Figure 8A] Characterizing the affinity-driven binding mechanism between BiP063N2 and FGFR3b Figure 8A is a cartoon presenting two models of avidity-driven binding. [Figure 8B] Characterizing the affinity-driven binding mechanism between BiP063N2 and FGFR3b Figure 8B is a table summarizing the calculated molecular weights for the indicated combinations of BiP063N2 and FGFR3b-mmh peptides. [Figure 8C] Characterizing the affinity-driven binding mechanism between BiP063N2 and FGFR3b. Figure 8C shows an exemplary plot of asymmetric flow field separation coupled with MALS (A4F-MALS) of samples containing the indicated molar ratios of BiP063N2 and FGFR3b-mmh. [Figure 8D] Characterizing the affinity-driven binding mechanism between BiP063N2 and FGFR3b Figure 8D is a table summarizing the calculated molecular weights for each combination of 1+1 N-scFv-Fab and FGFR3b-mmh peptide. [Figure 8E]Characterizing the affinity-driven binding mechanism between BiP063N2 and FGFR3b, Figure 8E is an exemplary size-exclusion chromatography (SEC) MALS plot of a sample containing the indicated molar ratios of 1+1 N-scFv-Fab and FGFR3b-mmh. [Figure 9A] Figure 9 shows the pharmacokinetic (PK) profiles of BiP063N2 and its parent antibody. Figure 9A is a cartoon depiction of the antibody capture and detection procedure using the Gyros immunoassay. [Figure 9B] Figure 9B shows the pharmacokinetic (PK) profiles of BiP063N2 and its parental antibody. Figure 9B shows the PK profiles of BiP063N2 and its parental bivalent antibody as determined by capture with human FGFR3b. Arrows indicate the days of antibody dosing. [Figure 9C] 9A and 9B show the pharmacokinetic (PK) profiles of BiP063N2 and its parent antibody. Figure 9C shows the PK plot of BiP063N2 as determined by capture by human or mouse FGFR3b or human FGFR3c. The arrows indicate the days of antibody dosing. [Figure 9D] Figure 9D shows the pharmacokinetic (PK) profiles of BiP063N2 and its parent antibodies. Figure 9D is a table listing the binding affinities of BiP063N2 and its parent antibodies to different FGFR3 isoforms. [Figure 9E] Figure 9E shows the pharmacokinetic (PK) profiles of BiP063N2 and its parent antibody. Figure 9E is a table of PK parameters corresponding to Figure 9B. [Figure 9F] Figure 9F shows the pharmacokinetic (PK) profiles of BiP063N2 and its parent antibody. Figure 9F is a table of PK parameters corresponding to Figure 9C. [Figure 10A] Figure 10A shows the affinity and pharmacokinetics of antibodies with forced pairing of light chains. Figure 10A is a cartoon illustrating the generation of two antibodies by switching the pairing between heavy and light chains. [Figure 10B]Figure 10B shows the affinity and pharmacokinetics of antibodies with forced-paired light chains. Figure 10B is a table summarizing the binding affinity and pharmacokinetic properties of mAb117 forced-paired with the light chain of mAb063 (mAb117-VL063) compared to the parent mAb117. [Figure 11A] 11A and 11B are graphs showing dose-dependent inhibition of cancer cell spheroid growth by 2+2 Fab AF antibodies. Figure 11A illustrates the inhibition of UMUC14 cell growth by 2+2 N-Fab antibodies compared to 2+2-N-ScFv AF antibody BiP063N2. [Figure 11B] Figure 11B is a graph showing the dose-dependent inhibition of cancer cell spheroid growth by 2+2 Fab AF antibodies. Figure 11B illustrates the inhibition of RT4 cell growth by 2+2 N-Fab antibodies compared to 2+2-N-ScFv AF antibody BiP063N2. [Figure 11C] Figure 11C is a graph showing the dose-dependent inhibition of cancer cell spheroid growth by 2+2 Fab AF antibody. Figure 11C illustrates the inhibition of UMUC14 cell growth by 2+2 C-Fab antibody compared to BiP063N2. [Figure 11D] Figure 11D illustrates the dose-dependent inhibition of cancer cell spheroid growth by 2+2 Fab AF antibody. Figure 11D illustrates the inhibition of RT4 cell growth by 2+2 C-Fab antibody compared to BiP063N2. [Figure 12A] Figure 12 illustrates the influence of IgG backbone on the activity of exemplary antibodies. Figure 12A is a graph showing the effect of IgG subclass on the anti-proliferative activity of BiP063N2 in a UMUC14 spheroid proliferation assay. [Figure 12B] Figure 12B illustrates the effect of IgG backbone on the activity of exemplary antibodies. Figure 12B is a graph showing the effect of IgG subclass on the anti-proliferative activity of BiP063N2 in an RT4 spheroid proliferation assay. [Figure 12C] Figure 12C illustrates the effect of IgG backbone on the activity of exemplary antibodies. Figure 12C is a graph showing the effect of IgG subclass on the anti-proliferative activity of mAb063 in a UMUC14 spheroid proliferation assay. [Figure 12D] Figure 12D illustrates the effect of IgG backbone on the activity of exemplary antibodies. Figure 12D is a graph showing the effect of IgG subclass on the anti-proliferative activity of mAb063 in an RT4 spheroid proliferation assay. [Figure 12E] 12E illustrates the effect of IgG backbone on the activity of exemplary antibodies. Figure 12E is a graph showing the effect of IgG subclass on the anti-proliferative activity of mAb117 in a UMUC14 spheroid proliferation assay. [Figure 12F] Figure 12F illustrates the effect of IgG backbone on the activity of exemplary antibodies. Figure 12F is a graph showing the effect of IgG subclass on the anti-proliferative activity of mAb117 in an RT4 spheroid proliferation assay. [Figure 13A] Figure 13 illustrates the influence of IgG backbone on the antiproliferative activity of 2+2 N-Fab AF antibodies. Figure 13A is a graph showing the effect of IgG subclass on the antiproliferative activity of 2+2 N-Fab(8)AF antibodies in a UMUC14 spheroid proliferation assay. [Figure 13B] Figure 13B illustrates the influence of IgG backbone on the antiproliferative activity of 2+2 N-Fab AF antibodies. Figure 13B is a graph showing the effect of IgG subclass on the antiproliferative activity of 2+2 N-Fab(8)AF antibodies in an RT4 spheroid proliferation assay. [Figure 13C] Figure 13C illustrates the effect of IgG backbone on the antiproliferative activity of 2+2 N-Fab(9)AF antibodies. Figure 13C is a graph showing the effect of IgG subclass on the antiproliferative activity of 2+2 N-Fab(9)AF antibodies in a UMUC14 spheroid proliferation assay. [Figure 13D] Figure 13D illustrates the effect of IgG backbone on the antiproliferative activity of 2+2 N-Fab AF antibodies. Figure 13D is a graph showing the effect of IgG subclass on the antiproliferative activity of 2+2 N-Fab(9)AF antibodies in an RT4 spheroid proliferation assay. [Figure 14A]Figure 14 shows the inhibitory effect of BiP063N2 on heparin-dependent FGFR3 S249C signaling in BaF3 cells. Figure 14A is a Western blot image showing the effect of BiP063N2 on hFGF1 / heparin-induced MAPK phosphorylation in BaF3 cells expressing FGFR3 S249C. [Figure 14B] Figure 14B shows the inhibitory effect of BiP063N2 on heparin-dependent FGFR3 S249C signaling in BaF3 cells. Figure 14B is a graph showing the effects of BiP063N2, parent antibody mAb063, and tyrosine kinase inhibitor AZD4547 and erdafitinib on the proliferation of BaF3 cells expressing FGFR3 S249C. [Figure 15A] Figure 15A shows the inhibition of BiP063N2-mediated signal transduction in TKI-resistant FGFR3. Figure 15A shows the viability of BaF3 cells expressing the double mutant FGFR3 S249C+V557L after 72 hours of incubation with BiP063N2, mAb063, or AZD4547. [Figure 15B] Figure 15B shows the inhibition of BiP063N2-mediated signal transduction in TKI-resistant FGFR3. Figure 15B shows the viability of BaF3 cells expressing the double mutant FGFR3 S249C+V557M after 72 hours of incubation with BiP063N2, mAb063, or AZD4547. [Figure 15C] Figure 15C shows the inhibition of BiP063N2-mediated signaling in TKI-resistant FGFR3. Figure 15C shows the viability of BaF3 cells expressing FGFR3 V557L after 72 hours of incubation with BiP063N2, mAb063, or AZD4547. [Figure 15D] Figure 15D shows the inhibition of BiP063N2-mediated signaling in TKI-resistant FGFR3. Figure 15D shows the viability of BaF3 cells expressing FGFR3 V557M after 72 hours of incubation with BiP063N2, mAb063, or AZD4547. [Figure 15E]Figure 15E shows the inhibition of BiP063N2-mediated signal transduction in TKI-resistant FGFR3. Figure 15E shows Western blot images showing the effects of BiP063N2 and AZD4547 on the dimerization and phosphorylation of mutant FGFR3. [Figure 16A] Figure 16 shows the effect of BiP063N2 on MAPK phosphorylation and proliferation in BaF3 cells expressing mutant FGFR3. Figure 16A is a Western blot showing the inhibitory effect of BiP063N2 on FGF / heparin-induced MAPK phosphorylation in BaF3 cells expressing WT hFGFR3b. [Figure 16B] Figure 16B shows the effect of BiP063N2 on MAPK phosphorylation and proliferation in BaF3 cells expressing mutant FGFR3. Figure 16B is a Western blot showing the inhibitory effect of BiP063N2 on FGF / heparin-induced MAPK phosphorylation in BaF3 cells expressing the hFGFR3b Y375C mutation. [Figure 16C] Figure 16C shows the effect of BiP063N2 on MAPK phosphorylation and proliferation in BaF3 cells expressing mutant FGFR3. Figure 16C shows the inhibitory effect of BiP063N2 on proliferation of BaF3 cells expressing the hFGFR3b S248C mutation. DETAILED DESCRIPTION OF THE INVENTION

[0017] 6. MODE FOR CARRYING OUT THE INVENTION 6.1. Definition About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before a numerical value to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also be a disclosure of "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0018] Agonist: As used herein, the term "agonist" with respect to a polypeptide, antibody, or binding molecule (e.g., a multispecific binding molecule) refers to the ability of the polypeptide, antibody, or binding molecule to increase signaling, activation, or activity of the protein to which it binds. For example, an agonistic FGFR3 binding molecule (e.g., an agonistic anti-FGFR3 antibody or multispecific binding molecule or FGFR3-binding fragment thereof) describes an FGFR3 binding molecule that 1) upon contact with cellular FGFR3 molecules, increases FGFR3 signaling by at least 10%, as measured by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis of phospho-ERK1 / 2 levels, and / or 2) increases cell proliferation in FGFR3-positive cancer cells by at least 10%, as measured by the cell proliferation assay described in Section 8.1.3. In some embodiments, the agonist FGFR3 binding molecule increases FGFR signaling in FGFR3-positive cancer cells and / or increases cell proliferation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 400%.

[0019] The term "agonist" as used herein with respect to the ABD of a binding molecule refers to an ABD derived from an agonist antibody. That is, an "agonist ABD" is an ABD such that when the ABD is present as the antigen-binding domain of a monospecific antibody in the format of a natural IgG antibody having a wild-type human IgG1 Fc sequence, the antibody is an agonist antibody. Thus, an "agonist FGFR3 ABD" describes an ABD derived from an agonist anti-FGFR3 antibody.

[0020] And / Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (selection of A is mutually exclusive from B, A OR B) and the selection of conjunctive features (selection of both A and B, A OR B). In several places in the text, the term "and / or" is used interchangeably and should not be construed to mean that "or" is used in reference to mutually exclusive alternatives.

[0021] Antagonist: As used herein, the term "antagonist" with respect to a polypeptide, antibody, or binding molecule (e.g., a multispecific binding molecule) refers to the ability of the polypeptide, antibody, or binding molecule to reduce the signaling, activation, or activity of a protein to which it binds. For example, antagonistic FGFR3 binding molecules (e.g., antagonistic anti-FGFR3 antibodies or multispecific binding molecules) are described that: 1) upon contact with cellular FGFR3 molecules, reduce FGFR3 dimerization by at least 10% as measured by non-reducing SDS-polyacrylamide gel electrophoresis followed by Western blot analysis of FGFR3 dimer levels; 2) upon contact with cellular FGFR3 molecules, reduce FGFR3 signaling by at least 10% as measured by SDS-polyacrylamide gel electrophoresis followed by Western blot analysis of phospho-ERK1 / 2 levels; and / or 3) reduce cell proliferation in FGFR3-positive cancer cells by at least 10% as measured by the cell proliferation assay described in Section 8.1.3. In some embodiments, the antagonist FGFR3 binding molecule reduces dimerization, reduces FGFR signaling, and / or reduces cell proliferation in FGFR3-positive cancer cells by at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0022] The term "antagonist" as used herein with reference to the ABD of a binding molecule refers to an ABD derived from an antagonist antibody. That is, an "antagonist ABD" is an ABD such that when the ABD is present as the antigen-binding domain of a monospecific antibody in the format of a natural IgG antibody having a wild-type human IgG1 Fc sequence, the antibody is an antagonist antibody. Thus, an "antagonist FGFR3 ABD" describes an ABD derived from an antagonist anti-FGFR3 antibody.

[0023] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties, such as, for example, secretion, placental transport, Fc receptor binding, and complement fixation. By convention, the numbering of constant region domains increases as they become more distant from the antigen-binding domain or amino-terminus of the antibody. At the N-terminus are the variable regions, at the C-terminus are the constant regions, and the CH3 and CL domains represent the carboxy-termini of the heavy and light chains of native antibodies, respectively. For convenience, and unless the context dictates otherwise, reference to an antibody also refers to antibody fragments and engineered antibodies containing non-native antigen-binding domains and / or antigen-binding domains with non-native configurations.

[0024] Antigen-binding domain: As used herein, the term "antigen-binding domain" or "ABD" refers to a portion of a binding molecule (e.g., a multispecific binding molecule, an antibody, or an antibody fragment) that has the ability to non-covalently, reversibly, and specifically bind to a target molecule (e.g., an antigen such as FGFR3). Examples of antibody fragments that can contain an ABD include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting of the VH domain and the CH1 domain, Fv fragments consisting of the VL domain and the VH domain of a single antibody arm, dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies (e.g., Fab fragments and F(ab)2 fragments) and engineered proteins containing one or more portions of antibodies (e.g., scFvs). Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136). Examples of multispecific binding molecules that can include ABDs include the FGFR3 binding molecules of the present disclosure. In some embodiments, the multispecific binding molecules of the present disclosure include one, two, three, four, or more antigen-binding domains, such as a first ABD (ABD1), a second ABD (ABD2), a third ABD (ABD3), and a fourth ABD (ABD4).

[0025] Antigen-binding fragment of anti-FGFR3 antibody: The term "antigen-binding fragment of anti-FGFR3 antibody" refers to a portion of a native immunoglobulin or other antibody that can bind to FGFR3. In some embodiments, the antigen-binding fragment of an FGFR3 antibody can be in the form of Fab, Fv, or scFv.

[0026] Associated: In the context of FGFR3-binding molecules, the term "associated" refers to a functional relationship between two or more polypeptide chains or portions of polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks, to generate a functional FGFR3-binding molecule. Examples of associations that may exist in the FGFR3-binding molecules of the present disclosure include, but are not limited to, the association between homodimeric or heterodimeric Fc domains within an Fc region, the association between VH and VL regions within an Fab or scFv, the association between CH1 and CL within an Fab, and the association between CH3 and CH3 within a domain-substituted Fab.

[0027] Bivalent: As used herein with respect to a multispecific binding molecule (e.g., an FGFR3-binding molecule), the term "bivalent" refers to a multispecific binding molecule having two antigen-binding sites. In some embodiments, the two antigen-binding sites bind to the same epitope on the same target (e.g., FGFR3). In other embodiments, the two antigen-binding sites specifically bind to different epitopes on the same target molecule. In other embodiments, the two antigen-binding sites specifically bind to different epitopes on two different target molecules. Thus, a bivalent antigen-binding molecule can be monospecific or bispecific.

[0028] With respect to an FGFR3-binding molecule, "bivalent" means that the FGFR3-binding molecule has two FGFR3 ABDs (e.g., two antigen-binding fragments of an anti-FGFR3 antibody). In some embodiments, the two FGFR3 ABDs specifically bind to the same epitope on FGFR3. In some embodiments, the two FGFR3 ABDs specifically bind to different epitopes on FGFR3. In certain embodiments, an FGFR3-binding molecule of the present disclosure comprises a first FGFR3 ABD that specifically binds to an epitope comprising a sequence present in D3 and / or D2 of FGFR3, and a second FGFR3 ABD that specifically binds to an epitope comprising a sequence present in D1 of FGFR3.

[0029] Complementarity determining region or CDR: The term "complementarity determining region" or "CDR" as used herein refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary rules that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, the ABD definition, and the IMGT definition. See, e.g., Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (Kabat numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme), Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABD numbering scheme), and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). For example, for the classical format, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues in the VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).Combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH, and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) ("Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align. Public databases are available to identify CDR sequences within antibodies.

[0030] Dimerization moiety: The term "dimerization moiety" refers to a polypeptide chain or amino acid sequence that can promote the association between two polypeptide chains to form a dimer. A first dimerization moiety can associate with an identical second dimerization moiety, or with a second dimerization moiety that is different from the first dimerization moiety. In some embodiments, the dimerization moiety is an Fc domain, and the association of two Fc domains forms an Fc region. Thus, the Fc region can be a homodimer or a heterodimer.

[0031] EC50: The term "EC50" refers to the half maximal effective concentration of a molecule, such as a multispecific binding molecule (e.g., an FGFR3-binding molecule), that induces a response midway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of a molecule at which 50% of its maximal effect is observed. Thus, the greater the EC50 or half maximal effective concentration value, the less binding or weakening is observed. The EC50 values ​​of the MBMs of the present disclosure, in some embodiments, are about 10 -5M or less (e.g., 10 -5 Under M, 10 -6 Under M, 10 -7 Under M, 10 -8 Less than M or 10 -9 In certain embodiments, the EC50 value is equal to the concentration of FGFR3 binding molecule that provides half-maximal activation in a cell proliferation assay.

[0032] Epitope: An epitope, or antigenic determinant, is a portion of an antigen (e.g., FGFR3) that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational. As described herein, epitopes can be described as "comprising the sequence" of a specific region (e.g., a protein domain) of an antigen (e.g., FGFR3). Such descriptions include both linear and conformational epitopes and describe epitopes that include at least one amino acid present in a specific region of an antigen. Such epitopes may or may not include additional amino acids not present in the specific region.

[0033] Fab: The term "Fab" in the context of the multispecific binding molecules (e.g., FGFR3-binding molecules) of the present disclosure refers to a pair of polypeptide chains, where the first polypeptide chain comprises the antibody N-terminal variable heavy chain (VH) domain through to the first constant domain (referred to herein as C1), and the second polypeptide chain comprises the antibody N-terminal variable light chain (VL) domain through to the second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and the VL is located at the N-terminus of the constant domain (CL) of the light chain. The Fabs of the present disclosure can be oriented according to their natural orientation or can include domain substitutions or exchanges that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in the Fab can be replaced with a CH3 domain pair to promote correct altered Fab-chain pairing in a heterodimeric molecule. The CH1 and CL can also be reversed, so that CH1 is attached to VL and CL is attached to VH, a configuration commonly known as a crossmab (a type of "domain-swapped" configuration). Alternatively, or in addition to the use of substituted or exchanged constant domains, correct chain pairing can be achieved by the use of a universal light chain that can pair with both variable regions of the heterodimeric MBMs of the present disclosure. The term "Fab" encompasses single-chain Fabs.

[0034] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be identical to one another or different. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, e.g., via knob-in-hole interactions, and / or to enable purification, e.g., via a star mutation.

[0035] FGFR3: "FGF receptor 3," "FGFR3," and similar terms, unless otherwise indicated, refer to any native fibroblast growth factor receptor 3 (FGFR3) from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). The term encompasses "full-length," native FGFR3, as well as any form of FGFR3 resulting from intracellular processing. The term also encompasses naturally occurring variants of FGFR3, such as splice variants or allelic variants, including FGFR3b and FGFR3c. Exemplary amino acid sequences of human FGFR3 molecules include the following:

[0036] mFGFR3c (mature): (query number 22) mFGFR3b(mature): (query number 1) hFGFR3c(mature): (query number 23) hFGFR3b(mature): (SEQ ID NO: 2) FGFR3 binding molecule: The term "FGFR3 binding molecule" refers to a molecule that comprises at least one FGFR3 ABD. Generally, an FGFR3 binding molecule is a molecule that is composed of one or more polypeptide chains (for example, one, two, three, or four polypeptide chains) that together comprise at least one FGFR3 ABD (for example, one, two, three, four or more FGFR3 ABD). In the context of the FGFR3 binding molecule of the present disclosure, the term "FGFR3 binding molecule" can refer to the core component of the molecule, i.e., one or more FGFR3 ABDs, and can also refer to dimerization moieties such as Fc domains and / or associated linker molecules. Unless otherwise indicated by the context, the term "FGFR3 binding molecule" should also be understood to include molecules that comprise additional features, such as one or more stabilizing moieties, one or more dimerization moieties, one or more linker moieties, and any combination of the above.

