Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes

Abstract

The present invention provides, in part, a protein-drug conjugate comprising an anti-fibroblast growth factor receptor 3 (FGFR3) (e.g., human FGFR3) antigen-binding protein (e.g., scFv, Fab) conjugated to a molecular cargo (e.g., a polynucleotide, a polypeptide, a liposome, or a lipid nanoparticle), for delivering the molecular cargo to a target tissue (e.g., the brain). Methods are provided for treating various diseases or disorders, such as neurological disorders, with the conjugate.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 383,673, filed November 14, 2022, and U.S. Provisional Application No. 63 / 587,585, filed October 3, 2023, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy, created on November 6, 2023, is named 250298_000565_SL.xml and is 476,394 bytes in size.

[0003] The present invention relates to protein-drug conjugates comprising an anti-fibroblast growth factor receptor 3 (FGFR3) antigen binding protein conjugated to a molecular cargo, and methods of treating disease with such protein-drug conjugates. [Background technology]

[0004] A lack of understanding of the molecular signatures of disease, coupled with a limited toolbox of robust model systems, has contributed to the failure to develop disease-modifying therapies targeting various neurological diseases and / or disorders. Such neurological disorders range from progressive neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease) to neurodevelopmental disorders (e.g., Alexander disease, multiple sulfatase deficiency) and can include those associated with physical injury (e.g., traumatic brain injury, spinal cord injury, and stroke). While progress has been made on many fronts to identify agents that can prevent, slow, or halt disease progression, current therapies offer little relief. Thus, there remains a need to develop therapies that can interrupt the onset and / or course of neurological diseases, particularly to improve the quality of life for those affected by such diseases. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides, inter alia, protein-drug conjugates that specifically bind to fibroblast growth factor receptor 3 (FGFR3) and include an antigen-binding protein conjugated to a molecular cargo.

[0006] In one aspect, provided herein is a protein-drug conjugate comprising an antigen-binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3) and is conjugated to a molecular cargo.

[0007] In some embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof. In some embodiments, the protein-drug conjugate comprises a heavy chain variable region (HCVR or V H ) and / or light chain variable region (LCVR or V L In some embodiments, the antigen binding protein is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain fragment variable region (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

[0008] In some embodiments, the antigen binding protein comprises a fragment antigen binding region (Fab).

[0009] In some embodiments, the antigen binding protein is a single-chain fragment variable region (scFv). In some embodiments, the scFv comprises domains arranged, from N-terminus to C-terminus, in the following orientation: HCVR-LCVR. In some embodiments, the scFv comprises domains arranged, from N-terminus to C-terminus, in the following orientation: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS) n -(SEQ ID NO: 321), wherein n is 1 to 10.

[0010] In various embodiments, the antigen binding protein specifically binds to FGFR3b and / or FGFR3c. In some embodiments, the antigen binding protein specifically binds to FGFR3b. In some embodiments, the antigen binding protein specifically binds to FGFR3c. In some embodiments, the antigen binding protein specifically binds to FGFR3b and FGFR3c. In some embodiments, FGFR3b is monomeric and / or dimeric FGFR3b. In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3b. In some embodiments, the antigen binding protein specifically binds to dimeric FGFR3b. In some embodiments, the antigen binding protein specifically binds to monomeric and dimeric FGFR3b. In some embodiments, FGFR3c is monomeric and / or dimeric FGFR3c. In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3c. In some embodiments, the antigen binding protein specifically binds to dimeric FGFR3c. In some embodiments, the antigen binding protein specifically binds to monomeric and dimeric FGFR3c.

[0011] In various embodiments, the antigen binding protein specifically binds to human FGFR3b and / or FGFR3c. In some embodiments, the antigen binding protein specifically binds to human FGFR3b. In some embodiments, the antigen binding protein specifically binds to human FGFR3c. In some embodiments, the antigen binding protein specifically binds to human FGFR3b and FGFR3c.

[0012] In some embodiments, the antigen binding protein is about 1 x 10 -7 K of M D or higher affinity. In some embodiments, the antigen binding protein binds to human FGFR3b with an affinity of about 1 x 10 -8 K of M D or higher affinity. In some embodiments, the antigen binding protein binds to human FGFR3b with an affinity of about 1 x 10 -9 K of M D or binds to human FGFR3b with higher affinity.

[0013] In some embodiments, the antigen binding protein is about 1 x 10 -7 K of M D or higher affinity. In some embodiments, the antigen binding protein binds to human FGFR3c with an affinity of about 1 x 10 -8 K of M D or binds to human FGFR3c with higher affinity.

[0014] In some embodiments, the antigen binding protein is (i) an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 140, 159, 169, 179, 199, or 219 (or a variant thereof); and / or (ii) An LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 148, 187, 207, or 227 (or a variant thereof).

[0015] In some embodiments, the antigen binding protein is (1) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (2) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 30; (3) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 50; (4) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (5) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); or (6) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (7) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (8) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (9) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (10) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (11) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (12) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); or (13) An HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).

[0016] In some embodiments, the antigen binding protein is (a) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (b) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36 (or a variant thereof); (c) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 (or a variant thereof); (d) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76 (or a variant thereof); (e) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96 (or a variant thereof); (f) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 116 (or a variant thereof); (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof); (h) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 146 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (i) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 161 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (j) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 171 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (k) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 181 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 191 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof); (l) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof); and / or (m) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 221 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and It comprises an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76 (or a variant thereof).

[0017] In some embodiments, the antigen binding protein is (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); or (xiii) Comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).

[0018] In some embodiments, the antigen binding protein is (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 138; (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; or (m) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0019] In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3b. In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3b. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 138; (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (m) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0020] In some embodiments, the antigen binding protein specifically binds to dimeric FGFR3b. In some embodiments, the antigen binding protein specifically binds to dimeric FGFR3b. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 138; (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0021] In some embodiments, the antigen binding protein specifically binds to monomeric and dimeric FGFR3b.

[0022] In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3c. In some embodiments, the antigen binding protein specifically binds to monomeric FGFR3c. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0023] In some embodiments, the antigen binding protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:18 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:20.

[0024] In some embodiments, the antigen binding protein binds to the same epitope on FGFR3 as an antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1-1.

[0025] In some embodiments, the antigen binding protein competes for binding to FGFR3 with an antibody comprising an HCVR / LCVR amino acid sequence pair set forth in Table 1-1.

[0026] In one aspect, there is provided a protein-drug conjugate comprising an antigen binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3), wherein the antigen binding protein is conjugated to a molecular cargo and comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to: a. an epitope comprising the sequence GPTVWVK (SEQ ID NO: 378), and / or an epitope comprising the sequence TQR, b. an epitope comprising the sequence ADVR (SEQ ID NO: 376), and / or an epitope comprising the sequence IGVAEK (SEQ ID NO: 377), c. an epitope comprising the sequence HCKVY (SEQ ID NO: 379), and / or an epitope comprising the sequence KSWISE (SEQ ID NO: 380), and / or an epitope comprising the sequence ADVR (SEQ ID NO: 376), e. the sequence GPTVWVK (SEQ ID NO: 378) Provided herein are protein-drug conjugates that bind to one or more epitopes of FGFR3 selected from: f. an epitope contained within or overlapping with the sequence TQR, and / or an epitope contained within or overlapping with the sequence ADVR (SEQ ID NO: 376), and / or an epitope contained within or overlapping with the sequence IGVAEK (SEQ ID NO: 377), and g. an epitope contained within or overlapping with HCKVY (SEQ ID NO: 379), and / or an epitope contained within or overlapping with the sequence KSWISE (SEQ ID NO: 380), and / or an epitope contained within or overlapping with the sequence ADVR (SEQ ID NO: 376). In some embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of FGFR3 selected from: a. an epitope consisting of the sequence GPTVWVK (SEQ ID NO: 378), and / or an epitope consisting of the sequence TQR; b. an epitope consisting of the sequence ADVR (SEQ ID NO: 376), and / or an epitope consisting of the sequence IGVAEK (SEQ ID NO: 377); and c. an epitope consisting of the sequence HCKVY (SEQ ID NO: 379), and / or an epitope consisting of the sequence KSWISE (SEQ ID NO: 380), and / or an epitope consisting of the sequence ADVR (SEQ ID NO: 376).

[0027] In one aspect there is provided a protein-drug conjugate comprising an antigen binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3), wherein the antigen binding protein is conjugated to a molecular cargo and comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a. an epitope comprising the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363), and / or an epitope comprising the sequence YSCRQRLTQRVL (SEQ ID NO: 364), b. an epitope comprising the sequence LLAVPAAN (SEQ ID NO: 365), and / or an epitope comprising the sequence VLERSPHRPILQAG (SEQ ID NO: 366), and / or an epitope comprising the sequence YVTVLKSWISE (SEQ ID NO: 367), and / or an epitope comprising the sequence ADVRLR (SEQ ID NO: 368), and / or an epitope comprising the sequence LCRATNFIGVAE an epitope comprising the sequence VLVGPQRL (SEQ ID NO: 371); d. an epitope comprising the sequence VLERSPHRPILQAG (SEQ ID NO: 372) and / or an epitope comprising the sequence HCKVYSDAQP (SEQ ID NO: 373) and / or an epitope comprising the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO: 374) and / or an epitope comprising the sequence LCRATNFIGVAEKAF (SEQ ID NO: 375); e. an epitope contained within or overlapping with the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363) and / or an epitope contained within or overlapping with the sequence YSCRQRLTQRVL (SEQ ID NO: 364); f.an epitope contained within or overlapping with the sequence LLAVPAAN (SEQ ID NO: 365), and / or an epitope contained within or overlapping with the sequence VLERSPHRPILQAG (SEQ ID NO: 366), and / or an epitope contained within or overlapping with the sequence YVTVLKSWISE (SEQ ID NO: 367), and / or an epitope contained within or overlapping with the sequence ADVRLR (SEQ ID NO: 368), and / or an epitope contained within or overlapping with the sequence LCRATNFIGVAEKAFW (SEQ ID NO: 369); g. an epitope contained within or overlapping with the sequence GQQEQLVFGSGDAVE (SEQ ID NO: 370), and / or an epitope contained within or overlapping with the sequence VL Provided herein are protein-drug conjugates that bind to one or more epitopes of FGFR3 selected from an epitope contained within or overlapping with VGPQRL (SEQ ID NO: 371), and an epitope contained within or overlapping with the sequence VLERSPHRPILQAG (SEQ ID NO: 372), and / or an epitope contained within or overlapping with the sequence HCKVYSDAQP (SEQ ID NO: 373), and / or an epitope contained within or overlapping with the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO: 374), and / or an epitope contained within or overlapping with the sequence LCRATNFIGVAEKAF (SEQ ID NO: 375). In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: a. an epitope consisting of the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363), and / or an epitope consisting of the sequence YSCRQRLTQRVL (SEQ ID NO: 364); b. an epitope consisting of the sequence LLAVPAAN (SEQ ID NO: 365), and / or an epitope consisting of the sequence VLERSPHRPILQAG (SEQ ID NO: 366), and / or an epitope consisting of the sequence YVTVLKSWISE (SEQ ID NO: 367), and / or an epitope consisting of the sequence ADVRLR (SEQ ID NO: 368), and / or an epitope consisting of the sequence LCRATNFIGVAEKAFW (SEQ ID NO: 369); c.d. Binds to one or more epitopes of FGFR3 selected from the epitope consisting of the sequence GQQEQLVFGSGDAVE (SEQ ID NO: 370), and / or the epitope consisting of the sequence VLVGPQRL (SEQ ID NO: 371), and d. the epitope consisting of the sequence VLERSPHRPILQAG (SEQ ID NO: 372), and / or the epitope consisting of the sequence HCKVYSDAQP (SEQ ID NO: 373), and / or the epitope consisting of the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO: 374), and / or the epitope consisting of the sequence LCRATNFIGVAEKAF (SEQ ID NO: 375).

[0028] In various embodiments of the protein-drug conjugates described herein, the antigen-binding protein is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain fragment variable region (scFv), a bis-scFv, an (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

[0029] In various embodiments of the protein-drug conjugates described herein, the molecular cargo comprises (i) the HCVR of the antigen binding protein, (ii) the LCVR of the antigen binding protein, (iii) the heavy chain of the antigen binding protein, and / or (iv) the light chain of the antigen binding protein.

[0030] In some embodiments, the molecular cargo is conjugated to the antigen binding protein via a glutamine and / or lysine residue.

[0031] In some embodiments, the glutamine residue (i) is introduced at the N-terminus and / or C-terminus of the heavy chain of the antigen binding protein, (ii) is introduced at the N-terminus and / or C-terminus of the light chain of the antigen binding protein, (iii) is naturally present in the CH2 domain or CH3 domain of the antigen binding protein, (iv) is introduced into the antigen binding protein by modifying one or more amino acids, and / or (v) is Q295 or is mutated from N297 to Q297 (N297Q).

[0032] In some embodiments, the antigen binding protein comprises a glutamine-containing tag, and the molecular cargo is conjugated to the antigen binding protein via a glutamine residue of the glutamine-containing tag.

[0033] In some embodiments, the glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO: 290), LLQG (SEQ ID NO: 291), LSLSQG (SEQ ID NO: 292), GGGLLQGG (SEQ ID NO: 293), GLLQG (SEQ ID NO: 294), LLQ, GSPLAQSHGG (SEQ ID NO: 295), GLLQGGG (SEQ ID NO: 296), GLLQGG (SEQ ID NO: 297), GLLQ (SEQ ID NO: 298), LLQLLQGA (SEQ ID NO: 299), LLQGA (SEQ ID NO: 300), LLQYQGA (SEQ ID NO: 301), LLQGSG (SEQ ID NO: 302), LLQYQG (SEQ ID NO: 303), LLQLLQG (SEQ ID NO: 304), SLLQG (SEQ ID NO: 305), LLQLQ (SEQ ID NO: 306), LLQLLQ (SEQ ID NO: 307), and LLQGR (SEQ ID NO: 308).

[0034] In some embodiments, the antigen binding protein and the molecular cargo are conjugated via a linker.

[0035] In some embodiments, the molecular cargo comprises a polynucleotide molecule, a polypeptide molecule, a carrier, a viral particle, a viral capsid protein, or a small molecule.

[0036] In some embodiments, molecular cargo comprises polynucleotide molecule.In some embodiments, polynucleotide molecule is interference nucleic acid molecule, guide RNA, ribozyme, aptamer, mixomer, multimer or mRNA.In some embodiments, interference nucleic acid molecule is siRNA, shRNA, miRNA, antisense oligonucleotide or gapmer.

[0037] In some embodiments, the interfering nucleic acid molecule is siRNA.In some embodiments, the siRNA comprises a sense strand of 21 nucleotides in length.In some embodiments, the siRNA comprises an antisense strand of 23 nucleotides in length.In some embodiments, the siRNA comprises two phosphorothioate bonds in the first and second internucleoside linkages at the 5'-end of the sense strand.In some embodiments, the siRNA comprises two phosphorothioate bonds in the first and second internucleoside linkages at the 3'-end and / or 5'-end of the antisense strand.

[0038] In some embodiments, the interfering nucleic acid molecule is an antisense oligonucleotide.

[0039] In some embodiments, the polynucleotide molecule is a guide RNA.

[0040] In various embodiments, the polynucleotide molecule targets a gene or gene product associated with a neurological disease and / or disorder. In some embodiments, the gene is APOE4, GFAP, MECP2, AQP4, or STAT3.

[0041] In various embodiments, the polynucleotide molecule comprises one or more modified nucleotides.

[0042] In some embodiments, the molecular cargo comprises a polypeptide molecule. In some embodiments, the polypeptide molecule is an enzyme that binds to a target other than FGFR3, a neuroprotective molecule, or an antigen-binding protein. In some embodiments, the polypeptide molecule is associated with a neurological disease and / or disorder. In some embodiments, the polypeptide molecule is a protective ApoE isoform or variant, ATPase 13A2 (encoded by ATP13A2), sulfatase modifier 1 (encoded by SUMF1), fragile X messenger ribonucleoprotein (FMRP) (encoded by FMR1), or glutamate transporter 1 (encoded by GLT1). In some embodiments, the protective ApoE isoform or variant is ApoE2, ApoE Christchurch, or ApoE Jacksonville. In some embodiments, the polypeptide molecule is a neurotrophic factor, an antibody or antibody fragment, an antibody-receptor fusion protein, or a cytokine signaling inhibitor. In some embodiments, the neurotrophic factor is ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), or insulin-like growth factor 1 (IGF). In some embodiments, the antibody receptor fusion protein is an anti-amyloid beta Gas6 fusion protein. In some embodiments, the suppressor of cytokine signaling is suppressor of cytokine signaling 3 (Socs3).

[0043] In some embodiments, the molecular cargo is conjugated to the antigen binding protein at a drug-to-antibody ratio (DAR) of at least 1 to at least 10.

[0044] In some embodiments, the molecular cargo is conjugated to the antigen binding protein with a DAR of 1, 2, 3, or 4.

[0045] In some embodiments, the molecular cargo is conjugated to the antigen binding protein with a DAR of 2.

[0046] In some embodiments, the molecular cargo is conjugated to the antigen binding protein with a DAR of 4.

[0047] In some embodiments, provided herein is a protein-drug conjugate for use in the treatment or prevention of a neurological disease or disorder.

[0048] In various embodiments, the neurological disease or disorder is a neurodegenerative disease, a neurodevelopmental disease, a physical injury, a neuropsychiatric disease, or brain cancer. In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or a prion disease (transmissible spongiform encephalopathy). In some embodiments, the neurodevelopmental disease is Alexander disease, multiple sulfatase deficiency, autism, epilepsy, Rett syndrome, or fragile X. In some embodiments, the physical injury is traumatic brain injury, spinal cord injury, stroke, or cerebral edema. In some embodiments, the neuropsychiatric disease or disorder is major depressive disorder, anxiety disorder, or bipolar disorder. In some embodiments, the brain cancer is a glioma. In some embodiments, the glioma is an astrocytoma.

[0049] In some embodiments, the molecular cargo comprises a carrier. In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex.

[0050] In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP). In some embodiments, the LNP further comprises a polynucleotide molecule and / or a polypeptide molecule. In some embodiments, the LNP comprises one or more components of a gene editing system. In some embodiments, the LNP comprises (a) a Cas nuclease or a nucleic acid encoding a Cas nuclease, and / or (b) a guide RNA or one or more DNAs encoding the guide RNA. In some embodiments, the Cas nuclease is a Cas9 protein. In some embodiments, the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in mammalian cells. In some embodiments, the nucleic acid encoding the Cas nuclease is codon-optimized for expression in human cells. In some embodiments, the nucleic acid encoding the Cas nuclease is mRNA. In various embodiments, the guide RNA is a single guide RNA (sgRNA). In some embodiments, the LNP comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

[0051] In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a helper lipid, a stealth lipid, or any combination thereof. In some embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the helper lipid is cholesterol. In some embodiments, the stealth lipid is PEG2k-DMG.

[0052] In another aspect, provided herein is a pharmaceutical composition comprising a protein-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0053] In another aspect, provided herein is a composition or kit comprising a protein-drug conjugate described herein or a pharmaceutical composition described herein and an additional therapeutic agent.

[0054] In another aspect, provided herein is a complex comprising a protein-drug conjugate described herein bound to fibroblast growth factor receptor 3 (FGFR3).

[0055] In another aspect, there is provided a method of making a protein-drug conjugate described herein, comprising: (a) contacting an antigen-binding protein with a molecular cargo under conditions favorable for conjugation of the antigen-binding protein to the molecular cargo; (b) optionally isolating the protein-drug conjugate produced in step (a).

[0056] In another aspect, there is provided a method of making a protein-drug conjugate described herein, wherein the molecular cargo comprises a polypeptide molecule; (a) culturing a host cell containing a polynucleotide encoding a protein-drug conjugate under conditions that allow expression of the protein-drug conjugate; (b) optionally isolating the protein-drug conjugate produced in step (a).

[0057] In another aspect, provided herein is a protein-drug conjugate produced by or obtainable by the above-described method.

[0058] In another aspect, provided herein is a vascular or injection device comprising a protein-drug conjugate described herein.

[0059] In another aspect, provided herein is a method for administering to a subject a protein-drug conjugate described herein, the method comprising introducing the protein-drug conjugate into the body of the subject.

[0060] In another aspect, the present specification provides a method for delivering molecular cargo to tissues or cell types that express FGFR3 in a subject's body, comprising administering to the subject a protein-drug conjugate described herein or a pharmaceutical composition described herein.In some embodiments, the tissue is the brain, spinal cord, or eye.In some embodiments, the cell type is an astrocyte or an astrocyte-derived tumor cell.

[0061] In another aspect, provided herein is a method for treating or preventing a neurological disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the protein-drug conjugates described herein. In some embodiments, the neurological disease or disorder is associated with astrocytes. In some embodiments, the neurological disease or disorder is a neurodegenerative disease, a neurodevelopmental disease, a physical injury, a neuropsychiatric disease, or brain cancer. In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or a prion disease (transmissible spongiform encephalopathy). In some embodiments, the neurodevelopmental disease is Alexander disease, multiple sulfatase deficiency, autism, epilepsy, Rett syndrome, or fragile X. In some embodiments, the physical injury is a traumatic brain injury, spinal cord injury, stroke, or cerebral edema. In some embodiments, the neuropsychiatric disease or disorder is major depressive disorder, anxiety disorder, or bipolar disorder. In some embodiments, the brain cancer is glioma. In some embodiments, the glioma is an astrocytoma. In some embodiments, the method further comprises administering an additional treatment to the subject.

[0062] In various embodiments of the methods described herein, the protein-drug conjugate is administered to the subject's body and administered to the central nervous system via intrathecal, intracisternal, intracerebroventricular, or intraparenchymal administration.

[0063] In various embodiments of the methods described herein, the protein-drug conjugate is administered to the subject via intravitreal or intraocular administration to the eye.