[0037] FGFR3 ABD: The term "FGFR3 antigen-binding domain" or "FGFR3 ABD" refers to a portion of a binding molecule capable of binding to FGFR3. In some embodiments, the FGFR3 ABD comprises or consists of an antigen-binding fragment of an anti-FGFR3 antibody. The FGFR3-binding fragment of an anti-FGFR3 antibody can be in the form of a Fab, Fv, or scFv. FGFR3 ABDs are further described in Section 6.2.1.

[0038] Fv: The term "Fv" refers to the smallest antibody fragment derivable from an immunoglobulin that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In many cases, six CDRs confer target binding specificity to an antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three CDRs specific for a target) may have the ability to recognize and bind to a target. Reference herein to a VH-VL dimer is not intended to convey any particular configuration. When present on a single polypeptide chain (e.g., scFv), the VH and the N- or C-terminus of the VL are present.

[0039] Half antibody: The term "half antibody" refers to a molecule that contains at least one Fc domain and can associate with another molecule containing Fc, for example, through a disulfide bridge or molecular interaction. A half antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., the two polypeptide chains of a Fab). An example of a half antibody is a molecule containing the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule containing a first polypeptide containing a VL domain and a CL domain, and a second polypeptide containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain form an ABD. Yet another example of a half antibody is a polypeptide containing an scFv domain, a CH2 domain, and a CH3 domain. The term "half antibody" is intended solely for descriptive purposes and does not imply a particular configuration or method of production. Descriptions of half antibodies as "first," "second," "left," "right," etc., are solely for convenience and descriptive purposes.

[0040] Host cell or recombinant host cell: The terms "host cell" and "recombinant host cell," as used herein, refer to a cell that has been genetically engineered, for example, by the introduction of heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A host cell can harbor heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing multispecific binding molecules (e.g., FGFR3-binding molecules), host cells can be cell lines of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or their derivatives and / or engineered variants. Engineered variants include, for example, modified glycan profiles and / or site-specific integration site derivatives.

[0041] Monomer: As used herein, the term "monomer" refers to a molecule comprising a first polypeptide chain that (a) comprises at least one FGFR3 ABD and is capable of associating with a second polypeptide chain, (b) comprises a dimerization moiety (e.g., an Fc domain) and is capable of associating with a corresponding dimerization moiety (e.g., another Fc domain) on a second polypeptide chain, or (c) comprises a combination of both (a) and (b). A monomer can associate with another monomer through a pair of dimerization moieties (e.g., an Fc domain). In some embodiments, one or more associations between monomers are stabilized through a hinge sequence or other portion of the Fc domain. Thus, a monomer of the present disclosure can associate with another monomer to form a dimer. A dimer can be a homodimer, in which each constituent monomer is identical, or a heterodimer, in which each constituent monomer is different. As used herein, reference to a "monomer" is for convenience and does not exclude the presence of one or more additional polypeptide chains, e.g., one or more light chains of one or more Fab domains. Thus, a "dimer" of two monomers may contain more than two polypeptide chains, e.g., three, four, or more polypeptide chains, and reference to a monomer or dimer is not intended to imply any temporal order of association between the polypeptide chains.

[0042] Multispecific Binding Molecule or MBM: As used herein, the term "multispecific binding molecule" or "MBM" refers to a molecule (e.g., an assembly of multiple polypeptide chains) that comprises two half antibodies and specifically binds to at least two different epitopes (and in some cases, three, four, or more different epitopes). MBMs of the present disclosure can be bivalent, trivalent, tetravalent, or otherwise multivalent, and can be monospecific, bispecific, or otherwise multispecific. MBMs of the present disclosure can specifically bind to epitopes on one, two, three, four, or more different antigens. In certain embodiments, MBMs of the present disclosure specifically bind to two or more epitopes on a single antigen and are tetravalent.

[0043] Multivalent: As used herein with respect to a multispecific binding molecule, the term "multivalent" means that the binding molecule has two or more targeting moieties (e.g., two, three, four, or more targeting moieties). With respect to an FGFR3 binding molecule, "multivalent" means that the FGFR3 binding molecule has two or more FGFR3 ABDs (e.g., two, three, four, or more antigen-binding fragments of an anti-FGFR3 antibody). In some embodiments, all FGFR3 ABDs of a multivalent FGFR3 binding molecule specifically bind to the same epitope on FGFR3. In some embodiments, all FGFR3 ABDs of a multivalent FGFR3 binding molecule specifically bind to different epitopes on FGFR3. In some embodiments, one or more FGFR3 ABDs of a multivalent FGFR3 binding molecule bind to one epitope on FGFR3, and the remaining FGFR3 ABDs bind to different epitopes on FGFR3. In certain embodiments, an FGFR3 binding molecule of the present disclosure comprises one or more FGFR3 ABDs that specifically bind to an epitope comprising a sequence present in D3 and / or D2 of FGFR3, and one or more FGFR3 ABDs that specifically bind to an epitope comprising a sequence present in D1 of FGFR3.

[0044] Non-antagonist: As used herein, the term "non-antagonist" in reference to a polypeptide, antibody, or binding molecule (e.g., a multispecific binding molecule) refers to a polypeptide, antibody, or binding molecule that is not an antagonist. A non-antagonist FGFR3 binding molecule may also be an agonist.

[0045] The term "non-antagonist" as used herein with reference to the ABD of a binding molecule refers to an ABD derived from a non-antagonist antibody. That is, a "non-antagonist ABD" is an ABD such that when the ABD is present as the antigen-binding domain of a monospecific antibody in the format of a natural IgG antibody having a wild-type human IgG1 Fc sequence, the antibody is a non-antagonist antibody. Thus, a "non-antagonist FGFR3 ABD" describes an ABD derived from a non-antagonist anti-FGFR3 antibody.

[0046] Operably linked: As used herein, the term "operably linked" refers to the functional relationship between two or more regions of a polypeptide chain, where the two or more regions are linked to produce a functional polypeptide or two or more nucleic acid sequences, for example, to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are linked to produce a functional polypeptide. For example, in the context of the FGFR3 binding molecules of the present disclosure, separate components (e.g., a first FGFR3 ABD and a second FGFR3 ABD) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as a monomer of the FGFR3 binding molecule of the present disclosure, "operably linked" means that the two nucleic acids are joined so that the amino acid sequences encoded by the two nucleic acids remain in frame. In the context of transcriptional regulation, the term refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0047] Polypeptide, Peptide, and Protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0048] Single-chain Fab or scFab: As used herein, the term "single-chain Fab" or "scFab" refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linker are arranged in one of the following orders, from N- to C-terminus: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is suitably a non-cleavable linker of at least 30 amino acids, preferably 32-50 amino acids. Single-chain Fab fragments are typically stabilized via a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to the Kabat numbering).

[0049] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315.

[0050] Subject: The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. In certain embodiments, the subject is a human. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0051] Tetravalent: As used herein, the term "tetravalent" refers to a multispecific binding molecule having four antigen-binding sites. In certain embodiments, all four of the antigen-binding sites bind to the same epitope. In some embodiments, two of the antigen-binding sites bind to the same epitope, and the other two antigen-binding sites bind to different epitopes, whether to the same or different target molecules. In other embodiments, two of the antigen-binding sites bind to the same epitope, the third antigen-binding site binds to a different epitope, and the fourth antigen-binding site binds to yet another different epitope. In still other embodiments, all four epitopes bind to different epitopes, whether to the same or different target molecules. Thus, a tetravalent multispecific binding molecule can be monospecific, bispecific, trispecific, or tetraspecific.

[0052] With respect to an FGFR3-binding molecule, "tetravalent" means that the FGFR3-binding molecule has four FGFR3 ABDs (e.g., four antigen-binding fragments of an anti-FGFR3 antibody). In some embodiments, all of the FGFR3 ABDs of a tetravalent FGFR3-binding molecule specifically bind to the same epitope on FGFR3. In some embodiments, all of the FGFR3 ABDs of a tetravalent FGFR3-binding molecule specifically bind to different epitopes on FGFR3. In some embodiments, two of the FGFR3 ABDs of a tetravalent FGFR3-binding molecule bind to one epitope on FGFR3, while the other two FGFR3 ABDs bind to different epitopes on FGFR3. In certain embodiments, a tetravalent FGFR3 binding molecule of the present disclosure comprises two FGFR3 ABDs that specifically bind to an epitope comprising a sequence present in D3 and / or D2 of FGFR3, and two FGFR3 ABDs that specifically bind to an epitope comprising a sequence present in D1 of FGFR3.

[0053] Treat, treatment, treating: As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression of a disease or condition, the severity and / or duration of a disease or condition, and / or the amelioration of one or more symptoms (preferably one or more discernible symptoms) resulting from the administration of one or more multispecific binding molecules (e.g., FGFR3 binding molecules) of the present disclosure.

[0054] In some embodiments, the disease or condition is a proliferative disorder. With respect to a proliferative disorder, the terms "treat," "treatment," and "treating" refer to a shortening or amelioration of the progression, severity, and / or duration of the proliferative disorder, or an improvement in one or more symptoms (preferably one or more discernible symptoms) of the proliferative disorder resulting from the administration of one or more multispecific binding molecules (e.g., FGFR3-binding molecules) of the present disclosure. In certain embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of the proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of the progression of the proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0055] Trivalent: As used herein, the term "trivalent" refers to a multispecific binding molecule having three antigen-binding sites. In certain embodiments, all three of the antigen-binding sites bind to the same epitope. In some embodiments, two of the antigen-binding sites bind to the same epitope, and the other antigen-binding site binds to a different epitope, whether on the same target molecule or on different target molecules. In other embodiments, all three of the antigen-binding sites bind to different epitopes, whether on the same target molecule or on any combination of two or more different target molecules. Thus, a trivalent multispecific binding molecule can be monospecific, bispecific, or trispecific.

[0056] With respect to an FGFR3 binding molecule, "trivalent" means that the FGFR3 binding molecule has three FGFR3 ABDs (e.g., three antigen-binding fragments of an anti-FGFR3 antibody). In some embodiments, all FGFR3 ABDs of a trivalent FGFR3 binding molecule specifically bind to the same epitope on FGFR3. In some embodiments, all FGFR3 ABDs of a trivalent FGFR3 binding molecule specifically bind to different epitopes on FGFR3. In some embodiments, two of the FGFR3 ABDs of a trivalent FGFR3 binding molecule bind to one epitope on FGFR3, and the other FGFR3 ABD binds to a different epitope on FGFR3. In certain embodiments, a trivalent FGFR3 binding molecule of the present disclosure comprises two FGFR3 ABDs that specifically bind to an epitope comprising a sequence present in D3 and / or D2 of FGFR3, and one FGFR3 ABD that specifically binds to an epitope comprising a sequence present in D1 of FGFR3.

[0057] Universal light chain, ULC: As used herein, the term "universal light chain" or "ULC" refers to a light chain variable region (VL) that can pair with more than one heavy chain variable region (VL). In the context of FGFR3 ABD, the term "universal light chain" or "ULC" refers to a light chain polypeptide that can pair with the heavy chain region of FGFR3 ABD and can also pair with other heavy chain regions. The ULC may also contain a constant domain, such as the CL domain of an antibody. The universal light chain is also known as a "common light chain."

[0058] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

[0059] 6.2. FGFR3 binding molecules Aspects of the present disclosure relate to multispecific binding molecules ("MBMs"), in particular FGFR3 binding molecules. Generally, an FGFR3 binding molecule binds to at least two different epitopes of FGFR3.

[0060] Typically, the MBM of the present disclosure comprises two half antibodies. In some embodiments, one half antibody comprises at least one FGFR3 antigen-binding domain that binds to a first epitope of FGFR3, and the other half antibody comprises at least one FGFR3 antigen-binding domain that binds to a second, different epitope of FGFR3. In certain aspects, the MBM of the present disclosure is a tetravalent FGFR3-binding molecule comprising two half antibodies, one of which comprises two FGFR3 antigen-binding domains and the other of which comprises two FGFR3 antigen-binding domains.

[0061] In other aspects, the MBM of the present disclosure is a tetravalent FGFR3 binding molecule comprising two half antibodies, one of which comprises one FGFR3 antigen-binding domain and the other of which comprises two FGFR3 antigen-binding domains.

[0062] The MBMs of the present disclosure specifically bind to at least two different epitopes (and in some cases three or more different epitopes) of FGFR3. Thus, certain MBMs of the present disclosure specifically bind to two, three, four, or more epitopes of FGFR3.

[0063] In certain embodiments, the MBM of the present disclosure is bispecific.For clarity, as used herein, the term "bispecific" refers to binding to any two different epitopes, whether on the same antigen or target molecule or on different antigens or target molecules.Therefore, "bispecific FGFR3 binding molecule" describes an FGFR3 binding molecule that has antigen binding domains that bind to two different epitopes of FGFR3.The bispecific FGFR3 binding molecule of the present disclosure can be bivalent, trivalent, or tetravalent.

[0064] In certain aspects, the present disclosure provides an MBM comprising: (a) an antigen binding domain 1 (ABD1) that specifically binds to a first epitope of FGFR3; and (b) an antigen binding domain 2 (ABD2) that specifically binds to a second epitope of FGFR3 that is different from the first epitope. In some embodiments, the first epitope comprises a sequence present in D3 of FGFR3. In some embodiments, the first epitope comprises a sequence present in D2 of FGFR3. In some embodiments, the first epitope comprises a sequence present in D2 and D3 of FGFR3. In some embodiments, the second epitope comprises a sequence present in D1 of FGFR3. Thus, in some embodiments, the present disclosure provides an MBM comprising: (a) an antigen-binding domain 1 (ABD1) that specifically binds to a first epitope comprising a sequence present in D3 and / or D2 of FGFR3; and (b) an antigen-binding domain 2 (ABD2) that specifically binds to a second epitope comprising a sequence present in D1 of FGFR3. In certain aspects, ABD1 is an antagonistic antigen-binding domain, and ABD2 is a non-antagonistic (e.g., agonist) antigen-binding domain. ABD1 and ABD2 may each optionally be scFv or Fab.

[0065] In certain aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab, to which it is operably linked, (ii) a second heavy chain region of a second Fab, to which it is operably linked, and (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab, to which it is operably linked, (ii) a fourth heavy chain region of a fourth Fab, to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with a third heavy chain region to form a third Fab; (f) a sixth polypeptide chain comprising a fourth light chain paired with a fourth heavy chain region to form a fourth Fab.

[0066] One, two, three, or all of the Fabs may bind to the same or different epitopes of FGFR3 (ie, two, three, or four different epitopes of FGFR3).

[0067] The FGFR3 binding molecule of the present disclosure may contain one, two, or more FGFR3 ABDs in addition to one of the additional targeting moieties. In certain embodiments, the FGFR3 binding molecule of the present disclosure contains one, two, three, four, or more FGFR3 ABDs, and does not contain any additional targeting moieties. Without being bound by theory, it is believed that the MBM of the present disclosure, which contains FGFR3 ABDs targeting two (or more) different regions of FGFR3, has advantages over monospecific anti-FGFR3 antibodies in inhibiting FGFR3 dimerization and activation and treating conditions associated with increased or constitutive FGFR3 activity, such as FGFR3-positive cancers (e.g., bladder cancer). Thus, in some embodiments, the FGFR3 binding molecule of the present disclosure can inhibit FGFR3 activity and FGFR3-positive cancer cell proliferation to a greater extent than monospecific anti-FGFR3 antibodies. For example, an FGFR3 binding molecule of the present disclosure may, in some embodiments, have a more potent EC50 value in a cell proliferation assay than a corresponding monospecific anti-FGFR3 antibody (e.g., as described in Section 8).

[0068] FGFR3 contains three extracellular domains: D1 (Ig-like domain I, IgG-I, Ig-I, or IgD1, also amino acids 25-119 of hFGFR3), D2 (Ig-like domain II, IgG-II, Ig-II, or IgD2, also amino acids 158-246 of hFGFR3), and D3 (Ig-like domain III, IgG-III, Ig-III, or IgD3, also amino acids 255-377 of hFGFR3). See Figure 1A (see also Babina et al., 2017, Nat Rev Cancer. 17(5):318-332, incorporated herein by reference). FGF binds to a region of FGFR3 that includes portions of D2 and D3 (see Figure 1D, Protein Data Bank code 1RY7). As described herein, certain FGFR3 MBMs of the present disclosure comprise a first FGFR3 ABD that binds to D2 and / or D3 of FGFR3 and a second FGFR3 ABD that binds to D1 of FGFR3. Unexpectedly, such FGFR3 binding molecules have the advantage of inhibiting FGFR3 activation over monospecific anti-FGFR3 antibodies that target D2 and / or D3 alone, despite the fact that many monospecific antibodies that bind to D1 of FGFR3 are agonists (i.e., activate FGFR3 signaling).

[0069] Certain exemplary FGFR3 binding molecules of the present disclosure are shown in Figures 2B-2P. FGFR3 ABD The FGFR3 binding molecule of the present disclosure comprises one or more FGFR3 antigen binding domains. In some embodiments, the FGFR3 binding molecule of the present disclosure comprises an ABD1 that binds to a first epitope of FGFR3 and an ABD2 that binds to a second epitope of FGFR3. In certain embodiments, the first epitope comprises a sequence present in D3 and / or D2 of FGFR3, and the second epitope comprises a sequence present in D1 of FGFR3. The FGFR3 binding molecule of the present disclosure may comprise an ABD3 that specifically binds to the first epitope. The FGFR3 binding molecule of the present disclosure may further comprise an ABD4 that specifically binds to the second epitope. Without being bound by theory, it is believed that the binding of FGFR3 binding molecules with ABD1, ABD2, ABD3 and ABD4 inhibits receptor dimerization, thereby antagonizing FGFR3 receptor complex, and thus inhibiting FGFR3 signal transduction in cells such as cancer cells.FGFR3 point mutation or fusion is present in about 15% of invasive bladder cancers, and FGFR3 isoform FGFR3b is preferentially expressed in bladder cancer.Therefore, certain FGFR3 binding molecules of the present disclosure comprise one or more antigen binding domains that preferentially bind to FGFR3b compared to FGFR3c.

[0070] The FGFR3 ABD of the present disclosure can be non-antagonist, agonist or antagonist.In certain embodiments, the FGFR3 binding molecule of the present disclosure comprises at least one non-antagonist (for example, agonist) ABD and at least one antagonist ABD.Without being bound by theory, it is believed that the FGFR3 binding molecule having one or more non-antagonist (for example, agonist) ABD and one or more antagonist ABD surprisingly has advantages over monospecific antagonist anti-FGFR3 antibody in inhibiting FGFR3 activation and FGFR3-positive cancer cell proliferation, either alone or in combination with additional non-antagonist anti-FGFR3 antibody. For example, an FGFR3 binding molecule of the present disclosure comprising two distinct FGFR3 ABDs may, in some embodiments, have a more potent EC50 value in a cell proliferation assay than a corresponding monospecific anti-FGFR3 antibody having the same FGFR3 ABD (e.g., as described in Section 8.3).

[0071] ABD1, ABD2, ABD3, and ABD4 can be derived from one or more suitable anti-FGFR3 antibodies or non-immunoglobulin-based antigen-binding domains. The antibody from which one or more of ABD1, ABD2, ABD3, and ABD4 are derived may be referred to herein as a "parent" antibody. The FGFR3 parent antibody can be a monoclonal antibody (e.g., a mouse or rabbit monoclonal antibody), a chimeric antibody, a humanized antibody, a human antibody, a primatized antibody, a bispecific antibody, a single-chain antibody, or the like. In various embodiments, the MBM of the present disclosure comprises all or a portion of a constant region derived from the parent. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM.

[0072] As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.

[0073] Monoclonal antibodies useful as a source of FGFR3 ABDs, including FGFR3 ABDs that bind to specific epitopes or domains of FGFR3 (e.g., D1, D2, and / or D3 of FGFR3), can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0074] The term "chimeric" antibody as used herein refers to an antibody having variable sequences derived from non-human immunoglobulins, such as rabbit, rat, or mouse antibodies, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for producing 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, which are incorporated herein by reference in their entirety.

[0075] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al.; EP 239400; PCT Publication No. WO 91 / 09967; U.S. Patent No. 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng., 7:805-814; Roguska et al. al., 1994, Proc. Natl. Acad. Sci. 91:969-973, as well as U.S. Pat. No. 5,565,332, all of which are incorporated herein by reference in their entireties.

[0076] "Human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from a human immunoglobulin library or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins. Human antibodies can be produced by various methods known in the art, including phage display methods, using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT Publication Nos. WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, WO 96 / 33735, U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, which are incorporated herein by reference in their entireties. Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

[0077] A "primatized antibody" comprises a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated by reference in their entireties.

[0078] In some embodiments, parent antibodies to the FGFR3 binding molecules of the present disclosure are generated using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®). A chimeric parent antibody with high affinity to FGFR3 (e.g., a specific region or domain of FGFR3, such as D1, D2, and / or D3) can first be isolated with a human variable region and a mouse constant region. VELOCIMMUNE® technology involves generating transgenic mice with genomes comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies comprising the human variable region and the mouse constant region in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0079] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express antibodies. The lymphocytes can be fused with a myeloma cell line to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0080] Targeted antibodies can also be isolated from mouse B cells. Briefly, splenocytes are collected from each mouse, and B cells are selected by FACS (for example, as described in US2007 / 0280945A1), using the target antigen as a selection reagent to bind and identify reactive antibodies (antigen-positive B cells).Various methods for identifying and selecting antigen-positive B cells, and various methods for constructing immunoglobulin gene expression cassettes by PCR to prepare cells expressing recombinant antibodies, are well known in the art.For example, see WO2014 / 1460741, US Patent No. 7884054B2, and Liao et al., 2009, J Virol Methods 158(1-2):171-9.

[0081] First, high-affinity chimeric antibodies with human variable regions and mouse constant regions are isolated. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies, such as wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the specific application, the high-affinity antigen-binding and target specificity characteristics reside in the variable regions.

[0082] Examples of publications disclosing parent anti-FGFR3 antibodies for use in MBM of the present disclosure include, but are not limited to, WO2002 / 102972A2, WO2006 / 048877A2, WO2010 / 002862A2, WO2010 / 048026A2, WO2010 / 111367A1, WO2016 / 134234A1, WO2022 / 040560A1, and WO2021 / 010326A1, each of which is incorporated herein by reference.

[0083] In some embodiments, FGFR3-binding agent sequences that can be incorporated into the MBMs of the present disclosure are identified in Tables T-1, T-2, T-3, and T-4 below.

[0084] [Table 1-1]

[0085] [Table 1-2]

[0086] [Table 1-3]

[0087] [Table 1-4]

[0088] [Table 1-5]

[0089] [Table 1-6]

[0090] [Table 1-7]

[0091] Table 1-8

[0092] Table 1-9

[0093] Table 1-10

[0094] Table 1-11

[0095] Table 1-12

[0096] Table 2-1

[0097] Table 2-2

[0098] Table 3-1

[0099] Table 3-2

[0100] Table 3-3

[0101] Table 3-4

[0102] Table 3-5

[0103] Table 3-6

[0104] Table 3-7

[0105] Table 4-1

[0106] Table 4-2

[0107] Table 4-3

[0108] Table 4-4

[0109] Table 4-5

[0110] Thus, an FGFR3 ABD of the present disclosure can include, for example, the CDR or VH and / or VL sequence of any of the aforementioned anti-FGFR3 antibodies, e.g., any of the anti-FGFR3 antibodies provided in Tables T-1, T-2, T-3, and T-4. Additional FGFR3 binding agents are known in the art, and their use is contemplated herein. In some embodiments, an MBM of the present disclosure includes (a) an anti-D1 ABD having the CDR or VH and / or VL sequence of any of the anti-D1 antibodies listed in Table T-4, and (b) (i) an anti-D2 ABD having the CDR or VH and / or VL sequence of any of the anti-D2 antibodies listed in Table T-2, and / or (ii) an anti-D3 ABD having the CDR or VH and / or VL sequence of any of the anti-D3 antibodies listed in Table T-3.

[0111] Thus, an FGFR3 ABD of the present disclosure can include, for example, any of the anti-FGFR3 antibodies provided in Tables T-1, T-2, T-3, and T-4, together with the VH sequence of any of the aforementioned anti-FGFR3 antibodies, e.g., a universal or common light chain. In some embodiments, an MBM of the present disclosure includes (a) an anti-D1 ABD having the VH sequence of any of the anti-D1 antibodies listed in Table T-4 and a universal or common light chain, and (b) (i) an anti-D2 ABD having the VH sequence of any of the anti-D2 antibodies listed in Table T-2 and a universal or common light chain, and / or (ii) an anti-D3 ABD having the VH sequence of any of the anti-D3 antibodies listed in Table T-3 and a universal or common light chain.

[0112] The antigen-binding site of the FGFR3 binding molecules of the present disclosure can be selected from immunoglobulin-based and non-immunoglobulin-based binding domains. In some embodiments, one or more of the ABDs are derived from an immunoglobulin, e.g., comprise or consist of a Fab (as described in Section 6.2.5), an scFv (as described in Section 6.2.4), or another immunoglobulin-based format, e.g., an Fv, a dsFv, a (Fab')2, a single domain antibody (SDAB), a VH domain or a VL domain, or a camelid VHH domain (also called a nanobody).

[0113] The ABD can be derived from a single domain antibody, which is composed of a single VH or VL domain that exhibits sufficient affinity for the target. In a specific embodiment, the single domain antibody is a camelid VHH domain (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38, WO94 / 04678).

[0114] In certain embodiments, one or more of the ABDs are derived from a non-antibody scaffold protein (including, but not limited to, designed ankyrin repeat proteins (DARPins), avimers (short for avidity-activated multimers), anticalins / lipocalins, centilins, Kunitz domains, adnexins, affilins, affitins (also known as nonphytins), knottins, pronectins, versabodies, duocalins, and phynomers), ligands, receptors, cytokines, or chemokines.

[0115] Non-immunoglobulin scaffolds that can be used in the MBMs of the present disclosure include those listed in Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48, Table 1 and Figure 1 of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83, and Table 1 and Box 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18. The contents of Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48; Table 1 and Figure 1 of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83; and Table 1 and Box 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18 (collectively, the "Scaffold Disclosures"). In certain embodiments, the scaffold disclosures are incorporated by reference for what they disclose in the context of adnexins. In other embodiments, the scaffold disclosures are incorporated by reference for what they disclose in the context of avimers. In other embodiments, the scaffold disclosures are incorporated by reference for what they disclose in the context of affibodies. In yet other embodiments, the scaffold disclosures are incorporated by reference for what they disclose in the context of anticalins. In yet other embodiments, the scaffold disclosures are incorporated by reference for what they disclose in the context of DARPins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to Kunitz domains. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to knottins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to Pronectins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to nanophytins.In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to affilins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to adnectins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to ABDs. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to adilons. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to affimers. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to alphabodies. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to armadillo repeat proteins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to atrimers / tetranectins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to O-bodies / OB-folds. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to sentinins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to lipibodies. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to anticalins. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to atrimers. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to bicyclic peptides. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to cis-knots. In yet another embodiment, scaffold disclosures are incorporated by reference for what they disclose in relation to Fn3 scaffolds (including adnectins, centrilin, pronectin, and Tn3).

[0116] 6.2.2. FGFR3 epitopes The antigen-binding domains (e.g., ABD1, ABD2, ABD3, and / or ABD4) of the MBMs of the present disclosure each specifically bind to fibroblast growth factor receptor 3 ("FGFR3"), e.g., human FGFR3 ("hFGFR3") and / or mouse FGFR3 ("mFGFR3"). The ABDs of the MBMs of the present disclosure may bind to the same or different epitopes on FGFR3. In certain embodiments, one or more ABDs bind to a first epitope on FGFR3, and one or more additional ABDs bind to a second epitope on FGFR3 that is different from the first epitope. If two or more of the ABDs bind to different epitopes on FGFR3, binding to FGFR3 is preferably non-competitive, i.e., the ABDs do not compete for binding to FGFR3 (as may occur, for example, if the epitopes overlap).

[0117] In some embodiments, one, two, or more ABDs of an FGFR3-binding molecule of the present disclosure bind to an epitope comprising a sequence present in D1 of FGFR3. D1 of FGFR3 (also known as the "D1 domain," "Ig-like domain I," "IgG-I," "Ig-I," and "IgD1") consists of amino acids 25-119 of human FGFR3 (hFGFR3) and amino acids 22-118 of mouse FGFR3 (mFGFR3). As used herein, an epitope comprising a sequence present in D1 of FGFR3 refers to a linear or conformational epitope of FGFR3 that contains at least one amino acid of D1 (i.e., at least one of amino acids 25-119 of hFGFR3 or at least one of amino acids 22-118 of mFGFR3). An epitope comprising a sequence present in D1 of FGFR3 may or may not further comprise a sequence present in any other region of FGFR3. In some embodiments, the ABD of the present disclosure binds to an epitope comprising a sequence present in D1 of FGFR3, and the epitope does not comprise a sequence present in D2 or D3 of FGFR3. Exemplary ABDs that bind to an epitope comprising a sequence present in D1 include, but are not limited to, those listed in Table T-4. As shown in Figure 1B, the D1 (Ig-I) domain is outside the region of FGF that binds to FGFR3, and therefore, an ABD that binds to an epitope comprising a sequence present in D1 may not interfere with the binding of FGF to FGFR3, and therefore may be a non-antagonist, or in some cases, an agonist.

[0118] In some embodiments, one, two, or more ABDs of an FGFR3-binding molecule of the present disclosure bind to an epitope comprising a sequence present in D2 of FGFR3. D2 of FGFR3 (also known as the "D2 domain," "Ig-like domain II," "IgG-II," "Ig-II," and "IgD2") consists of amino acids 158-246 of hFGFR3 and amino acids 152-240 of mFGFR3. As used herein, an epitope comprising a sequence present in D2 of FGFR3 refers to a linear or conformational epitope of FGFR3 that contains at least one amino acid of D2 (i.e., at least one of amino acids 158-246 of hFGFR3 or at least one of amino acids 152-240 of mFGFR3). An epitope comprising a sequence present in D2 of FGFR3 may or may not further comprise a sequence present in any other region of FGFR3. In some embodiments, the ABD of the present disclosure binds to an epitope comprising a sequence present in D2 of FGFR3, wherein the epitope further comprises a sequence present in D3 of FGFR3. In other embodiments, the ABD of the present disclosure binds to an epitope comprising a sequence present in D2 of FGFR3, wherein the epitope does not comprise a sequence present in D3 of FGFR3. Exemplary ABDs that bind to an epitope comprising a sequence present in D2 include, but are not limited to, those listed in Table T-2. As shown in FIG. 1B, FGF binds to a region of FGFR3 spanning the D2 (Ig-II) and D3 (Ig-III) domains; therefore, an ABD that binds to an epitope comprising a sequence present in D2 may interfere with the binding of FGF to FGFR3 and may therefore be an antagonist (e.g., by blocking the binding of FGF to FGFR3).

[0119] In some embodiments, one, two, or more ABDs of an FGFR3-binding molecule of the present disclosure bind to an epitope comprising a sequence present in D3 of FGFR3. D3 of FGFR3 (also known as the "D3 domain," "Ig-like domain III," "IgG-III," "Ig-III," and "IgD3") consists of amino acids 255-377 of hFGFR3c, amino acids 250-371 of mFGFR3c, amino acids 255-377 of hFGFR3b, and amino acids 250-371 of mFGFR3b. As used herein, an epitope comprising a sequence present in D3 of FGFR3 describes a linear or conformational epitope of FGFR3 that contains at least one amino acid of D3 (i.e., at least one of amino acids 255-377 of hFGFR3c, amino acids 255-377 of mFGFR3c, amino acids 250-371 of mFGFR3c, amino acids 255-377 of hFGFR3b, or amino acids 250-371 of mFGFR3b). An epitope comprising a sequence present in D3 of FGFR3 may or may not further comprise a sequence present in any other region of FGFR3. In some embodiments, the FGFR3 epitope comprises a sequence present in D3 of FGFR3b but not in D3 of FGFR3c. In other embodiments, the FGFR3 epitope comprises a sequence present in D3 of FGFR3b and also present in D3 of FGFR3c. In some embodiments, the ABD of the present disclosure binds to an epitope comprising a sequence present in D3 of FGFR3, wherein the epitope further comprises a sequence present in D2 of FGFR3. In other embodiments, the ABD of the present disclosure binds to an epitope comprising a sequence present in D3 of FGFR3, wherein the epitope does not comprise a sequence present in D2 of FGFR3. Exemplary ABDs that bind to an epitope comprising a sequence present in D3 include, but are not limited to, those set forth in Table T-3.As shown in Figure 1B, FGF binds to a region of FGFR3 spanning the D2 (Ig-II) and D3 (Ig-III) domains; therefore, an ABD that binds to an epitope containing a sequence present in D3 may interfere with the binding of FGF to FGFR3 and thus may be an antagonist (e.g., by blocking FGF binding to FGFR3).

[0120] The binding properties of FGFR3-binding agents, for example, whether they bind to epitopes in D1, D2, and / or D3, can be easily confirmed by those skilled in the art using methods known in the art.The identification of the binding site of FGFR3-binding antibodies on FGFR3 can be achieved through known techniques, including, for example, array-based oligopeptide scanning, cross-linking mass spectrometry, high-throughput shotgun mutagenesis epitope mapping, hydrogen-deuterium exchange, site-directed mutagenesis mapping, X-ray cocrystallography, and cryo-electron microscopy.Alternatively, the binding of FGFR3-binding agents to D1, D2, and / or D3 can be detected by immunoassays, such as enzyme-linked immunosorbent assay (ELISA), Luminix bead-based assay, mesoscale discovery (MSD), αLISA, and flow cytometry.

[0121] Assays for measuring binding competition between antibodies and antibody fragments are known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) assay, and surface plasmon resonance assay.Competition for binding to FGFR3 can be determined, for example, using real-time, label-free biolayer interferometry assay on Octet HTX biosensor platform (Pall ForteBio Corp.).In a specific embodiment of the assay, the entire assay is carried out in a buffer solution of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EBT buffer) at 25 ° C, and the plate is shaken at a speed of 1000 rpm. To assess whether two antibodies or antigen-binding fragments thereof can compete with each other for binding to their respective epitopes on their specific target antigens, a penta-His-tagged target antigen ("penta-His" (SEQ ID NO: 90)) is first captured onto an Octetet biosensor chip (Fortebio Inc, #18-5122) coated with an anti-penta-His antibody ("penta-His" (SEQ ID NO: 90)) by immersing the biosensor chip into a well containing the penta-His-tagged target antigen ("penta-His" (SEQ ID NO: 90)). The antigen-captured biosensor chip is then saturated with a first antibody or antigen-binding fragment thereof (hereinafter referred to as Ab-1) by immersion in a well containing a solution of Ab-1 (e.g., a 50 μg / mL solution). The biosensor chip is then subsequently immersed in a well containing a solution of a second antibody or antigen-binding fragment thereof (hereinafter referred to as Ab-2) (e.g., a 50 μg / mL solution). The biosensor chip is washed with HBS-EBT buffer between all assay steps. Real-time binding responses can be monitored throughout the assay, and the binding response can be recorded at the end of each step. The responses of Ab-2 binding to the target antigen precomplexed with Ab-1 can be compared, allowing the competitive / non-competitive behavior of different antibodies / antigen-binding fragments against the same target antigen to be determined.

[0122] 6.2.3. FGFR3 Binding Molecule Format In various embodiments, the FGFR3 binding molecules of the present disclosure comprise two half antibodies, one comprising one or two FGFR3 ABDs and the other comprising one or two FGFR3 ABDs, and the two half antibodies are paired via their Fc regions.

[0123] In one aspect, the first half antibody comprises two Fab domains and an Fc domain, and the second half antibody comprises an Fab domain and an Fc domain. The first and second half antibodies associate via the Fc domain to form an Fc region. In various embodiments, the second Fab domain in the first half antibody can be N-terminal to the first Fab domain (a configuration referred to as 2+1 N-Fab) or C-terminal to the Fc domain (a configuration referred to as 2+1 C-Fab). Examples of such configurations are shown in Figures 2C-2E.

[0124] In another aspect, the first half antibody comprises a Fab, scFv, and Fc domain, and the second half antibody comprises a Fab domain and an Fc domain. The first and second half antibodies associate via the Fc domain to form an Fc region. In various embodiments, the scFv domain of the first half antibody can be N-terminal to the Fab domain (a configuration referred to as 2+1 N-scFv) or C-terminal to the Fc domain (a configuration referred to as 2+1 C-scFv). Examples of such configurations are shown in Figures 2F and 2G.

[0125] In another aspect, the first half antibody comprises a Fab, scFv, and Fc domain, and the second half antibody comprises a Fab, scFv, and Fc domain. The first and second half antibodies associate via the Fc domain to form the Fc region. In various embodiments, the scFv domain can be N-terminal to the Fab domain (a configuration referred to as 2+2 N-scFv) or C-terminal to the Fc domain (a configuration referred to as 2+2 C-scFv). Examples of such configurations are shown in Figures 2L and 2M.

[0126] In another aspect, a first half antibody comprises a first Fab, a second Fab, and an Fc domain, and a second half antibody comprises a first Fab, a second Fab, and an Fc domain. The first and second half antibodies associate via the Fc domain to form an Fc region. In various embodiments, the first Fab domain can be N-terminal to the second Fab domain (a configuration referred to as 2+2 N-Fab) or C-terminal to the Fc domain (a configuration referred to as 2+2 C-Fab). Examples of such configurations are shown in Figures 2H-2K and 2N-2P.

[0127] In some embodiments, the FGFR3 binding molecule is or comprises antigen-binding portions arranged in a 2+2 N-scFv format. (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first scFv to which it is operably linked, (ii) a first heavy chain region of a first Fab to which it is operably linked, (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second scFv to which it is operably linked, (ii) a second heavy chain region of a second Fab to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab.

[0128] The scFv can be linked to the first heavy chain region via a linker, e.g., a peptide linker (a) at least 5, at least 6, or at least 7 amino acids in length, and optionally (b) up to 30, 40, 50, or 60 amino acids in length. In various embodiments, the linker is 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, 6 to 20, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids in length.

[0129] The peptide linker can include a multimer of GnS (SEQ ID NO: 91) or SGn (SEQ ID NO: 92), for example, a multimer where n is an integer from 1 to 7 (e.g., a multimer of G4S (SEQ ID NO: 93)), and / or a multimer of glycine (e.g., two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 94), five consecutive glycines (5Gly) (SEQ ID NO: 95), six consecutive glycines (6Gly) (SEQ ID NO: 96), seven consecutive glycines (7Gly) (SEQ ID NO: 97), eight consecutive glycines (8Gly) (SEQ ID NO: 98), or nine consecutive glycines (9Gly) (SEQ ID NO: 99)).

[0130] In some embodiments, the FGFR3 binding molecule is or comprises antigen-binding portions arranged in a 2+2 N-Fab format. (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab, to which it is operably linked, (ii) a second heavy chain region of a second Fab, to which it is operably linked, and (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab, to which it is operably linked, (ii) a fourth heavy chain region of a fourth Fab, to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with a third heavy chain region to form a third Fab; (f) a sixth polypeptide chain comprising a fourth light chain paired with a fourth heavy chain region to form a fourth Fab.

[0131] The first and second Fabs, e.g., the first heavy chain region of the first Fab and the second heavy chain region of the second Fab, are in some embodiments connected via a linker, e.g., (a) a peptide linker at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally (b) a peptide linker at most 30 amino acids, at most 40 amino acids, at most 45 amino acids, at most 50 amino acids, or at most 60 amino acids in length. In various embodiments, the linker is from 5 to 50 amino acids in length, from 5 to 45 amino acids in length, from 5 to 40 amino acids in length, from 5 to 35 amino acids in length, from 5 to 30 amino acids in length, from 5 to 25 amino acids in length, from 5 to 20 amino acids in length, from 6 to 50 amino acids in length, from 6 to 45 amino acids in length, from 6 to 40 amino acids in length, from 6 to 35 amino acids in length, from 6 to 30 amino acids in length, from 6 to 25 amino acids in length, from 6 to 20 amino acids in length, from 7 to 40 amino acids in length, from 7 to 35 amino acids in length, from 7 to 30 amino acids in length, from 7 to 25 amino acids in length, or from 7 to 20 amino acids in length. The peptide linker can include a multimer of GnS (SEQ ID NO: 91) or SGn (SEQ ID NO: 92), for example, a multimer where n is an integer from 1 to 7 (e.g., a multimer of G4S (SEQ ID NO: 93)), and / or a multimer of glycine (e.g., two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 94), five consecutive glycines (5Gly) (SEQ ID NO: 95), six consecutive glycines (6Gly) (SEQ ID NO: 96), seven consecutive glycines (7Gly) (SEQ ID NO: 97), eight consecutive glycines (8Gly) (SEQ ID NO: 98), or nine consecutive glycines (9Gly) (SEQ ID NO: 99)).

[0132] The third and fourth Fabs, e.g., the first heavy chain region of the first Fab and the second heavy chain region of the second Fab, are in some embodiments connected via a linker, e.g., (a) a peptide linker at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally (b) a peptide linker at most 30 amino acids, at most 40 amino acids, at most 45 amino acids, at most 50 amino acids, or at most 60 amino acids in length. In various embodiments, the linker is from 5 to 50 amino acids in length, from 5 to 45 amino acids in length, from 5 to 40 amino acids in length, from 5 to 35 amino acids in length, from 5 to 30 amino acids in length, from 5 to 25 amino acids in length, from 5 to 20 amino acids in length, from 6 to 50 amino acids in length, from 6 to 45 amino acids in length, from 6 to 40 amino acids in length, from 6 to 35 amino acids in length, from 6 to 30 amino acids in length, from 6 to 25 amino acids in length, from 6 to 20 amino acids in length, from 7 to 40 amino acids in length, from 7 to 35 amino acids in length, from 7 to 30 amino acids in length, from 7 to 25 amino acids in length, or from 7 to 20 amino acids in length. The peptide linker can include a multimer of GnS (SEQ ID NO: 91) or SGn (SEQ ID NO: 92), for example, a multimer where n is an integer from 1 to 7 (e.g., a multimer of G4S (SEQ ID NO: 93)), and / or a multimer of glycine (e.g., two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 94), five consecutive glycines (5Gly) (SEQ ID NO: 95), six consecutive glycines (6Gly) (SEQ ID NO: 96), seven consecutive glycines (7Gly) (SEQ ID NO: 97), eight consecutive glycines (8Gly) (SEQ ID NO: 98), or nine consecutive glycines (9Gly) (SEQ ID NO: 99)).