[0064] In various embodiments of the methods described herein, the protein-drug conjugate is administered to the subject via systemic administration. In one embodiment, the protein-drug conjugate is administered to the subject via intranasal administration. [Brief explanation of the drawings]

[0065] [Figure 1A] A schematic diagram of the general architecture of fibroblast growth factor receptor 3 (FGFR3) and its isoforms, FGFR3b and FGFR3c, is shown. A schematic diagram (top) showing the orientation of the FGFR dimer within the plasma membrane. The FGF3 receptor domain structure consists of an extracellular domain containing the ligand-binding site, a transmembrane domain, and an intracellular domain containing the salivary tyrosine kinase. The receptor is stabilized by the HS chains of heparin / heparan sulfate (HS) proteoglycans (HSPGs). A schematic diagram (bottom) of an FGF monomer illustrating the three IgG-like loops (Ig-I, Ig-II, and Ig-III) of the extracellular ligand-binding domain and the split kinase domain. The Ig-I, Ig-II, and Ig-II loops of the extracellular ligand-binding domain are encoded by exons 7–9. FGFR3b contains exon 8 but excludes exon 9, whereas FGFR3c contains exon 9 but excludes exon 8. Thus, inclusion / exclusion of exons 8 and 9 directs expression of the IIIb versus IIIc splice form. [Figure 1B]A schematic diagram of the general architecture of fibroblast growth factor receptor 3 (FGFR3) and its isoforms FGFR3b and FGFR3c is shown. A schematic diagram showing a structural comparison of the alternatively spliced ​​FGFR3b and FGFR3c isoforms. The Ig-I, Ig-II, and Ig-II loops of the extracellular ligand-binding domain are encoded by exons 7–9. FGFR3b includes exon 8 but excludes exon 9, whereas FGFR3c includes exon 9 but excludes exon 8. Therefore, the inclusion / exclusion of exons 8 and 9 dictates the expression of the IIIb versus IIIc splice form. [Figure 2A] FGFR3 is highly expressed on mouse and human astrocytes. Graph of RNA sequencing (RNASeq) data showing expression of FGFR1-FGFR4 transcripts in mouse neural and supporting cells. [Figure 2B] FGFR3 is highly expressed on mouse and human astrocytes. Graph of RNA sequencing (RNASeq) data showing expression of FGFR1-FGFR4 transcripts in human neural and supporting cells. [Figure 3] Figure 1 shows the expression of total FGFR3 and the FGFR3b and FGFR3c isoforms in mouse brain, primary mouse astrocytes, and primary human astrocytes. Data are plotted against the Ct values ​​of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to the relative expression of each isoform from mouse brain (½ΔΔCt, mean ± standard deviation [SD]). [Figure 4A] Figure 1 shows that FGFR3 antibodies are internalized into live astrocytes as evidenced by perinuclear speckles (puncta). Schematic showing an exemplary experimental timeline. [Figure 4B] Figure 1 shows that FGFR3 antibodies are internalized into live astrocytes, as evidenced by perinuclear speckles (puncta). Photomicrographs showing immunofluorescence detection of the actin cytoskeleton and perinuclear FGFR3 puncta in human astrocytes using the DEAD staining approach described herein. [Figure 4C] Figure 1 shows that FGFR3 antibodies are internalized in live astrocytes, as evidenced by perinuclear speckles (puncta). Micrographs show immunofluorescence detection of the vimentin (VIM) cytoskeleton and perinuclear FGFR3 puncta in human and mouse astrocytes using the LIVE staining approach described herein. The non-degradable (H4H30063P) FGFR3 antibody showed robust internalization, as evidenced by puncta that extended around the nucleus. [Figure 5A] 1 shows validation of FGFR3 expression and internalization in the U87-FGFR3b-FLuc-GFP line. Schematic showing an exemplary experimental timeline. [Figure 5B] Verification of FGFR3 expression and internalization in the U87-FGFR3b-FLuc-GFP strain. Photomicrographs showing immunofluorescence detection of FGFR3b and FelD1 (negative control) in GFP-positive U87 cells stained with DAPI. FGFR3 is visualized as fluorescent puncta surrounding the nucleus (DAPI staining), confirming FGFR3 expression and internalization in the U87-FGFR3b-FLuc-GFP strain. [Figure 5C] Graph showing percent normalized TaqMan FGFR3 expression (mean ± standard deviation [SD]) in U87 parental cells and U87 cell lines expressing human FGFR3b (U87-hFGFR3b) and human FGFR3c (U87-hFGFR3c) isoforms using hFGFR3b and hFGFR3c probes. [Figure 6A]GFP and luciferase siRNA knockdown of their respective targets in U87-FGFR3b / c-GFP-luciferase cells. Schematic showing the processing of U87-FGFR3b or U87-FGFR3c cell lines, including illustration of GFP-P2A-FLuc mRNA. The schematic shows, in particular, that GFP siRNA can knock down luciferase expression and / or luciferase siRNA can knock down GFP expression, since luciferase and GFP are present in a single, continuous mRNA. [Figure 6B] Figure 1 shows knockdown of GFP and luciferase siRNAs of their respective targets in U87-FGFR3b / c-GFP-luciferase cells. Graph showing the average GFP fluorescence intensity within two different siRNAs (GFP siRNA and luciferase siRNA) relative to a non-targeting siRNA, compared to an untreated control. Treating cells with parent siRNA (i.e., unmodified siRNA) revealed that GFP siRNA was more potent than luciferase siRNA (arrow). This finding motivated us to advance GFP siRNA via the introduction of cleavage modifications to this molecule, as described herein. [Figure 6C] GFP and luciferase siRNA knockdown of their respective targets in U87-FGFR3b / c-GFP-luciferase cells. A graph showing luciferase activity within two different siRNAs (GFP siRNA and luciferase siRNA) relative to a non-targeting siRNA, compared to an untreated control. Treating cells with the parent siRNA (i.e., unmodified siRNA) revealed that GFP siRNA was more potent than luciferase siRNA (arrow). This finding prompted us to advance GFP siRNA via the introduction of cleavage modifications to this molecule, as described herein. [Figure 7] Exemplary GFP and luciferase siRNA sequences are shown. [Figure 8A]1 shows modified GFP-targeted siRNA knockdown in U87-FGFR3b-GFP-luciferase cells. Schematic showing the processing of the U87-FGFR3b cell line, including illustration of GFP-P2A-FLuc mRNA. [Figure 8B] Graph showing luciferase activity in cells treated with 3'-modified (mod) GFP siRNA (3'-truncated siRNA) and 5'-modified (mod) GFP siRNA (5'-truncated siRNA) relative to GFP siRNA and untreated. [Figure 9A] Modified GFP-targeting siRNA molecules are shown. Chemical structure of the N6 linker (Horizon Discovery) (top) and the corresponding N6-modified GFP siRNA sequence based on the reference sequence described in Caplen et al., 2001 (bottom). [Figure 9B] 1 shows modified GFP-targeting siRNA molecules. Exemplary 5' modified GFP siRNA sequences (truncated) (top). Exemplary 3' modified GFP siRNA sequences (truncated) (bottom). [Figure 10A] Figure 1 shows that the internalized FGFR3b antibody H4H30105P2 colocalizes with early and late endosomes, but not with Rab4-positive recycling endosomes, and the internalized FGFR3b antibody H4H30063P colocalizes with early and late endosomes, lysosomes, and Rab-4-positive recycling endosomes. Schematic showing the treatment of U87-FGFR3b cell line. [Figure 10B]The internalized FGFR3b antibody H4H30105P2 colocalizes with early and late endosomes but not with Rab4-positive recycling endosomes. The internalized FGFR3b antibody H4H30063P colocalizes with early and late endosomes, lysosomes, and Rab4-positive recycling endosomes. Photographs show immunofluorescence detection of FGFR3b and the early endosome marker EEA1 (far left), recycling endosome marker Rab4 (middle left), late endosome marker Rab7 (middle right), and lysosomal marker Lamp1 (far right) in GFP-positive U87 cells stained with DAPI. Endosomal trafficking of the FGFR3b antibody H4H30105P2 (unconjugated) demonstrates robust internalization, as evidenced by perinuclear speckles (puncta). The FGFR3b antibody H4H30105P2 colocalizes with the early endosome marker EEA1 and the late endosome marker Rab7, but not with the recycling endosome marker Rab4. [Figure 10C] The internalized FGFR3b antibody H4H30105P2 colocalizes with early and late endosomes but not with Rab4-positive recycling endosomes. The internalized FGFR3b antibody H4H30063P colocalizes with early and late endosomes, lysosomes, and Rab4-positive recycling endosomes. Photographs show immunofluorescence detection of FGFR3b and the early endosome marker EEA1 (far left), recycling endosome marker Rab4 (middle left), late endosome marker Rab7 (middle right), and lysosomal marker Lamp1 (far right) in GFP-positive U87 cells stained with DAPI. Endosomal trafficking of the FGFR3b antibody H4H30063P (unconjugated) demonstrates robust internalization, as evidenced by perinuclear speckles (puncta). The FGFR3b antibody H4H30063P colocalizes with the early endosome marker EEA1, the late endosome marker Rab7, and the lysosomal marker Lamp1, as well as the recycling endosome marker Rab4. [Figure 11]Schematic diagram showing the potential fate(s) of the FGFR3 receptor after internalization by an FGFR3 antibody conjugated to a molecular cargo. It is not yet known which fate pathway (e.g., degradation or recycling) will be most beneficial for delivery of antibody-conjugated molecular cargo. [Figure 12A] 1 shows the screening of FGFR3 antibodies in the U87-FGFR3b-Fluc-GFP cell line. Photomicrographs showing immunofluorescence detection of FGFR3b in GFP-positive U87 cells stained with DAPI demonstrate strong internalization of FGFR3 antibodies in the U87-hFGFR3b cell line. [Figure 12B] Screening of FGFR3 antibodies in the U87-FGFR3b-Fluc-GFP cell line. Photomicrographs showing immunofluorescence detection of FGFR3 in GFP-positive U87 cells stained with DAPI show primarily surface staining with small, diffuse puncta in the U87-hFGFR3c cell line for FGFR3 antibodies, characteristic of best binders (i.e., strong surface binding), weak binders, and non-binders. [Figure 13] 1 shows the results of HDX epitope mapping of anti-FGFR3b H4H30117P2 and H4H30063P. [Figure 14] 1 shows the results of HDX epitope mapping of anti-FGFR3b H4H30045P and H4H30108P2. [Figure 15] 1 shows HDX protection by FGFR3 antibodies. Regions with greater than 20% and 25% HDX protection are indicated. [Figure 16] 1 shows HDX epitope mapping of FGFR3 antibodies. [Figure 17] 1 shows HDX epitope mapping of FGFR3 antibodies. [Figure 18] 1 shows the results of HDX epitope mapping of H4H30063P. The figure discloses SEQ ID NO: 238. [Figure 19] 1 shows the results of HDX epitope mapping of H4H30108P2. The figure discloses SEQ ID NO: 238. [Figure 20]1 shows the results of HDX epitope mapping of H4H30117P2. The figure discloses SEQ ID NO: 238. [Figure 21] 1 shows the results of HDX epitope mapping of H4H30045P. The figure discloses SEQ ID NO: 238. [Figure 22] 1 illustrates an exemplary hydrogen-deuterium exchange mass spectrometry experimental process. DETAILED DESCRIPTION OF THE INVENTION

[0066] Provided herein are antigen-binding proteins that specifically bind to fibroblast growth factor receptor 3 (FGFR3) or its antigen fragments conjugated to molecular cargoes. Such conjugates are useful, for example, for delivering molecular cargoes to various tissues (e.g., central nervous system (CNS) tissues, or eyes) and / or cells (e.g., astrocytes) in the body. The conjugates described herein have the ability to efficiently deliver molecular cargoes to the nervous system, including the brain and spinal cord, particularly astrocytes present therein, and can therefore be used to treat diseases and disorders, such as neurodegenerative and neurodevelopmental diseases and disorders.

[0067] In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein referred to as “Sambrook, et al., 1989”), DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985), Oligonucleotide Synthesis (MJ Gait ed.1984), Nucleic Acid Hybridization(BDHames&S.J.Higgins eds.(1985)), Transcription And Translation(BDHames&S.J.Higgins, eds.(1984)), Animal Cell Culture(RIFreshney, ed.(1986)), Immobilized Cells And Enzymes(IRL Press, (1986), B. Perbal, A Practical Guide To Molecular See Cloning (1984), FMA Usubel, et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0068] Polynucleotides include DNA and RNA. The present disclosure includes any polynucleotide described herein operably linked to a promoter or other expression control sequence.

[0069] The term "FGFR3" refers to fibroblast growth factor receptor 3. Fibroblast growth factor receptor 3 (FGFR3) belongs to a family of structurally related tyrosine kinase receptors that includes four distinct genes (FGFR1-4). These receptors have three glycosylated extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain, and an intracellular split tyrosine kinase domain. Ligand binding induces FGFR dimerization, leading to autophosphorylation of the kinase domain and interaction with and phosphorylation of effector signaling proteins. Alternative mRNA splicing mechanisms generate many different receptor isoforms with different ligand specificities. The isoforms FGFR3b and FGFR3c arise from mutually exclusive splicing events, and the second half of the third Ig-like domain is encoded by either the 151 nucleotides of exon 8 or the 145 nucleotides of exon 9. In some embodiments, the FGFR3 referred to herein is human FGFR3.

[0070] In one embodiment, the human FGFR3c isoform comprises the following amino acid sequence: ESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGE HRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPAEEELVEADEAGSVYAG (SEQ ID NO: 232)

[0071] In one embodiment, the human FGFR3b isoform comprises the following amino acid sequence: ESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEH RIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEADEAGSVYAG (SEQ ID NO: 233)

[0072] In one embodiment, the FGFR3 referred to herein comprises one or more of the following mutations: S249C, R248C, G372C, Y375C, K650E, or FGFR3-TACC3. See, for example, Singh et al., Transforming fusions of FGFR and TACC genes in human glioblastoma. Science (New York, NY) 2012;337:1231-1235.

[0073] FGFR3-binding protein conjugates The present disclosure provides FGFR3 binding protein-drug conjugates. The FGFR3 binding protein-drug conjugates include an optional signal peptide connected to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment of an antibody, such as a fragment antigen-binding region (Fab) or a single-chain fragment variable region (scFv)) that specifically binds to FGFR3 (preferably human FGFR3), which is conjugated (optionally via a linker) to a molecular cargo. The FGFR3 binding proteins described herein can deliver the conjugated molecular cargo to a desired tissue (e.g., neural tissue) and / or a desired cell type (e.g., astrocytes) in the body.

[0074] An antigen-binding protein that specifically binds to FGFR3 may have a specific binding affinity of about 1×10 at about 25° C., for example, in a surface plasmon resonance assay. -7 It can bind to FGFR3 or its fusion protein (e.g., a tag such as His6 (SEQ ID NO: 235) and / or myc fused to human FGFR3b or monkey FGFR3b) with a KD of at least M or with higher affinity. Such antigen-binding proteins can be referred to as "anti-FGFR3."

[0075] In one embodiment, the FGFR3 binding protein-drug conjugate comprises an anti-FGFR3 scFv comprising a variable region arrangement such as LCVR-HCVR or HCVR-LCVR, wherein the HCVR and LCVR are optionally connected by a linker, and the scFv is optionally connected by a linker to a molecular cargo (e.g., LCVR-(Gly4Ser)3-HCVR-molecular cargo ("GGGGSGGGGSGGGGS" disclosed as SEQ ID NO: 246) or LCVR-(Gly4Ser)3-HCVR-molecular cargo ("GGGGSGGGGSGGGGS" disclosed as SEQ ID NO: 246)).

[0076] The term "conjugate" refers to an entity in which two substances are covalently or non-covalently linked. The term "covalently linked" refers to the characteristic of at least two molecules being linked together by one or more covalent bonds. In various embodiments, the two molecules may be covalently linked together by a single bond (e.g., a disulfide bridge or disulfide bond) that functions as a linker between the molecules. In some embodiments, two or more molecules may be covalently linked together by a molecule that functions as a linker, linking at least two molecules together through multiple covalent bonds. In certain embodiments, the linker may be a cleavable linker or a non-cleavable linker. In a conjugate, the two substances may be directly linked or may be linked via a linker. In the present disclosure, one of the two substances is an antigen-binding protein, such as an antibody or antigen-binding fragment thereof, and the other is a drug (e.g., a polynucleotide, a polypeptide, a liposome, or an LNP, or a viral particle or viral capsid protein disclosed herein). In the present disclosure, the linker may be a cleavable linker or a non-cleavable linker. In some embodiments, two polypeptide molecules that are covalently linked, either directly or indirectly (eg, by a linker), can be expressed from one single polynucleotide molecule.

[0077] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a conjugate of an antibody or antigen-binding fragment thereof and a drug (e.g., a polynucleotide, polypeptide, liposome, or LNP, or a viral particle or viral capsid protein disclosed herein). Affinity for an antigen is imparted to the drug by linking the antibody or antigen-binding fragment thereof to the drug (e.g., a polynucleotide, polypeptide, liposome, or LNP, or a viral particle or viral capsid protein disclosed herein), thereby increasing the efficiency of delivering the drug to a target site in vivo. As used herein, "antibody-drug conjugate" or "ADC" also encompasses fusion proteins in which an antibody or antigen-binding fragment thereof is fused to another polypeptide molecule.

[0078] In one embodiment, the assignment of amino acids to each framework or CDR domain is based on the amino acid sequence of the amino acid sequence of interest described in Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; th ed.; NIH Publ. No. 91-3242 (1991), Kabat (1978), Adv. Prot. Chem. 32:1-75, Kabat et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia, et al., (1987) J. Mol. Biol. 196:901-917, or Chothia, et al., (1989) Nature 342:878-883. Therefore, V H CDR and V L Also included herein are antibodies and antigen-binding fragments comprising the CDRs of V H and V L comprises an amino acid sequence described herein (see, eg, the sequences in Table 1-1 or variants thereof), and the CDRs are as defined according to Kabat and / or Chothia.

[0079] The protein-drug conjugates described herein include antibodies that specifically bind to human FGFR3.

[0080] As used herein, the term "antibody" refers to an immunoglobulin molecule (e.g., IgG) comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds. In one embodiment, each antibody heavy chain (HC) comprises a heavy chain variable region ("HCVR" or "V"). H ") (e.g., SEQ ID NOs: 2, 22, 42, 62, 82, 102, or variants thereof) and a heavy chain constant region, and each antibody light chain (LC) comprises a light chain variable region ("LCVR" or "VVR"). L ") (e.g., SEQ ID NO: 10, 30, 50, 70, 90, 110, or a variant thereof) and a light chain constant region (CL). H and V L The region can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L comprises three CDRs and four FRs. The anti-FGFR3 antibodies described herein may also be conjugated to a molecular cargo.

[0081] In one embodiment, the assignment of amino acids to each framework or CDR domain follows the definitions in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia, et al., (1987) J Mol. Biol. 196:901-917, or Chothia, et al., (1989) Nature 342:878-883. Thus, the present disclosure includes antibodies and antigen-binding fragments comprising a VH CDR and a VL CDR, wherein the VH and VL comprise an amino acid sequence described herein (see, e.g., the sequences in Table 1-1 or variants thereof), and the CDRs are as defined according to Kabat and / or Chothia.

[0082] The FGFR3 binding protein described herein can be an antigen-binding fragment of an antibody that can be conjugated to a molecular cargo. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, as used herein, refers to an immunoglobulin molecule that binds to an antigen but does not contain the entire sequence of a complete antibody (preferably, the complete antibody is an IgG). Non-limiting examples of antigen-binding fragments include: (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, and (vi) dAb fragments, consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, one-arm antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, and small modular immunopharmaceuticals (SMIPs) are also encompassed by the term "antigen-binding fragment" as used herein.

[0083] In some embodiments, the anti-FGFR3 protein-drug conjugates described herein may comprise an scFv conjugated to a molecular cargo. The scFv (single-chain fragment variable region) comprises a heavy (V H ) domain and light (V L) domain variable regions (in either order), which are preferably linked together by a flexible linker (e.g., a peptide linker). The length of the flexible linker used to link both V regions can be important for ensuring correct folding of the polypeptide chain. Previously, it has been estimated that a peptide linker must span 3.5 nm (35 Å) between the carboxy terminus of one variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods in Enzymology. 1991;203:46-88). In one embodiment, the linker comprises an amino acid sequence of such length that it separates the variable domains by approximately 3.5 nm. In one embodiment of the present invention, the anti-FGFR3 scFv drug conjugate comprises an scFv comprising a variable region arrangement such as LCVR-HCVR or HCVR-LCVR, wherein the HCVR and LCVR are optionally connected by a linker, and the scFv is optionally connected by a linker to a molecular cargo (e.g., LCVR-(Gly4Ser)3 (SEQ ID NO: 246)-HCVR-molecular cargo, or LCVR-(Gly4Ser)3 (SEQ ID NO: 246)-HCVR-molecular cargo).

[0084] In some embodiments, the anti-FGFR3 protein-drug conjugates described herein may comprise a Fab conjugated to a molecular cargo.

[0085] In some embodiments, the anti-FGFR3 protein-drug conjugates described herein comprise a bivalent antibody conjugated to a molecular cargo.

[0086] In some embodiments, the anti-FGFR3 protein-drug conjugates described herein comprise a monovalent or "one-arm" antibody conjugated to a molecular cargo. As used herein, a monovalent or "one-arm" antibody refers to an immunoglobulin protein comprising a single variable domain. For example, a one-arm antibody may comprise a single variable domain within a Fab, which is linked to at least one Fc fragment. In certain embodiments, a one-arm antibody comprises a polypeptide comprising: (i) a heavy chain comprising a heavy chain constant region and a heavy chain variable region; (ii) a light chain comprising a light chain constant region and a light chain variable region; and (iii) an Fc fragment or a truncated heavy chain. In certain embodiments, the Fc fragment or truncated heavy chain contained in a separate polypeptide is a "dummy Fc," which refers to an Fc fragment not linked to an antigen-binding domain. The one-arm antibodies described herein may comprise any of the HCVR / LCVR pairs or CDR amino acid sequences listed in Table 1-1 herein. One-arm antibodies comprising a full-length heavy chain, a full-length light chain, and an additional Fc domain polypeptide can be constructed using standard methodology (see, e.g., WO2010 / 151792, incorporated herein by reference in its entirety), where the heavy chain constant region differs from the Fc domain polypeptide by at least two amino acids (e.g., H95R and Y96F according to the IMGT exon numbering system, H435R and Y436F according to the EU numbering system). Such modifications are useful for purifying monovalent antibodies (see, e.g., WO2010 / 151792).

[0087] An antigen-binding fragment of an antibody, in one embodiment, comprises at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in-frame with one or more framework sequences. L In an antigen-binding fragment having a VH domain associated with a V H Domains and V LThe domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may be formed by non-covalently linking monomeric V dimers. H and / or V L It may also include a domain.

[0088] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragments of antibodies described herein include: (i) a V H -CH1, (ii) V H -CH2, (iii) V H -CH3, (iv)V H -CH1-CH2, (v) V H -CH1-CH2-CH3, (vi) V H -CH2-CH3, (vii)V H -CL, (viii)V L -CH1, (ix)V L -CH2, (x)V L -CH3, (xi)V L -CH1-CH2, (xii)VL-CH1-CH2-CH3, (xiii)V L -CH2-CH3, and (xiv) V L-CL. In any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains can be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies described herein can be linked to each other and / or to one or more monomeric V H Or V L The present disclosure may include homodimers or heterodimers (or other multimers) of any of the above-listed variable and constant domain configurations non-covalently linked (e.g., by disulfide bond(s)) domains. The present disclosure includes antigen-binding fragments of antigen-binding proteins such as antibodies described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2).

[0089] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein. The present disclosure includes monospecific as well as multispecific (e.g., bispecific) antigen-binding fragments comprising one or more variable domains from the antigen-binding proteins specifically described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2).