[0133] In the foregoing embodiments, the Fab can be any Fab described in Section 6.2.5 and the scFv can be any scFv described in Section 6.2.4.

[0134] In some embodiments, the FGFR3 binding molecules of the present disclosure comprise an Fc heterodimer, e.g., as described in Section 6.3.2, and may also comprise one or more mutations that reduce effector function, e.g., as described in Section 6.3.1. Examples of Fc heterodimers include Fc regions with star mutations and / or knobs-in-hole mutations. In other embodiments, the MBMs of the present disclosure comprise an Fc homodimer.

[0135] In some embodiments, an FGFR3 binding molecule of the present disclosure has a pair of Fc domains as described in Section 6.3.4. 6.2.4 scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFv are the linkers identified in Section 6.2.6.

[0136] As used herein, unless otherwise specified, an scFv may have a VL variable region and a VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0137] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. To generate nucleic acids encoding scFvs, DNA fragments encoding the VH and VL can be operably linked to another fragment encoding a linker, for example, a fragment encoding any of the linkers described in Section 6.2.6 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 100)), and the VH and VL sequences can be expressed as VL and VH regions joined by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0138] Fab The MBMs of the present disclosure can comprise one or more Fab domains, and typically comprise at least one Fab domain in each half antibody, hi certain embodiments, the MBMs of the present disclosure comprise four Fab domains, with two Fab domains in each half antibody.

[0139] Fab domains have traditionally been generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the MBMs of the present disclosure, the Fab domains are typically recombinantly expressed as part of a larger molecule. The Fab domains can include constant and variable region sequences from any suitable species and thus may be murine, chimeric, human, or humanized.

[0140] The Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0141] For the MBMs of the present disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to enable correct association of Fab domains belonging to the same ABS and minimize aberrant pairing of Fab domains belonging to different ABSs. For example, the Fab heterodimerization strategy shown in Table F-1 below can be used.

[0142] [Table 5]

[0143] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is facilitated by exchanging the VL and VH domains of the Fab with one another, or by exchanging the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0144] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with other Fab components.

[0145] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0146] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of the structural and chemical match between the two interacting surfaces.

[0147] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0148] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0149] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0150] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0151] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0152] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with a T cell receptor constant domain and the CL domain with a T cell receptor b domain, pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0153] Instead of, or in addition to, using a Fab heterodimerization strategy to promote correct VH-VL pairing, a VL from a common light chain (also referred to as a universal light chain) can be used in each FabVL region of the MBMs of the present disclosure. In various embodiments, using a common light chain as described herein reduces the number of incorrect species of the MBM compared to using the original cognate VL. In various embodiments, the VL domain of the MBM is identified from a monospecific antibody that includes a common light chain. In various embodiments, the VH region of the MBM contains human heavy chain variable gene segments that are rearranged in vivo in mouse B cells that have been previously engineered to express a limited human light chain repertoire or a single human light chain, cognate to the human heavy chain, and in response to exposure to an antigen of interest, generates an antibody repertoire that includes one of two possible human VLs or multiple human VHs that are cognate to one of the human heavy chains, and this antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0154] Linker In certain aspects, the present disclosure provides MBMs in which two or more components of an ABD (e.g., the VH and VL of an scFv), two or more ABDs (e.g., the first Fab and second Fab of a half antibody), or an ABD and a non-ABD component (e.g., a Fab or scFv and an Fc domain) are connected to each other by a peptide linker. Such linkers are sometimes referred to herein as "ABD linkers."

[0155] Peptide linkers can range from 2 to 60 or more amino acids, and in certain embodiments, peptide linkers range from 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0156] In certain embodiments, the peptide linker, e.g., the peptide linker separating the Fab and heavy chain at their C-termini, is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0157] In some of the foregoing embodiments, the linker is between 5 and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other of the foregoing embodiments, the linker is between 6 and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In still other of the foregoing embodiments, the linker is between 7 and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0158] In certain embodiments, charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible ABD linkers that can be used in the MBMs of the present disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369, and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 116) or SG n (SEQ ID NO: 117), where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is, for example, (GGGGS) n (SEQ ID NO: 93), or comprises a monomer or multimer of repeats of G4S (SEQ ID NO: 93).

[0159] Polyglycine linkers are suitable for use in the MBMs of the present disclosure. In some embodiments, the peptide linker, e.g., the peptide linker separating the first and second Fab domains, comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO: 94), five consecutive glycines (5 Gly) (SEQ ID NO: 95), six consecutive glycines (6 Gly) (SEQ ID NO: 96), seven consecutive glycines (7 Gly) (SEQ ID NO: 97), eight consecutive glycines (8 Gly) (SEQ ID NO: 98), or nine consecutive glycines (9 Gly) (SEQ ID NO: 99).

[0160] 6.3. Fc area In certain embodiments, the MBMs of the present disclosure comprise a pair of Fc domains that associate to form an Fc region. In natural antibodies, Fc regions include a hinge region at their N-terminus to form the Fc domain. Throughout this disclosure, unless otherwise specified, reference to an Fc domain includes an Fc domain having a hinge domain at its N-terminus.

[0161] The Fc domain can be derived from any suitable species and operably linked to the ABD or its components. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the antigen-binding domain of the MBM of the present disclosure is fused to an IgG Fc molecule. The antigen-binding domain can be fused to the N-terminus, C-terminus, or both of the IgG Fc domain.

[0162] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0163] The two Fc domains within the Fc region can be identical or different from one another. In natural antibodies, the Fc domains are typically identical, but for purposes of producing multispecific binding molecules, such as the MBMs described herein, the Fc domains can advantageously be different to allow heterodimerization, as described in Section 6.3.2 below. In other embodiments, the two Fc domains of the MBMs disclosed herein are the same.

[0164] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0165] In the MBMs of the present disclosure, the Fc region, and / or Fc domains therein, can comprise heavy chain constant domains from one or more different classes of antibodies, for example, from one, two, or three different classes.

[0166] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG1. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG2.

[0167] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG3. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG4.

[0168] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from an IgG and a CH4 domain derived from an IgM.

[0169] It will be understood that the heavy chain constant domains used to produce the Fc region for the MBMs of the present disclosure can include variants of the naturally occurring constant domains described above. Such variants can include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0170] IgM and IgA naturally occur in humans as shared multimers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated and as a hexamer when the J chain is absent. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the MBM of the present disclosure does not include a tail.

[0171] The Fc domain incorporated into the MBMs of the present disclosure may contain one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.3.1.

[0172] The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric MBMs, for example, by enabling heterodimerization, the preferential pairing of non-identical Fc domains with identical Fc domains. Heterodimerization allows for the production of MBMs in which different polypeptide components are connected to each other by Fc regions that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.3.2.

[0173] It will be understood that any of the above modifications can be combined in any suitable manner to achieve desired functional properties and / or combined with other modifications to alter the properties of the MBM.

[0174] Exemplary Fc domain sequences are provided in Table H-1 below.

[0175] [Table 6-1]

[0176] [Table 6-2]

[0177] [Table 6-3]

[0178] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the sequences disclosed in Table H-1.

[0179] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 3. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 3 (e.g., 90%-99% sequence identity to SEQ ID NO: 3), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.3.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.3.2).

[0180] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 4. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 4 (e.g., 90%-99% sequence identity to SEQ ID NO: 4), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.3.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.3.2).

[0181] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 5. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 5 (e.g., 90%-99% sequence identity to SEQ ID NO: 5), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.3.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.3.2).

[0182] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 6. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 6 (e.g., 90%-99% sequence identity to SEQ ID NO: 6), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.3.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.3.2).

[0183] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:7.

[0184] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:8.

[0185] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:9.

[0186] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:10.

[0187] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:11.

[0188] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:12.

[0189] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:13.

[0190] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:14.

[0191] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:15.

[0192] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:16.

[0193] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:17.

[0194] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:18.

[0195] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:19.

[0196] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:20.

[0197] 6.3.1. Fc Domains with Altered Effector Functions In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

[0198] In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0199] In one embodiment, the Fc domain (e.g., the Fc domain of an MBM half antibody) or Fc region (e.g., one or both Fc domains of an MBM that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In more specific embodiments, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In more specific embodiments, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”).

[0200] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering).

[0201] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU numbering) to reduce effector function.

[0202] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table H-2 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below:

[0203] [Table 7-1]

[0204] [Table 7-2]

[0205] [Table 7-3]

[0206] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, and is sometimes referred to herein as IgG4s or hIgG4s.

[0207] For heterodimeric Fc regions, it is possible to incorporate combinations of the above-mentioned variant IgG4 Fc sequences, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or a bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or a bolded portion thereof).

[0208] 6.3.2. Fc Heterodimerization Variants Certain MBMs differ from natural immunoglobulins in that they involve dimerization between two Fc domains operably linked to non-identical N- or C-terminal regions. Insufficient heterodimerization of two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a challenge for purification. Various approaches available in the art can be used to enhance the dimerization of the Fc domains present in the MBMs of the present disclosure, as disclosed in, for example, EP1870459A1, U.S. Patent No. 5,582,996, U.S. Patent No. 5,731,168, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441, U.S. Patent No. 7,183,076, U.S. Patent Application Publication No. 2006 / 204493A1, and PCT Publication No. WO2009 / 089004A1.

[0209] In some embodiments, the present disclosure provides MBMs comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably the IgG (lgG1, lgG2, lgG3, and lgG4) class, as described in the preceding section.

[0210] In specific embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and prevent homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0211] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0212] In certain such embodiments, in a first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In further embodiments, the first Fc domain additionally has a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), and the second Fc domain additionally has a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU index numbering).

[0213] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of a first Fc domain and a second Fc domain of an Fc region.

[0214] Alternatively, or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide contains a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that results in a heterodimeric protein. See, e.g., U.S. Patent No. 8,586,713. As such, the MBM comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the MBM to Protein A compared to a corresponding MBM lacking the amino acid difference. In one embodiment, the first CH3 domain binds to Protein A and the second CH3 domain contains a mutation / modification, e.g., an H95R modification (according to IMGT exon numbering, H435R according to EU numbering), that reduces or eliminates Protein A binding. The second CH3 may further comprise a Y96F modification (Y436F by EU, by IMGT). This class of modifications is referred to herein as a "star" mutation.

[0215] In some embodiments, the Fc may contain one or more mutations to promote heterodimerization (eg, knob and hole mutations) and a star mutation to facilitate purification.

[0216] 6.3.3. Hinge Domain The MBMs of the present disclosure can include an Fc domain comprising a hinge domain at its N-terminus. The hinge region can be a natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge domain," unless otherwise indicated by context, refers to a naturally occurring or non-naturally occurring hinge sequence, which can be a monomeric hinge domain in the context of a single or monomeric polypeptide chain, or can include two associated hinge sequences on separate polypeptide chains in the context of a dimeric polypeptide (e.g., a homodimeric or heterodimeric MBM formed by the association of two Fc domains). The two associated hinge sequences are sometimes referred to as "hinge regions." In certain embodiments of the MBMs of the present disclosure, additional repeats of the hinge region can be incorporated into the polypeptide sequence.

[0217] A native hinge region is typically the hinge region found between the Fab and Fc domains of naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions can include complete hinge regions derived from antibodies of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion or repeat units of a native hinge, with each repeat unit derived from a native hinge region. In a further alternative, the native hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0218] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0219] In one embodiment, the MBM of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge domain at their N-terminus. In various embodiments, positions 233-236 in the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered according to EU numbering.

[0220] In some embodiments, the MBMs of the present disclosure comprise a modified hinge region that reduces binding affinity to Fcγ receptors compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0221] In one embodiment, an MBM of the present disclosure comprises an Fc region in which each Fc domain has an intact hinge domain at its N-terminus, the Fc domain and hinge domain each being derived from IgG4, and each hinge domain containing the modified sequence CPPC (SEQ ID NO: 106). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 107), compared to IgG1, which contains the sequence CPPC (SEQ ID NO: 106). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within an IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows for the complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0222] 6.3.3.1. Chimeric hinge sequences The hinge domain can be a chimeric hinge domain. For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0223] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 108) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 109) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may be suitably linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.3.1).

[0224] 6.3.3.2. Hinge Sequences with Reduced Effector Function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016 / 161010A2, the entire contents of which are incorporated herein by reference. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering (as shown in Figure 1 of WO2016 / 161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an empty position.

[0225] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016 / 161010A2).

[0226] Hinge modifications within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively (SEQ ID NO: 106).

[0227] Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO:110) (previously disclosed as SEQ ID NO:1 in WO2016 / 161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:111) (previously disclosed as SEQ ID NO:2 in WO2016 / 161010A2), CPPCPAPG---GPSVF (SEQ ID NO:112) (previously disclosed as SEQ ID NO:3 in WO2016 / 161010A2), or CPPCPAP----GPSVF (SEQ ID NO:113) (previously disclosed as SEQ ID NO:4 in WO2016 / 161010A2).

[0228] The modified hinge regions described above can be incorporated into heavy chain constant regions, which typically include a CH2 domain and a CH3 domain and may have additional hinge segments (e.g., upper hinges) adjacent to the designated regions. Such additional constant region segments are typically of the same isotype, preferably a human isotype, but can be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can also be human IgG1, IgG2, or IgG3, or hybrids thereof, in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 to 4 of WO2016 / 161010A2.

[0229] In certain embodiments, a modified hinge sequence can be linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domain to reduce effector function, e.g., as described in Section 6.3.1).

[0230] 6.3.4. Chimeric Fc Domains In some embodiments, the Fc domain may be "chimeric," comprising Fc domain sequences from two or more immunoglobulin isotypes. In some embodiments, the chimeric Fc domain has sequences from different IgG isotypes (e.g., any two of IgG1, IgG2, IgG3, and IgG4). A "chimeric" Fc domain includes, for example, an Fc domain comprising a chimeric hinge sequence as described in Section 6.3.3.1.

[0231] An exemplary chimeric Fc domain is referred to herein as the "IgG1PVA" isotype or similar terminology and comprises an IgG1 upper hinge domain, an IgG1 core hinge domain, and an IgG1 lower hinge domain with a substitution / deletion mutation ELLG (SEQ ID NO: 114) → PVA- (or "PVA-deleted") ("ELLG" (SEQ ID NO: 114)) at amino acid positions 233-236 (EU numbering), an IgG1 CH2 domain, and an IgG1 CH3 domain. The ELLG (SEQ ID NO: 114) → PVA- (or "PVA-deleted") ("ELLG" (SEQ ID NO: 114)) modification incorporates an IgG2 sequence into an IgG1. In certain embodiments, the chimeric Fc domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 8 (hIgG1 PVA).

[0232] The chimeric Fc domain can be further modified, for example, to further alter effector function (e.g., as described in Section 6.3.1) and / or to facilitate correct pairing or purification with the asymmetric half-antibodies of MBM (e.g., as described in Section 6.3.2).

[0233] In certain embodiments, there is provided an MBM of two Fc domains of the present disclosure that form an Fc heterodimer, wherein the two Fc domains comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 8 (hIgG1 PVA Fc domain); Both Fc domains contain a PVA-deletion sequence at amino acids 233-236 (EU numbering), one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L368A, and Y407V; Optionally, one or both Fc domains comprise star mutations H435R and Y436F; Both Fc domains contain the disulfide structural mutation S354C or E356C, or neither Fc domain contains it.

[0234] In certain embodiments, the MBMs of the present disclosure comprise two Fc domains that form an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 13, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)) and the knob mutation T366W; and a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17, where the amino acid sequence has less than 100% identity to SEQ ID NO: 17, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)) and hole mutations T366S, L368A, and Y407V.

[0235] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 13, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)) and the knob mutation T366W; a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, where the amino acid sequence has less than 100% identity to SEQ ID NO: 18, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), hole mutations T366S, L368A, and Y407V, and star mutations H435R and Y436F.

[0236] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 14, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), knob mutation T366W, and star mutations H435R and Y436F; and a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17, where the amino acid sequence has less than 100% identity to SEQ ID NO: 17, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)) and hole mutations T366S, L368A, and Y407V.

[0237] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 14, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), knob mutation T366W, and star mutations H435R and Y436F; a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18, where the amino acid sequence has less than 100% identity to SEQ ID NO: 18, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), hole mutations T366S, L368A, and Y407V, and star mutations H435R and Y436F.

[0238] In another specific embodiment, the MBM of the present disclosure comprises two Fc domains comprising an Fc heterodimer, wherein the two Fc domains are a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 15, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), a disulfide structure mutation S354C, and a knob mutation T366W; a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15, where the amino acid sequence has less than 100% identity to SEQ ID NO: 15, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), disulfide structure mutation S354C, and hole mutations T366S, L368A, and Y407V.

[0239] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 15, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), a disulfide structural mutation S354C (or alternatively, the structural mutation S354C is replaced with a disulfide structural mutation E356C), and a knob mutation T366W; and a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20, where the amino acid sequence has less than 100% identity to SEQ ID NO:20, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), disulfide structural mutation S354C (or alternatively, structural mutation S354C is replaced with disulfide structural mutation E356C), hole mutations T366S, L368A, and Y407V, and star mutations H435R and Y436F.

[0240] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 16, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), a disulfide structural mutation S354C (or alternatively, the structural mutation S354C is replaced with a disulfide structural mutation E356C), a knob mutation T366W, and star mutations H435R and Y436F; and a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19, where the amino acid sequence has less than 100% identity to SEQ ID NO: 19, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), disulfide structural mutation S354C (or alternatively, structural mutation S354C is replaced with disulfide structural mutation E356C), and hole mutations T366S, L368A, and Y407V.

[0241] In another specific embodiment, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains are: a first Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, where if the amino acid sequence has less than 100% identity to SEQ ID NO: 16, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), a disulfide structural mutation S354C (or alternatively, the structural mutation S354C is replaced with a disulfide structural mutation E356C), a knob mutation T366W, and star mutations H435R and Y436F; and a second Fc domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20, where the amino acid sequence has less than 100% identity to SEQ ID NO:20, the sequence retains a PVA modification in the hinge (a PVA- deletion at amino acid positions 233-236 (EU numbering)), disulfide structural mutation S354C (or alternatively, structural mutation S354C is replaced with disulfide structural mutation E356C), hole mutations T366S, L368A, and Y407V, and star mutations H435R and Y436F.

[0242] In still further embodiments, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity to SEQ ID NO: 11 (also referred to as hIgG1 N180G, hIgG1 N297G); Both Fc domains contain N180G / N297G amino acid substitutions; one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L368A, and Y407V; Optionally, one or both Fc domains comprise star mutations H435R and Y436F; Both Fc domains contain the disulfide structural mutation S354C or E356C, or neither Fc domain contains it.

[0243] In still further embodiments, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity to SEQ ID NO: 21 (also referred to as hIgG4 S108P, hIgG4 S228P); Both Fc domains contain S108P / S228P amino acid substitutions, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L368A, and Y407V; Optionally, one or both Fc domains comprise star mutations H435R and Y436F; Both Fc domains contain the disulfide structural mutation S354C or E356C, or neither Fc domain contains it.

[0244] In still further embodiments, the two Fc domain MBM of the present disclosure comprises an Fc heterodimer, wherein the two Fc domains comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% sequence identity to SEQ ID NO: 7 (variant IgG4s); Both Fc domains contain S108P / S228P amino acid substitutions, one Fc domain contains the knob mutation T366W and the other Fc domain contains the hole mutations T366S, L368A, and Y407V; Optionally, one or both Fc domains comprise star mutations H435R and Y436F; Both Fc domains contain the disulfide structural mutation S354C or E356C, or neither Fc domain contains it.

[0245] 6.4. Antibody-Drug Conjugates The MBMs of the present disclosure can be conjugated to a drug moiety, for example, via a linker, particularly when the MBMs are intended for use as cancer therapeutics. For convenience, such conjugates are referred to herein as antibody-drug conjugates (or "ADCs").

[0246] In certain aspects, the drug moiety exerts cytotoxic or cytostatic activity. In one embodiment, the drug moiety is selected from the group consisting of maytansinoids, kinesin-like protein KIF11 inhibitors, V-ATPase (vacuolar H+-ATPase) inhibitors, pro-apoptotic agents, Bcl2 (B-cell lymphoma 2) inhibitors, MCL1 (myeloid cell leukemia 1) inhibitors, HSP90 (heat shock protein 90) inhibitors, IAP (inhibitors of apoptosis) inhibitors, mTOR (mechanistic target of rapamycin) inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins, dolastatins, MetAP (methionine aminopeptidase), CRM1 (chromosome maintenance 1) inhibitors, DP The inhibitor is selected from PIV (dipeptidyl peptidase IV) inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphoryl transfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 (cyclin-dependent kinase 2) inhibitors, CDK9 (cyclin-dependent kinase 9) inhibitors, kinin inhibitors, HDAC (histone deacetylase) inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor group binders, RNA polymerase inhibitors, topoisomerase inhibitors, or DHFR (dihydrofolate reductase) inhibitors.

[0247] In some embodiments, the cytotoxic agent is a maytansinoid having the following structure:

[0248] [ka]

[0249] In some embodiments, the cytotoxic agent is a maytansinoid having the following structure:

[0250] [ka]

[0251] In some embodiments, the ADC comprises an MBM of the present disclosure,

[0252] [ka]

[0253] During the ceremony,

[0254] [ka]

[0255] is the binding to MBM. In some embodiments, the antibody-drug conjugate comprises an MBM of the present disclosure,

[0256] [ka]

[0257] During the ceremony,

[0258] [ka]

[0259] is the binding to MBM. In some embodiments, the ADC comprises an MBM of the present disclosure,

[0260] [ka]

[0261] or a mixture thereof, During the ceremony,

[0262] [ka]

[0263] is binding to the MBM of the present disclosure. In some embodiments, the bond is linked to the MBM via the sulfur moiety of a cysteine ​​residue.

[0264] In some embodiments, the bond is linked to the MBM via the nitrogen moiety of a lysine residue. In the ADCs of the present disclosure, cytotoxic and / or cytostatic agents are linked to the MBM by an ADC linker. The ADC linker linking the cytotoxic and / or cytostatic agent to the MBM of the ADC can be short, long, hydrophobic, hydrophilic, flexible, or rigid, or can be composed of segments each independently possessing one or more of the above-mentioned properties, such that the linker can contain segments with different properties. Linkers can be multivalent, covalently linking two or more agents to a single site on the MBM, or monovalent, covalently linking a single agent to a single site on the MBM.

[0265] In certain aspects, the linker is selected from a cleavable linker, a non-cleavable linker, a hydrophilic linker, a pro-charged linker, or a dicarboxylic acid-based linker. As will be appreciated by those skilled in the art, the ADC linker links the cytotoxic and / or cytostatic agent to the MBM by forming a covalent linkage to the cytotoxic and / or cytostatic agent at one location and to the MBM at another location, where the covalent linkages are formed by reaction between functional groups on the ADC linker and functional groups on the agent and MBM.

[0266] The ADC linker is preferably chemically stable to conditions outside the cell, but need not be; it can be designed to cleave, break, and / or otherwise specifically degrade inside the cell. Alternatively, an ADC linker that is not designed to specifically cleave or degrade inside the cell may be used. The choice of stable versus unstable ADC linker may depend on the toxicity of the cytotoxic and / or cytostatic agent. For drugs that are toxic to normal cells, a stable linker is preferred. Selective or targeted drugs with lower toxicity to normal cells may be utilized, in which case the chemical stability of the ADC linker to the extracellular environment is less important. A wide variety of ADC linkers useful for linking drugs to MBMs in the context of ADCs are known in the art. Any of these ADC linkers, as well as other ADC linkers, can be used to link cytotoxic and / or cytostatic agents to the MBMs of the ADCs of the present disclosure.

[0267] Exemplary multivalent ADC linkers that can be used to link multiple cytotoxic and / or cytostatic agents to a single MBM molecule are described, for example, in WO2009 / 073445, WO2010 / 068795, WO2010 / 138719, WO2011 / 120053, WO2011 / 171020, WO2013 / 096901, WO2014 / 008375, WO2014 / 093379, WO2014 / 093394, and WO2014 / 093640, the contents of which are incorporated herein by reference in their entireties. For example, the Fleximer linker technology developed by Mersana et al. has the potential to enable high-DAR ADCs with favorable physicochemical properties. Mersana's technology is based on incorporating drug molecules into a solubilizing polyacetal backbone via an array of ester bonds, a methodology that results in highly loaded ADCs (up to 20 DAR) while maintaining favorable physicochemical properties.

[0268] Exemplary monovalent ADC linkers that can be used are described, for example, in Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Ducrry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623, U.S. Patent No. 7,223,837, U.S. Patent No. 8,568,728, U.S. Patent No. 8,535,678, and WO2004 / 010957, each of which is incorporated herein by reference.

[0269] By way of example and not limitation, some cleavable and non-cleavable ADC linkers that can be included in the ADCs of the present disclosure are listed below. In certain embodiments, the selected ADC linker is cleavable in vivo. Cleavable ADC linkers may contain chemically or enzymatically unstable or degradable linkages. Cleavable ADC linkers generally rely on intracellular processes to release the drug, such as reduction in the cytoplasm, exposure to the acidic conditions in lysosomes, or the action of specific proteases or other enzymes within the cell. Cleavable ADC linkers generally incorporate one or more chemical bonds that are cleavable either chemically or enzymatically, while the remainder of the ADC linker is non-cleavable. In certain embodiments, the ADC linker contains a chemically labile group, such as a hydrazone group and / or a disulfide group. Linkers containing chemically labile groups take advantage of the different properties between plasma and some cytoplasmic compartments. The intracellular conditions that promote drug release for hydrazone-containing ADC linkers are the acidic environment of endosomes and lysosomes, while disulfide-containing ADC linkers are reduced in the cytosol, which contains high thiol concentrations, such as glutathione. In certain embodiments, the phenotypic stability of ADC linkers containing chemically labile groups can be increased by introducing steric hindrance using substituents near the chemically labile group.

[0270] A cleavable ADC linker may contain a non-cleavable moiety or segment, and / or a cleavable segment or moiety may be included in an otherwise non-cleavable ADC linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For example, a polyethylene glycol or polymer ADC linker may contain one or more cleavable groups, such as a disulfide, hydrazone, or dipeptide.

[0271] Other degradable linkages that can be included in ADC linkers include ester linkages formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a biologically active agent; such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of amines and aldehydes, phosphate ester linkages formed by the reaction of alcohols with phosphate groups, acetal linkages that are the reaction products of aldehydes and alcohols, orthoester linkages that are the reaction products of formates and alcohols, and phosphoramidite groups, including but not limited to, at the termini of polymers and oligonucleotide linkages formed by the 5' hydroxyl group of an oligonucleotide.

[0272] In certain embodiments, the ADC linker comprises an enzymatically cleavable peptide moiety, e.g., a tripeptide or dipeptide. In certain embodiments, the dipeptide is selected from Val-Cit, Cit-Val, Ala-Ala, Ala-Cit, Cit-Ala, Asn-Cit, Cit-Asn, Cit-Cit, Val-Glu, Glu-Val, Ser-Cit, Cit-Ser, Lys-Cit, Cit-Lys, Asp-Cit, Cit-Asp, Ala-Val, Val-Ala, Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, 111-Cit, Phe-Arg, and Trp-Cit. In certain embodiments, the dipeptide is selected from Cit-Val and Ala-Val.

[0273] In any of the various embodiments of ADCs described above or discussed herein, the ADC can have a drug:antibody ratio (or, in this case, drug:MBM ratio) ranging from 1 to 20, more typically from 2 to 10.

[0274] 6.5. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the MBM of the present disclosure. In some embodiments, the MBM is encoded by a single nucleic acid. In other embodiments, the MBM is encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0275] A single nucleic acid can encode a MBM comprising a single polypeptide chain, an MBM comprising two or more polypeptide chains, or a portion of an MBM comprising three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an MBM comprising three, four or more polypeptide chains, or three polypeptide chains of an MBM comprising four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains can be placed under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0276] In some embodiments, an MBM comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the MBM can be equal to or less than the number of polypeptide chains of the MBM (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0277] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA). In another aspect, the present disclosure provides host cells and vectors comprising the nucleic acids of the present disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0278] Vectors The present disclosure provides vectors comprising nucleotide sequences encoding one or two of the polypeptide chains of an MBM or MBM component described herein, e.g., a half antibody. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0279] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, eastern equine encephalitis virus, and flaviviruses.

[0280] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0281] When the DNA sequence containing expression vector or construct is prepared for expression, expression vector can be transfected or introduced into suitable host cell.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.The method and conditions for culturing the obtained transfected cells and recovering expressed polypeptide are known to those skilled in the art, and can be changed or optimized according to the specific expression vector and mammalian host cell used based on this specification.

[0282] 6.5.2. Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0283] In one embodiment, the host cell is genetically engineered by using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host that is compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for effecting expression, such as an inducible promoter, can also be used.

[0284] The present disclosure also provides host cells comprising the vectors described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0285] 6.6. Pharmaceutical Compositions 6.6.1. Pharmaceutical Compositions Comprising MBM The MBM of the present disclosure may be in the form of a composition comprising the MBM and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the MBM and, in the case of therapeutic applications, the mode of administration.

[0286] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to a patient). The pharmaceutical composition may be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0287] The pharmaceutical composition can be conveniently presented in a unit dosage form containing a predetermined amount of MBM of the present disclosure per dose.The amount of MBM contained in a unit dosage will depend on the disease being treated and other factors well known in the art.Such a unit dosage can be in the form of a lyophilized dry powder containing an amount of MBM suitable for a single administration, or in liquid form.The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent, and / or other components useful for administration.A unit dosage in liquid form can be conveniently provided in the form of a syringe pre-filled with an amount of MBM suitable for a single administration.

[0288] The pharmaceutical composition may also be supplied in bulk, as it contains an amount of MBM suitable for multiple administration. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing MBM of the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington, The Science and Practice of Pharmacy, 23rd edition (Adejare, ed. 2020). Such additives should be nontoxic to recipients at the dosages and concentrations used.

[0289] Buffering agents help maintain pH in a range close to physiological conditions. They can be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium glyconate mixtures, etc.), Examples of buffers include oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) may be used.

[0290] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," may be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives, and help to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents, Stabilizers can be selected from the following: stabilizers (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate), low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as xylose, mannose, fructose, and glucose, disaccharides such as lactose, maltose, sucrose, and trehalose, trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers can be present in amounts ranging from 0.5 to 10% by weight per weight of MBM.

[0291] Nonionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the glycoprotein and to protect it from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable nonionic surfactants include polysorbates (e.g., 20, 80), polyoxamers (e.g., 184, 188), and pluronic polyols. The nonionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, e.g., about 0.07 mg / mL to about 0.2 mg / mL.

[0292] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0293] 6.6.2. Pharmaceutical Compositions for Delivery of Nucleic Acids Encoding MBM The MBM of the present disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or via a viral vector encoding the MBM under the control of a suitable promoter.

[0294] Exemplary gene therapy vectors include adenovirus or AAV-based therapeutics.Non-limiting examples of adenovirus- or AAV-based therapeutics for use in the methods, uses, or compositions herein include, for example, rAd-p53 (also known as Gendicine®, Genkaxin®), a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53, for use in the treatment of cancer; Ad5 d11520 (also known as H101 or ONYX-015), an adenovirus lacking the E1B gene to inactivate host p53; see, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670); AD5-D24-GM-CSF (Cerullo et al., 2010, Cancer Res. 70:4297; e.g., rAd-HSVtk, a replication-deficient adenovirus carrying the HSV thymidine kinase gene for the treatment of cancer (Cerepro®, developed by Ark Therapeutics, see, e.g., U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak™; International PCT Application No. WO 2005 / 049094), a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter for the treatment of cancer (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7, for example, Ad-IFNβ (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther. 18:852-860), an adenovirus serotype 5 vector for cancer treatment in which the E1 and E3 genes have been deleted and which expresses the human interferon beta gene under the direction of the cytomegalovirus (CMV) immediate-early promoter.

[0295] The nucleic acid molecule (e.g., mRNA) or virus can be formulated as the only pharmaceutically active ingredient in the pharmaceutical composition, or can be combined with other active agents for the specific disease being treated. Optionally, other medicinal agents, pharmaceuticals, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives, antioxidants, chelating agents, and inert gases can also be present in the composition. Exemplary other agents and excipients that may be included in the compositions include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid, and the like.

[0296] 6.7. Treatment Indications and Methods The MBM of the present disclosure can be used to treat any proliferative disorder (e.g., cancer) that expresses FGFR3. In certain embodiments, the proliferative disorder is a cancer that expresses (e.g., overexpresses) FGFR3. Exemplary cancers that can be treated using the MBM of the present disclosure include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial cancer, Burkitt's lymphoma, cancer of unknown primary cancer, cardiac cancer, cervical cancer, spinal cord tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, and cutaneous T-cell lymphoma. , ductal carcinoma, embryonal tumor, endometrial cancer, epithelioma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoma-like tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytoma, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, epithelial lobular Cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract cancer with Nat gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer In some embodiments, the cancer is bladder cancer, but is not limited to pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.In some embodiments, the bladder cancer is FGFR3-mutated bladder cancer. In some embodiments, the bladder cancer is FGFR3-TACC3 fusion bladder cancer. In some embodiments, the bladder cancer is FGFR3-S249C bladder cancer. In some embodiments, the bladder cancer is FGFR3-R248C bladder cancer. In some embodiments, the bladder cancer is FGFR3-G372C bladder cancer. In some embodiments, the bladder cancer is FGFR3-G370C bladder cancer. In some embodiments, the bladder cancer is FGFR3-Y375C bladder cancer. In some embodiments, the bladder cancer is FGFR3-Y373C bladder cancer. In some embodiments, the bladder cancer is FGFR3-K650E bladder cancer.

[0297] In certain embodiments, disclosed is a method for treating FGFR3-positive bladder cancer using the FGFR3 MBM of the present disclosure.For example, certain embodiments are directed to administering to a subject who needs treatment for bladder cancer (for example, FGFR3-positive or FGFR3-mutated bladder cancer) a pharmaceutical composition comprising an FGFR3 binding molecule comprising four ABDs, wherein one or more ABDs bind to D1 of FGFR3, and one or more ABDs bind to D2 and / or D3 of FGFR3.The MBM (for example, FGFR3 binding molecule) of the present disclosure can be administered by itself or in any suitable pharmaceutical composition.

[0298] In one aspect, the MBM of the present disclosure is provided for use as a medicine.In another aspect, the MBM of the present disclosure is provided for use in treating disease.In certain embodiments, the MBM of the present disclosure is provided for use in treatment method.In one embodiment, the present disclosure provides the MBM described herein for use in treating disease in a subject who needs to be treated with the disease.

[0299] In certain embodiments, the present disclosure provides an MBM for use in a method of treating a subject having cancer, the method comprising administering to the individual a therapeutically effective amount of the MBM (e.g., an FGFR3-binding molecule). In certain embodiments, the disease being treated is cancer. In certain embodiments, the disease is bladder cancer. In certain embodiments, the bladder cancer is FGFR3-mutated bladder cancer. An "individual" or "subject" according to any of the above embodiments is a mammal, for example, a human.

[0300] In a further aspect, the present disclosure provides use of an MBM of the present disclosure (e.g., an FGFR3 binding molecule) in the manufacture or preparation of a medicament for treating a subject in need of such treatment. In one embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to a subject having the disease. In certain embodiments, the disease being treated is cancer. In certain embodiments, the disease is bladder cancer. In certain embodiments, the bladder cancer is FGFR3-mutated bladder cancer. An "individual" or "subject" according to any of the above embodiments may be a mammal, preferably a human.

[0301] In a further aspect, the present disclosure provides a method for inhibiting FGFR3 dimerization and / or FGFR3 activity in a subject, comprising administering to the subject an effective amount of an MBM (e.g., an FGFR3-binding molecule) of the present disclosure. In some embodiments, the MBM of the present disclosure can prevent the formation of FGFR3 dimers and / or reduce the amount of FGFR3 dimers present on the surface of cells. In some embodiments, the MBM of the present disclosure can prevent the formation of FGFR3 signal activation and / or reduce the amount of FGFR3 signal activity present in cells.

[0302] The appropriate dosage of the MBM of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the route of administration, the patient's weight, the particular MBM, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the MBM, and the judgment of the attending physician. In any event, the medical professional responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0303] MBM is suitably administered to patients at one time or over a series of treatments. For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of MBM can be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion. Typical daily dosages can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment will generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of MBM would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, dosages can also include from about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges derivable from the numerical values ​​recited herein include ranges based on the above numerical values, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that a patient receives about two to about 20, or, for example, about six, doses of MBM). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0304] The MBM of the present disclosure will generally be used in an amount effective to achieve its intended purpose.For use in treating or preventing disease conditions, the MBM of the present disclosure or its pharmaceutical composition will be administered or applied in a therapeutically effective amount.Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0305] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The EC 50 Doses can be formulated in animal models to achieve a circulating concentration range including, but not limited to, 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0306] Initial dosages can also be estimated from in vivo data, for example, animal models, using techniques that are well known in the art. Those skilled in the art will readily be able to optimize human administration based on animal data.

[0307] Dosage and interval can be individually adjusted to provide plasma levels of MBM sufficient to maintain therapeutic efficacy. Typical patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by ELISA or HPLC.

[0308] In cases of local administration or selective uptake, the effective local concentration of MBM may not be related to plasma concentration. One skilled in the art will be able to optimize the therapeutically effective local dosage without undue experimentation.

[0309] The therapeutically effective dose of MBM described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of MBM can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Using cell culture assays and animal studies, LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of ED . MBMs that exhibit a large therapeutic index are preferred. In one embodiment, MBMs according to the present disclosure exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. Dosages are preferably within the ED range with little or no toxicity. 50 The circulating concentration range includes the range of 0.01 to 0.01 mg / kg of the active ingredient. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, which is incorporated herein by reference in its entirety.)

[0310] The attending physician of a patient treated with the MBM of the present disclosure will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is not adequate (excluding toxicity), the attending physician will also know how to adjust treatment to higher levels. The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0311] 6.8. Combination Therapy The MBM of the present disclosure can be administered in combination with one or more other agents in therapy. For example, the MBM of the present disclosure can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in a subject in need of such treatment. Such additional therapeutic agents can include any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, an immunotherapeutic agent (e.g., an immune checkpoint inhibitor), or other cancer therapeutic agent.

[0312] Such other agents are preferably present in combination in amounts that are effective for the intended purpose. The effective amount of such other agents will depend on the amount of MBM used, the type of disorder or treatment, and other factors discussed above. MBM will generally be used in the same dosages and by any route of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0313] Such combination therapy as described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of the MBM of the present disclosure may occur before, simultaneously with, and / or after administration of the additional therapeutic agent.

[0314] 7. Specific embodiments, references While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s), which is exemplified by the numbered embodiments set forth below.

[0315] 1. A multispecific binding molecule (MBM), comprising: (a) an antigen-binding domain 1 (ABD1) that specifically binds to the first epitope of FGFR3; (b) an antigen-binding domain 2 (ABD2) that specifically binds to a second epitope of FGFR3 that is different from the first epitope.

[0316] 2. The MBM of embodiment 1, wherein the first epitope comprises a sequence present in D3 of FGFR3. 3. The MBM of embodiment 1 or 2, wherein the first epitope comprises a sequence present in D2 of FGFR3.

[0317] 4. An MBM according to any one of embodiments 1 to 3, wherein the second epitope comprises a sequence present in D1 of FGFR3. 5. An MBM according to any one of embodiments 1 to 4, wherein ABD2 is an FGFR3 non-antagonist antigen-binding domain.

[0318] 6. The MBM of embodiment 5, wherein ABD2 is an FGFR3 agonist antigen-binding domain. 7. An MBM according to any one of embodiments 1 to 4, wherein ABD2 is an FGFR3 antagonist antigen-binding domain.

[0319] 8. An MBM according to any one of embodiments 1 to 5, wherein ABD1 is an FGFR3 antagonist antigen-binding domain. 9. A multispecific binding molecule (MBM), comprising: (a) an antigen-binding domain 1 (ABD1) that specifically binds to a first epitope comprising a sequence present in D3 of FGFR3; (b) an antigen-binding domain 2 (ABD2) that specifically binds to a second epitope comprising a sequence present in D1 of FGFR3.

[0320] 10. The MBM of embodiment 9, wherein the first epitope further comprises a sequence present in D2 of FGFR3. 11. The MBM of embodiment 9 or 10, wherein ABD1 is an FGFR3 antagonist antigen-binding domain.

[0321] 12. An MBM according to any one of embodiments 9 to 11, wherein ABD2 is an FGFR3 non-antagonist antigen-binding domain. 13. The MBM of embodiment 12, wherein ABD2 is an FGFR3 agonist antigen-binding domain.

[0322] 14. An MBM according to any one of embodiments 9 to 11, wherein ABD2 is an FGFR3 antagonist antigen-binding domain. 15. A multispecific binding molecule (MBM), comprising: (a) an FGFR3 antagonist antigen-binding domain 1 (ABD1) that specifically binds to a first epitope of FGFR3; (b) an FGFR3 non-antagonist antigen-binding domain 2 (ABD2) that specifically binds to a second epitope of FGFR3.

[0323] 16. The MBM of embodiment 15, wherein ABD2 is an FGFR3 agonist antigen-binding domain. 17. The MBM of embodiment 15 or 16, wherein the first epitope comprises a sequence present in D3 of FGFR3.

[0324] 18. An MBM described in any one of embodiments 15 to 17, wherein the first epitope comprises a sequence present in D2 of FGFR3. 19. An MBM described in any one of embodiments 15 to 18, wherein the second epitope comprises a sequence present in D1 of FGFR3.

[0325] 20. A multispecific binding molecule (MBM), comprising: (a) an antigen binding domain 1 (ABD1) comprising a first means for binding FGFR3; (b) an antigen binding domain 2 (ABD2) comprising a second means for binding FGFR3.

[0326] 21. The MBM of embodiment 20, wherein ABD1 comprises a means for binding to D3 of FGFR3. 22. The MBM of embodiment 20, wherein ABD1 comprises a means for binding to D2 of FGFR3.