[0090] The terms "specifically binds" or "binds specifically" refer to an affinity of at least about 10 as measured by a real-time label-free biolayer interference assay (e.g., at 25°C or 37°C, e.g., Octet® HTX biosensor), by surface plasmon resonance (e.g., BIACORE™), or by solution affinity ELISA. -9 M (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 nM) of (K D "Anti-FGFR3" refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that has binding affinity to an antigen, such as a human FGFR3 protein (e.g., FGFR3b and / or FGFR3c isoform), a mouse FGFR3 protein (e.g., FGFR3b and / or FGFR3c isoform), or a cynomolgus monkey FGFR3 protein (e.g., FGFR3b and / or FGFR3c isoform). The present disclosure includes antigen-binding proteins that specifically bind to an FGFR3 protein (e.g., FGFR3b and / or FGFR3c isoform). "Anti-FGFR3" refers to an antigen-binding protein (or other molecule), such as an antibody or antigen-binding fragment thereof, that specifically binds to FGFR3 (e.g., FGFR3b and / or FGFR3c isoform).

[0091] "Isolated" antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof), polypeptides, polynucleotides, and vectors are at least partially free from other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other materials such as cell debris and growth medium. Isolated antigen binding proteins may also be at least partially free from expression system components such as biological molecules from the host cell or its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules (e.g., trace or insignificant amounts of impurities may remain), or the absence of water, buffers or salts, or components of a pharmaceutical formulation that comprises the antigen binding protein (e.g., antibody or antigen-binding fragment).

[0092] The present disclosure includes antibodies or antigen-binding fragments that bind to the same epitope as an antigen binding protein, e.g., an antigen binding protein described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2).

[0093] An antigen is a molecule, such as, for example, a peptide (e.g., FGFR3 or a fragment thereof (antigenic fragment)) to which an antibody or its antigen-binding fragment binds. The specific region on an antigen that an antibody recognizes and binds to is called an epitope. Antigen-binding proteins (e.g., antibodies) described herein that specifically bind to such antigens are part of the present disclosure.

[0094] The term "epitope" refers to a specific antigen-binding site of an antigen-binding protein, e.g., an antigenic determinant (e.g., on FGFR3b and / or FGFR3c) that interacts with the variable region of an antibody, known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" may also refer to the site on an antigen to which B and / or T cells respond and / or the region of an antigen bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may be linear or conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. The epitope to which the antigen-binding proteins described herein bind may be contained in a fragment of FGFR3 (e.g., human FGFR3b and / or FGFR3c), e.g., its extracellular domain. Antigen-binding proteins (e.g., antibodies) described herein that bind to such epitopes are also contemplated.

[0095] Methods for determining the epitope of an antigen-binding protein, such as an antibody or fragment or polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallography, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify the amino acid in a polypeptide with which an antigen-binding protein (e.g., an antibody or fragment or polypeptide) interacts is hydrogen / deuterium exchange detected by mass spectrometry. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259, Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0096] The present disclosure includes antigen binding proteins that compete with an antigen binding protein discussed herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2) for binding to FGFR3 (e.g., the FGFR3b and / or FGFR3c epitopes discussed herein). As used herein, the term "compete" refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., FGFR3) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) to the antigen. Unless otherwise stated, this term also includes competition between two antigen-binding proteins, e.g., antibodies, in both orientations, i.e., a first antibody that binds to an antigen and blocks binding by a second antibody, and vice versa. Thus, in one embodiment, competition occurs in one such orientation. In certain embodiments, the first antigen-binding protein (e.g., an antibody) and the second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but overlapping or non-overlapping, epitopes, such that the binding of one inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, for example, by real-time label-free biolayer interferometry assays. Binding competition between FGFR3 antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can also be determined using real-time label-free biolayer interferometry assays with an Octet RED384 biosensor (Pall ForteBio Corp.).

[0097] Typically, antibodies or antigen-binding fragments described herein that have been modified in some way retain the ability to specifically bind to FGFR3 (e.g., FGFR3b and / or FGFR3c), e.g., retain at least 10% of their FGFR3 binding activity (compared to the parent antibody), when activity is expressed on a molar basis. Preferably, antibodies or antigen-binding fragments described herein retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100%, or more, of the FGFR3 binding affinity of the parent antibody. It is also intended that the antibodies or antigen-binding fragments described herein may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of antibodies) that do not substantially alter their biological activity.

[0098] The FGFR3 binding proteins described herein can be monoclonal antibodies or antigen-binding fragments of monoclonal antibodies that can be conjugated to molecular cargo. The present disclosure includes monoclonal FGFR3 binding proteins, such as antibodies and antigen-binding fragments thereof (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2), as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody from a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring variations that may be present in minor amounts. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., in amino acid sequence except for possible naturally occurring variations that may be present in minor amounts, as discussed above) antibodies and fragments that exceeds the concentration normally present in nature, e.g., in the blood of a host organism such as a mouse or human.

[0099] In one embodiment, the FGFR3 binding protein, e.g., an antibody or antigen-binding fragment (optionally conjugated to a molecular cargo), comprises a heavy chain constant domain, e.g., of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM type. In one embodiment, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises a light chain constant domain, e.g., of the kappa or lambda type. In one embodiment, the VFGFR3 binding protein described herein comprises a heavy chain constant domain, e.g., of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM type. H is linked to a human heavy chain constant domain (e.g., IgG) and L is linked to a human light chain constant domain (e.g., kappa). The present disclosure includes antigen binding proteins comprising a variable domain described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2), which is linked to a heavy chain constant domain and / or a light chain constant domain described herein.

[0100] The present disclosure includes human FGFR3 binding proteins that can be conjugated to molecular cargoes. The term "human" antigen-binding protein (e.g., antibody or antigen-binding fragment), as used herein, includes antibodies and fragments having variable and constant regions derived from human germline immunoglobulin sequences, whether in human cells or grafted into non-human cells (e.g., mouse cells). See, for example, U.S. Patent Nos. 8,502,018, 6,596,541, or 5,789,215. The anti-FGFR3 human mAbs described herein may contain amino acid residues (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation) that are not encoded by human germline immunoglobulin sequences, for example, in the CDRs, and particularly in the CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences. This term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. This term is not intended to include natural antibodies directly isolated from human subjects. The present disclosure includes human antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof such as H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2).

[0101] The present disclosure includes anti-FGFR3 chimeric antigen-binding proteins, such as antibodies and antigen-binding fragments thereof (which can be conjugated to molecular cargo), and methods of using them. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first antibody and the second antibody are derived from different species (see, for example, US4816567, and Morrison et al., (1984) Proc.Natl.Acad.Sci.USA 81:6851-6855). The present disclosure includes chimeric antibodies comprising a variable domain described herein (e.g., from H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2) and a non-human constant domain.

[0102] The term "recombinant" FGFR3-binding protein, such as an antibody or antigen-binding fragment thereof (which may be conjugated to a molecular cargo), refers to such molecules that are produced, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. This term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, such as transgenic mice), or host cells (e.g., Chinese hamster ovary (CHO) cells) or cell expression systems, or antibodies isolated from recombinant combinatorial human antibody libraries. The present disclosure includes recombinant antigen-binding proteins such as the antibodies and antigen-binding fragments described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2).

[0103] Antigen-binding fragments of antibodies, in one embodiment, include antibodies that are less than the complete antibody, but still specifically bind to an antigen, e.g., FGFR3, and that include, for example, at least one variable domain. The variable domain may be of any size or amino acid composition and generally includes at least one (e.g., three) CDR(s) adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may be formed by non-covalently linking monomeric V dimers. H and / or V L It may contain domains.

[0104] Immunoglobulin chains (e.g., V of H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2, including the amino acid sequences specifically described herein) H , V L, HC, or LC, or CDRs thereof) is a polypeptide that has a match of at least about 70 to 99.9% (e.g., at least 70, 72, 74, 75, 76, 77, 78, 79, 80, 82, 90, 98, 100, 102, 110, 118, or 120) with a reference amino acid sequence described herein (e.g., any of SEQ ID NOs: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, 60, 62, 70, 78, 80, 82, 90, 98, 100, 102, 110, 118, or 120) when the comparison is made using the BLAST algorithm and the algorithm parameters are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 3, maximum match within query range: 0, BLOSUM 62 matrix, gap cost: presence 11, extension 1, conditional composition score matrix adjustment). 96, 97, 98, 99, 99.5, or 99.9%) identical or similar amino acid sequence and / or polypeptides containing an amino acid sequence with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations (e.g., point mutations, insertions, truncations, and / or deletions).

[0105] Additionally, a variant of a polypeptide can comprise the amino acid sequence of a reference polypeptide specifically described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, e.g., one or more missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions, of an immunoglobulin chain (e.g., V4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2). H , V L, HC, or LC, or CDRs thereof). See Table 1-1. For example, the present disclosure provides an immunoglobulin light chain (or VH chain) comprising the amino acid sequence set forth in SEQ ID NO: 10, but having one or more of such mutations. L ) variants, and / or immunoglobulin heavy chains (or V) comprising the amino acid sequence set forth in SEQ ID NO:2 but having one or more of such mutations. H In one embodiment, the FGFR3 binding protein comprises an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3, in which one or more (e.g., one, or two, or three) of such CDRs have one or more of such mutations (e.g., conservative substitutions), and / or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3, in which one or more (e.g., one, or two, or three) of such CDRs have one or more of such mutations (e.g., conservative substitutions).

[0106] The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al. al.,(1993)Comput.Chem.17:149-163, Hancock,JMet al.,(1994)Comput.Appl.Biosci.10:67-70,ALIGNMENT SCORING SYSTEMS:Dayhoff,MO,et al.,”A model of evolutionary change in proteins.”in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.MODayhoff(ed.),pp.345-352,Natl.Biomed.Res.Found.,Washington,DC,Schwartz,RM,et al.,“Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure,(1978)vol.5,suppl.3.“MODayhoff(ed.),pp.353-358, Natl.Biomed.Res.Found.,Washington,DC,Altschul,SF,(1991)J.Mol.Biol.219:555-565,States,DJ,et al.,(1991)Methods 3:66-70,Henikoff,S.,et al.,(1992)Proc.Natl.Acad.Sci.USA 89:10915-10919, Altschul,SF,et al.,(1993)J.Mol.Evol.36:290-300,ALIGNMENT STATISTICS:Karlin,S.,et al.,(1990)Proc.Natl.Acad.Sci.USA 87:2264-2268,Karlin,S.,et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, Dembo, A., et al., (1994) Ann. Prob.22:2022-2039, and Altschul, SF “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, NY. .

[0107] For example, "conservatively modified variants" or "conservative substitutions" of immunoglobulin chains described herein refer to variants in which one or more amino acids in a polypeptide are replaced with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.). Such changes can be frequently made without significantly disrupting the biological activity of antibodies or fragments. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are unlikely to significantly disrupt biological activity. The present disclosure includes FGFR3-binding proteins comprising such conservatively modified variant immunoglobulin chains.

[0108] Examples of groups of amino acids with side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445.

[0109] "H4H30063P", "H4H30066P", "H4H30071P", "H4H30089P2", "H4H30093P2", "H4H30076P", "H4H30105P2", "H4H30108P2", "H4H30117P2", "H4H30045P", "H4H30061P", "H4H30095P2", and "H4H30102P2" comprise or represent the amino acid sequence pairs specifically set forth in SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 62 and 70, 82 and 90, and 102 and 110 (or any variant of said sequences), respectively, unless otherwise stated. Each of the immunoglobulins comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC) comprising the amino acid sequence pair specifically set forth in SEQ ID NOs: 18 and 20, 38 and 40, 58 and 60, 78 and 80, 90 and 100, 118 and 120, 136 and 138, 155 and 157, 167 and 157, 177 and 157, 195 and 197, 215 and 217, or 229 and 231 (or a variant of any of said sequences), or a heavy chain (or V) comprising its CDRs (CDR-H1 (or a variant thereof), CDR-H2 (or a variant thereof), and CDR-H3 (or a variant thereof)). H), and / or a light chain (or V) comprising its CDRs (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof), and CDR-L3 (or a variant thereof)). L ), including an immunoglobulin heavy chain variable region (V H ) and immunoglobulin light chain variable region (V L ) refers to FGFR3 binding proteins, e.g., antibodies and antigen-binding fragments thereof (including multispecific antigen-binding proteins). In one embodiment, V H is linked to an IgG constant heavy chain domain, e.g., a human IgG constant heavy chain domain (e.g., IgG1 or IgG4 (e.g., containing an S228P and / or S108P mutation)), and / or V L is linked to a light chain constant domain, e.g., a human light chain constant domain (e.g., a lambda or kappa constant light chain domain). Any such immunoglobulin chain (e.g., V H , V L Polynucleotides encoding one or more of the HC, HC, and / or LC form part of the present disclosure.

[0110] The antibodies and antigen-binding fragments described herein (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2) comprise immunoglobulin chains that include the amino acid sequences (and variants thereof) specifically described herein, as well as cellular and in vitro post-translational modifications to the antibodies or fragments. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to FGFR3, comprising the heavy and / or light chain amino acid sequences described herein, as well as antibodies and fragments in which one or more asparagine, serine, and / or threonine residues are glycosylated, one or more asparagine residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal glutamine is pyroglutamic acid (pyroE), and / or the C-terminal lysine or other amino acid is deleted.

[0111] In one embodiment, the FGFR3 protein-drug conjugate (e.g., scFv, Fab, or other antibody or antigen-binding fragment thereof format) may exhibit one or more of the following characteristics: an affinity (K) of about 16 nM or greater (e.g., about 16 nM, 12 nM, 10 nM, 7 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.22 nM, 0.2 nM, 0.19 nM, 0.14 nM, 0.1 nM) at 25°C (e.g., in a surface plasmon resonance assay); D ) and binds to monomeric human FGFR3b (e.g., C-terminally tagged with myc-myc-His6); an affinity (K ) of about 20 nM or greater (e.g., about 20 nM, 16 nM, 15 nM, 10 nM, 8 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.65 nM, 0.3 nM, 0.28 nM, 0.2 nM, 0.15 nM, 0.1 nM) at 25°C (e.g., in a surface plasmon resonance assay); D) and binds to monomeric cynomolgus monkey FGFR3b (e.g., C-terminally tagged with myc-myc-His6); an affinity (K) of about 70 nM or greater (e.g., about 70 nM, 20 nM, 17 nM, 12 nM, 10 nM, 9 nM, 8 nM, 0.1 nM) at 25°C (e.g., in a surface plasmon resonance assay); D ) and binds to monomeric mouse FGFR3b (e.g., C-terminally tagged with myc-myc-His6); • does not significantly bind to monomeric human FGFR3c (e.g., C-terminally tagged with myc-myc-His6) at 25°C (e.g., in a surface plasmon resonance assay); • binds to dimeric human FGFR3b (e.g., C-terminally tagged with mouse Fc (mFc)) with a higher affinity of about 0.6 nM (e.g., about 0.58 nM, 0.17 nM, 0.11 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, 0.023 nM, 0.061 nM, 0.031 nM, 0.016 nM, 0.034 nM, 0.027 nM) at 25°C (e.g., in a surface plasmon resonance assay); • Blocks the binding of acidic FGF1 to human FGFR3b-mFc by approximately 68% or more (e.g., 90% or 0.5%) at 200 nM antibody; The binding of monomeric cynomolgus monkey and monomeric mouse FGFR3b to monomeric or dimeric human FGFR3b was investigated using K D K within approximately 0.1 nM D Combine with; IC at a concentration of approximately 15 nM or less 50 (e.g., IC of about 1, 2, or 3 nM 50 ) that blocks the binding of 4 nM human FGFR3b-mFc to human acidic FGF1 protein; or • Competes for binding to hFGFR3b.mmH with another anti-FGFR3 antibody described in Table 5-1 herein.

[0112] Table 1-1 below summarizes the amino acid sequences of domains in the FGFR3-binding proteins of the conjugates of the present disclosure. For example, anti-FGFR3 antibodies and antigen-binding fragments thereof (e.g., scFv and Fab) comprising the HCVR and LCVR or CDRs of the molecules in Table 1-1 conjugated to molecular cargos form part of the present disclosure. [Table 1-1] *In one embodiment, the heavy chain lacks a C-terminal lysine.