[0327] 23. An MBM according to any one of embodiments 20 to 22, wherein ABD2 comprises a means for binding to D1 of FGFR3. 24. A multispecific binding molecule (MBM) according to any one of embodiments 1 to 23, which inhibits interactions between FGFR3 molecules.

[0328] 25. The MBM of any one of embodiments 1 to 24, wherein the MBM has at least 100-fold greater selectivity for FGFR3b compared to FGFR3c. 26. An MBM described in any one of embodiments 1 to 25, wherein the MBM is bispecific.

[0329] 27. The MBM of any one of embodiments 1 to 26, wherein the MBM is bivalent. 28. The MBM of any one of embodiments 1 to 26, wherein the MBM is trivalent. 29. The MBM of any one of embodiments 1 to 26, wherein the MBM is tetravalent.

[0330] 30. An MBM according to any one of embodiments 1 to 29, wherein ABD1 and / or ABD2 specifically bind to mouse FGFR3. 31. The MBM of embodiment 30, wherein ABD1 and / or ABD2 specifically bind to an FGFR3 molecule comprising SEQ ID NO: 1.

[0331] 32. An MBM according to any one of embodiments 1 to 29, wherein ABD1 and / or ABD2 specifically bind to human FGFR3. 33. The MBM of embodiment 32, wherein ABD1 and / or ABD2 specifically bind to an FGFR3 molecule comprising SEQ ID NO: 2.

[0332] 34. An MBM described in any one of embodiments 1 to 33, wherein ABD1 is an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single domain antibody (SDAB), VH domain or VL domain, or Camelidae VHH domain.

[0333] 35. The MBM of embodiment 34, wherein ABD1 is an scFv. 36. The MBM of embodiment 34, wherein ABD1 is Fab. 37. An MBM according to any one of embodiments 34 to 36, wherein the light chain of ABD1 is a universal light chain.

[0334] 38. An MBM according to any one of embodiments 34 to 36, wherein the light chain constant region and the first heavy chain constant region (CH1) of ABD1 are in a domain-swapped configuration. 39. An MBM described in any one of embodiments 1 to 38, wherein ABD2 is an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single domain antibody (SDAB), VH domain or VL domain, or Camelidae VHH domain.

[0335] 40. The MBM of embodiment 39, wherein ABD2 is an scFv. 41. The MBM of embodiment 39, wherein ABD2 is Fab. 42. An MBM according to any one of embodiments 39 to 41, wherein the light chain of ABD2 is a universal light chain.

[0336] 43. The MBM of embodiment 42, wherein the light chain constant region and the first heavy chain constant region (CH1) of ABD2 are in a domain-swapped configuration. 44. An MBM described in any one of embodiments 1 to 43, further comprising an antigen binding domain 3 (ABD3) that specifically binds to the first epitope.

[0337] 45. The MBM of embodiment 44, wherein ABD3 is an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single domain antibody (SDAB), VH domain or VL domain, or Camelidae VHH domain.

[0338] 46. ​​The MBM of embodiment 45, wherein ABD3 is an scFv. 47. The MBM of embodiment 45, wherein ABD3 is Fab. 48. An MBM according to any one of embodiments 45 to 47, wherein the light chain of ABD3 is a universal light chain.

[0339] 49. An MBM according to any one of embodiments 45 to 47, wherein the light chain constant region and the first heavy chain constant region (CH1) of ABD3 are in a domain-swapped configuration. 50. An MBM described in any one of embodiments 45 to 49, wherein ABD3 has the same amino acid sequence as ABD1.

[0340] 51. An MBM according to any one of embodiments 45 to 49, wherein ABD3 and ABD1 are identical. 52. An MBM described in any one of embodiments 1 to 51, further comprising an antigen binding domain 4 (ABD4) that specifically binds to a second epitope.

[0341] 53. The MBM of embodiment 52, wherein ABD4 is an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single domain antibody (SDAB), VH domain or VL domain, or Camelidae VHH domain.

[0342] 54. The MBM of embodiment 53, wherein ABD4 is an scFv. 55. The MBM of embodiment 53, wherein ABD4 is Fab. 56. An MBM according to any one of embodiments 53 to 55, wherein the light chain of ABD4 is a universal light chain.

[0343] 57. An MBM according to any one of embodiments 53 to 55, wherein the light chain constant region and the first heavy chain constant region (CH1) of ABD4 are in a domain-swapped configuration. 58. An MBM described in any one of embodiments 53 to 57, wherein ABD4 has the same amino acid sequence as ABD2.

[0344] 59. An MBM according to any one of embodiments 53 to 57, wherein ABD4 and ABD2 are identical. 60. An MBM described in any one of embodiments 1 to 59, wherein the MBM comprises an Fc heterodimer.

[0345] 61. The MBM of embodiment 60, wherein the Fc domain in the Fc heterodimer comprises a knob-in-hole mutation compared to the wild-type Fc domain. 62. An MBM according to embodiment 60 or 61, wherein the Fc domain in the Fc heterodimer comprises a star mutation compared to the wild-type Fc domain.

[0346] 63. MBM, (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first scFv to which it is operably linked, (ii) a first heavy chain region of a first Fab to which it is operably linked, (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second scFv to which it is operably linked, (ii) a second heavy chain region of a second Fab to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab.

[0347] 64. The MBM of embodiment 63, wherein ABD1 is the first scFv. 65. The MBM of embodiment 63, wherein the first scFv comprises ABD1. 66. An MBM described in any one of embodiments 63 to 65, wherein ABD2 is the first Fab.

[0348] 67. An MBM described in any one of embodiments 63 to 66, wherein ABD3 is a second scFv. 68. An MBM described in any one of embodiments 63 to 66, wherein the second scFv comprises ABD3.

[0349] 69. An MBM described in any one of embodiments 63 to 68, wherein ABD4 is the second Fab. 70. An MBM described in any one of embodiments 63 to 68, wherein the second Fab comprises ABD4.

[0350] 71. An MBM described in any one of embodiments 63 to 69, wherein the first scFv is linked to the first heavy chain region via a linker. 72. An MBM described in any one of embodiments 63 to 71, wherein the second scFv is linked to the second heavy chain region via a linker.

[0351] 73. An MBM described in embodiment 71 or 72, wherein the linker is 5 to 50 amino acids in length. 74. MBM, (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (ii) a first heavy chain region of a first Fab, operably linked to (iii) an Fc domain, operably linked to (iii) a first scFv; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second heavy chain region of a second Fab, operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab.

[0352] 75. The MBM of embodiment 74, wherein ABD1 is the first scFv. 76. The MBM of embodiment 74, wherein the first scFv comprises ABD1. 77. An MBM described in any one of embodiments 74 to 76, wherein ABD2 is the first Fab.

[0353] 78. An MBM described in any one of embodiments 74 to 77, wherein ABD3 is the second scFv. 79. An MBM described in any one of embodiments 74 to 77, wherein the second scFv comprises ABD3.

[0354] 80. An MBM described in any one of embodiments 74 to 79, wherein ABD4 is the second Fab. 81. The MBM of any one of embodiments 74 to 79, wherein the second Fab comprises ABD4.

[0355] 82. An MBM described in any one of embodiments 74 to 81, wherein the first scFv is linked to the Fc domain via a linker. 83. An MBM described in any one of embodiments 74 to 82, wherein the second scFv is linked to the Fc domain via a linker.

[0356] 84. The MBM of embodiment 83, wherein the linker is between 5 amino acids and 50 amino acids in length. 85. MBM, (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab, to which it is operably linked, (ii) a second heavy chain region of a second Fab, to which it is operably linked, and (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab, to which it is operably linked, (ii) a fourth heavy chain region of a second Fab, to which it is operably linked, and (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with a third heavy chain region to form a third Fab; (f) a sixth polypeptide chain comprising a fourth light chain paired with a fourth heavy chain region to form a fourth Fab.

[0357] 86. The MBM of embodiment 85, wherein two of the light chains are identical. 87. The MBM of embodiment 85 or 86, wherein three of the light chains are identical. 88. The MBM of any one of embodiments 85 to 87, wherein the first, second, third, and fourth light chains are identical.

[0358] 89. The MBM of any one of embodiments 85 to 88, wherein ABD1 is the first Fab. 90. The MBM of any one of embodiments 85 to 88, wherein the first Fab comprises ABD1.

[0359] 91. An MBM described in any one of embodiments 85 to 90, wherein ABD2 is the second Fab. 92. The MBM of any one of embodiments 85-90, wherein the second Fab comprises ABD2.

[0360] 93. An MBM described in any one of embodiments 85 to 92, wherein ABD3 is the third Fab. 94. The MBM of any one of embodiments 85 to 92, wherein the third Fab comprises ABD3.

[0361] 95. The MBM of any one of embodiments 85-94, wherein ABD4 is the fourth Fab. 96. The MBM of any one of embodiments 85-94, wherein the fourth Fab comprises ABD4.

[0362] 97. An MBM described in any one of embodiments 85 to 96, wherein the first heavy chain region is linked to the second heavy chain region via a linker. 98. An MBM described in any one of embodiments 85 to 97, wherein the third heavy chain region is linked to the fourth heavy chain region via a linker.

[0363] 99. The MBM of embodiment 98, wherein the linker is 5 to 50 amino acids in length. 100. MBM, (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second heavy chain region of a second Fab, operably linked to (iii) an Fc domain, operably linked to (iii) a first heavy chain region of a first Fab; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (ii) a fourth heavy chain region of a second Fab, operably linked to (i) an Fc domain, operably linked to (iii) a third heavy chain region of a third Fab; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with a third heavy chain region to form a third Fab; (f) a sixth polypeptide chain comprising a fourth light chain paired with a fourth heavy chain region to form a fourth Fab.

[0364] 101. The MBM of embodiment 100, wherein two of the light chains are identical. 102. The MBM of embodiment 100 or 101, wherein three of the light chains are identical.

[0365] 103. An MBM described in any one of embodiments 100 to 102, wherein the first, second, third, and fourth light chains are identical. 104. An MBM according to any one of embodiments 100 to 103, wherein ABD1 is the first Fab.

[0366] 105. An MBM described in any one of embodiments 100 to 103, wherein the first Fab comprises ABD1. 106. An MBM according to any one of embodiments 100 to 105, wherein ABD2 is a second Fab.

[0367] 107. The MBM of any one of embodiments 100 to 105, wherein the second Fab comprises ABD2. 108. An MBM according to any one of embodiments 100 to 107, wherein ABD3 is the third Fab.

[0368] 109. An MBM described in any one of embodiments 100 to 107, wherein the third Fab comprises ABD3. 110. An MBM according to any one of embodiments 100 to 109, wherein ABD4 is the fourth Fab.

[0369] 111. An MBM described in any one of embodiments 100 to 109, wherein the fourth Fab comprises ABD4. 112. An MBM described in any one of embodiments 100 to 110, wherein the first heavy chain region is linked to the Fc domain via a linker.

[0370] 113. An MBM described in any one of embodiments 100 to 112, wherein the third heavy chain region is linked to the Fc domain via a linker. 114. The MBM of embodiment 113, wherein the linker is 5 to 50 amino acids in length.

[0371] 115. A multispecific binding molecule (MBM), comprising: (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first scFv to which it is operably linked, (ii) a first heavy chain region of a first Fab to which it is operably linked, (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second scFv to which it is operably linked, (ii) a second heavy chain region of a second Fab to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain paired with a second heavy chain region to form a second Fab.

[0372] 116. The MBM of embodiment 115, wherein the first scFv specifically binds to a first epitope of FGFR3. 117. The MBM of embodiment 116, wherein the first epitope comprises a sequence present in D3 of FGFR3 and a sequence present in D2 of FGFR3.

[0373] 118. The MBM of embodiment 116 or 117, wherein the second scFv specifically binds to the first epitope. 119. The MBM of any one of embodiments 115-118, wherein the first Fab specifically binds to a second epitope of FGFR3 that is different from the first epitope.

[0374] 120. The MBM of embodiment 119, wherein the second epitope comprises a sequence present in D1 of FGFR3. 121. The MBM of embodiment 119 or 120, wherein the second Fab specifically binds to a second epitope.

[0375] 122. A multispecific binding molecule (MBM): (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab, to which it is operably linked, (ii) a second heavy chain region of a second Fab, to which it is operably linked, and (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab, to which it is operably linked, (ii) a fourth heavy chain region of a fourth Fab, to which it is operably linked, (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with a first heavy chain region to form a first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with a second heavy chain region to form a second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with a third heavy chain region to form a third Fab; (f) a sixth polypeptide chain comprising a fourth light chain paired with a fourth heavy chain region to form a fourth Fab.

[0376] 123. The MBM of embodiment 122, wherein the first Fab specifically binds to a first epitope of FGFR3. 124. The MBM of embodiment 123, wherein the first epitope comprises a sequence present in D3 of FGFR3 and a sequence present in D2 of FGFR3.

[0377] 125. The MBM of embodiment 123 or 124, wherein the third Fab specifically binds to the first epitope. 126. The MBM of any one of embodiments 123-125, wherein the second Fab specifically binds to a second epitope of FGFR3 that is different from the first epitope.

[0378] 127. The MBM of embodiment 126, wherein the second epitope comprises a region of D1 of FGFR3. 128. The MBM of embodiment 126 or 127, wherein the fourth Fab specifically binds to a second epitope.

[0379] 129. A nucleic acid or nucleic acids encoding the MBM of any one of embodiments 1 to 128. 130. A cell engineered to express the MBM described in any one of embodiments 1 to 128.

[0380] 131. A cell comprising a nucleic acid or nucleic acids described in embodiment 129. 132. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding an MBM described in any one of embodiments 1 to 128 under the control of one or more promoters.

[0381] 133. A method for producing MBM, comprising: (a) culturing a cell described in any one of embodiments 130 to 132 under conditions sufficient to express MBM; and (b) recovering MBM from the cell.

[0382] 134. A pharmaceutical composition comprising the MBM described in any one of embodiments 1 to 128 and an excipient. 135. A method comprising administering to a subject an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0383] 136. The method of embodiment 135, wherein the MBM or pharmaceutical composition is administered to the subject in an amount effective to treat FGFR3-positive cancer. 137. The method of embodiment 135 or 136, wherein the subject has bladder cancer.

[0384] 138. A method for treating bladder cancer in a subject in need thereof, comprising administering to the subject an effective amount of MBM described in any one of embodiments 1 to 128 or a pharmaceutical composition described in embodiment 104.

[0385] 139. The method of embodiment 138, wherein the bladder cancer is metastatic bladder cancer. 140. The method of embodiment 138 or 139, wherein the bladder cancer is FGFR3-mutated bladder cancer.

[0386] 141. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-TACC3 fusion bladder cancer. 142. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-S249C bladder cancer.

[0387] 143. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-R248C bladder cancer. 144. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-G372C bladder cancer.

[0388] 145. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-Y375C bladder cancer. 146. The method of embodiment 140, wherein the FGFR3 mutant bladder cancer is FGFR3-K650E bladder cancer.

[0389] 147. The method of embodiment 138 or 139, wherein the bladder cancer is not FGFR3 mutant bladder cancer. 148. The method of any one of embodiments 138 to 147, wherein administering MBM or the pharmaceutical composition treats bladder cancer in the subject.

[0390] 149. A method for treating FGFR3-positive tumor cells in a subject in need thereof, comprising administering to the subject an effective amount of an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0391] 150. The method of embodiment 149, wherein the FGFR3-positive tumor is a bladder cancer tumor. 151. The method of embodiment 149 or 150, wherein administration of MBM or the pharmaceutical composition reduces the tumor burden of an FGFR3-positive tumor in the subject.

[0392] 152. A method for sensitizing FGFR3-positive tumor cells in a subject to cancer therapy, the method comprising administering to the subject an effective amount of an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0393] 153. The method of embodiment 152, wherein the FGFR3-positive tumor cells are bladder cancer tumor cells. 154. The method of embodiment 152 or 153, wherein the cancer therapy is chemotherapy, immunotherapy, or radiation therapy.

[0394] 155. The method of claim 154, wherein the cancer therapy is immunotherapy. 156. The method of any one of embodiments 152 to 155, wherein administration of MBM or the pharmaceutical composition improves the efficacy of cancer therapy.

[0395] 157. A method for inhibiting metastasis of FGFR3-positive tumor cells in a subject in need thereof, the method comprising administering to the subject an effective amount of an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0396] 158. The method of embodiment 157, wherein the FGFR3-positive tumor cells are bladder cancer tumor cells. 159. The method of embodiment 157 or 158, wherein administration of MBM or the pharmaceutical composition suppresses metastasis of FGFR3-positive tumor cells in the subject.

[0397] 160. A method for reducing the viability of FGFR3-positive cancer cells, the method comprising administering to a subject an effective amount of an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0398] 161. The method of embodiment 157, wherein the FGFR3-positive cancer cells are bladder cancer cells. 162. The method of embodiment 157 or 158, wherein administration of MBM or the pharmaceutical composition reduces the viability of FGFR3-positive cancer cells.

[0399] 163. A method for reducing FGFR3 signaling in FGFR3-positive cells, the method comprising administering to a subject an effective amount of an MBM described in any one of embodiments 1 to 128, or a pharmaceutical composition described in embodiment 134.

[0400] 164. The method of embodiment 163, wherein the method is an in vitro method. 165. The method of embodiment 163, wherein the method is an in vivo method. 166. The method of embodiment 163, wherein the FGFR3-positive cells are in cell culture.

[0401] 167. The method of embodiment 163, wherein the FGFR3-positive cells are in a subject. 168. The method of embodiment 167, wherein the subject is a human subject. 169. The method of any one of embodiments 163 to 168, wherein the FGFR3-positive cells are cancer cells.

[0402] 170. The method of embodiment 169, wherein the FGFR3-positive cells are bladder cancer cells. [Example]

[0403] 8. Working Example 8.1. Materials and Methods 8.1.1. Construction of Anti-FGFR3 Alternative Format (AF) Antibodies Monospecific bivalent N-Fabs were constructed by connecting a single Fab fragment to the N-terminal end of an Fc fragment. Bispecific bivalent N-Fabs were constructed in a similar manner, with "knobs-in-holes" mutations in the Fc region to promote Fc heterodimerization. Trivalent 2+1 N-Fab AF antibodies were constructed by heterodimerizing an Fc fragment connected at their N-termini to one Fab fragment on one side and two Fab fragments connected to each other via a (G4S)4 linker (SEQ ID NO: 115) on the other side. Trivalent 2+1 N-scFv AF antibodies were constructed by heterodimerizing an Fab fragment linked to an scFv fragment on one side via a (G4S)4 linker (SEQ ID NO: 115) and an Fc fragment connected at its N-terminus to one Fab fragment on the other side. Tetravalent 2+2 N-Fab AF antibodies were constructed by dimerization or heterodimerization of Fc fragments, each linked to two Fab fragments at their N-terminus. Tetravalent 2+2 N-scFv AF antibodies were constructed by dimerization or heterodimerization of Fc fragments, each linked to a Fab fragment at its N-terminus linked to an scFv fragment. Tetravalent 2+2 C-scFv AF antibodies were constructed by dimerization or heterodimerization of Fc fragments connected to a Fab fragment at their N-terminus and an scFv fragment at their C-terminus via a (G4S)3 linker (SEQ ID NO: 100). Tetravalent 2+2 C-Fab fragments were constructed by dimerization or heterodimerization of Fc fragments connected to a Fab fragment at their N-terminus and a Fab fragment at their C-terminus via a (G4S)3 linker (SEQ ID NO: 100). The pairing between the variable heavy and light chains of the Fab fragment in any of the described AF antibodies can be natural or forced. Additionally, the Fc portion can include wild-type IgG1, IgG2, or mutated variants of IgG4. Exemplary AF antibody structures are shown in Figures 2A-2P.

[0404] 8.1.2. Expression and Purification of Anti-FGFR3 AF Antibodies Mammalian expression vectors for individual heavy and light chains were generated by DNA synthesis and cloning into ready-to-use constructs in the pcDNA3.4 Topo Expression System from Life Technologies (Carlsbad, CA). To express the molecules, heavy and corresponding light chain DNAs were co-transfected into Expi293 cells (ThermoFisher Scientific) according to the manufacturer's protocol. 50 ml of cell culture medium was harvested and processed for purification via HiTrap Protein A FF or Mab Select SuRe column (GE Healthcare). For functional validation, selected MBMs were scaled up to 2 L and subjected to a series of purification steps, including two-step Mab-Select SuRe column and size-exclusion chromatography as the final step.

[0405] 8.1.3. Cell proliferation assay Two human bladder cancer cell lines, UMUC14 (Sigma-Aldrich) and RT4 (ATCC), expressing either the S249C mutation of FGFR3 or the TACC3-FGFR3 fusion mutation, were maintained in culture according to the supplier's recommendations.

[0406] 7,500 UMUC14 or 5,000 RT4 bladder cancer cells were seeded into U-bottom low-attachment 96-well spheroid plates (Corning) in culture medium (MEM medium containing 10% FBS, 1% non-essential amino acids, and Pen / Strep). Cells were cultured at 37°C and 5% CO2 for 24–48 hours to allow tumor spheroid formation. Tumor spheroids were treated with antibodies at concentrations ranging from 100 nM to 15.2 pM in 1:3 serial dilutions, as indicated. Cells were cultured for 5–6 days and then subjected to the CellTiter-Glo 3D Viability Assay (Promega) according to the manufacturer's protocol. Luminescence signals were read on a PE Envision plate reader. Cell proliferation was expressed as a percentage of the untreated control. Data were analyzed using GraphPad Prism software using a three-parameter nonlinear curve fit.

[0407] 8.1.4. Cell proliferation of BaF3 cell lines expressing FGFR3 mutations cDNAs expressing FGFR3 mutations (S249C, S248C, Y375C, V555M, V555L, S249C+V555M, or S249C+V555L) were synthesized and cloned into the pLVX lentiviral vector (Thermo Fisher Scientific). Viral particles were generated by Lipofectamine transfection of LentiX 293T cells using Lenti-X Packaging Single Shot according to the manufacturer's protocol (Clontech / Takara), concentrated using a Lenti-X concentrator, and titered using the Lenti-X p24 Rapid Titer Kit (Clontech / Takara Scientific). BaF3 cells were transduced with the virus at approximately 0.3 MOI. Expression of the FGFR3 receptor was confirmed by FACS analysis and Western blot.

[0408] For cell proliferation assays, engineered BaF3 / hFGFR3 cells grown in complete medium were washed and plated in IL-3-free medium ± 5 μg / ml heparin and 1 nM human FGF1. Cells were plated at 1 × 10 5 Cells / well were plated out in 96-well plates, followed by the addition of 1:3 serially diluted antibodies as indicated. After antibody addition, cells were incubated at 37°C for 72 hours, followed by the addition of CellTiter-Glo™ (Promega) reagent. Luminescent signals were detected by an Envison plate reader (PerkinElmer). Data were analyzed using Prism software (GraphPad).

[0409] Western Blot For receptor dimerization assessment, UMUC14 bladder cancer cells or BaF3 cells were seeded in 6-well tissue culture plates (Corning) in complete medium (MEM medium with 10% FBS, 1% non-essential amino acids, glutamine, Pen / Strep) and cultured overnight at 37°C with 5% CO2. Cells were then serum-starved overnight in starvation medium (MEM medium with 0.5% FBS, Pen / Strep) followed by antibody treatment at the indicated concentrations for 3 hours. Equal amounts of cell lysates were analyzed by either reducing or non-reducing SDS-PAGE. Membranes were blotted with anti-FGFR3 primary antibody (Santa Cruz) and developed with SuperSignal West Pico substrate (Pierce), and luminescence images were captured with a C300 imager (Azure Biosystems).

[0410] To assess receptor phosphorylation and downstream signaling, cells were serum-starved overnight in the presence or absence of the indicated antibodies, followed by ligand stimulation (100 ng / ml human FGF1 and 10 μg / ml heparin) for 15 minutes at 37°C. Equal amounts of cell lysates were analyzed by SDS-PAGE. Blots were incubated with pMAPK, MAPK, or actin antibodies, followed by incubation with an HRP secondary antibody. For FGFR3 phosphorylation, equal amounts of cell lysates were incubated overnight with pY100 Sepharose beads, and nonspecific binding was removed by washing with lysis buffer. Immunoprecipitated proteins were analyzed by reducing or nonreducing SDS-PAGE and blotted with FGFR3 antibodies.

[0411] For receptor degradation assessment, cells were treated with 33 nM of the indicated antibody for 16 hours. Cells were then washed with pre-chilled PBS and collected in lysis buffer containing protease inhibitors (Cell Signaling Technology). For protein separation, equal amounts of cell lysates were loaded onto either reducing or non-reducing SDS-PAGE. PVDF membranes with transferred proteins were blocked in Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.5% Tween 20 and incubated overnight with anti-FGFR3 primary antibody (Santa Cruz). The membranes were washed three times with TBST, incubated with HRP-conjugated goat anti-rabbit secondary antibody (Seracare), and developed with SuperSignal West chemiluminescent substrate (Thermo Scientific). Images were captured with a C300 imager (Azure Biosystems).

[0412] 8.1.6. In Vivo Tumor Growth Inhibition Assay Tumor cells (5 × 10 in 50% Matrigel 6 UMUC14 or RT112 were implanted subcutaneously (sc) into the right flank of 6- to 8-week-old female SCID mice (Jackson Laboratory). After tumors were established (approximately 200 mm 3 Tumor volume was calculated in mm2 using the following formula: 3 Expressed as: V = 0.5 × a × b 2 , where a and b were the long and short diameters of the tumor, respectively. Tumor size was monitored twice weekly. All procedures were performed in accordance with the guidelines of the Regeneron Institutional Animal Care and Use Committee. All data were analyzed using GraphPad Prism, and tumor size was graphed as mean + SEM.

[0413] 8.1.7. Biacore Analysis of Antibody Binding to FGFR3 Isoforms The binding affinity and mechanism of action of anti-FGFR3 antibodies to FGFR3 isoforms were determined by Biacore analysis. Briefly, in-house generated monoclonal anti-human Fc antibodies were immobilized on the surface of a CM-5 sensor chip. Different concentrations of FGFR3 antibodies were injected at 50 μL / min for 4 min, and the assay was performed at 25°C. The mAb binding response was monitored, and the steady-state binding equilibrium was calculated for the low-affinity receptor. The data were processed using Scrubber 2.0 curve-fitting software, and the kinetic association (ka) and dissociation (kd) rate constants were determined by fitting to a 1:1 binding model. The binding-dissociation equilibrium constant (KD) and dissociation half-life (t 1 / 2 ) using the formula: KD(M)=kd / ka and t 1 / 2 K values ​​were calculated from the kinetic rate constants using (min) = (In² / (60*kd). Some K values ​​were derived using the steady-state equilibrium dissociation constant; if a K value was not derived, it was marked as NB (no binding observed) or IC (uncertain affinity determination due to low specific RU signal).

[0414] 8.1.8. Asymmetric flow field separation coupled to multi-angle laser light scattering (A4F-MALS) The A4F-MALS system consisted of an Eclipse™ DualTec A4F separations system coupled to an Agilent 1200 Series HPLC system equipped with an ultraviolet (UV) diode array detector, a Wyatt Technology Dawn laser light scattering (LS) detector, and an Optilab® T-rEX differential refractometer (RI) detector. The detectors were connected sequentially in the following order: UV-LS-RI. The LS and RI detectors were calibrated according to the instructions provided by Wyatt Technology.

[0415] Defined amounts of the 2+2-N-scFv anti-FGFR3 AF antibody, BiP063N2, or its 1+1 N-scFv-Fab variant were each combined with hFGFR3b-mmh and diluted with 1X DPBS, pH 7.4, to obtain the ratios defined in Figures 8C and 8E. All samples were incubated at ambient temperature for 2 hours, kept unfiltered at 4°C, and then injected into an Eclipse™ short channel equipped with a W350 spacer foil (spacer thickness 350 μm, spacer width 2.2 cm) using a 10 kDa MWCO regenerated cellulose membrane. Before injection of each sample, the channel was pre-equilibrated with mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Bovine serum albumin (BSA, 2 mg / mL, 10 μg sample load) was injected separately and included as a system suitability control.

[0416] The separation method consisted of four steps: injection, focusing, elution, and a channel "washout" step. A4F-MALS mobile phase buffer was used throughout the separation method. Each sample (7 μg) was injected for 1 min at a flow rate of 0.2 mL / min, followed by 2 min of focusing at a focusing flow rate of 1.5 mL / min. Samples were eluted over 45 min with a linear gradient crossflow from 3.0 mL / min to 0 mL / min at a channel flow rate of 1.0 mL / min. Finally, the crossflow was held at 0 mL / min for an additional 5 min to wash out the channel. BSA was separated using the same parameter settings.

[0417] Data were analyzed using ASTRA V software (version 7.3.2, Wyatt Technology). Data were fitted to an equation relating excess scattered light to solute concentration and weight-average molar mass, Mw (Kendrick et al., 2001, Anal Biochem. 299(2):136-46; Wyatt, 1993, Anal. Chim. Acta 272(1):1-40):

[0418]

number

[0419] where c is the solute concentration, R(q,c) is the excess Raley ratio from the solute as a function of scattering angle and concentration, Mw is the molar mass, P(q) describes the angular dependence of the scattered light (approximately 1 for particles with a radius of gyration <50 nm), A2 is the second virial coefficient in the osmotic expansion (which can be neglected since the measurements are made in dilute solutions), and

[0420]

number

[0421] (wherein n0 represents the refractive index of the solvent, and N A is Avogadro's number, λ0 is the wavelength of the incident light in a vacuum, and dn / dc represents the relative refractive index increment of the solute). The molar mass of the BSA monomer served to evaluate the calibration constants of the light scattering and refractive index detectors during data collection (system suitability check). The relative standard deviation (RSD%) of the mean molar mass of BSA determined from the UV and RI detectors was less than 5.0%.

[0422] The light scattering detector normalization factor, interdetector delay, and band broadening term were calculated from the BSA chromatogram collected for the A4F-MALS conditions used, and these values ​​were applied to the data files collected for all other samples to correct for these aspects.

[0423] The dn / dc values ​​and extinction coefficients at 215 nm were experimentally determined using the protein conjugate analysis provided by the Astra software. All protein-protein complex samples were analyzed using the corrected extinction coefficients and dn / dc values.

[0424] 8.1.9. Antibody Pharmacokinetic Screening The Gyrolab xPlore instrument was used to measure the concentrations of BiP063N2 (total and specific components), mAb117, mAb063, or a control mAb (REGN1945) in mouse serum. Gyros' technology uses an affinity flow-through format for automated immunoassays with laser-induced fluorescence detection. Samples are loaded onto a compact disc (CD) containing multiple radially arranged nanoliter-scale affinity capture columns. Liquid flow is controlled by centrifugal and capillary forces. Antibody plasma concentrations are determined using the immunoassay.

[0425] In this assay, biotinylated human or mouse FGFR3b-ecto.mmH, human FGFR3c-ecto.mmH, or Fel-d1.1mmH at a concentration of 20 μg / mL was added to a Gyrolab Bioaffy 200CD containing an affinity column preloaded with streptavidin-coated beads. The standards used for calibration in this assay were BiP063N2, mAb117, mAb063, or REGN1945 at concentrations ranging from 0.488 to 2000 ng / mL. Serial dilutions of standards and samples were prepared in dilution buffer consisting of phosphate-buffered saline (PBS) containing 1% normal mouse serum plus 0.5% bovine serum albumin (BSA).

[0426] Diluted serum sample singlets and standard duplicates were applied to a capture antigen-coated affinity column at room temperature. Captured human IgG was detected using 0.5 μg / mL of Alexa Fluor®-647-conjugated mouse anti-human IgG1 / IgG4-specific monoclonal antibody (REGN2567) diluted in Rexxip F buffer, and the resulting fluorescent signal was recorded in response units by a Gyrolab xPlore instrument. The lower limit of quantitation (LLOQ) of the assay (0.098 μg / mL) was defined as the lowest concentration on the respective standard curve at which quality control samples consistently deviated by less than 25% from the expected concentration. Sample concentrations were determined by interpolation from the standard curve constructed using a four-parameter logistic curve fit in Gyrolab Evaluator software. The average concentration from two replicate experiments was reported.

[0427] PK parameters were determined by non-compartmental analysis (NCA) using Phoenix® WinNonlin® software version 6.3 (Certara, LP, Princeton, NJ) and an extravascular dosing model. The respective mean concentration values ​​(total drug) for each antibody were used to calculate the maximum observed serum concentration (C max ), the estimated observed half-life (t 1 / 2 ), area under the concentration curve versus time to last measurable concentration (AUC last ), and all PK parameters, including antibody clearance rate (Cl), were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

[0428] 8.2. Example 1: 2+2 scFv Alternative Format mAb Screening An exemplary set of 27 anti-FGFR3 2+2 alternative format (AF) antibodies was constructed and produced as described in Sections 8.1.1 and 8.1.2. Three antibodies (anchor mAbs) with different biological activities and epitopes were selected and paired with an additional scFv arm. Paired scFv arms attached either to the N-terminus of the anchor Fab arm (2+2 N-scFv) or to the C-terminus of the Fc domain (2+2 C-scFv) were generated from a group of nine parent anti-FGFR3 antibodies with various epitopes. Details regarding the parent antibodies used to construct the anchor Fab domain and alternative arm scFv domains of the AF antibodies are provided in Table 1.

[0429] [Table 8]

[0430] The activity of 2+2AF antibodies is influenced by both the epitope and the orientation of their Fab and / or scFv portions. Details about exemplary 2+2AF antibodies used in the screening evaluation are provided in Table 2.

[0431] [Table 9-1]

[0432] [Table 9-2]

[0433] In one evaluation, the anti-proliferative effects of the 272+2N-scFv and 2+2C-scFv AF antibodies were screened in the bladder cancer cell lines UMUC14 and RT4, as described in Section 8.1.3. The activity of the 2+2AF antibody was affected by the pairing of the scFv arms, as demonstrated by the different anti-proliferative effects of mAb063 linked to various scFv arms (e.g., BiP063N1-N8 in Figure 3B and Figure 3C). The activity of the 2+2AF antibody was also affected by the anchor mAb, as demonstrated by the different effects of the same scFv when linked to different anchor mAbs (e.g., BiP063N1, BiP108N1 vs. BiP076N1 in Figure 3B and Figure 3C). Furthermore, the activity of the 2+2AF antibody was affected by the orientation of the scFv, as demonstrated by the different antiproliferative effects of BiP063N versus BiP063CAF antibodies (FIGS. 3B and 3C).

[0434] 8.3. Example 2: In Vitro Activity of 2+2 AF Antibodies Against Oncogenic FGFR3 Mutations The dose-dependent inhibitory effects of antibodies on tumor cell proliferation were evaluated as described in section 8.1.3. Briefly, UMUC14 and RT4 tumor cell spheroids were treated for 5–6 days with a subset of 2+2 AF antibodies at the indicated concentrations. AF antibodies exhibited varying levels of antiproliferative activity (Figure 4A and Figure 4B). BiP063N2 demonstrated potent activity against the proliferation of both UMUC14 cells endogenously expressing the FGFR3 S249C point mutation (Figure 4A) and RT4 cells endogenously expressing the FGFR3-TACC3 fusion mutation (Figure 4B).

[0435] In the next evaluation, the antiproliferative activity of BiP063N2 was compared with that of its parent mAb, other conventional anti-FGFR3 antibodies, and an isotype control, as described in section 8.1.3. In proliferation assays, parent mAb 063 exhibited potent inhibitory activity against the FGFR3 S249C mutation (Figure 4C) and moderate inhibitory activity against the FGFR3-TACC3 fusion mutation (Figure 4D). Parent mAb 117 exhibited agonistic activity against the FGFR3 S249C mutation (Figure 4C) and minimal activity against the FGFR3-TACC3 fusion mutation (Figure 4D). In UMUC14 spheroid proliferation assays, BiP063N2 exhibited enhanced antiproliferative activity with an approximately 3- to 10-fold increase in IC50 compared to parent mAb 063 (Figure 4C). In the RT4 cell proliferation assay, BiP063N2 demonstrated a 25% increase in maximal inhibition compared to the parental mAb063 (Figure 4D).

[0436] In the next evaluation, the effect of antibody combinations was tested in a cell proliferation assay, as described in Section 8.1.3. Equal amounts of the parent antibodies mAb063 and mAb117 were combined, and cells were incubated with mAb063 alone, mAb117 alone, or mAb063 + mAb117 at the indicated concentrations for 5–6 days. Antibody combination treatment did not result in superior antiproliferative activity compared to mAb063 or mAb117 alone (Figure 4E and Figure 4F), suggesting that the superior activity of BiP063N2 requires the proper molecular configuration of its scFv and mAb arms.

[0437] 8.4. Example 3: In vivo activity of BiP063N2 against oncogenic FGFR3 mutations The activity in tumor growth inhibition was evaluated in a mouse xenograft tumor model as described in Section 8.1.6. In the first evaluation, mice bearing established UMUC14 tumors endogenously expressing the FGFR3 S249C mutation were administered either 10 mg / kg and 3 mg / kg of the parent antibody mAb063, or 13.3 mg / kg and 4 mg / kg of BiP063N2 at the same molar concentrations. Treatment with both mAb063 and BiP063N2 resulted in UMUC14 tumor regression (Figure 5A) at a level superior to the anti-tumor activity of the anti-FGFR3 antibody, REGN6331 (i.e., "R3Mab" described in U.S. Patent No. 8,710,189, which is incorporated herein by reference).

[0438] To determine the minimum dose required to achieve UMUC14 tumor regression, mice bearing established UMUC14 tumors were treated with 5, 3, or 1 mg / kg mAb063 or 6.65, 4, or 1.33 mg / kg BiP063N2 at equivalent molar concentrations. At 4 mg / kg, BiP063N2 antibody treatment resulted in tumor stabilization, whereas at 1.33 mg / kg, BiP063N2 antibody treatment delayed tumor growth compared to the isotype control (Figure 5B). Tumor growth was monitored after dosing was discontinued. After discontinuation of antibody dosing on day 32, groups treated with 6.65 mg / kg BiP063N2 or 5 mg / kg mAb063 showed sustained tumor growth suppression through day 46 (Figure 5B).

[0439] To evaluate the antitumor activity of the 2+2 AF antibody BiP063N2 against the FGFR3-TACC3 fusion mutation, mice bearing established RT112 tumors endogenously expressing the FGFR3-TACC3 fusion mutation were administered 15 mg / kg of either the parental antibody, mAb063, or mAb108, or 20 mg / kg of BiP063N2 at the same molar concentration. Both mAb108 and BiP063N2 treatment resulted in RT112 tumor growth inhibition (Figure 5C). BiP063N2 treatment showed approximately 25% improvement in tumor growth inhibition compared to the parental mAb063 antibody (Figure 5C).

[0440] To assess the antitumor activity of BiP063N2 relative to that achieved by its parental antibody at lower doses, mice bearing established UMUC14 tumors were treated twice weekly with 2 mg / kg of isotype antibodies, mAb063, or mAb117, 2.66 mg / kg of BiP063N2, or 2 mg / kg each of mAb063 and mAb117 in combination. Even at the suboptimal dose of 2.66 mg / kg, BiP063N2 was more effective than its parental antibody alone or in combination (Figure 5D).

[0441] Next, the effect of BiP063N2 on FGFR3 downstream signaling was assessed in tumor lysates from mice bearing established UMUC14 tumors treated once with 6.65 mg / kg BiP063N2, 5 mg / kg isotype control antibody or mAb063, or 25 mg / kg tyrosine kinase inhibitor (TKI) AZD4547. MAPK phosphorylation was analyzed by Western blot 48 or 72 hours after treatment. Total MAPK levels correlated with actin levels 72 hours after treatment, indicating no treatment-induced changes in MAPK expression (Figure 5E). BiP063N2 treatment potently inhibited MAPK phosphorylation compared to the inhibition achieved by the maximally effective dose of AZD4547 at both 48 and 72 hours (Figure 5E-G), suggesting robust inhibition of FGFR3 S249C signaling by BiP063N2.

[0442] We next evaluated the ability of BiP063N2 to inhibit the growth of the patient-derived lung squamous cell carcinoma model LU-0813. Mice bearing established LU-0813 tumors, which also harbor the FGFR3 S249C mutation, were treated twice weekly by oral gavage with either an isotype control antibody or mAb063 at 10 mg / kg, BiP063N2 at 13.3 mg / kg, or the FDA-approved pan-FGFR TKI, erdafitinib, at 25 mg / kg daily. Both mAb063 and BiP063N2 potently inhibited tumor growth, nearly to the same extent as erdafitinib (Figure 5H).

[0443] In summary, BiP063N2 exhibited potent in vivo antitumor activity against both the S249C point mutation and the FGFR3-TACC3 fusion mutation, with levels higher than those of the parent antibody mAb063 and the anti-FGFR3 antibody REGN6331.

[0444] 8.5. Example 4: Effects of BiP063N2 on FGFR3 dimerization, degradation and downstream signaling To understand the mechanism of BiP063N2's antiproliferative activity, the antibody's effects on FGFR3 receptor dimerization, degradation, and downstream signaling were assessed by Western blot, as described in Section 8.1.5. UMUC14 cells endogenously expressing the FGFR3 S249C mutation were treated with the indicated antibodies at 33 nM for 3 hours. After treatment, FGFR3 monomers and disulfide-linked dimers were separated via non-reducing SDS-polyacrylamide gel (Figure 6A, top). The mobilities of monomeric and dimeric FGFR3 are shown. In control cell lysates, both monomeric and dimeric FGFR3 S249C were detected. mAb063 and BiP063N2 potently inhibited receptor dimerization, as demonstrated by a shift in band density from the band corresponding to the receptor dimer to that of the monomeric receptor. The parent antibody, mAb117, did not inhibit receptor dimerization, and the combination of mAb063 and mAb117 resulted in partial inhibition of receptor dimerization (FIG. 6A).

[0445] To evaluate the effect of antibodies on receptor degradation, UMUC14 cells were treated with the indicated antibodies at 33 nM for 16 hours. Total FGFR3 receptor levels were analyzed by Western blot. Actin levels were shown to demonstrate equal loading of cell lysates. Only parental mAb117 treatment induced FGFR3 receptor degradation, consistent with its agonist activity. Treatment with mAb063 or BiP063N2 did not induce receptor degradation (Figure 6B).