[0113] The sequences of the domains or chains of the antibodies or antigen-binding fragments (e.g., Fab or scFv molecules) in the protein-drug conjugates described herein are shown below. The present disclosure includes any antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR having the amino acid sequences set forth below, or an HCVR and an LCVR having the HCDRs and LCDRs thereof, respectively. H4H30063P HCVR nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATTAATAGTTACTTCTGGAGCTGGATCCGGCAGTTGCCAGGGAAGGAACTGGAGTGGATTGGCCATATCTATTCTAGTGGGAGTACCA GATACAACCCCTCCCTCCAGAGTCGAGTCACCATATCAATAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTATATTACTGTGCGAGGGGCGCCAGCGCAGTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 1) HCVR amino acid sequence [ka] (SEQ ID NO: 2) HCDR1 nucleotide sequence GGT GAC TCC ATT AAT AGT TAC TTC (SEQ ID NO: 3) HCDR1 amino acid sequence GDSINSYF (SEQ ID NO: 4) HCDR2 nucleotide sequence ATC TAT TCT AGT GGG AGT ACC (SEQ ID NO: 5) HCDR2 amino acid sequence IYSSGST (SEQ ID NO: 6) HCDR3 nucleotide sequence GCG AGG GGC GCC AGC GCA GTT GAC TAC (SEQ ID NO: 7) HCDR3 amino acid sequence ARGASAVDY (SEQ ID NO: 8) LCVR nucleotide sequence GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGACCAGTCAGAGTATTAGCAGCGGCTATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGA AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGACTTTGTAGTGTATTACTGTCAACAATATGGTAGCTCACCATACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 9) LCVR amino acid sequence [ka] (SEQ ID NO: 10) LCDR1 nucleotide sequence CAG AGT ATT AGC AGC GGC TAT (SEQ ID NO: 11) LCDR1 amino acid sequence QSISSGY (SEQ ID NO: 12) LCDR2 nucleotide sequence GGT GCA TCC (SEQ ID NO: 13) LCDR2 amino acid sequence GAS (SEQ ID NO: 14) LCDR3 nucleotide sequence CAA CAA TAT GGT AGC TCA CCA TAC ACT (SEQ ID NO: 15) LCDR3 amino acid sequence QQYGSSPYT (SEQ ID NO: 16) Heavy chain nucleotide sequence (SEQ ID NO: 17) Heavy chain amino acid sequence [ka] (SEQ ID NO: 18) Light chain nucleotide sequence GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGACCAGTCAGAGTATTAGCAGCGGCTATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGA AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGACTTTGTAGTGTATTACTGTCAACAATATGGTAGCTCACCATACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 19) Light chain amino acid sequence [ka] (SEQ ID NO: 20) H4H30066P HCVR nucleotide sequence CAGGTGCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGTTTCCGGATACACCCTCACTGAATTATCCATGCACTGGGTGCGACAAGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGATGGTGAAATAATCTACGCACAGAAG TTCCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGACCTGAGCAGTCTGACATCTGAAGACACGGCCGTGTATTACTGTGCAACGGAGAAGCAGCAACTGGTACGAAAATACTACTTCTACTACGGTTTGGCCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 21) HCVR amino acid sequence [ka] (SEQ ID NO: 22) HCDR1 nucleotide sequence GGA TAC ACC CTC ACT GAA TTA TCC (SEQ ID NO: 23) HCDR1 amino acid sequence GYTLTELS (SEQ ID NO: 24) HCDR2 nucleotide sequence TTT GAT CCT GAA GAT GGT GAA ATA (SEQ ID NO: 25) HCDR2 amino acid sequence FDPEDGEI (SEQ ID NO: 26) HCDR3 nucleotide sequence GCA ACG GAG AAG CAG CAA CTG GTA CGA AAA TAC TAC TTC TAC TAC GGT TTG GCC GTC (SEQ ID NO: 27) HCDR3 amino acid sequence ATEKQQLVRKYYFYYGLAV (SEQ ID NO: 28) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 29) LCVR amino acid sequence [ka] JPEG2025538220000008.jpg469 (sequence number 30) LCDR1 nucleotide sequence CAG AGC ATT AGC AGT TAT (SEQ ID NO: 31) LCDR1 amino acid sequence QSISSY (SEQ ID NO: 32) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 33) LCDR2 amino acid sequence AAS (SEQ ID NO: 34) LCDR3 nucleotide sequence CAA CAG AGT TAC AGT CCC CCA TTC ACT (SEQ ID NO: 35) LCDR3 amino acid sequence QQSYSPPFT (SEQ ID NO: 36) Heavy chain nucleotide sequence (SEQ ID NO: 37) Heavy Chain Amino Acids [ka] JPEG2025538220000010.jpg10161 (sequence number 38) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGTCCCTAAGCTCCTGATCTATGCTGCATCCAGTTT GCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 39) Light Chain Amino Acids [ka] (SEQ ID NO: 40) H4H30071P HCVR nucleotide sequence CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGGAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGTGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTTTTATG ATTATGCATATCTGTGAAAAGTCGAATAACCGTCAACCCAGACACATCCAAGAACCAATTCTCCCTTCACCTGAACTCTGTGACTCCCGAAGACACGGCTGTCTATTACTGTGCGAGAGGCTACGGTGGCTACGAGGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 41) HCVR amino acid sequence [ka] (SEQ ID NO: 42) HCDR1 nucleotide sequence GGG GAC AGT GTC TCT AGG AAC AGT GCT GCT (SEQ ID NO: 43) HCDR1 amino acid sequence GDSVSRNSAA (SEQ ID NO: 44) HCDR2 nucleotide sequence ACA TAC TAC AGG TCC AAG TGG TTT TAT (SEQ ID NO: 45) HCDR2 amino acid sequence TYYRSKWFY (SEQ ID NO: 46) HCDR3 nucleotide sequence GCG AGA GGC TAC GGT GGC TAC GAG GAC TAC TTT GAC TAC (SEQ ID NO: 47) HCDR3 amino acid sequence ARGYGGYEDYFDY (SEQ ID NO: 48) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAACAGGGTAGCAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 49) LCVR amino acid sequence [ka] JPEG2025538220000014.jpg569 (sequence number 50) LCDR1 nucleotide sequence CAG GGT ATT AGC AGC TGG (SEQ ID NO: 51) LCDR1 amino acid sequence QGISSW (SEQ ID NO: 52) LCDR2 nucleotide sequence GGT GCA TCC (SEQ ID NO: 53) LCDR2 amino acid sequence GAS (SEQ ID NO: 54) LCDR3 nucleotide sequence CAA CAG GGT AGC AGT TTC CCG TAC ACT (SEQ ID NO: 55) LCDR3 amino acid sequence QQGSSFPYT (SEQ ID NO: 56) Heavy chain nucleotide sequence (SEQ ID NO: 57) Heavy Chain Amino Acids [ka] (SEQ ID NO: 58) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTT GCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAACAGGGTAGCAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 59) Light Chain Amino Acids [ka] (SEQ ID NO: 60) H4H30089P2 HCVR nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTATAGTCTCTGGATTCATCTTCAGTAGTTATGAAATGAGCTGGCTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGATTTCATATATTAGTAGTAGTGGTCGTGTCATATACT ATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAATTCACTGTATCTGGAAATGAATAGTCTGAGAGCCGAAGACACGGCTATATATTATTGTACGAGAAAGTGGGATAGTAGTGGCCCATTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 61) HCVR amino acid sequence [ka] (SEQ ID NO: 62) HCDR1 nucleotide sequence GGA TTC ATC TTC AGT AGT TAT GAA (SEQ ID NO: 63) HCDR1 amino acid sequence GFIFSSYE (SEQ ID NO: 64) HCDR2 nucleotide sequence ATT AGT AGT AGT GGT CGT GTC ATA (SEQ ID NO: 65) HCDR2 amino acid sequence ISSSGRVI (SEQ ID NO: 66) HCDR3 nucleotide sequence ACG AGA AAG TGG GAT AGT AGT GGC CCA TTT GAC TTC (SEQ ID NO: 67) HCDR3 amino acid sequence TRKWDSSGPFDF (SEQ ID NO: 68) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 69) LCVR amino acid sequence [ka] (SEQ ID NO: 70) LCDR1 nucleotide sequence CAG AGC ATT AGC AGC TAT (SEQ ID NO: 71) LCDR1 amino acid sequence QSISSY (SEQ ID NO: 72) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 73) LCDR2 amino acid sequence AAS (SEQ ID NO: 74) LCDR3 nucleotide sequence CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 75) LCDR3 amino acid sequence QQSYSTPPIT (SEQ ID NO: 76) Heavy chain nucleotide sequence (SEQ ID NO: 77) Heavy Chain Amino Acids [ka] (SEQ ID NO: 78) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 79) Light Chain Amino Acids [ka] (SEQ ID NO: 80) H4H30093P2 HCVR nucleotide sequence CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTAAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATGACTACCCAATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTACTAGCAGTAGTGGTAGTACCATATACTACGCAG ACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGTTGTAGTGGCTACGATTGGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 81) HCVR amino acid sequence [ka] (SEQ ID NO: 82) HCDR1 nucleotide sequence GGA TTC ACC TTC AAT GAC TAC CCA (SEQ ID NO: 83) HCDR1 amino acid sequence GFTFNDYP (SEQ ID NO: 84) HCDR2 nucleotide sequence ATT ACT AGC AGT AGT GGT AGT ACC ATA (SEQ ID NO: 85) HCDR2 amino acid sequence ITSSSGSTI (SEQ ID NO: 86) HCDR3 nucleotide sequence GCG AGA GAG GTT GTA GTG GCT ACG ATT GGG GGC TAC TAC GGT ATG GAC GTC (SEQ ID NO: 87) HCDR3 amino acid sequence AREVVVATIGGYYGMDV (SEQ ID NO: 88) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 89) LCVR amino acid sequence [ka] (SEQ ID NO: 90) LCDR1 nucleotide sequence CAG AGC ATT AGC AGC TAT (SEQ ID NO: 91) LCDR1 amino acid sequence QSISSY (SEQ ID NO: 92) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 93) LCDR2 amino acid sequence AAS (SEQ ID NO: 94) LCDR3 nucleotide sequence CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 95) LCDR3 amino acid sequence QQSYSTPPIT (SEQ ID NO: 96) Heavy chain nucleotide sequence (SEQ ID NO: 97) Heavy Chain Amino Acids [ka] (SEQ ID NO: 98) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 99) Light Chain Amino Acids [ka] (SEQ ID NO: 100) H4H30102P2 HCVR nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAATAGTGGTTCTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCGAGACCTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGAGGGATGGGCGCATATTGTGCTGGTGATTGCTATTCTGGTTTTGATATTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 101) HCVR amino acid sequence [ka] (SEQ ID NO: 102) HCDR1 nucleotide sequence GGA TTC ACC TTC AGT AGT TAT GAA (SEQ ID NO: 103) HCDR1 amino acid sequence GFTFSSYE (SEQ ID NO: 104) HCDR2 nucleotide sequence ATT AGT AAT AGT GGT TCT ACC ATA (SEQ ID NO: 105) HCDR2 amino acid sequence ISNSGSTI (SEQ ID NO: 106) HCDR3 nucleotide sequence GCG AGA GAG GGA TGG GGC GCA TAT TGT GCT GGT GAT TGC TAT TCT GGT TTT GAT ATT (SEQ ID NO: 107) HCDR3 amino acid sequence AREGWGAYCAGDCYSGFDI (SEQ ID NO: 108) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 109) LCVR amino acid sequence [ka] (SEQ ID NO: 110) LCDR1 nucleotide sequence CAG AGC ATT AGC AGC TAT (SEQ ID NO: 111) LCDR1 amino acid sequence QSISSY (SEQ ID NO: 112) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 113) LCDR2 amino acid sequence AAS (SEQ ID NO: 114) LCDR3 nucleotide sequence CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 115) LCDR3 amino acid sequence QQSYSTPPIT (SEQ ID NO: 116) Heavy chain nucleotide sequence (SEQ ID NO: 117) Heavy Chain Amino Acids [ka] (SEQ ID NO: 118) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 119) Light Chain Amino Acids [ka] (SEQ ID NO: 120) H4H30076P HCVR nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGCGGTGGTCACACATACTACT CAGACTCCGTGAAGGCCGATTCACCATCTCCAGACACAATTCCAAGAACACTCTGTATCTTCAAATGAACAGCCTGAGAGGTGGGGACACGGCCGTGTATTACTGTGCGAGAGGGTATACCAGTGGCTGGTACGGATTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 121) HCVR amino acid sequence [ka] (SEQ ID NO: 122) HCDR1 nucleotide sequence GGG TTC ACC GTC AGT AGC AAC TAC (SEQ ID NO: 123) HCDR1 amino acid sequence GFTVSSNY (SEQ ID NO: 124) HCDR2 nucleotide sequence ATT TAT AGC GGT GGT CAC ACA (SEQ ID NO: 125) HCDR2 amino acid sequence IYSGGHT (SEQ ID NO: 126) HCDR3 nucleotide sequence GCG AGA GGG TAT ACC AGT GGC TGG TAC GGA TTT GAC TTC (SEQ ID NO: 127) HCDR3 amino acid sequence ARGYTSGWYGFDF (SEQ ID NO: 128) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCGTCAAGATTCAGCGGCACTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTATTGTCAGCAGACTAACAGTTTCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 129) LCVR amino acid sequence [ka] (SEQ ID NO: 130) LCDR1 nucleotide sequence CAG GGT ATT AGC ACC TGG (SEQ ID NO: 131) LCDR1 amino acid sequence QGISTW (SEQ ID NO: 132) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 33) LCDR2 amino acid sequence AAS (SEQ ID NO: 34) LCDR3 nucleotide sequence CAG CAG ACT AAC AGT TTC CCG TGG ACG (SEQ ID NO: 133) LCDR3 amino acid sequence QQTNSFPWT (SEQ ID NO: 134) Heavy chain nucleotide sequence (SEQ ID NO: 135) Heavy chain amino acid sequence [ka] (SEQ ID NO: 136) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTT GCAAAGTGGGGTCCCGTCAAGATTCAGCGGCACTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTATTGTCAGCAGACTAACAGTTTCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 137) Light chain amino acid sequence [ka] (SEQ ID NO: 138) H4H30105P2 HCVR nucleotide sequence GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGAT ACAGCCCGTCCTTCCAAGGCCAGGTCACCATGTCAGCCGACAAGTCCATCAGGATCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTATTGTGCGAGACTTGATTATAGCGGCAGCTGGTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 139) HCVR amino acid sequence [ka] (SEQ ID NO: 140) HCDR1 nucleotide sequence GGA TAC AGC TTT ACC AGC TAC TGG (SEQ ID NO: 141) HCDR1 amino acid sequence GYSFTSYW (SEQ ID NO: 142) HCDR2 nucleotide sequence ATC TAT CCT GGT GAC TCT GAT ACC (SEQ ID NO: 143) HCDR2 amino acid sequence IYPGDSDT (SEQ ID NO: 144) HCDR3 nucleotide sequence GCG AGA CTT GAT TAT AGC GGC AGC TGG TTT GAC TAC (SEQ ID NO: 145) HCDR3 amino acid sequence ARLDYSGSWFDY (SEQ ID NO: 146) LCVR nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG (SEQ ID NO: 147) LCVR amino acid sequence [ka] (SEQ ID NO: 148) LCDR1 nucleotide sequence CAG TCA GTC TCT AGC TCT TAT (SEQ ID NO: 149) LCDR1 amino acid sequence QSVSSSY (SEQ ID NO: 150) LCDR2 nucleotide sequence GGG GCA AGT (SEQ ID NO: 151) LCDR2 amino acid sequence GAS (SEQ ID NO: 14) LCDR3 nucleotide sequence CAA CAG TAC GGA AGC AGC CCG TGG ACG (SEQ ID NO: 152) LCDR3 amino acid sequence QQYGSSPWT (SEQ ID NO: 153) Heavy chain nucleotide sequence (SEQ ID NO: 154) Heavy chain amino acid sequence [ka] (SEQ ID NO: 155) Light chain nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 156) Light chain amino acid sequence [ka] (SEQ ID NO: 157) H4H30108P2 HCVR nucleotide sequence CAGGTCCAGCTGGTGCAGTCTGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCAACAGTTATGATGTCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTCACAGTGGTAACACAGGCTACGCA CAGAAGTTCCAGGGCAGAGTCACTATGACCAGGGACACCTCCACAAGCACAAGCTATATGGAGTTGAGCAGCCTGACATCTGAGGACACGGCCGTATATTACTGTGCGAGAGGCCCTTTTTCTCTACTTTCAGCTGAATTCTTCCAGCACTGGGCCAGGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 158) HCVR amino acid sequence [ka] (SEQ ID NO: 159) HCDR1 nucleotide sequence GGA TAC ACC TTC AAC AGT TAT GAT (SEQ ID NO: 160) HCDR1 amino acid sequence GYTFNSYD (SEQ ID NO: 161) HCDR2 nucleotide sequence ATG AAC CCT CAC AGT GGT AAC ACA (SEQ ID NO: 162) HCDR2 amino acid sequence MNPHSGNT (SEQ ID NO: 163) HCDR3 nucleotide sequence GCG AGA GGC CCT TTT TCT CTA CTT TCA GCT GAA TTC TTC CAG CAC (SEQ ID NO: 164) HCDR3 amino acid sequence ARGPFSLLSAEFFQH (SEQ ID NO: 165) LCVR nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG (SEQ ID NO: 147) LCVR amino acid sequence [ka] (SEQ ID NO: 148) LCDR1 nucleotide sequence CAG TCA GTC TCT AGC TCT TAT (SEQ ID NO: 149) LCDR1 amino acid sequence QSVSSSY (SEQ ID NO: 150) LCDR2 nucleotide sequence GGG GCA AGT (SEQ ID NO: 151) LCDR2 amino acid sequence GAS (SEQ ID NO: 14) LCDR3 nucleotide sequence CAA CAG TAC GGA AGC AGC CCG TGG ACG (SEQ ID NO: 152) LCDR3 amino acid sequence QQYGSSPWT (SEQ ID NO: 153) Heavy chain nucleotide sequence (SEQ ID NO: 166) Heavy chain amino acid sequence [ka] (SEQ ID NO: 167) Light chain nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 156) Light chain amino acid sequence [ka] (SEQ ID NO: 157) H4H30117P2 HCVR nucleotide sequence CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGACACCTTCAGTAACTATGTTATCGGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAACAAACT ACGCACAGCAGTTCCAGGGCAGAGTCACGATTACCACGGACGAATCCACGAGCACGGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATGGGAACTACGGTGACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 168) HCVR amino acid sequence [ka] (SEQ ID NO: 169) HCDR1 nucleotide sequence GGA GAC ACC TTC AGT AAC TAT GTT (SEQ ID NO: 170) HCDR1 amino acid sequence GDTFSNYV (SEQ ID NO: 171) HCDR2 nucleotide sequence ATC ATC CCT ATC TTT GGT ACA ACA (SEQ ID NO: 172) HCDR2 amino acid sequence IIPIFGTT (SEQ ID NO: 173) HCDR3 nucleotide sequence GCG AGA GAT GGG AAC TAC GGT GAC TAC TTT GAC TAC (SEQ ID NO: 174) HCDR3 amino acid sequence ARDGNYGDYFDY (SEQ ID NO: 175) LCVR nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG (SEQ ID NO: 147) LCVR amino acid sequence [ka] (SEQ ID NO: 148) LCDR1 nucleotide sequence CAG TCA GTC TCT AGC TCT TAT (SEQ ID NO: 149) LCDR1 amino acid sequence QSVSSSY (SEQ ID NO: 150) LCDR2 nucleotide sequence GGG GCA AGT (SEQ ID NO: 151) LCDR2 amino acid sequence GAS (SEQ ID NO: 14) LCDR3 nucleotide sequence CAA CAG TAC GGA AGC AGC CCG TGG ACG (SEQ ID NO: 152) LCDR3 amino acid sequence QQYGSSPWT (SEQ ID NO: 153) Heavy chain nucleotide sequence (SEQ ID NO: 176) Heavy chain amino acid sequence [ka] JPEG2025538220000044.jpg10160 (sequence number 177) Light chain nucleotide sequence GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 156) Light chain amino acid sequence [ka] (SEQ ID NO: 157) H4H30045P HCVR nucleotide sequence CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGACTTCTGGTTACAGATTCGTCAACTATGGTTTCAGCTGGGTGCGCCAGGCCCCTGGACAAGGCCTTGAATGGATGGGATGGATCAGCCTTATAATGGTAACACAA ACTATATACAGAATCTCCAGGACAGAATCACCATGACCACAGACACCTCTACGAACACAGCCTACATGGAACTGACGAACCTGAGATCTGACGACACGGCCGTATATTACTGTGCGACCTTAACTGGGGTTCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 178) HCVR amino acid sequence [ka] (SEQ ID NO: 179) HCDR1 nucleotide sequence GGT TAC AGA TTC GTC AAC TAT GGT (SEQ ID NO: 180) HCDR1 amino acid sequence GYRFVNYG (SEQ ID NO: 181) HCDR2 nucleotide sequence ATC AGC CCT TAT AAT GGT AAC ACA (SEQ ID NO: 182) HCDR2 amino acid sequence ISPYNGNT (SEQ ID NO: 183) HCDR3 nucleotide sequence GCG ACC TTA ACT GGG GTT CAC TTT GAC TAC (SEQ ID NO: 184) HCDR3 amino acid sequence ATLTGVHFDY (SEQ ID NO: 185) LCVR nucleotide sequence GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAATCTCCTGATCTTTGAAACATCTCGTT TACAAAGTGGGGTCCCTTCGAGGTTCAGCGGCAGTGGTTCTGGCACAGATTTCACTCTCACCATCAACAGCCTTCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTACAATTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 186) LCVR amino acid sequence [ka] (SEQ ID NO: 187) LCDR1 nucleotide sequence CAG GGC ATT AGA AAT GAT (SEQ ID NO: 188) LCDR1 amino acid sequence QGIRND (SEQ ID NO: 189) LCDR2 nucleotide sequence GAA ACA TCT (SEQ ID NO: 190) LCDR2 amino acid sequence ETS (SEQ ID NO: 191) LCDR3 nucleotide sequence CTA CAA GAT TAC AAT TAC CCG TGG ACG (SEQ ID NO: 192) LCDR3 amino acid sequence LQDYNYPWT (SEQ ID NO: 193) Heavy chain nucleotide sequence (SEQ ID NO: 194) Heavy chain amino acid sequence [ka] (SEQ ID NO: 195) Light chain nucleotide sequence GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAATCTCCTGATCTTTGAAACATCTCGTTT ACAAAGTGGGGTCCCTTCGAGGTTCAGCGGCAGTGGTTCTGGCACAGATTTCACTCTCACCATCAACAGCCTTCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTACAATTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 196) Light chain amino acid sequence [ka] (SEQ ID NO: 197) H4H30061P HCVR nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCCCAGCCTGGGGGGTCCCTGAAACTCTCTTGTGAAGCCTCTGGATTCACGTTCAGTGATTCTGCAATGCACTGGGTCCGCCAGGCTTCCGGAAAAGGGCTGGAGTGGGTTGGTCGTATTAGAAGCAAAGCTAATAGTTACG CGACAGGATATGCTGCGTCGGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAGAACATGGCGTTTCTGGAAATGAACAGCCTGAAACCGAAGACACGGCCGTATATTACTGTCTCCGACAAACTTACGGTGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 198) HCVR amino acid sequence [ka] (SEQ ID NO: 199) HCDR1 nucleotide sequence GGA TTC ACG TTC AGT GAT TCT GCA (SEQ ID NO: 200) HCDR1 amino acid sequence GFTFSDSA (SEQ ID NO: 201) HCDR2 nucleotide sequence ATT AGA AGC AAA GCT AAT AGT TAC GCG ACA (SEQ ID NO: 202) HCDR2 amino acid sequence IRSKANSYAT (SEQ ID NO: 203) HCDR3 nucleotide sequence CTC CGA CAA ACT TAC GGT GAC CCC (SEQ ID NO: 204) HCDR3 amino acid sequence LRQTYGDP (SEQ ID NO: 205) LCVR nucleotide sequence GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCTTGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCTAAGCCTCGTATACAGTGATGAAACAACTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTACTTTATAAAGTT TTTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGAACACACTGGCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 206) LCVR amino acid sequence [ka] (SEQ ID NO: 207) LCDR1 nucleotide sequence CTA AGC CTC GTA TAC AGT GAT GGA AAC AAC TAC (SEQ ID NO: 208) LCDR1 amino acid sequence LSLVYSDGNNY (SEQ ID NO: 209) LCDR2 nucleotide sequence AAA GTT TTT (SEQ ID NO: 210) LCDR2 amino acid sequence KVF (SEQ ID NO: 211) LCDR3 nucleotide sequence ATG CAA GGA ACA CAC TGG CCG TGG ACG (SEQ ID NO: 212) LCDR3 amino acid sequence MQGTHWPWT (SEQ ID NO: 213) Heavy chain nucleotide sequence (SEQ ID NO: 214) Heavy chain amino acid sequence [ka] (SEQ ID NO: 215) Light chain nucleotide sequence GATGTTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCTTGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCTAAGCCTCGTATACAGTGATGGAAACAACTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTACTTTATAAA GTTTTTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGAACACACTGGCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAA ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 216) Light chain amino acid sequence [ka] (SEQ ID NO: 217) H4H30095P2 HCVR nucleotide sequence CAGGTTCAGCTGGTGCAGTCTGGAGTTTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAAGTTTTATGGTATCAGTTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGCTGGATCAGTGTTTACAATGGTAAAACAAAGTATGCA CAGAAGCTCCAGGGCAGAGTCACCATGACAACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTTTTGTGCGAGAGATGGGGACTATGAGAGTAGTGGTTATCCGTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 218) HCVR amino acid sequence [ka] (SEQ ID NO: 219) HCDR1 nucleotide sequence GGT TAC ACC TTT AAG TTT TAT GGT (SEQ ID NO: 220) HCDR1 amino acid sequence GYTFKFYG (SEQ ID NO: 221) HCDR2 nucleotide sequence ATC AGT GTT TAC AAT GGT AAA ACA (SEQ ID NO: 222) HCDR2 amino acid sequence ISVYNGKT (SEQ ID NO: 223) HCDR3 nucleotide sequence GCG AGA GAT GGG GAC TAT GAG AGT AGT GGT TAT CCG TTT GAC TAC (SEQ ID NO: 224) HCDR3 amino acid sequence ARDGDYESSGYPFDY (SEQ ID NO: 225) LCVR nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 226) LCVR amino acid sequence [ka] (SEQ ID NO: 227) LCDR1 nucleotide sequence CAG AGC ATT AGC AGT TAT (SEQ ID NO: 31) LCDR1 amino acid sequence QSISSY (SEQ ID NO: 32) LCDR2 nucleotide sequence GCT GCA TCC (SEQ ID NO: 33) LCDR2 amino acid sequence AAS (SEQ ID NO: 34) LCDR3 nucleotide sequence CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 75) LCDR3 amino acid sequence QQSYSTPPIT (SEQ ID NO: 76) Heavy chain nucleotide sequence (SEQ ID NO: 228) Heavy chain amino acid sequence [ka] JPEG2025538220000057.jpg17161 (sequence number 229) Light chain nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 230) Light chain amino acid sequence [ka] (SEQ ID NO: 231)

[0114] The present disclosure provides an anti-FGFR3 protein-drug conjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (e.g., monomeric or dimeric human FGFR3b and / or FGFR3c) or an antigenic fragment thereof, the antibody or antigen-binding fragment comprising: a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 140, 159, 169, 179, 199, or 219; and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 148, 187, 207, or 227 (e.g., fused to an IgG4 Fc having an S108P mutation).

[0115] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (e.g., monomeric or dimeric human FGFR3b and / or FGFR3c) or an antigen-binding fragment thereof, comprising: (a) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a light chain variable region (LCVR) comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22; (c) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 30; (c) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 50; (d) an amino acid sequence set forth in SEQ ID NO: 62. (e) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 70, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 72; (f) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102; and a light chain variable region (LCVR) comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 110; (g) a heavy chain variable region (HCVR) comprising an HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122; and a light chain variable region (LCVR) comprising an LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 130;(h) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 140, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148; (i) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 159, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148; (j) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 169, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148; (k) an HCVR HCD comprising the amino acid sequence set forth in SEQ ID NO: 179. Also provided is an anti-FGFR3 protein-drug conjugate comprising an isolated antibody or antigen-binding fragment thereof comprising: (1) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of said LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187; (2) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of said HCVR comprising the amino acid sequence set forth in SEQ ID NO: 199, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of said LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207; and / or (m) a heavy chain variable region (HCVR) comprising HCDR1, HCDR2, and HCDR3 of said HCVR comprising the amino acid sequence set forth in SEQ ID NO: 219, and a light chain variable region (LCVR) comprising LCDR1, LCDR2, and LCDR3 of said LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227.

[0116] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (e.g., monomeric or dimeric human FGFR3b and / or FGFR3c) or an antigen-binding fragment thereof, comprising: (a) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; (b) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; and (c) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, and an amino acid sequence set forth in SEQ ID NO: 46. (d) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76; (e) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96;(f) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 112, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 116; and / or (g) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 128, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134; (h) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 142, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 146, and an amino acid sequence set forth in SEQ ID NO: 150. (i) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 161, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 163, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153; (j) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 171, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153; (k) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 181, and an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183;and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185, and a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 191, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 193; (l) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205, and an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 209, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and Also provided is an anti-FGFR3 protein-drug conjugate comprising an isolated antibody or antigen-binding fragment thereof comprising: (m) a light chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 221, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225; and (m) a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76.

[0117] The present disclosure further provides an anti-FGFR3 protein-drug conjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable region comprising the amino acids set forth in SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 140, 159, 169, 179, 199, or 219, and a light chain variable region comprising the amino acids set forth in SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 148, 187, 207, or 227.

[0118] Additionally, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (e.g., monomeric or dimeric human FGFR3b and / or FGFR3c) or an antigenic fragment thereof, comprising: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 62 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 70; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90; (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 102 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 110 (e.g., the heavy chain variable region is an IgG4 Fc), and / or (g) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 122 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 130, (h) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 148, (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 159 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 148, (j) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 169 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 148, (k) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 179 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 187, (l) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 199 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 207, and / or (m) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 219 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 227.