[0446] To evaluate the effect of antibodies on FGFR3 downstream signaling, UMUC14 cells were treated with 100 nM of the indicated antibodies or AZD4547 for 24 hours. MAPK phosphorylation was analyzed by Western blot. Total MAPK protein levels were measured, demonstrating that equal amounts of cell lysates were loaded. BiP063N2 treatment potently inhibited MAPK phosphorylation with activity comparable to that of the tyrosine kinase inhibitor AZD4547 (Figure 6C), suggesting potent inhibition of FGFR3 signaling by BiP063N2.

[0447] 8.6. Example 5: Antigen Binding Affinity of BiP063N2 The binding affinities of BiP063N2 and its parent antibody to FGFR3 isoforms from different species were assessed via Biacore analysis as described in Section 8.1.7, and the results are summarized in Figure 7.

[0448] Both parent antibodies were shown to bind to human FGFR3b (hFGFR3b) and monkey FGFR3b. mAb063 was also shown to bind to monomeric mouse FGFR3b, but its binding to monomeric human FGFR3c (hFGFR3c) was not detected. In contrast, mAb117 was able to bind to monomeric hFGFR3c, but its binding to mouse FGFR3b was not detected. BiP063N2 exhibited 4- to 100-fold higher affinity for human or monkey FGFR3b than its parent antibodies, mAb063 and mAb117. BiP063N2 also maintained selective binding to the hFGFR3b isoform with approximately 400-fold higher affinity than FGFR3c (Figure 7).

[0449] 8.7. Example 6: Characterization of Avidity-Driven Binding of BiP063N2 to hFGFR3b BiP063N2 has a 4- to 100-fold higher affinity for hFGFR3b than its parent antibody. Given that BiP063N2's parent antibody has a different epitope on hFGFR3b, there are two possible models of BiP063N2-hFGFR3 binding (Figure 8A). In the cis model, each BiP063N2 arm binds to one hFGFR3b and extends to both the Fab and scFv domains, whereas in the trans model, each hFGFR3b binds to the Fab domain of one arm and the scFv domain of the other arm (Figure 8A). To determine whether the interaction between BiP063N2 and hFGFR3 involves cis or trans binding, asymmetric flow field separation coupled to multi-angle laser light scattering (A4F-MALS) was performed as described in section 8.1.8.

[0450] The size distribution of molecular complexes in various BiP062N2-hFGFR3b-mmh combinations was analyzed by A4F-MALS (Figure 8C). Based on the theoretical molar masses of different combinations of full-size BiP063N2 and hFGFR3b-mmh fragments in the 2+2 N-scFv format (Figure 8B), the results showed the formation of complexes with one BiP063N2 binding to up to two hFGFR3b fragments (Figure 8C).

[0451] According to the cis binding model, each arm of BiP063N2 binds to only one hFGFR3b fragment, whereas according to the trans binding model, each arm interacts with two hFGFR3b fragments. Therefore, additional evaluations were performed to evaluate samples containing various molar ratios of 1+1 N-scFv-Fab and hFGFR3b fragments (Figures 8D and 8E). In this case, the predominant protein complex corresponded to the size of a 1:1 ratio of 1+1 N-scFv-Fab and hFGFR3b fragments, but there were few or no protein complexes with individual 1+1 N-scFv-Fab arms interacting with two hFGFR3b fragments (Figure 8E). These observations provided evidence for the cis binding model of BiP063N2-hFGFR3b binding.

[0452] 8.8. Example 7: Pharmacokinetic Profile of BiP063N2 Mice were administered an sc injection of 6.65 mg / kg BiP063N2, or 5 mg / kg of one of its parent antibodies, or an isotype control. Serum samples were collected from the mice on the day of injection (6 hours) and on days 1, 2, 3, 4, 7, 10, 14, 21, and 30 post-injection. Pharmacokinetic profiling of BiP063N2 and its parent antibodies in the samples was performed as described in Section 8.1.9.

[0453] As seen in Figure 9B, BIP063N2 exhibited slower clearance compared to H4H30063P. BIP063N2 had a relatively faster clearance than mAb117 after 10 days, likely due to its ability to bind to mFGFR3b (Figures 9B and 9D). Furthermore, the presence of the mAb117scFv arm in BiP063N2 contributed to a better PK profile than that of the other parent antibody, mAb063 (Figure 9E). Serum capture of BiP063N2 using hFGFR3b, mFGFR3b, or hFGFR3c exhibited comparable profiles, and all samples revealed that BiP063N2 remained intact for at least 30 days after injection (Figures 9C and 9F).

[0454] 8.9. Example 8: Design of Forced-Pairing Antibodies A new antibody was designed to generate an antibody with lower immunogenicity and / or better development potential by forced pairing of the light chains of the parent antibody, mAb117, and mAb063 (Figure 10A). The Biacore binding affinity and pharmacokinetic profile of the forced paired antibody (mAb117-VL063) with the mAb117 heavy chain paired with the mAb063 light chain were comparable to those of its parent antibody, mAb117 (Figure 10B).

[0455] 8.10. Example 9: In vitro activity of 2+2 Fab antibodies derived from BiP063N2 against oncogenic FGFR3 mutations Three 2+2 N-Fab and three 2+2 C-Fab antibodies were designed with the properties presented in Table 3.

[0456] [Table 10]

[0457] All 2+2 N-Fab antibodies inhibited UMUC14 cell proliferation with potency comparable to that of BiP063N2 (Figure 11A). The same antibody set also showed effective inhibition of RT4 proliferation (Figure 11B).

[0458] The 2+2 C-Fab antibodies exhibited variable levels of inhibitory activity against UMUC14 and RT4 cells, but none of them was superior to BiP063N2 (Figures 11C and 11D). Conversion of BiP063N2 to a 2+2 N-Fab antibody by forced light chain pairing maintained its activity.

[0459] 8.11. Example 10: Effect of IgG Backbone on Antibody Activity To assess the effect of different IgG subclasses on antibody activity, antibodies with identical CDRs were constructed using IgG1, IgG2, and IgG4 scaffolds as described in Section 8.1.1. The following antibodies were evaluated: mAb063, mAb117, BiP063N2, 2+2 N-Fab (8), and 2+2 N-Fab (9). The effect of the resulting antibodies on the proliferation of UMUC14 and RT4 cells was assessed as described in Section 8.1.3.

[0460] In an assay using UMUC14 cells, the IgG subclass did not affect the inhibitory activity of BiP063N2 against cell proliferation (Figure 12A). BiP063N2 maintained potent activity across all IgG scaffolds. The activity of the mAb063 antibody was affected by different IgG scaffolds, with partial or complete loss of activity observed with IgG4 and IgG1 scaffolds, respectively (Figure 12B). The activity of mAb117 was not affected by the IgG subclass, as moderate agonist activity was observed across all IgG subclasses (Figure 12C).

[0461] In the RT4 cell proliferation assay, the IgG subclass did not affect the inhibitory activity of BiP063N2 (Fig. 12D), but had a moderate effect on the IC50 of mAb063 activity (Fig. 12E). The agonistic activity was comparable to that of mAb117 with IgG1, IgG4S108P, and IgG4S backbones, whereas mAb117 with IgG2 backbone showed moderate inhibitory activity (Fig. 12F).

[0462] The IgG backbone had no effect on the inhibitory activity of 2+2 N-Fab(8) on UMUC14 cell proliferation (Figure 13A) and little to no effect on RT4 cell proliferation (Figure 13B). The 2+2 N-Fab(9) construct with an IgG1 backbone exhibited comparable levels of inhibition of UMUC14 and RT4 cell proliferation as the 2+2 N-Fab(9) with an IgG4(S108P) backbone (Figures 13C and 13D, respectively).

[0463] 8.12. Example 11: Inhibitory Effect of BiP063N2 on Heparin-Dependent FGFR3 S249C Activity Heparin is involved in disulfide formation between FGFR3 S249C monomers. To assess whether FGFR3 S249C dimerization and signaling are heparin-dependent, BaF3 cells stably expressing FGFR3 S249C were serum-starved overnight in the absence or presence of 100 nM BiP063N2 and then treated for 15 min with 10 μg / ml heparin alone, 100 ng / ml hFGF1 alone, or 100 ng / ml hFGF1 + 10 μg / ml heparin. Phospho-MAPK (pMAPK) and total MAPK levels were determined by Western blot as described in section 8.1.5. Next, BaF3 cells expressing FGFR3 S249C were cultured in the presence of hFGF1 and heparin for 72 hours, and the effects of BiP063N2, mAb063, AZD4547, and erdafitinib on the proliferation of BaF3 cells expressing FGFR3 S249C were determined as described in Section 8.1.4.

[0464] In the absence of BiP063N2, cotreatment with hFGF1 and heparin was associated with increased levels of pMAPK compared to cells treated with either hFGF1 or heparin. The presence of BiP063N2 was associated with similar levels of pMAPK in cells under all treatment conditions (Figure 14A), suggesting that BiP063N2 was able to inhibit heparin / FGF1-induced MAPK phosphorylation. Both BiP063N2 and mAb063 were able to inhibit the proliferation of hFGF1 / heparin-treated BaF3 cells at comparable levels (Figure 14B).

[0465] 8.13. Example 12: Inhibitory Effect of BiP063N2 on Cell Proliferation Driven by TKI-Resistant FGFR3V557M / L Mutation To further evaluate the efficacy of BiP063N2 in the context of secondary FGFR3 mutations that confer resistance to FGFR TKIs, growth assays were performed as described in section 8.1.4 using BaF3 cell lines expressing FGFR3 S249C with or without the TKI resistance-associated mutations V557M or V557L in the presence of FGF1 / heparin.

[0466] The pan-FGFR TKIs AZD4547 and erdafitinib were ineffective in cells expressing FGFR3 with either the V557M or V557L mutation alone or together with the S249C mutation (Figures 15A-15D). In contrast, both BiP063N2 and mAb063 robustly inhibited the growth of BaF3 double-mutant FGFR3-expressing cells. Consistent with these effects on cell growth, BiP063N2 completely inhibited the dimerization and phosphorylation of the double-mutant FGFR3 S249C / V557M or FGFR3 S249C / V557L receptors (Figure 15E).

[0467] 8.14. Example 13: Inhibition of signal transduction in BaF3 cells expressing FGFR3 Y375C or S248C. BaF3 cells expressing either WT hFGFR3b or Y375C hFGFR3b were serum starved overnight in the absence or presence of 100 nM isotype control antibody, mAb063, mAb117, or BiP063N2, followed by stimulation with 100 nM FGF1 and 10 μg / ml heparin for 15 min, and lysates were analyzed for pMAPK and MAPK as described in Section 8.1.5.

[0468] In BaF3 cells expressing WT hFGFR3b, FGF1 / heparin stimulation increased pMAPK levels in control cells that were not treated with antibody. A similar increase in pMAPK was observed in cells treated with either an isotype control antibody or mAb117. In contrast, both mAb063 and BiP063N2 were associated with no increase in pMAPK (Figure 16A), suggesting that mAb063 and BiP063N2 were able to completely inhibit MAPK phosphorylation. Results obtained in BaF3 cells expressing Y375C hFGFR3b were similar in the no-antibody, isotype control antibody, and mAb117 treatment conditions. In contrast to the results obtained with WT hFGFR3b cells, FGF1 / heparin stimulation was associated with a slight increase in pMAPK levels in cells treated with mAb063, which appeared to be further reduced in cells treated with BiP063N2 (Fig. 16B ), suggesting that BiP063N2 was more effective in inhibiting signaling associated with Y375C hFGFR3b.

[0469] BaF3 cells expressing S248C hFGFR3b were then used to culture in medium with 1 nM FGF1 and 5 μg / ml heparin in the absence (control) or presence of mAb063 or BiP063N2 as described in Section 8.1.4.

[0470] Compared to control conditions, both mAb063 and BiP063N2 reduced cell viability in a dose-dependent manner. The decrease in cell viability was greater when cells were treated with BiP063N2.

[0471] Taken together, these results suggest that BiP063N2 is more effective than the parent antibody mAb063 in inhibiting signaling and cell proliferation mediated by FGFR3 harboring the Y375C or S248C mutation.

[0472] All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. A multispecific binding molecule (MBM), comprising: (a) an antigen-binding domain 1 (ABD1) that specifically binds to a first epitope of FGFR3; (b) an antigen binding domain 2 (ABD2) that specifically binds to a second epitope of FGFR3 that is different from the first epitope.

2. The MBM of claim 1, wherein the first epitope comprises a sequence present in D3 of FGFR3.

3. The MBM of claim 1 or 2, wherein the first epitope comprises a sequence present in D2 of FGFR3.

4. The MBM of any one of claims 1 to 3, wherein the second epitope comprises a sequence present in D1 of FGFR3.

5. The MBM of any one of claims 1 to 4, wherein ABD2 is an FGFR3 non-antagonist antigen-binding domain.

6. 6. The MBM of claim 5, wherein ABD2 is an FGFR3 agonist antigen-binding domain.

7. The MBM of any one of claims 1 to 4, wherein ABD2 is an FGFR3 antagonist antigen-binding domain.

8. The MBM of any one of claims 1 to 5, wherein ABD1 is an FGFR3 antagonist antigen-binding domain.

9. The multispecific binding molecule (MBM) of any one of claims 1 to 8, which inhibits interactions between FGFR3 molecules.

10. The MBM of any one of claims 1 to 9, wherein said MBM has at least 100-fold higher selectivity for FGFR3b compared to FGFR3c.

11. The MBM of any one of claims 1 to 10, wherein the MBM is bispecific.

12. The MBM of any one of claims 1 to 11, wherein the MBM is tetravalent.

13. 13. The MBM of any one of claims 1 to 12, wherein ABD1 and / or ABD2 specifically bind to human FGFR3, optionally an FGFR3 molecule comprising the amino acid sequence of SEQ ID NO:

2.

14. The MBM of any one of claims 1 to 13, wherein ABD1 is an scFv.

15. The MBM of any one of claims 1 to 13, wherein ABD1 is Fab.

16. The MBM of any one of claims 1 to 15, wherein ABD2 is an scFv.

17. The MBM of any one of claims 1 to 15, wherein ABD2 is Fab.

18. 18. The MBM of any one of claims 1 to 17, wherein the MBM comprises an Fc heterodimer, and optionally an Fc domain in the Fc heterodimer comprises a knob-in-hole mutation compared to a wild-type Fc domain and / or a star mutation compared to a wild-type Fc domain.

19. The MBM of any one of claims 1 to 18, further comprising an antigen binding domain 3 (ABD3) that specifically binds to the first epitope.

20. 20. The MBM of claim 19, wherein ABD3 is an scFv.

21. 20. The MBM of claim 19, wherein ABD3 is Fab.

22. The MBM of any one of claims 1 to 21, further comprising an antigen binding domain 4 (ABD4) that specifically binds to the second epitope.

23. 23. The MBM of claim 22, wherein ABD4 is an scFv.

24. 23. The MBM of claim 22, wherein ABD4 is Fab.

25. The MBM is (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first scFv operably linked to (ii) a first heavy chain region of a first Fab operably linked to (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second scFv, operably linked to (ii) a second heavy chain region of a second Fab, operably linked to (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab; Optionally, (i) ABD1 is the first scFv; (ii) ABD2 is the first Fab; (iii) ABD3 is the second scFv; (iv) ABD4 is the second Fab; or (v) The MBM of any one of claims 22 to 24, which is any combination of two, three, or all four of (i), (ii), (iii), and (iv).

26. The MBM is (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (ii) a first heavy chain region of a first Fab, operably linked to (iii) an Fc domain, operably linked to (iii) a first scFv; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second heavy chain region of a second Fab, operably linked to (ii) an Fc domain, operably linked to (iii) a second scFv; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab; Optionally, (i) ABD1 is the first scFv; (ii) ABD2 is the first Fab; (iii) ABD3 is the second scFv; (iv) ABD4 is the second Fab; or (v) The MBM of any one of claims 22 to 24, which is any combination of two, three, or all four of (i), (ii), (iii), and (iv).

27. The MBM is (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab, to which is operably linked (ii) a second heavy chain region of a second Fab, to which is operably linked (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab, operably linked to (ii) a fourth heavy chain region of a fourth Fab, operably linked to (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with the third heavy chain region to form the third Fab; (f) a sixth polypeptide chain comprising a fourth light chain that pairs with the fourth heavy chain region to form the fourth Fab; Optionally, (i) ABD1 is the first Fab; (ii) ABD2 is the second Fab; (iii) ABD3 is the third Fab; (iv) ABD4 is the fourth Fab; or (v) The MBM of any one of claims 22 to 24, which is any combination of two, three, or all four of (i), (ii), (iii), and (iv).

28. 28. The MBM of claim 27, wherein two, three, or all four of the light chains are identical.

29. The MBM is (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second heavy chain region of a second Fab, operably linked to (iii) an Fc domain, operably linked to (iii) a first heavy chain region of a first Fab; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (ii) a fourth heavy chain region of a fourth Fab, operably linked to (i) an Fc domain, operably linked to (iii) a third heavy chain region of a third Fab; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with the third heavy chain region to form the third Fab; (f) a sixth polypeptide chain comprising a fourth light chain that pairs with the fourth heavy chain region to form the fourth Fab; Optionally, (i) ABD1 is the first Fab; (ii) ABD2 is the second Fab; (iii) ABD3 is the third Fab; (iv) ABD4 is the fourth Fab; or (v) The MBM of any one of claims 22 to 24, which is any combination of two, three, or all four of (i), (ii), (iii), and (iv).

30. 30. The MBM of claim 29, wherein two, three, or all four of the light chains are identical.

31. A multispecific binding molecule (MBM), comprising: (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first scFv that specifically binds to a first epitope of FGFR3, operably linked to (ii) a first heavy chain region of a first Fab that specifically binds to a second epitope of FGFR3 different from the first epitope, operably linked to (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a second scFv that specifically binds to the first epitope, operably linked to (ii) a second heavy chain region of a second Fab that specifically binds to the second epitope, operably linked to (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab.

32. A multispecific binding molecule (MBM), comprising: (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a first heavy chain region of a first Fab that specifically binds to a first epitope of FGFR3, operably linked to (ii) a second heavy chain region of a second Fab that specifically binds to a second epitope of FGFR3 different from the first epitope, operably linked to (iii) an Fc domain; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, (i) a third heavy chain region of a third Fab that specifically binds to the first epitope, operably linked to (ii) a fourth heavy chain region of a fourth Fab that specifically binds to the second epitope, operably linked to (iii) an Fc domain; (c) a third polypeptide chain comprising a first light chain that pairs with the first heavy chain region to form the first Fab; (d) a fourth polypeptide chain comprising a second light chain that pairs with the second heavy chain region to form the second Fab; (e) a fifth polypeptide chain comprising a third light chain that pairs with the third heavy chain region to form the third Fab; (f) a sixth polypeptide chain comprising a fourth light chain that pairs with the fourth heavy chain region to form the fourth Fab.

33. A nucleic acid or nucleic acids encoding the MBM of any one of claims 1 to 32.

34. 33. A cell engineered to express the MBM of any one of claims 1 to 32, optionally transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the MBM of any one of claims 1 to 32 under the control of one or more promoters.

35. 34. A cell comprising the nucleic acid or nucleic acids of claim 33.

36. A method for producing MBM, comprising: (a) culturing the cells of claim 34 or 35 under conditions sufficient to express the MBM; and (b) recovering the MBM from the cells.

37. A pharmaceutical composition comprising the MBM of any one of claims 1 to 32 and an excipient.

38. A method comprising administering to a subject the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37.

39. 38. A method of treating bladder cancer, optionally metastatic bladder cancer, in a subject in need thereof, comprising administering to the subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37.

40. 40. The method of claim 39, wherein the bladder cancer is an FGFR3 mutant bladder cancer.

41. 41. The method of claim 40, wherein the FGFR3 mutant bladder cancer is FGFR3-TACC3 fusion bladder cancer, FGFR3-S249C bladder cancer, FGFR3-R248C bladder cancer, FGFR3-G372C bladder cancer, FGFR3-Y375C bladder cancer, or FGFR3-K650E bladder cancer.

42. 40. The method of claim 39, wherein the bladder cancer is not an FGFR3 mutant bladder cancer.

43. 38. A method of treating an FGFR3-positive tumor in a subject in need thereof, comprising administering to the subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37, optionally wherein the FGFR3-positive tumor is a bladder cancer tumor.

44. 38. A method of sensitizing FGFR3-positive tumor cells in a subject to cancer therapy, optionally chemotherapy, immunotherapy, or radiation therapy, said method comprising administering to said subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37, optionally wherein said FGFR3-positive tumor cells are bladder cancer tumor cells.

45. 38. A method of inhibiting metastasis of FGFR3-positive tumor cells in a subject in need thereof, said method comprising administering to said subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37, optionally wherein said FGFR3-positive tumor cells are bladder cancer tumor cells.

46. 38. A method for reducing the viability of FGFR3-positive cancer cells, the method comprising administering to the subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37, optionally wherein the FGFR3-positive tumor cells are bladder cancer tumor cells.

47. 38. A method for reducing FGFR3 signaling in FGFR3-positive cells, the method comprising administering to the subject an effective amount of the MBM of any one of claims 1 to 32, or the pharmaceutical composition of claim 37.

48. 48. The method of claim 47, wherein the FGFR3 positive cells are in cell culture.

49. 48. The method of claim 47, wherein the FGFR3 positive cells are in a subject, optionally a human subject.

50. 50. The method of any one of claims 47 to 49, wherein the FGFR3 positive cells are cancer cells, optionally bladder cancer cells.

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