[0119] The present disclosure provides an anti-FGFR3 protein-drug conjugate comprising: (a) an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 (e.g., monomeric or dimeric human FGFR3b and / or FGFR3c) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, 38, 58, 78, 98, 118, 136, 155, 167, 177, 195, 215, or 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20, 40, 60, 80, 100, 120, 138, 157, 197, 217, or 231.

[0120] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 or an antigen-binding fragment thereof, comprising: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 118 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 120; and / or (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 138. (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (m) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0121] The present disclosure provides an anti-FGFR3 protein-drug conjugate comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 or an antigenic fragment thereof, the antibody or antigen-binding fragment comprising a heavy chain comprising the amino acids set forth in SEQ ID NO: 18, 38, 58, 78, 98, 118, 136, 155, 167, 177, 195, 215, or 229, and a light chain comprising the amino acids set forth in SEQ ID NO: 20, 40, 60, 80, 100, 120, 138, 157, 197, 217, or 231.

[0122] The present disclosure also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR3 or an antigen-binding fragment thereof, comprising: (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; or (m) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 231.

[0123] In some embodiments, the antigen binding protein binds to the same epitope on FGFR3 (eg, FGFR3b or FGFR3c) as an antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1-1.

[0124] In some embodiments, the antigen binding protein competes for binding to FGFR3 (eg, FGFR3b or FGFR3c) with an antibody comprising an HCVR / LCVR amino acid sequence pair set forth in Table 1-1.

[0125] As discussed, an anti-FGFR3 protein-drug conjugate may comprise an anti-FGFR3 scFv (e.g., comprising a VL and a VH, optionally connected by a linker), optionally including a signal peptide (e.g., an mROR signal sequence), connected to an scFv, optionally including a linker, and connected to a molecular cargo. In various embodiments, the optional signal peptide is a signal peptide from Mus musculus Ror1 (e.g., comprising or consisting of the amino acids MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 245)).

[0126] In some embodiments, the anti-FGFR3 scFv described herein comprises an amino acid sequence set forth in Table 1-1 in the format VL-(Gly4Ser)3 (SEQ ID NO: 246)-VH. In other embodiments, the present disclosure comprises an scFv in the format VH-(Gly4Ser)3 (SEQ ID NO: 246)-VL. Optionally, the anti-FGFR3 scFv of the present disclosure further comprises a tag sequence LLQGSG (SEQ ID NO: 247) and / or HHHHHH (SEQ ID NO: 235). The tag sequence LLQGSG (SEQ ID NO: 247) or HHHHHH (SEQ ID NO: 235) may be included at the N-terminus and / or C-terminus of the anti-FGFR3 scFv. In one embodiment, the anti-FGFR3 scFv of the present invention further comprises an N-terminal LLQGSG (SEQ ID NO: 247) and / or a C-terminal HHHHHH (SEQ ID NO: 235).

[0127] In some embodiments, the FGFR3 binding proteins described herein comprise a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof (e.g., a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelized antibody or antigen-binding fragment thereof, a single heavy-chain antibody, a bispecific antibody or binding fragment thereof (e.g., a bis-scFv, or a bispecific T-cell engager (BiTE)), a triabody (e.g., a F(ab)'3 fragment or a triabody), or a chemically modified derivative thereof.

[0128] As used herein, the term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences or otherwise modified to increase their similarity to antibody variants naturally produced in humans.

[0129] In some cases, the FGFR3 binding protein is an antibody comprising one or more mutations in a framework region, such as the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations are for stabilizing the antibody and / or increasing half-life. In some embodiments, the one or more mutations are for modulating Fc receptor interaction, reducing or eliminating Fc effector function, such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional embodiments, the one or more mutations are for modulating glycosylation.

[0130] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1), and / or the CH3 domain (residues 341-447 of human IgG1), and / or hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat) of an antibody described herein, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In some embodiments, One, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0131] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment), to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo, see, e.g., PCT Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, as well as U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745. In some embodiments, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutation comprises L234 and L235. In some embodiments, the mutation comprises L234A and L235A.

[0132] The anti-FGFR3 antibodies and antigen-binding fragments described herein may be post-translationally modified (eg, glycosylation).

[0133] For example, the antibodies and antigen-binding fragments described herein may be glycosylated (e.g., N-glycosylated and / or O-glycosylated) or aglycosylated. Typically, antibodies and antigen-binding fragments are glycosylated at the conserved residue N297 of the IgG Fc domain. Some antibodies and fragments contain one or more additional glycosylation sites in the variable region. In one embodiment, the glycosylation site is located in one of the following situations: FN 297 S or YN 297 It's in S.

[0134] In one embodiment, the glycosylation is any one or more of the three different N-glycan types: high mannose, complex, and / or hybrid, which are found on IgG with their respective linkages. Complex and hybrid types exist with core fucosylation, the addition of a fucose residue to the innermost N-acetylglucosamine, and without core fucosylation.

[0135] In some cases, the anti-FGFR3 antigen binding protein is an aglycosylated antibody, i.e., an antibody that does not contain a glycosylation sequence that may interfere with transglutamination, for example, an antibody that does not have a sugar group at N297 (according to the EU numbering system) on one or more heavy chains. In certain embodiments, the antibody heavy chain has an N297 mutation. In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation. The N-linked glycan found at position 297 can be found as a core structure common to all IgGs found in humans and rodents. Antibodies containing such above-mentioned mutations can be prepared by site-directed mutagenesis to remove or disable the glycosylation sequence, or by site-directed mutagenesis to insert a glutamine residue at a site away from any interfering glycosylation site or any other interfering structure. Such antibodies can also be isolated from natural or artificial sources. Glycosylated antibodies also include antibodies containing T299 or S298P or other mutations, or combinations of mutations that result in the absence of glycosylation.

[0136] In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody in which sugar groups have been removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N297 according to the EU numbering system. In some embodiments, removal of sugar groups is achieved enzymatically, including, but not limited to, via PNGase.

[0137] In one embodiment, the antibody or fragment described herein is defucosylated.

[0138] The antibodies and antigen-binding fragments described herein may also be post-translationally modified in other ways, including, for example, Glu or Gln cyclization at the N-terminus, loss of the N-terminal positive charge, Lys variants at the C-terminus, deamidation (Asn to Asp), isomerization (Asp to isoAsp), deamidation (Gln to Glu), oxidation (Cys, His, Met, Tyr, Trp), and / or disulfide bond heterogeneity (shuffling, thioether, and trisulfide formation).

[0139] In some embodiments, the antibodies disclosed herein comprise Q295, which may be naturally occurring in the antibody heavy chain sequence. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295 and the amino acid substitution N297D.

[0140] According to certain embodiments of the present disclosure, anti-FGFR3 (e.g., monomeric or dimeric FGFR3b and / or FGFR3c) antibodies and antigen-binding fragments (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2) are provided, including an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2). For example, the present disclosure includes anti-FGFR3 antibodies containing mutations in the CH2 or CH3 region of the Fc domain, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes at pHs ranging from about 5.5 to about 6.0). Such mutations can result in an extended serum half-life of the antibody when administered to an animal.

[0141] Non-limiting examples of such Fc modifications include, for example, modifications at the following positions: 250th place (e.g., E or Q), positions 250 and 428 (e.g., L or F), 252nd place (e.g., L / Y / F / W or T), Position 254 (e.g., S or T), and / or Modification at position 256 (e.g., S / R / Q / E / D, or T), and / or location: positions 428 and / or 433 (e.g., H / L / R / S / P / Q, or K), and / or a modification at position 434 (e.g., A, W, H, F, or Y), and / or location: 250th and / or 428th place, and / or location: Modifications at positions 307 or 308 (e.g., 308F, V308F), and / or 434.

[0142] In one embodiment, the modification is 428L (e.g., M428L position) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and / or · Containing 307 and / or 308 modifications (e.g., 308F or 308P).

[0143] For example, the present disclosure provides: 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), 428L and 434S (e.g., M428L and N434S), and · Anti-FGFR3 antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 433K and 434F (e.g., H433K and N434F).

[0144] In yet another embodiment, the modifications include a 265A (eg, D265A) and / or a 297A (eg, N297A) modification.

[0145] In one embodiment, the heavy chain constant domain is gamma 4 containing an S228P and / or S108P mutation. See Angal et al., A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Mol Immunol. 1993 Jan;30(1):105-108.

[0146] All possible combinations of the aforementioned Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0147] The anti-FGFR3 antibodies described herein may comprise a modified Fc domain with reduced effector function. As used herein, a "modified Fc domain with reduced effector function" refers to any Fc portion of an immunoglobulin that has been modified, mutated, truncated, or the like, compared to a wild-type, naturally occurring Fc domain, such that the molecule comprising the modified Fc exhibits a reduced severity or extent of at least one effect selected from the group consisting of cell-killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, compared to a comparator molecule comprising a wild-type, naturally occurring version of the Fc portion. In certain embodiments, a "modified Fc domain with reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcγR).

[0148] In certain embodiments, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present disclosure may comprise a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region is substituted with the corresponding amino acid in an IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present disclosure may comprise a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region is substituted with the corresponding amino acid in an IgG2 Fc hinge region. Non-limiting exemplary modified Fc regions that may be used in the context of the present disclosure are described in U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions described therein.

[0149] The present disclosure also includes antigen-binding proteins, antibodies, or antigen-binding fragments comprising the HCVRs and chimeric heavy chain constant (CH) regions described herein, where the chimeric CH region comprises segments derived from CH regions of multiple immunoglobulin isotypes. For example, an antibody of the present disclosure may comprise a chimeric CH region comprising part or all of the CH2 domain from a human IgG1, human IgG2, or human IgG4 molecule combined with part or all of the CH3 domain from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, an antibody of the present disclosure comprises a chimeric CH region with a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, IgG2, or IgG4 hinge region (amino acid residues 216-227 according to EU numbering) combined with a "lower hinge" sequence derived from a human IgG1, IgG2, or IgG4 hinge region (amino acid residues 228-236 according to EU numbering). According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. Antibodies comprising the chimeric CH regions described herein, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO2014 / 022540.)

[0150] Other modified Fc domains and Fc modifications that may be used in the context of the present disclosure include any of the modifications described in US2014 / 0171623, US8,697,396, US2014 / 0134162, WO2014 / 043361, the disclosures of which are incorporated herein by reference in their entireties. Methods for constructing antibodies or other antigen-binding fusion proteins comprising modified Fc domains described herein are known in the art.

[0151] In some cases, the anti-FGFR3 antibody may contain one or more mutations in the framework region, for example, the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations are for stabilizing the antibody and / or increasing half-life. In some embodiments, the one or more mutations are for modulating Fc receptor interaction, reducing or eliminating Fc effector function such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional embodiments, the one or more mutations are for modulating glycosylation.

[0152] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1), and / or the CH3 domain (residues 341-447 of human IgG1), and / or hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat) of an antibody described herein, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In some embodiments, One, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0153] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment), to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo, see, e.g., PCT Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, as well as U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745. In some embodiments, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutation comprises L234 and L235. In some embodiments, the mutation comprises L234A and L235A.

[0154] The present disclosure provides a container (e.g., a plastic or glass vial with a cap or chromatography column, a hollow needle or syringe cylinder) containing an anti-FGFR3 protein-drug conjugate, e.g., an FGFR3 binding protein-drug conjugate or an anti-FGFR3 Fab-drug conjugate described herein.

[0155] The present disclosure also provides an injection device comprising an anti-FGFR3 protein-drug conjugate described herein, for example, an anti-FGFR3 scFv-drug conjugate or an anti-FGFR3 Fab-drug conjugate, or a pharmaceutical composition thereof. The injection device can be packaged in a kit. An injection device is a device for introducing a substance into a subject's body via a parenteral route, for example, intrathecally, intracisternally (e.g., cisterna magna), intracerebroventricularly, intraparenchyma, intraocularly, intravitreally, intramuscularly, subcutaneously, or intravenously. For example, the injection device can be a syringe or auto-injector (e.g., pre-filled with a pharmaceutical formulation) that includes a cylinder or barrel for holding the fluid to be injected (e.g., containing an antibody or fragment thereof or a pharmaceutical formulation thereof), a needle for penetrating the skin, blood vessel, or other tissue for injecting the fluid, and a plunger for forcing the liquid from the cylinder through the hole of the needle into the subject's body.

[0156] The present disclosure provides a method for administering an anti-FGFR3 (e.g., monomeric or dimeric FGFR3b and / or FGFR3c) antigen binding protein, e.g., an antibody or antigen-binding fragment thereof (e.g., H4H30063P, H4H30089P2, H4H30071P, H4H30066P, H4H30102P2, H4H30076P, H4H30105P2, H4H30108P2, H4H30117P2, H4H30045P, H4H30061P, H4H30095P2, or H4H30093P2) to a subject, the method comprising introducing the protein or a pharmaceutical formulation thereof into the subject's body. For example, in one embodiment, the method comprises puncturing the subject's body, e.g., with the needle of a syringe, and injecting the antigen binding protein or a pharmaceutical formulation thereof into the subject's body (e.g., into the subject's eye, vein, artery, muscle tissue, or subcutaneous tissue).

[0157] The present disclosure further provides a method for delivering a molecular cargo, e.g., conjugated to an antigen-binding protein described herein, e.g., an anti-FGFR3 scFv or anti-FGFR3 Fab described herein, to a target tissue (e.g., neural tissue of the central nervous system, eye) or target cell (e.g., astrocyte) of a subject, the method comprising introducing the protein-drug conjugate into the body of a subject (e.g., a human) parenterally (e.g., via intrathecal, intraventricular, intracisternal (e.g., cisterna magna), or intraparenchymal injection). For example, the method comprises puncturing the body of the subject with a syringe needle and injecting the protein-drug conjugate into the subject's body (e.g., the subject's brain or spinal cord). For example, the protein-drug conjugate can be introduced into the central nervous system of a subject via intrathecal, intracisternal (e.g., cisterna magna), intraventricular, or intraparenchymal injection.

[0158] The present disclosure further provides cell lines useful for screening the FGFR3 binding proteins or anti-FGFR3 protein-drug conjugates described herein. The cell lines described herein express FGFR3b and / or FGFR3c on the cell surface, and optionally further comprise exogenous nucleic acids for expressing one or more reporter proteins. In some embodiments, the cell line is modified from a brain cell line, such as a glioblastoma cell line. In one embodiment, the cell line is modified from a U87 glioblastoma cell line.

[0159] In some embodiments, the cell line contains exogenous nucleic acids (e.g., mRNA) for expressing two reporter proteins. Non-limiting examples of reporter proteins that can be used in the present application include fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), or luminescent proteins such as firefly luciferase, Renilla luciferase, or Nanoluc luciferase. In some embodiments, the cell line contains exogenous nucleic acids for expressing both GFP and firefly luciferase. The genes encoding the two reporter proteins are optionally separated by a sequence encoding a self-cleaving peptide or an internal ribosome entry site (IRES). The self-cleaving peptide may be a 2A peptide, such as a T2A, P2A, E2A, or F2A peptide.

[0160] In some embodiments, cell lines can be used to evaluate the binding and / or internalization properties of the FGFR3 binding proteins or anti-FGFR3 protein-drug conjugates described herein. Cell lines allow for high-throughput screening of FGFR3 binding proteins or anti-FGFR3 protein-drug conjugates to identify the best candidates for therapeutic delivery and / or allow for testing of the most efficient siRNA modifications, linker chemistries, and LNP chemistries.

[0161] In some embodiments, cell lines can be used to screen interfering nucleic acids (e.g., siRNAs) or gRNAs to identify genes or factors that can further promote endosomal escape of FGFR3 protein-drug conjugates (or alleviate the burden of lack of endosomal escape), allowing better delivery of the cargo to target cells.

[0162] Molecular Cargo In some aspects, the present disclosure includes methods and compositions for delivering conjugated molecular cargo to cells or tissues. In certain aspects, antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., scFvs) that specifically bind to fibroblast growth factor receptor 3 (FGFR3) disclosed herein can be conjugated (e.g., covalently conjugated) to molecular cargo.

[0163] As used herein, the term "molecular cargo" refers to a molecule that operates to produce a biological result. As non-limiting examples, molecular cargo may operate to regulate transcription of a DNA sequence, regulate protein expression or protein activity, delete or disrupt an endogenous gene (or fragment thereof), achieve an enzymatic activity, supplement or replace a missing endogenous protein, insert an exogenous gene (or fragment thereof), or replace an endogenous gene (or fragment thereof) with an exogenous gene (or fragment thereof). In various embodiments, the molecular cargo may comprise a polynucleotide. In various embodiments, the molecular cargo may comprise a polypeptide. In various embodiments, the molecular cargo comprises a lipid nanoparticle, liposome, or non-lipid nanoparticle described herein, optionally comprising one or more polynucleotide and / or protein molecules. In various embodiments, the molecular cargo may comprise a small molecule. In various embodiments, the molecular cargo may comprise a viral particle (e.g., AAV) or viral capsid protein.

[0164] In some embodiments, the anti-FGFR3 antibodies or antigen-binding fragments thereof disclosed herein can be used to deliver conjugated molecular cargos to cells or tissues expressing FGFR3 (e.g., the brain or spinal cord), for example, to diagnose and / or treat diseases (e.g., neurological diseases). In some embodiments, molecular cargos conjugated to anti-FGFR3 antibodies or antigen-binding fragments thereof can be taken up by astrocytes, for example, via binding to FGFR3. FGFR3 can be endocytosed, for example, via clathrin-mediated endocytosis, or via a clathrin- and dynamin-independent pathway (Haugsten et al., PLoS One. 2011;6(7):e21708). In some embodiments, the anti-FGFR3 antibodies or antigen-binding fragments thereof described herein can exhibit excellent activity, for example, in delivering molecular cargos to target tissues (e.g., the brain or spinal cord) or target cells (e.g., astrocytes).

[0165] In some embodiments, the molecular cargo comprises a polynucleotide molecule. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to multimeric compounds comprising nucleosides or nucleoside analogs (including traditional RNA, DNA, mixed RNA-DNA, and polymers of their analogs) with nitrogenous heterocyclic bases or base analogs linked together along the backbone. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA, PCT No. 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with optional substitutions (e.g., 2' methoxy or 2' halide substitutions). In some embodiments, polynucleotides up to about 30 nucleotides in length can be referred to herein as "oligonucleotides." Oligonucleotides can be of various different lengths, depending, for example, on the form. In some embodiments, the spacer sequence is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length, hi some embodiments, the oligonucleotide is 8-30, 10-15, 10-20, 15-25, or 21-23 nucleotides in length.

[0166] In some embodiments, the molecular cargo comprises a polypeptide molecule. The terms "polypeptide" and "protein," used interchangeably herein, encompass natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. A polypeptide or protein may be a monomer or a polymer.

[0167] In some embodiments, the molecular cargo described herein may comprise a carrier such as a liposome or lipid nanoparticle (LNP). Lipid particles, such as liposomes or lipid nanoparticles disclosed herein, may comprise lipid formulations that can be used to deliver therapeutic nucleic acids (e.g., gRNA) to a desired target site (e.g., a cell, tissue, organ, etc.). Without wishing to be bound by theory, carriers may be used, for example, as a means for delivering the polynucleotides disclosed herein and / or the proteins disclosed herein. In some embodiments, carriers (e.g., liposomes or LNPs) may be useful for delivering nucleic acids (e.g., DNA or RNA), proteins (e.g., RNA-guided DNA binders), or combinations thereof. As a non-limiting example, carriers (e.g., liposomes or LNPs) may be used to deliver various components of a gene editing system, such as a CRISPR / Cas system or additional gene editing systems described herein.

[0168] In some embodiments, the molecular cargo comprises a small molecule. Small molecules (SMs) have a low molecular weight (typically up to about 1 kDa) and can easily enter cells. Once inside the cell, they can affect other molecules, such as proteins including apolipoprotein (apo)E risk alleles (e.g., ApoE4), glial fibrillary acidic protein (Gfap), methyl-CpG binding protein 2 (MeCp2), aquaporin 4 (Aqp4), and signal transduction and activator of transcription 3 (Stat3). This differs from many high-molecular-weight molecules, such as antibodies. As an example, a small molecule can be conjugated to an FGFR3-binding protein to form an anti-FGFR3:SM conjugate. SMs for delivery via anti-FGFR3-mediated delivery may be suitable for targeting, for example, the pathological consequences of neurodegenerative diseases, neurodevelopmental diseases, physical injuries, or diseases or disorders of neuropsychiatric origin (e.g., cell death).

[0169] Exemplary molecular cargoes are described in further detail herein, although it should be understood that the exemplary molecular cargoes provided herein are not intended to be limiting.

[0170] Polynucleotide molecules Non-limiting examples of polynucleotide molecules useful as molecular cargos in the protein-drug conjugates of the present disclosure include, but are not limited to, interfering nucleic acids (e.g., shRNA, siRNA, microRNA, antisense oligonucleotides), gapmers, mixmers, ribozymes, morpholino phosphorodiamidites, peptide nucleic acids, aptamers, and guide nucleic acids (e.g., Cas9 guide RNA), mRNA, etc. In various embodiments, the polynucleotide may comprise one or more modified nucleotides. In various embodiments, the polynucleotide may comprise one or more modified internucleotide linkages. The polynucleotide may be single-stranded or double-stranded.

[0171] In some embodiments, the molecular cargo comprises at least one polynucleotide molecule, hi some embodiments, the molecular cargo comprises at least two, at least three, at least four, at least five, or at least 10 polynucleotide molecules.

[0172] In some embodiments, the polynucleotide molecule is DNA. In some embodiments, the polynucleotide molecule is RNA.

[0173] In various embodiments, a polynucleotide (e.g., an interfering nucleic acid or a guide RNA) described herein can comprise a region of complementarity to a target nucleic acid that can range from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In certain embodiments, the region of complementarity of a polynucleotide to a target nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity can be complementary to at least 10 consecutive nucleotides of the target nucleic acid. In some embodiments, a polynucleotide may contain 1, 2, 3, 4, or 5 base mismatches compared to a portion of consecutive nucleotides of a target nucleic acid. In some embodiments, a polynucleotide may have up to 3 mismatches over 15 bases, or up to 4 mismatches over 10 bases. In some embodiments, a polynucleotide is complementary (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100%) to a target sequence of any one of the polynucleotides described herein. In various embodiments, such a target sequence may be 100% complementary to a polynucleotide described herein. In some embodiments, any one or more of the thymine bases (T) in any one of the polynucleotides described herein may be a uracil base (U), and / or any one or more of the Us may be a T. The target sequences described herein include sequences of nucleic acids in a target gene that have complementarity to a guide sequence of a gRNA. Interaction between the target sequence and the guide sequence directs an RNA-guided DNA-binding agent (e.g., a Cas protein) to bind and potentially (depending on the activity of the agent) form a nick or cleave within the target sequence.

[0174] The polynucleotides described herein may be modified, for example, by incorporating modified nucleotides, modified internucleoside linkages, and / or modified sugar moieties, or a combination thereof. Additionally, the polynucleotides may have one or more of the following properties: improved cellular uptake compared to unmodified polynucleotides; non-toxic to cells or immunostimulatory to mammals; avoiding pattern recognition receptors so as not to mediate alternative splicing; nuclease resistance; improved endosomal escape within cells; or minimal TLR stimulation. Any of the various modified chemical properties or forms of the polynucleotides disclosed herein may be combined together. By way of non-limiting example, one, two, three, four, five, six, seven, eight, or more different types of modifications may be included within the same polynucleotide.

[0175] In various embodiments, specific nucleotide modifications may be used that make the polynucleotide incorporating the modifications more resistant to nuclease digestion than natural oligoribonucleotide or oligodeoxynucleotide molecules, and such modified polynucleotides survive intact for longer periods of time than unmodified polynucleotides. Exemplary modified polynucleotides include those containing modified backbones, such as methylphosphonates, phosphotriesters, phosphorothioates, short alkyl or cycloalkyl intersugar linkages, heterocyclic intersugar linkages, or short heteroatoms. Thus, the polynucleotides described herein can be stabilized against nucleic acid degradation, for example, through the incorporation of modifications (e.g., nucleotide modifications).

[0176] In various embodiments, a polynucleotide may be up to 50 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, or 2 to 45 nucleotides of the polynucleotide may be modified nucleotides. A polynucleotide may be 8 to 30 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, or 2 to 30 nucleotides of the polynucleotide may be modified nucleotides. In some embodiments, a polynucleotide may be up to 8 to 15 nucleotides in length, and 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, or 2 to 14 nucleotides of the polynucleotide may be modified nucleotides. In some embodiments, a polynucleotide may have all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides modified.

[0177] In various embodiments, the polynucleotides disclosed herein may contain at least one nucleoside modified, for example, at the 2'-position of the sugar. In some embodiments, all of the nucleosides in the polynucleotide are 2'-modified nucleosides. In some embodiments, the polynucleotide contains at least one 2'-modified nucleoside.

[0178] In various embodiments, the polynucleotides disclosed herein may include one or more non-bicyclic 2'-modified nucleosides, such as 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-methyl (2'-O-Me), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-methoxyethyl (2'-MOE), 2'-deoxy, 2'-ON-methylacetamide (2'-O-NMA) modified nucleosides, 2'-fluoro (2'-F), 2'-O-aminopropyl (2'-O-AP), or 2'-O-dimethylaminopropyl (2'-O-DMAP).

[0179] In some embodiments, the polynucleotides described herein may comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring may comprise a bridging moiety, for example, connecting two atoms within the ring (e.g., connecting the 2'-O atom to the 4'-C atom via an ethylene (ENA) bridge, a methylene (LNA) bridge, or an (S)-constrained ethyl (cEt) bridge). Non-limiting examples of ENA are disclosed in PCT Publication No. WO2005 / 042777, Morita et al. Nucleic Acid Res., Suppl 1:241-242, 2001, Koizumi, Curr.Opin.Mol.Ther., 8:144-149, 2006, Surono et al., Hum.Gene Ther., 1 5:749-757, 2004, and Horie et al., Nucleic Acids Symp.Ser(Oxf), 49:171-172, 2005 (these disclosures are incorporated herein by reference in their entirety).Non-limiting examples of LNA are disclosed in PCT Patent Application Publication No. WO2008 / 043753 (the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of cEts are disclosed in US Pat. Nos. 7,569,686, 7,101,993, and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0180] In various embodiments, the polynucleotides described herein may include modified nucleosides as disclosed, for example, in U.S. Pat. Nos. 8,022,193, 7,569,686, 7,399,845, 7,741,457, 7,335,765, 7,816,333, 8,957,201, and 7,314,923, the entire contents of each of which are incorporated herein by reference for all purposes.

[0181] In various embodiments, a polynucleotide comprises at least one modified nucleoside, which results in an increase in the Tm of the polynucleotide in the range of 1° C. to 10° C. compared to a polynucleotide that does not have at least one modified nucleoside. A polynucleotide may have multiple modified nucleosides, which results in an overall increase in the Tm of the polynucleotide in the range of 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., or more, compared to a polynucleotide that does not have any modified nucleosides.

[0182] In some embodiments, a polynucleotide may contain a mixture of different types of nucleosides. A polynucleotide may contain a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. A polynucleotide may contain a mixture of 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. A polynucleotide may contain a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). A polynucleotide may contain a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. A polynucleotide can comprise a mixture of 2'-fluoro and 2'-O-Me modified nucleosides.

[0183] In various embodiments, oligonucleotides can alternately comprise different types of nucleosides.In certain embodiments, oligonucleotides can alternately comprise deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides.In certain embodiments, polynucleotides can alternately comprise 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides.In certain embodiments, oligonucleotides can alternately comprise 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides.In certain embodiments, oligonucleotides can alternately comprise 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. In certain embodiments, oligonucleotides may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0184] In various embodiments, the polynucleotides described herein may contain one or more abasic residues, 5-vinylphosphonate modifications, and / or one or more inverted abasic residues.

[0185] In various embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleoside linkages. In various embodiments, the oligonucleotide may contain phosphorothioate internucleoside linkages. In various embodiments, the oligonucleotide contains phosphorothioate internucleoside linkages between at least two nucleotides. In various embodiments, the oligonucleotide contains phosphorothioate internucleoside linkages between all nucleotides. As a non-limiting example, in certain embodiments, the oligonucleotide contains modified internucleoside linkages at the first, second, and / or (e.g., and) third internucleoside linkages at the 5'-end or 3'-end of the nucleotide sequence.

[0186] Non-limiting examples of phosphorus-containing linkages include aminoalkyl phosphotriester phosphorothioates, chiral phosphorothioates, phosphotriesters, phosphorodithioates, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionoalkyl phosphonates, thionophosphoramidates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. U.S. Patent Nos. 5,625,050, 4,469,863, 4,476,301, 5,023,243, 5,550,111, 5,177,196, 5,587,361, 5,188,897, 5,264,423, 5,276,019, 5,519,126, 5,278,302, 5,286,71 See Nos. 7, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,536,821, 5,541,306, 5,563,253, 5,571,799, and 3,687,808.

[0187] In various embodiments, the polynucleotides described herein may have a heteroatom backbone, e.g., a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are bound directly or indirectly to aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497), a morpholino backbone (see Summerton and Weller, U.S. Pat. No. 5,034,506), an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374), or an MMI or methylene (methylimino) backbone.

[0188] The nitrogenous base may be a conventional base (A, G, C, T, U), an analog thereof (e.g., a modified uridine such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine), a derivative of inosine, purine, or pyrimidine (e.g., N4-methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), purine bases having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, 6-O-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and 4-O-alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT Publication No. WO 93 / 13121). For a general discussion, see Adams et al., The Biochemistry of the Nucleic Acids, 5-36, 11th ed., 1992. Nucleic acids can contain one or more "abasic" residues, in which the backbone does not contain a nitrogenous base at one or more positions in the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases, and linkages, or can contain both conventional building blocks and substitutions (e.g., conventional nucleosides with 2'-methoxy substitutions, or polymers containing both conventional nucleotides and one or more nucleotide analogs). Nucleic acids include "locked nucleic acids" (LNAs), which are analogs containing one or more LNA nucleotide monomers that have a bicyclic furanose unit locked into an RNA-mimetic sugar conformation, enhancing hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or its analogs in RNA and thymine or its analogs in DNA.

[0189] Interfering nucleic acids In some embodiments, the conjugated molecular cargo may comprise a polynucleotide molecule(s) capable of modifying the expression of one or more genes in a target cell (e.g., inhibiting gene expression and / or translation, regulating RNA splicing, or inducing exon skipping). In some embodiments, the polynucleotide molecule may be, for example, an interfering nucleic acid molecule, such as an siRNA, shRNA, miRNA, or antisense oligonucleotide (ASO), that targets RNA (e.g., mRNA).

[0190] In some embodiments, the interfering nucleic acid molecule may modulate the expression of one or more genes associated with neurological diseases and / or disorders listed in Tables 1-4. In some embodiments, the interfering nucleic acid molecule may inhibit the expression of one or more genes encoding apolipoprotein (apo) E risk alleles (e.g., ApoE4), glial fibrillary acidic protein (Gfap), methyl-CpG binding protein 2 (MeCp2), aquaporin 4 (Aqp4), or signal transduction and activator of transcription 3 (Stat3).

[0191] In certain embodiments, interference nucleic acid molecules that selectively target and inhibit the activity or expression of target gene products (e.g., mRNA products) are used in the compositions and methods described herein.Interference nucleic acid molecules can inhibit the expression or activity of at least one target gene product (e.g., mRNA product) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.The agents disclosed herein can comprise a nucleic acid base sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to at least the target gene product (e.g., mRNA product). Without wishing to be bound by theory, "complementarity" of a nucleic acid can mean that a nucleotide sequence on one strand of a nucleic acid forms hydrogen bonds with another sequence on the opposite strand of the nucleic acid due to the orientation of its nucleobase groups. In DNA, complementary bases are usually A and T, C and G. In RNA, they are usually C and G, and U and A. Complementarity can be complete or substantial / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, and all bases in the duplex are bound to complementary bases by Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposite strand, but sufficient binding occurs between the bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by predicting the Tm (melting temperature) of the hybridized strands using the sequences and standard mathematical calculations, or by empirically determining the Tm using routine methods. The Tm comprises the temperature at which the population of hybridization complexes formed between two nucleic acid strands becomes 50% denatured (i.e., half of the population of double-stranded nucleic acid molecules dissociates into single strands).At temperatures below the Tm, the formation of a hybridization complex is favored, while at temperatures above the Tm, melting or separation of the strands in the hybridization complex is favored. While other known Tm calculations take into account the structural characteristics of nucleic acids, the Tm can be estimated for a nucleic acid with a known G+C content in 1 M aqueous NaCl solution, for example, by using Tm = 81.5 + 0.41 (% G+C).

[0192] An interfering nucleic acid can comprise a sequence of circular subunits, each having a base-pairing portion, linked by intersubunit linkages that enable the base-pairing portions to hybridize to a target sequence of nucleic acid (typically RNA) by Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence.

[0193] Typically, at least 17, 18, 19, 20, 21, 22, or 23 nucleotides of the complement of the target mRNA sequence are sufficient to mediate inhibition of the target transcript. Perfect complementarity is not required. In some embodiments, the interfering nucleic acid molecule is single-stranded RNA. In some embodiments, the interfering nucleic acid molecule is double-stranded RNA. The double-stranded RNA molecule may have a 1-3 nucleotide 3' overhang and / or a 5' overhang on either the sense strand and / or the antisense strand. In some embodiments, the double-stranded RNA molecule has a 2-nucleotide 3' overhang. In some embodiments, the two RNA strands are connected via a hairpin structure to form an shRNA molecule. The shRNA molecule may comprise a hairpin derived from a microRNA molecule.

[0194] The interfering nucleic acid molecules described herein can comprise RNA bases, non-RNA bases, or a mixture of RNA bases and non-RNA bases.For example, the interfering nucleic acid molecules described herein can be mainly composed of RNA bases or modified RNA bases, but also comprise DNA bases, modified DNA bases, and / or non-naturally occurring nucleotides.The term "ribonucleotide" or "nucleotide" can also refer to modified nucleotide or surrogate replacement moiety at one or more positions in the case of modified RNA or nucleotide surrogate.

[0195] In some embodiments, the interfering nucleic acid molecule is a small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. Such siRNA molecules typically contain a region of sufficient homology to the target region and are of sufficient length, in terms of nucleotides, so that the siRNA molecule downregulates the target nucleic acid. While perfect complementarity between the siRNA molecule and the target is not required, the correspondence must be sufficient to allow the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand only needs to be sufficiently complementary to the antisense strand to maintain the overall double-stranded character of the molecule.

[0196] The specificity of an siRNA molecule can be measured by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are often less than 30-35 base pairs in length, for example, to prevent stimulation of nonspecific RNA interference pathways in cells due to the interferon response, although longer siRNAs may also be effective. In various embodiments, the siRNA molecule is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In various embodiments, the siRNA molecule is about 35 to more than about 70 base pairs in length. In some embodiments, the siRNA molecule is more than 70 base pairs in length. In some embodiments, the siRNA molecule is 8 to 40 base pairs in length, 10 to 20 base pairs in length, 10 to 30 base pairs in length, 15 to 20 base pairs in length, 19 to 23 base pairs in length, or 21 to 24 base pairs in length. In some embodiments, the sense strand and antisense strand of the siRNA molecule are each independently about 19 to about 24 nucleotides in length. In some embodiments, the sense strand of the siRNA molecule is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand of the siRNA molecule are 21 nucleotides in length.

[0197] After selecting a suitable target RNA sequence, siRNA molecules can be designed and prepared using suitable methods, comprising the nucleotide sequence (i.e., antisense sequence) complementary to all or part of the target sequence (see, for example, US Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791, and PCT Publication No. WO2004 / 016735).In some embodiments, siRNA molecules can be single-stranded (i.e., ssRNA molecules that comprise only antisense strands) or double-stranded (i.e., dsRNA molecules that comprise antisense strands and complementary sense strands that hybridize to form dsRNA).In various embodiments, siRNA molecules can comprise duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structures, which comprise self-complementary sense strands and / or antisense strands.

[0198] In various embodiments, the antisense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the antisense strand of the siRNA molecule is about 35 to about 70 nucleotides in length. In various embodiments, the antisense strand of the siRNA molecule is greater than 70 nucleotides in length. In some embodiments, the antisense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.

[0199] In some embodiments, the sense strand of the siRNA molecule is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In various embodiments, the sense strand of the siRNA molecule is about 30 to about 70 nucleotides in length. In various embodiments, the sense strand of the siRNA molecule is greater than 70 nucleotides in length. In some embodiments, the sense strand is 8 to 40 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 20 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 24 nucleotides in length.

[0200] In various embodiments, siRNA molecule can comprise an antisense strand that comprises a complementary region to the target region in target mRNA.In some embodiments, the complementary region is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the target region in target mRNA.In some embodiments, the target region can comprise a region of consecutive nucleotides in target mRNA.In some embodiments, the complementary region may not need to be 100% complementary to the region of its target to be able to specifically hybridize or be specific to the target RNA sequence.

[0201] In some embodiments, the siRNA molecules disclosed herein may comprise an antisense strand, which comprises a region of complementarity to a target RNA sequence, wherein the region of complementarity is 8 to 20, 8 to 35, 8 to 45, 10 to 50, 5 to 55, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of the target RNA sequence. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence containing no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches compared to a portion of consecutive nucleotides of the target RNA sequence. In some embodiments, the siRNA molecule comprises a nucleotide sequence having up to 3 mismatches over 15 bases or up to 4 mismatches over 10 bases with the target sequence. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence having up to 0, 1, 2, or 3 mismatches over 15-22 bases with the target sequence. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with zero, one, or two mismatches with the target sequence over a 15-22 base range. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with zero or one mismatch with the target sequence over a 15-22 base range.In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence with 0 mismatches over 15-22 bases with the target sequence.

[0202] In various embodiments, the siRNA molecule may comprise an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% complementary to the target RNA sequence of the antisense oligonucleotide disclosed herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or 100% identical to any of the antisense oligonucleotides provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, or more consecutive nucleotides of any of the antisense oligonucleotides provided herein.

[0203] In some embodiments, double-stranded siRNAs can contain sense and antisense RNA strands of different lengths or the same length. In some embodiments, double-stranded siRNA molecules can also be generated from a single oligonucleotide in a stem-loop structure. The self-complementary sense and antisense regions of siRNA molecules with a stem-loop structure can be linked by a nucleic acid-based or non-nucleic acid-based linker. In some embodiments, siRNAs with a stem-loop structure comprise a circular single-stranded RNA with two or more loop structures and a stem comprising self-complementary sense and antisense strands. In some embodiments, the circular RNA can be processed in vivo or in vitro to produce active siRNA molecules capable of mediating RNAi. Therefore, short hairpin RNA (shRNA) molecules are also contemplated herein. Such molecules can contain a specific antisense sequence and a reverse complementary (sense) sequence, which in some instances can be separated by a spacer or loop sequence. The reverse complement described herein can include a sequence that is the complementary sequence of a reference sequence, where the complementary sequence is written in reverse. Due to redundancy in codon usage, a reverse complement may deviate from a reference sequence that encodes the same polypeptide. As used herein, "reverse complement" also includes a sequence that is, for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reverse complement of a reference sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, which can then anneal to form a dsRNA molecule. In various embodiments, an additional optional processing step can result in the removal or addition of 1, 2, 3, 4, 5, or more nucleotides from the 3' and / or 5' ends of one or both strands. The spacer may be of a suitable length to allow the antisense and sense sequences to anneal and form a double-stranded structure or stem prior to cleavage of the spacer.In certain embodiments, subsequent optional processing steps may result in the removal or addition of 1, 2, 3, 4, 5, or more nucleotides from the 3' and / or 5' ends of one or both strands. In some embodiments, the spacer sequence may be an unrelated nucleotide sequence, which may be located between two complementary nucleotide sequence regions that, for example, may comprise an shRNA when annealed to a double-stranded nucleic acid.

[0204] The length of an siRNA molecule can vary from about 10 to about 120 nucleotides depending on the type of siRNA molecule designed. Generally, these about 10 to about 55 nucleotides can be complementary to the RNA target sequence. For example, when the siRNA is a double-stranded siRNA or a single-stranded siRNA, its length can vary from about 10 to about 55 nucleotides, but when the siRNA is an shRNA or a circular molecule, its length can vary from about 30 to about 110 nucleotides.

[0205] In various embodiments, the siRNA molecule may comprise a 3' overhang at one end of the molecule. In some embodiments, the other end may be blunt-ended or may also comprise an overhang (e.g., 5' and / or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the lengths of the overhangs may be different or the same. In some embodiments, the siRNA molecules described herein may comprise a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on both the sense strand and the antisense strand. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on the antisense strand. In some embodiments, the siRNA molecule may comprise a 3' overhang of about 1 to about 3 nucleotides on the sense strand.

[0206] In various embodiments, the siRNA molecule comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) modified nucleotides. In some embodiments, all of the nucleotides in the sense strand and / or antisense strand of the siRNA molecule are modified. In certain embodiments, the siRNA molecule can comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, the siRNA molecule can comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule. In some embodiments, the siRNA molecule can comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule. In some embodiments, the siRNA molecule may comprise modified internucleoside linkages at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule and at the first and second internucleoside linkages at the 5'-end and 3'-end of the antisense strand of the siRNA molecule.

[0207] In some embodiments, modified nucleotides may contain modified sugar moieties (e.g., 2'-modified nucleotides). In some embodiments, siRNA molecules may contain one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide of an siRNA molecule may be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, an siRNA molecule may contain one or more morpholino phosphorodiamidates. In some embodiments, each nucleotide of an siRNA molecule consists of a morpholino phosphorodiamidate.

[0208] In various embodiments, siRNA molecules may contain phosphorothioate or other modified internucleoside linkages. In various embodiments, siRNA molecules may contain, for example, phosphorothioate internucleoside linkage(s). In some embodiments, siRNA molecules may contain phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, siRNA molecules may contain phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first, second, and / or third internucleoside linkages at the 5'-end or 3'-end of the siRNA molecule. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first and second internucleoside linkages at the 5'-end and / or 3'-end of the siRNA molecule. In some embodiments, siRNA molecules may contain modified internucleoside linkages at the first and second internucleoside linkages at the 5'-end of the sense strand of the siRNA molecule. In some embodiments, siRNA molecules can comprise modified internucleoside bonds at the first and second internucleoside bonds at the 5'-end and 3'-end of the antisense strand of siRNA molecules.In some embodiments, siRNA molecules can comprise modified internucleoside bonds at the first and second internucleoside bonds at the 5'-end of the sense strand of siRNA molecules and at the first and second internucleoside bonds at the 5'-end and 3'-end of the antisense strand of siRNA molecules.In some embodiments, siRNA molecules can comprise the first internucleoside bond at the 5'-end and 3'-end of the sense strand of siRNA molecules, the first, second and third internucleoside bonds at the 5'-end of the antisense strand of siRNA molecules, and the first internucleoside bond at the 3'-end of the antisense strand of siRNA molecules.

[0209] In various embodiments, modified internucleotide linkages can comprise phosphorus-containing linkages.In some embodiments, the phosphorus-containing linkages that can be used in the methods or compositions described herein include, but are not limited to, chiral phosphorothioates, phosphorothioates, phosphorodithioates, aminoalkylphosphotriesters, phosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), phosphinates, thionoalkylphosphonates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity (where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'). U.S. Patent Nos. 5,625,050, 3,687,808, 4,469,863, 4,476,301, 5,177,196, 5,455,233, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,93 See Nos. 9, 5,519,126, 5,453,496, 5,466,677, 5,476,925, 5,536,821, 5,023,243, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,188,897.

[0210] Any of the various modification types or chemicals of the siRNA molecules disclosed herein can be combined together. For example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included in the same siRNA molecule.

[0211] In various embodiments, the antisense strand may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) modified nucleotides. In some embodiments, the antisense strand may comprise one or more modified nucleotides and / or one or more modified internucleotide linkage(s). In some embodiments, the modified nucleotides may comprise modified sugar moieties (e.g., 2'-modified nucleotides). In some embodiments, the antisense strand comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide of the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand can comprise one or more morpholino phosphorodiamidates. In some embodiments, the antisense strand is composed of morpholino phosphorodiamidate oligomers (PMOs).

[0212] In some embodiments, the antisense strand comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage(s). In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the antisense strand may comprise a phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, the antisense strand may comprise a modified internucleotide linkage at the first, second, and / or third nucleotide at the 5'-end or 3'-end of the antisense strand. In some embodiments, the antisense strand may comprise a modified internucleotide linkage at the first and second nucleotide positions (e.g., between the first and second nucleotides, and between the second and third nucleotides) at the 5'-end and 3'-end of the antisense strand.

[0213] In various embodiments, modified internucleotide bond can comprise the phosphorus-containing bond of antisense strand.In some embodiments, the phosphorus-containing bond that can be used in the methods and compositions described herein includes but is not limited to chiral phosphorothioate, phosphorothioate, phosphorodithioate, aminoalkylphosphotriester, phosphotriester, methyl and other alkylphosphonate (including 3'-alkylenephosphonate and chiral phosphonate), phosphoramidate (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphinate, thionoalkylphosphonate, thionophosphoramidate, thionoalkylphosphotriester, and boranophosphate with normal 3'-5' bond, their 2'-5' bond analogues, and those with reverse polarity (where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'). U.S. Patent Nos. 5,625,050, 3,687,808, 4,469,863, 4,476,301, 5,177,196, 5,455,233, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,93 See Nos. 9, 5,519,126, 5,453,496, 5,466,677, 5,476,925, 5,536,821, 5,023,243, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,188,897.

[0214] Any of the antisense strand modification types or chemicals disclosed herein can be combined together. For example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included in the same antisense strand.

[0215] In some embodiments, the sense strand comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, or more) modified nucleotides. In some embodiments, the antisense strand may comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, the modified nucleotides may comprise modified sugar moieties (e.g., 2'-modified nucleotides). In some embodiments, the antisense strand comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In various embodiments, each nucleotide of the antisense strand can be a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand can comprise one or more morpholino phosphorodiamidates. In some embodiments, the antisense strand is composed of morpholino phosphorodiamidate oligomers (PMOs).

[0216] In some embodiments, the sense strand comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage(s). In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the sense strand may comprise a phosphorothioate internucleoside linkage between all nucleotides. For example, in some embodiments, the sense strand comprises a modified internucleotide linkage at the first, second, and / or third nucleotide at the 5'-end or 3'-end of the sense strand. In some embodiments, the sense strand may comprise a modified internucleotide linkage at the first and second nucleotide positions (e.g., between the first and second nucleotides, and between the second and third nucleotides) at the 5'-end of the sense strand.

[0217] In various embodiments, modified internucleotide bond can comprise the phosphorus-containing bond of the sense strand.In some embodiments, the phosphorus-containing bond that can be used in the methods and compositions described herein includes but is not limited to chiral phosphorothioate, phosphorothioate, phosphorodithioate, aminoalkylphosphotriester, phosphotriester, methyl and other alkylphosphonate (including 3'-alkylenephosphonate and chiral phosphonate), phosphoramidate (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphinate, thionoalkylphosphonate, thionophosphoramidate, thionoalkylphosphotriester, and boranophosphate with normal 3'-5' bond, their 2'-5' bond analogues, and those with reverse polarity (where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'). U.S. Patent Nos. 5,625,050, 3,687,808, 4,469,863, 4,476,301, 5,177,196, 5,455,233, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,93 See Nos. 9, 5,519,126, 5,453,496, 5,466,677, 5,476,925, 5,536,821, 5,023,243, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,188,897.

[0218] Any of the sense strand modification chemistries or types described herein can be combined together, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different types of modifications can be included in the same sense strand.

[0219] In various embodiments, the antisense and / or sense strands of the siRNA molecule may contain one or more modifications that can, for example, enhance or reduce RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule may contain one or more modifications that can enhance RISC loading. In various embodiments, the sense strand of the siRNA molecule may contain one or more modifications that can reduce RISC loading and / or reduce off-target effects. In various embodiments, the antisense strand of the siRNA molecule may contain a 2'-O-methoxyethyl (2'-MOE) modification. In some embodiments, for example, the addition of a 2'-O-methoxyethyl (2'-MOE) group at the cleavage site can improve the silencing activity and / or specificity of the siRNA, for example, by promoting the orientation of the modified strand into the RNA-induced silencing complex (RISC), as disclosed, for example, in Song et al., (2017) Mol Ther Nucleic Acids 9:242-250 (incorporated herein by reference in its entirety). In various embodiments, the antisense strand of the siRNA molecule may comprise a 2'-O-Me-phosphorodithioate modification. In some embodiments, the 2'-O-Me-phosphorodithioate modification may increase RISC loading, as disclosed, for example, in Wu et al., (2014) Nat Commun 5:3459 (incorporated herein by reference in its entirety).

[0220] In various embodiments, the sense strand of the siRNA molecule may comprise a 5'-nitroindole modification. In some embodiments, the 5'-nitroindole modification may reduce the RNAi potency of the sense strand and / or reduce off-target effects, as disclosed, for example, in Zhang et al., (2012) Chembiochem 13(13):1940-1945 (incorporated herein by reference in its entirety). In various embodiments, the sense strand may comprise a 2'-O-methyl (2'-O-Me) modification. In some embodiments, the 2'-O-Me modification may reduce RISC loading and / or reduce off-target effects of the sense strand, as disclosed, for example, in Zheng et al., FASEB (2013) 27(10):4017-4026 (incorporated herein by reference in its entirety). In various embodiments, the sense strand of the siRNA molecule may be fully substituted with morpholino, 2'-MOE, and / or 2'-O-Me residues and may not be recognized by RISC, as disclosed, for example, in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, which is incorporated herein by reference in its entirety.

[0221] In various embodiments, the sense strand of the siRNA molecule may include a 5'-morpholino modification. In various embodiments, the 5'-morpholino modification may reduce RISC loading of the sense strand and / or improve RNAi activity and / or antisense strand selection, as disclosed, for example, in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142 (incorporated herein by reference in its entirety). In various embodiments, the sense strand of the siRNA molecule may be modified with a synthetic RNA-like high-affinity nucleotide analog called a locked nucleic acid (LNA), which may reduce RISC loading of the sense strand and promote incorporation of the antisense strand into RISC, as disclosed, for example, in Elman et al., (2005) Nucleic Acids Res. 33(1):439-447 (incorporated herein by reference in its entirety). In various embodiments, the sense strand of the siRNA molecule may include a 5'-unlocked nucleic acid (UNA) modification. In various embodiments, 5' unlocked nucleic acid (UNA) modifications may reduce RISC loading of the sense strand and / or improve the silencing capacity of the antisense strand, as disclosed, for example, in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103, which is incorporated herein by reference in its entirety.

[0222] In some embodiments, the antisense strand of the siRNA molecule may include a 2'-MOE modification, and / or the sense strand may include a 2'-O-Me modification (see, e.g., Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least five, at least eight, at least nine, at least ten or more) siRNA molecule may be covalently conjugated to, for example, an FGFR3 binding protein described herein. In some embodiments, the FGFR3 binding protein may be conjugated to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the FGFR3 binding protein may be conjugated to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the FGFR3 binding protein may be internally conjugated to the sense strand of the siRNA molecule. In some embodiments, FGFR3 binding protein can be conjugated to the 5' end of the antisense strand of siRNA molecule.In some embodiments, FGFR3 binding protein can be conjugated to the 3' end of the antisense strand of siRNA molecule.In some embodiments, FGFR3 binding protein is internally conjugated to the antisense strand of siRNA molecule.

[0223] In addition, siRNA molecule can be modified or comprise nucleoside surrogate.The single-stranded region of siRNA molecule can be modified or comprise nucleoside surrogate, for example, the unpaired region (or regions) of the region that connects two complementary regions, such as hairpin structure, can have modification or nucleoside surrogate.For example, modification is also useful for stabilizing one or more 3'-end or 5'-end of siRNA molecule against exonuclease, or for making antisense siRNA agent easier to enter RISC. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (e.g., C3-C12 (e.g., C3, C6, C9, C12), abasic, triethylene glycol, hexaethylene glycol), biotin, or fluorescein reagents (which are provided as phosphoramidites and have another DMT-protected hydroxyl group, allowing for multiple couplings during RNA synthesis).

[0224] In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long, and the nucleotide positions at the 3' and 5' ends of the sense strand are inverted abasic residues. The inverted abasic residues at the 3' and 5' ends of the sense strand can be overhangs. The inverted abasic residues can be linked via a 3'-3' phosphodiester bond. In some embodiments, the antisense strand of the siRNA molecule contains one to two phosphorothioate linkages at the 3' and / or 5' ends. In some embodiments, the antisense strand contains two or three phosphorothioate internucleotide linkages at the 5' end and one phosphorothioate internucleotide linkage at the 3' end. The siRNA molecule can be linked to a targeting moiety at the 5' or 3' end of the sense strand.

[0225] In some embodiments, the sense strand is 21 nucleotides long, the antisense strand is 23 nucleotides long, and the antisense strand comprises a 2-nucleobase 3' overhang. In some embodiments, the antisense strand of the siRNA molecule comprises one to three phosphorothioate linkages at the 3' and 5' ends, and the sense strand of the siRNA molecule comprises one to two phosphorothioate linkages at the 5' end. In some embodiments, the antisense strand of the siRNA molecule comprises two to three phosphorothioate linkages at the 5' end and two phosphorothioate linkages at the 3' end, and the sense strand of the siRNA molecule comprises two phosphorothioate linkages at the 5' end. The siRNA molecule can be linked to a targeting moiety at the 5' or 3' end of the sense strand.

[0226] In some embodiments, the siRNA molecules described herein can be conjugated to a moiety that directs delivery to the CNS, e.g., a lipophilic ligand, optionally a C16 ligand, as described in WO 2021 / 119226 A1 (incorporated herein by reference in its entirety). In one embodiment, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, vomeol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0227] In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C6-C18 hydrocarbon chain. In one embodiment, the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain. In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6-position from the 5' end of the chain.

[0228] In one embodiment, lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in internal position(s) or double-stranded region.In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic moiety based on serinol backbone or diethanolamine backbone.In one embodiment, lipophilic moiety is conjugated to double-stranded siRNA agent via a linker that includes ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, product of click reaction, or carbamate. In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0229] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized monosaccharides, or oligosaccharides (galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof).

[0230] In one embodiment, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0231] In some embodiments, the interfering nucleic acid molecule is a short hairpin RNA (shRNA).The "small hairpin RNA" or "short hairpin RNA" or "shRNA" described herein can comprise a short RNA sequence that creates a tight hairpin turn, which can be used to silence gene expression through RNA interference.The shRNA provided herein can be chemically synthesized or transcribed from a transcription cassette in a DNA plasmid.The shRNA hairpin structure can be cleaved into siRNA by cellular machinery, and then the siRNA is bound to the RNA-induced silencing complex (RISC).

[0232] Non-limiting examples of shRNAs include double-stranded polynucleotide molecules assembled from single-stranded molecules, in which the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker, and double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure containing about 1 to about 25 nucleotides, about 2 to about 20 nucleotides, about 4 to about 15 nucleotides, about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

[0233] Further embodiments relating to shRNAs, as well as methods for designing and synthesizing such shRNAs, are described in U.S. Patent Publication No. 2011 / 0071208, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0234] In some embodiments, the interfering nucleic acid molecule is a microRNA (miRNA). miRNAs represent a large group of small RNAs naturally produced in organisms, some of which regulate the expression of target genes. miRNAs are short hairpin RNAs, approximately 18 to 25 nucleotides in length, that function in RNA silencing and post-translational regulation of gene expression. Typically, miRNAs are generated from larger RNA precursors (called pri-miRNAs), which are processed in the nucleus into approximately 70-nucleotide pre-miRNAs that fold into imperfect stem-loop structures. These pre-miRNAs typically undergo additional processing steps in the cytoplasm, where mature miRNAs, approximately 18 to 25 nucleotides in length, are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer. miRNAs are not translated into proteins but instead bind to specific messenger RNAs, thereby blocking translation. In some embodiments, miRNAs inhibit translation by imprecisely base-pairing with their targets.

[0235] The miRNAs described herein can include fragments of pri-miRNA, pre-miRNA, mature miRNA, or variants thereof that retain the biological activity of the mature miRNA. In some embodiments, the size range of the miRNA can be 21 nucleotides to 170 nucleotides. In one embodiment, the size range of the miRNA is 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of 21 to 25 nucleotides in length can be used.

[0236] In certain embodiments, the interfering nucleic acid molecule is an antisense oligonucleotide (ASO). ASOs can downregulate targets by inducing RNase H endonuclease cleavage of target RNA, steric hindrance of ribosomal activity, inhibiting 5' cap formation, or alternative splicing. ASOs can be, but are not limited to, gapmers or morpholinos. Antisense oligonucleotides typically contain short nucleotide sequences that are substantially complementary to the target nucleotide sequence of a pre-mRNA molecule, heterogeneous nuclear RNA (hnRNA), or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that, under physiological conditions, the molecule containing the antisense sequence can form a stable double-stranded hybrid with the target nucleotide sequence of the RNA molecule. Antisense oligonucleotides are often synthesized and chemically modified.

[0237] Antisense oligonucleotide can be 100% complementary to target sequence, or can contain mismatches, for example, to improve the selective targeting of the allele containing disease-related mutation, as long as the heteroduplex formed between oligonucleotide and target sequence is stable enough to withstand the action of cellular nuclease and other types of degradation that can occur in vivo.Therefore, some oligonucleotide can have about 70% or at least about 70% (for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence complementarity between oligonucleotide and target sequence. The oligonucleotide backbone discussed herein is less susceptible to nuclease cleavage.When mismatch exists, it is typically less unstable at the terminal region than at the center of hybrid double strand.The number of mismatches that can be allowed will depend on the length of oligonucleotide, the percentage of G:C base pair in double strand and the position of mismatch(es) in double strand, according to the well-understood principle of double strand stability.

[0238] In some embodiments, the interfering nucleic acid molecules described herein are gapmers. A "gapmer" is a modified oligonucleotide containing an internal region having multiple nucleosides that support RNase H cleavage, separated by external regions having one or more nucleosides, where the nucleosides comprising the internal region are chemically distinct from the nucleosides comprising the external regions. The internal region may also be referred to as the "gap," and the external regions may also be referred to as the "wings." Gapmers can have 5' and 3' wings, each having 2 to 6 nucleotides, and a gap having 7 to 12 nucleotides. In some embodiments, gapmers can have a 3-10-3 or 5-10-5 configuration.

[0239] Gapmers generally have the formula 5'-XYZ-3', where X and Z are flanking regions around gap region Y. In some embodiments, flanking region X of formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5'-wing region X, or 5'-wing segment. In some embodiments, flanking region Z of formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3'-wing region Z, or 3'-wing segment. In some embodiments, gap region Y of formula 5'-XYZ-3' is also referred to as Y region, Y segment, gap segment Y, gap segment, or gap region. In some embodiments, each nucleoside in gap region Y is a 2'-deoxyribonucleoside, and neither 5'-wing region X nor 3'-wing region Z contains any 2'-deoxyribonucleosides.

[0240] In some embodiments, the gap region of a gapmer polynucleotide may contain modified nucleotides known to tolerate efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-constituent nucleotides. In some embodiments, the gap region contains one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, or at least five, or more nucleotides. In some embodiments, each internucleoside linkage in the gap segment comprises a phosphorothioate linkage. In some embodiments, the gap region and two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, or at least five, or more nucleotides. In some embodiments, each internucleotide linkage in the 5' wing region or the 3' wing region comprises a phosphorothioate linkage. In some embodiments, each internucleotide linkage in a gapmer comprises a phosphorothioate linkage.

[0241] In some embodiments, the Y region can comprise a contiguous stretch of nucleotides, e.g., a region of five or more DNA nucleotides, that can recruit an RNase, including, but not limited to, RNase H. In some embodiments, a gapmer can bind to a target nucleic acid such that an RNase is recruited to cleave the target nucleic acid. In some embodiments, the Y region can be flanked on both the 5' and 3' ends by regions X and Z that comprise high-affinity modified nucleosides, e.g., 1 to 10 high-affinity modified nucleosides. Exemplary high-affinity modified nucleosides include, but are not limited to, 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA) and 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F). In some embodiments, the flanking sequences X and Z can be 1 to 30 nucleotides, 1 to 20 nucleotides, 1 to 10 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z can be sequences of similar or dissimilar lengths. In some embodiments, flanking sequences X and Z are each 5 nucleotides in length. In some embodiments, flanking sequences X and Z are each 3 nucleotides in length. In some embodiments, gap segment Y can be a nucleotide sequence of 5 to 30 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides in length. In some embodiments, the gap segment is 10 nucleotides in length.

[0242] Gapmers can be produced using suitable methods. The preparation of gapmers is described, for example, in U.S. Patent Nos. 10,260,069, 10,017,764, 9,695,418, 9,428,534, 9,428,534, 9,045,754, 8,580,756, 8,580,756, 7,750,131, and 7,683,036. , No. 7,569,686, No. 7,432,250, No. 7,399,845, No. 7,101,993, No. 7,015,315, No. 5,898,0 No. 31, No. 5,700,922, No. 5,652,356, No. 5,652,355, No. 5,623,065, No. 5,565,350, No. 5,49 Nos. 1,133, 5,403,711, 5,366,878, 5,256,775, 5,220,007, 5,149,797, and 5,013,830; U.S. Patent Publication Nos. US2010 / 0197762, US2005 / 0074801, US2009 / 0221685, U.S. Patent Publication Nos. S2009 / 0286969, and US2011 / 0112170, PCT Publication Nos. WO2005 / 023825, WO2004 / 069991, WO2008 / 049085, and WO2009 / 090182 (each of which is incorporated by reference herein in its entirety).

[0243] In some embodiments, gapmers are 10 to 50 nucleosides in length. For example, gapmers can be 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 40, 25 to 35, 25 to 30, 30 to 40, 30 to 35, or 35 to 40 nucleosides in length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length. In some embodiments, the gapmer is about 16 to about 20 nucleosides in length. In some embodiments, the gapmer is 16 nucleotides in length. In some embodiments, the gapmer is 20 nucleotides in length.

[0244] In some embodiments, the 5' and 3' wing regions of a gapmer are independently 1 to 20 nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) in length. For example, the 5' and 3' wing regions of a gapmer can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, or 15 to 20 nucleosides in length. In some embodiments, the 5' and 3' wing regions of a gapmer are the same length. In some embodiments, the 5' and 3' wing regions of a gapmer are different lengths. In some embodiments, the 5' wing region is longer than the 3' wing region of the gapmer. In some embodiments, the 5' wing region is shorter than the 3' wing region of the gapmer.

[0245] In some embodiments, the gap region of a gapmer is 5 to 20 nucleosides in length. For example, gap region Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the gap region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, one or more nucleosides in gap region Y are 2'-deoxyribonucleosides. In some embodiments, all nucleotides in the gap region are deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region are modified nucleosides (e.g., 2'-modified nucleosides such as those described herein). In some embodiments, one or more cytosines in gap region Y are 5-methyl-cytosines. In some embodiments, all cytosines in the gap region Y are 5-methyl-cytosines. In some embodiments, all cytosines in the gapmer are 5-methyl-cytosines.

[0246] In some embodiments, one or more nucleosides in the 5' or 3' wing region of a gapmer are modified nucleotides. In some embodiments, the modified nucleotides can be 2'-modified nucleosides, such as 2'-4' bicyclic nucleosides or non-bicyclic 2'-modified nucleosides. In some embodiments, the nucleoside can be a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)). In some embodiments, each nucleotide in the wing region is a modified nucleotide. In some embodiments, all nucleotides in the wing region are 2'-MOE, LNA, or cET nucleotides.

[0247] In some embodiments, the gapmers described herein may contain one or more modified nucleoside linkages in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage may comprise a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently comprises a combination of phosphodiester and phosphorothioate linkages. In some embodiments, each internucleoside linkage in gap region Y may be a phosphorothioate linkage, 5' wing region X comprises a combination of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a combination of phosphorothioate and phosphodiester linkages.

[0248] In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a 2'-MOE nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages.

[0249] In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is an LNA nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a cET nucleotide. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide and each nucleotide in the wing regions is a cET nucleotide, and all cytosines in the gapmer are 5-methyl-cytosines. In some embodiments, each nucleotide in the gap region of the gapmer is a deoxyribonucleotide, each nucleotide in the wing regions is a cET nucleotide, all cytosines in the gapmer are 5-methyl-cytosines, and all internucleotide linkages are phosphorothioate linkages.

[0250] Interfering nucleic acid can use various oligonucleotide chemistries.Examples of oligonucleotide chemistries include, but are not limited to, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate, 2'-O-Me modified oligonucleotide, and morpholino chemistry (including any combination of the above).Generally, PNA and LNA chemistry have relatively high target binding strength compared to 2'-O-Me oligonucleotide, so they can utilize shorter targeting sequences.Phosphorothioate and 2'-O-Me modified chemistry are often combined to produce 2'-O-Me modified oligonucleotide with phosphorothioate backbone.See, for example, PCT Publication No. WO / 2013 / 112053 and PCT Publication No. WO / 2009 / 008725 (their entirety is incorporated by reference).

[0251] Peptide nucleic acids (PNAs) are analogs of DNA whose backbone is structurally isomorphous with a deoxyribose backbone consisting of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine or purine bases hybridize to complementary oligonucleotides according to the Watson-Crick base-pairing rules, mimicking DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The PNA backbone is formed by peptide bonds rather than phosphodiester bonds, making it highly suitable for antisense applications. The backbone is uncharged, resulting in PNA / DNA or PNA / RNA duplexes that exhibit higher thermal stability than normal. PNAs are not recognized by nucleases or proteases.

[0252] Despite their radical structural changes relative to their native structure, PNAs can bind to DNA or RNA in a helical form with sequence specificity. PNA characteristics include high binding affinity to complementary DNA or RNA, destabilizing effects caused by single-base mismatches, resistance to nucleases and proteases, salt-independent hybridization with DNA or RNA, and triplex formation with homopurine DNA. PANAGENE™ has developed a proprietary Bts PNA monomer (Bts; benzothiazole-2-sulfonyl group) and a proprietary oligomerization process. PNA oligomerization using the Bts PNA monomer consists of repeated cycles of deprotection, coupling, and capping. PNAs can be produced synthetically using any technique known in the art. See, for example, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006, and 7,179,896. For the preparation of PNAs, see also U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the foregoing is incorporated herein by reference in its entirety.

[0253] The interfering nucleic acids described herein may also contain "locked nucleic acid" subunits (LNAs). "LNAs" are members of a class of modifications called bridged nucleic acids (BNAs). BNAs are characterized by a covalent bond that locks the conformation of the ribose ring to the C30-endo (northern) sugar pucker. In the case of LNAs, the bridge consists of a methylene between the 2'-O and 4'-C positions. LNAs enhance backbone preorganization and base stacking, increasing hybridization and thermal stability.

[0254] The structure of LNA can be found, for example, in Wengel, et al., Chemical Communications (1998) 455, Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301), Obika, et al., Tetrahedron Letters (1997) 38:8735 (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230. The compounds provided herein may incorporate one or more LNAs, and in some cases, the compounds may be composed entirely of LNAs. Methods for synthesizing individual LNA nucleoside subunits and incorporating them into oligonucleotides are described, for example, in U.S. Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461 (each of which is incorporated by reference in its entirety).Typical intersubunit linkers include phosphodiester moieties and phosphorothioate moieties.Alternatively, non-phosphorus-containing linkers may be used.In some embodiments, antisense oligonucleotides comprise LNA-containing compounds, and each LNA subunit is separated by a DNA subunit.Certain compounds are composed of alternating LNA and DNA subunits, and the intersubunit linker is phosphorothioate.

[0255] "Phosphorothioates" (or S-oligos) are variants of normal DNA in which one of the non-bridging oxygens is replaced by sulfur. Sulfuration of the internucleotide bond reduces the action of endo- and exonucleases, including 5'-3' and 3'-5' DNA POL 1 exonucleases, nucleases SI and PI, RNases, serum nucleases, and snake venom phosphodiesterases. Phosphorothioates are generated by two main routes: by the action of a solution of elemental sulfur in carbon disulfide on the phosphonate hydrogen, or by sulfurization of a phosphite triester with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter method avoids the problems of the insolubility of elemental sulfur in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield phosphorothioates of higher purity.

[0256] "2'O-Me oligonucleotide" molecules carry a methyl group at the 2'-OH residue of the ribose molecule. 2'-O-Me-RNA behaves the same as (or similar to) DNA, but is protected against nuclease degradation. 2'-O-Me-RNA can also be combined with phosphothioate oligonucleotides (PTO) for further stabilization. 2'-O-Me oligonucleotides (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (see, for example, Yoo et al., Nucleic Acids Res. 32:2008-16, 2004).

[0257] Interfering nucleic acid molecules can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA with RNase III or Dicer. They can be introduced into cells by transfection, electroporation, or other methods known in the art. Hannon,GJ,2002, Nature 418:244-251, Bernstein E et al.,2002,RNA 7:1509-1521, Hutvagner G et al.,Curr.Opin.Genetics&Development 12:225-232, Brummelkamp,2002, Science 296:550-553, Lee NS,et al.2002.Nature Biotechnol.20:500-505, Miyagishi M,Taira K.2002.Nature Biotechnol.20:497-500, Paddison PJ, et al.,2002. Genes&Dev.16:948-958, Paul CP,et al.,2002. Nature Biotechnol.20:505-508, Sui G et See Yu JY et al., 2002. Proc. Natl. Acad. Sci. USA 99(9):6047-6052, each of the foregoing is incorporated herein by reference in its entirety.

[0258] guide RNA In some embodiments, the conjugate molecule cargo comprises a guide RNA or DNA encoding the guide RNA. A "guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (e.g., a Cas9 protein) and targets the Cas protein to a specific location within a target DNA. A guide RNA can comprise two segments: a "DNA-targeting segment" (also called a "guide sequence") and a "protein-binding segment." A "segment" comprises a section or region of a molecule, such as a contiguous stretch of nucleotides in an RNA. Some gRNAs, such as those of Cas9, can comprise two separate RNA molecules: an "activator RNA" (e.g., tracrRNA) and a "targeter RNA" (e.g., CRISPR RNA or crRNA). Other gRNAs are single RNA molecules (single RNA polynucleotides), which may also be called "single-molecule gRNA," "single guide RNA," or "sgRNA." See, for example, WO2013 / 176772, WO2014 / 065596, WO2014 / 089290, WO2014 / 093622, WO2014 / 099750, WO2013 / 142578, and WO2014 / 131833 (each of which is incorporated herein by reference in its entirety for all purposes). Guide RNA refers to either CRISPR RNA (crRNA) or a combination of crRNA and trans-activating CRISPR RNA (tracrRNA). The crRNA and tracrRNA can be associated as a single RNA molecule (single guide RNA or sgRNA) or can be associated in two separate RNA molecules (dual guide RNA or dgRNA). For example, in the case of Cas9, the single guide RNA can include a crRNA fused to a tracrRNA (e.g., via a linker). For example, in the case of Cpf1 and CasΦ, only the crRNA is required to achieve binding to the target sequence. The terms "guide RNA" and "gRNA" include both double-molecule (i.e., modular) gRNAs and single-molecule gRNAs.In some of the methods and compositions disclosed herein, the gRNA is a S. pyogenes Cas9 gRNA or its equivalent. In some of the methods and compositions disclosed herein, the gRNA is a S. aureus Cas9 gRNA or its equivalent.

[0259] Exemplary bimolecular gRNAs include a crRNA-like ("CRISPR RNA" or "targeter RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-activating CRISPR RNA" or "activator RNA" or "tracrRNA") molecule. The crRNA includes both the DNA-targeting segment (single strand) of the gRNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the gRNA. An example of a crRNA tail located downstream (3') of the DNA-targeting segment (e.g., for use with S. pyogenes Cas9) comprises, consists essentially of, or consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 248) or GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 249). Any of the DNA-targeting segments disclosed herein can be linked to the 5' end of SEQ ID NO: 248 or 249 to form a crRNA.

[0260] The corresponding tracrRNA (activator-RNA) contains a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. The stretch of nucleotides in the crRNA is complementary to the stretch of nucleotides in the tracrRNA and hybridizes to form the dsRNA duplex of the protein-binding domain of the gRNA. Thus, each crRNA can be said to have a corresponding tracrRNA. Exemplary tracrRNA sequences (e.g., for use with S. pyogenes Cas9) comprise, consist essentially of, or consist of any one of AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 250), AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 251), or GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 252).

[0261] In systems requiring both a crRNA and a tracrRNA, the crRNA and the corresponding tracrRNA hybridize to form a gRNA. In systems requiring only a crRNA, the crRNA may be the gRNA. The crRNA also provides a single-stranded DNA targeting segment that hybridizes to the complementary strand of the target DNA. When used for intracellular modification, the precise sequence of a given crRNA or tracrRNA molecule can be designed to be specific for the species in which the RNA molecule is used. See, for example, Mali et al. (2013) Science 339(6121):823-826, Jinek et al. (2012) Science 337(6096):816-821, Hwang et al. (2013) Nat. Biotechnol. 31(3):227-229, Jiang et al. (2013) Nat. Biotechnol. 31(3):233-239, and Cong et al. (2013) Science 339(6121):819-823 (each of which is incorporated by reference in its entirety for all purposes).

[0262] The DNA-targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to a sequence on the complementary strand of the target DNA, as described in more detail below. The DNA-targeting segment of a gRNA interacts with the target DNA in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the nucleotide sequence of the DNA-targeting segment may vary and determines the location within the target DNA where the gRNA and target DNA interact. The DNA-targeting segment of a given gRNA can be modified to hybridize to any desired sequence within the target DNA. Naturally occurring crRNAs vary depending on the CRISPR / Cas system and organism, but often contain a targeting segment 21-72 nucleotides long flanked by two direct repeats (DRs) 21-46 nucleotides in length (see, e.g., WO2014 / 131833, incorporated herein by reference in its entirety for all purposes). In the case of S. pyogenes, the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long. The 3'-located DR is complementary to and hybridizes with the corresponding tracrRNA, which then binds to the Cas protein.

[0263] A DNA-targeting segment can have a length of, for example, at least about 12, at least about 15, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 nucleotides. Such a DNA-targeting segment can have a length of, for example, about 12 to about 100, about 12 to about 80, about 12 to about 50, about 12 to about 40, about 12 to about 30, about 12 to about 25, or about 12 to about 20 nucleotides. For example, a DNA-targeting segment can be about 15 to about 25 nucleotides (e.g., about 17 to about 20 nucleotides, or about 17, 18, 19, or 20 nucleotides). See, e.g., US2016 / 0024523 (incorporated by reference in its entirety for all purposes). For Cas9 from S. pyogenes, a typical DNA-targeting segment is 16 to 20 nucleotides in length, or 17 to 20 nucleotides in length. For Cas9 from S. aureus, a typical DNA targeting segment is 21-23 nucleotides in length. For Cpf1, a typical DNA targeting segment is at least 16 nucleotides in length or at least 18 nucleotides in length.

[0264] In one example, the DNA targeting segment can be about 20 nucleotides long. However, shorter and longer sequences can be used for the targeting segment (e.g., 15 to 25 nucleotides long, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long). The degree of identity between the DNA targeting segment and the corresponding guide RNA target sequence (or the degree of complementarity between the DNA targeting segment and the other strand of the guide RNA target sequence) can be, for example, about 75%, about 80%, about 85%, about 90%, about 95%, or 100%. The DNA targeting segment and the corresponding guide RNA target sequence can contain one or more mismatches. For example, the DNA-targeting segment of a guide RNA and the corresponding guide RNA target sequence can include 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4 mismatches (e.g., the total length of the guide RNA target sequence is at least 17, at least 18, at least 19, or at least 20, or more nucleotides). For example, the DNA-targeting segment of a guide RNA and the corresponding guide RNA target sequence can include 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4 mismatches (total length of the guide RNA target sequence, 20 nucleotides).

[0265] TracrRNA can be in any form (e.g., full-length tracrRNA or active portion tracrRNA) and of different lengths. These include primary transcripts or processed forms. For example, tracrRNA (as part of a single guide RNA or as a separate molecule as part of a bimolecular gRNA) can comprise, consist essentially of, or consist of a wild-type tracrRNA sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracrRNA sequence). Examples of wild-type tracrRNA sequences from S. pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and 65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature 471(7340):602-607, WO2014 / 093661 (each of which is incorporated by reference in its entirety for all purposes). Examples of tracrRNA within a single guide RNA (sgRNA) include the tracrRNA segments found within the +48, ​​+54, +67, and +85 versions of the sgRNA, where "+n" indicates that up to +n nucleotides of the wild-type tracrRNA are included in the sgRNA. See US8,697,359 (incorporated by reference in its entirety for all purposes).

[0266] The percent complementarity between the DNA-targeting segment of the guide RNA and the complementary strand of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%). The percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be at least 60% over approximately 20 consecutive nucleotides. As an example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over 14 consecutive nucleotides at the 5' end of the complementary strand of the target DNA and can be as low as 0% over the remainder. In such a case, the DNA-targeting segment can be considered to be 14 nucleotides in length. As another example, the percent complementarity between the DNA-targeting segment and the complementary strand of the target DNA can be 100% over 7 consecutive nucleotides at the 5' end of the complementary strand of the target DNA and can be as low as 0% over the remainder. In such cases, the DNA targeting segment can be considered to be 7 nucleotides long. In some guide RNAs, at least 17 nucleotides in the DNA targeting segment are complementary to the complementary strand of the target DNA. For example, the DNA targeting segment can be 20 nucleotides long and contain 1, 2, or 3 mismatches with the complementary strand of the target DNA. In one example, the mismatch is not adjacent to the region of the complementary strand corresponding to the protospacer adjacent motif (PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g., the mismatch is at the 5' end of the DNA targeting segment of the guide RNA, or the mismatch is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region of the complementary strand corresponding to the PAM sequence).

[0267] The protein-binding segment of a gRNA can contain two stretches of nucleotides that are complementary to each other. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of a target gRNA interacts with a Cas protein, and the gRNA directs the bound Cas protein to a specific nucleotide sequence within the target DNA via the DNA-targeting segment.

[0268] A single guide RNA can include a DNA-targeting segment and a scaffold sequence (i.e., the protein-binding sequence or Cas-binding sequence of the guide RNA). For example, such a guide RNA can have a 5' DNA-targeting segment linked to a 3' scaffold sequence. (For example, S. pyogenes Exemplary scaffold sequences (for use with Cas9) comprise, consist essentially of, or consist of the following: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (Version 1; SEQ ID NO: 253); GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (Version 2; SEQ ID NO: 254); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (Version 3; SEQ ID NO: 255); and GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGC (version 4; SEQ ID NO: 256); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (version 5; SEQ ID NO: 257); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (version 6; SEQ ID NO: 258); GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (version 7; SEQ ID NO: 259); or GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGGCACCGAGUCGGUGC (version 8; SEQ ID NO: 260).In some guide sgRNAs, none of the four terminal U residues of version 6 are present. In some sgRNAs, only one, two, or three of the four terminal U residues of version 6 are present. A guide RNA targeting any of the guide RNA target sequences disclosed herein can include, for example, a DNA-targeting segment at the 5' end of the guide RNA fused to any of the exemplary guide RNA scaffold sequences at the 3' end of the guide RNA. That is, any of the DNA-targeting segments disclosed herein can be linked to the 5' end of any one of the above scaffold sequences to form a single guide RNA (chimeric guide RNA).

[0269] Guide RNAs can include modifications or sequences that provide additional desirable characteristics (e.g., modified or modulated stability; intracellular targeting; tracking by fluorescent labeling; binding sites for proteins or protein complexes, etc.) That is, guide RNAs include one or more modified nucleosides or nucleotides, or one or more non-naturally occurring and / or naturally occurring components or structures, used in place of or in addition to the standard A, G, C, and U residues. Examples of such modifications include, for example, a 5' cap (e.g., a 7-methylguanylate cap (m7G)), a 3' polyadenylation tail (i.e., a 3' poly(A) tail), a riboswitch sequence (e.g., allowing for regulation of stability and / or regulation of accessibility by proteins and / or protein complexes), a stability control sequence, a sequence that forms a dsRNA duplex (i.e., a hairpin), a modification or sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), a modification or sequence that provides binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA methylases, histone acetyltransferases, histone deacetylases, etc.), and combinations thereof. Other examples of modifications include an engineered stem-loop duplex structure, an engineered bulge region, an engineered hairpin 3' of a stem-loop duplex structure, or any combination thereof. See, e.g., US2015 / 0376586 (incorporated herein by reference in its entirety for all purposes). The bulge can be an unpaired region of nucleotides within a duplex consisting of a crRNA-like region and a minimal tracrRNA-like region. The bulge can comprise an unpaired 5'-XXXY-3' (wherein X is any purine and Y can be a nucleotide that can form a wobble pair with a nucleotide on the opposite strand and an unpaired nucleotide on the opposite side of the duplex) on one side of the duplex.

[0270] In some cases, a guide RNA can be used for a transcription activation system comprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1. The guide RNA for such a system can be designed with an aptamer sequence added to the sgRNA tetraloop and stem loop 2, designed to bind to the dimerized MS2 bacteriophage coat protein. See, for example, Konermann et al. (2015) Nature 517(7536):583-588 (incorporated herein by reference in its entirety for all purposes).

[0271] Guide RNAs can include modified nucleosides and nucleotides, including, for example, one or more of the following: (1) modification or substitution of one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens in the phosphodiester backbone linkages (exemplary backbone modifications); (2) modification or substitution of a component of the ribose sugar, such as modification or substitution of the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (3) replacement (e.g., large substitution) of the phosphate moiety with a dephosphorylation linker (exemplary backbone modifications); (4) modification or substitution of naturally occurring nucleobases, including non-canonical nucleobases (exemplary base modifications); (5) substitution or modification of the ribose-phosphate backbone (exemplary base modifications). (6) modification of the 3' or 5' end of the oligonucleotide (e.g., removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (7) sugar modification or replacement (exemplary sugar modifications). Other possible guide RNA modifications include modification or replacement of uracil or polyuracil tracts. See, e.g., WO2015 / 048577 and US2016 / 0237455 (each of which is incorporated by reference herein in its entirety for all purposes). Similar modifications can be made to Cas-encoding nucleic acids, such as Cas mRNA. For example, Cas mRNA can be modified by depleting uridines using synonymous codons.

[0272] Chemical modifications such as those listed above can be combined to provide modified gRNAs and / or mRNAs containing residues (nucleosides and nucleotides) that can have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, each base of the gRNA is modified (e.g., all bases have modified phosphate groups such as phosphorothioates). For example, all or substantially all phosphate groups of the gRNA can be replaced with phosphorothioate groups. Alternatively or additionally, the modified gRNA can include at least one modified residue at or near the 5' end. Alternatively or additionally, the modified gRNA can include at least one modified residue at or near the 3' end.

[0273] Some gRNAs contain one, two, three, or more modified residues, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the positions in the modified gRNA are modified nucleosides or nucleotides.

[0274] Unmodified nucleic acids may be prone to degradation. Exogenous nucleic acids may also induce innate immune responses. Modifications may help to introduce stability and reduce immunogenicity. Some gRNAs described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. Some modified gRNAs described herein may exhibit reduced innate immune responses when introduced into a cell population.

[0275] The gRNAs described herein can include backbone modifications, which can involve replacing one or more of the phosphate groups of the modified residue with different oxygen substituents. Modifications can include large-scale replacement of unmodified phosphate moieties with modified phosphate groups, as described herein. Backbone modifications of the phosphate backbone can also include modifications that result in either uncharged linkers or charged linkers with asymmetric charge distribution.

[0276] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphate atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the atoms or groups of atoms listed above can make the phosphorus atom chiral. The stereogenic phosphorus atom can have either the "R" configuration (Rp) or the "S" configuration (Sp). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at either or both of the bonded oxygens.

[0277] The phosphate group can be replaced by a non-phosphorus-containing linking group in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0278] Nucleic acid-mimicking scaffolds can also be constructed in which phosphate linkers and ribose sugars are replaced with nuclease-resistant nucleoside or nucleotide substitutes. Such modifications can include backbone modifications and sugar modifications. In some embodiments, nucleobases can be anchored by surrogate backbones. Examples include, but are not limited to, morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleobase surrogates.

[0279] Modified nucleosides and nucleotides can include one or more modifications to the sugar (sugar modifications). For example, the 2' hydroxyl group (OH) can be modified (e.g., replaced with several different oxy or deoxy substituents). Modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid because the hydroxyl can no longer be further deprotonated to form a 2'-alkoxide ion.

[0280] Examples of 2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), O(CH2CHO) nExamples of suitable 2' hydroxyl group modifications include CH2CH2OR, where R is, for example, H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20). The 2' hydroxyl group modification can be 2'-O-Me. Similarly, the 2' hydroxyl group modification can be a 2'-fluoro modification, where the 2'-hydroxyl group is replaced with fluorine. 2' hydroxyl group modifications can include locked nucleic acids (LNAs), in which the 2' hydroxyl can be connected to the 4' carbon of the same ribose sugar, for example, by a C1-6 alkylene or C1-6 heteroalkylene bridge, exemplary bridges include methylene, propylene, ether, or amino bridges; O-amino (amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino); and aminoalkoxy, O(CH2)n-amino (amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, 2' hydroxyl group modifications can include "unlocked" nucleic acids (UNAs), in which the ribose ring lacks a C2'-C3' bond. The 2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CH2OCH3, eg, a PEG derivative).

[0281] Deoxy 2' modifications can include hydrogen (i.e., the deoxyribose sugar, e.g., in an overhanging portion of a partial dsRNA), halo (e.g., bromo, chloro, fluoro, or iodo), amino (wherein amino can be, e.g., NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), NH(CHCHNH)CHCH-amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, optionally substituted with, e.g., amino, as described herein.

[0282] Sugar modifications can include sugar groups that contain one or more carbons and have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acids can include nucleotides containing, for example, arabinose as the sugar. Modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. Modified nucleic acids can also include one or more sugars that are in the L-form (e.g., L-nucleosides).

[0283] The modified nucleosides and modified nucleotides described herein that can be incorporated into modified nucleic acids can contain modified bases, also referred to as nucleobases. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or completely replaced to result in modified residues that can be incorporated into modified nucleic acids. The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. In some embodiments, the nucleobases can include, for example, naturally occurring bases and synthetic derivatives of bases.

[0284] In dual guide RNAs, each of the crRNA and tracrRNA can contain modifications. Such modifications may be present at one or both ends of the crRNA and / or tracrRNA. In sgRNAs, one or more residues at one or both ends of the sgRNA may be chemically modified, and / or internal nucleosides may be modified, and / or the entire sgRNA may be chemically modified. Some gRNAs contain 5'-end modifications. Some gRNAs contain 3'-end modifications.

[0285] The guide RNAs disclosed herein may comprise one of the modification patterns disclosed in WO2018 / 107028A1 (incorporated herein by reference in its entirety for all purposes). The guide RNAs disclosed herein may also comprise one of the structure / modification patterns disclosed in US2017 / 0114334 (incorporated herein by reference in its entirety for all purposes). The guide RNAs disclosed herein may also comprise one of the structure / modification patterns disclosed in WO2017 / 136794, WO2017 / 004279, US2018 / 0187186, or US2019 / 0048338 (each of which is incorporated herein by reference in its entirety for all purposes).

[0286] As one example, the nucleotides at the 5' or 3' end of the guide RNA can include phosphorothioate linkages (e.g., the base can have a modified phosphate group that is a phosphorothioate group). For example, the guide RNA can include phosphorothioate linkages between the two, three, or four terminal nucleotides at the 5' or 3' end of the guide RNA. As another example, the nucleotides at the 5' and / or 3' end of the guide RNA can have 2'-O-methyl modifications. For example, the guide RNA can include 2'-O-methyl modifications in the two, three, or four terminal nucleotides at the 5' and / or 3' end (e.g., the 5' end) of the guide RNA. See, for example, WO 2017 / 173054A1 and Finn et al. (2018) Cell Rep. 22(9):2227-2235 (each of which is incorporated by reference in its entirety for all purposes). Other possible modifications are described in more detail elsewhere herein. In a specific example, the guide RNA comprises 2'-O-methyl analogs and 3' phosphorothioate internucleotide linkages at the first three 5'-end and 3'-end RNA residues.Such chemical modifications provide the guide RNA with greater stability and protection from exonucleases, allowing them to persist in cells longer than unmodified guide RNAs.Such chemical modifications can also protect against, for example, the natural intracellular immune response, which can actively degrade RNA or trigger an immune cascade that leads to cell death.

[0287] As an example, any of the guide RNAs described herein can include at least one modification. In one example, the at least one modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide, a phosphorothioate (PS) internucleotide bond, a 2'-fluoro (2'-F) modified nucleotide, or a combination thereof. For example, the at least one modification can include a 2'-O-methyl (2'-O-Me) modified nucleotide. Alternatively or additionally, the at least one modification can include a phosphorothioate (PS) internucleotide bond. Alternatively or additionally, the at least one modification can include a 2'-fluoro (2'-F) modified nucleotide. In one example, the guide RNAs described herein include one or more 2'-O-methyl (2'-O-Me) modified nucleotides and one or more phosphorothioate (PS) internucleotide bonds.

[0288] The modification can occur anywhere in the guide RNA. For example, the guide RNA comprises a modification in one or more of the first five nucleotides at the 5' end of the guide RNA, and the guide RNA comprises a modification in one or more of the last five nucleotides at the 3' end of the guide RNA, or a combination thereof. For example, the guide RNA can comprise a phosphorothioate bond between the first four nucleotides of the guide RNA, a phosphorothioate bond between the last four nucleotides of the guide RNA, or a combination thereof. Alternatively or additionally, the guide RNA can comprise 2'-O-Me modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and 2'-O-Me modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, or a combination thereof.

[0289] In one example, a modified gRNA can comprise the following sequence: mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmCmAmCmCmGmAmGmUmCmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 261), where "N" can be any natural or unnatural nucleotide. The entire N residues can comprise a DNA-targeting segment described herein. The terms "mA," "mC," "mU," and "mG" refer to 2'-O-Me modified nucleotides (A, C, U, and G, respectively). The symbol "*" indicates a phosphorothioate modification. In certain embodiments, A, C, G, U, and N independently represent a ribose sugar (i.e., 2'-OH). In certain embodiments, in the context of a modified sequence, A, C, G, U, and N represent a ribose sugar, i.e., 2'-OH. A phosphorothioate linkage or bond refers to a phosphodiester bond, e.g., a bond between nucleotide bases, in which a sulfur is replaced with one non-bridging phosphate oxygen. When phosphorothioates are used to generate the oligonucleotide, the modified oligonucleotide may also be referred to as an S-oligo. The terms A*, C*, U*, or G* refer to a nucleotide linked to the adjacent (e.g., 3') nucleotide by a phosphorothioate bond. The terms "mA*," "mC*," "mU*," and "mG*" refer to a nucleotide (A, C, U, and G, respectively) substituted with 2'-O-Me and linked to the next (e.g., 3') nucleotide by a phosphorothioate bond.

[0290] Another chemical modification that has been shown to affect the nucleotide sugar ring is halogen substitution. For example, 2'-fluoro (2'-F) substitution on the nucleotide sugar ring can increase oligonucleotide binding affinity and nuclease stability. Abasic nucleotides are those that lack a nitrogenous base. Inverted bases are those that have a linkage that is inverted from the normal 5' to 3' linkage (i.e., either 5' to 5' or 3' to 3' linkage).

[0291] The abasic nucleotide can be added via an inverted bond. For example, the abasic nucleotide can be added to the terminal 5' nucleotide via a 5' to 5' bond, or the abasic nucleotide can be added to the terminal 3' nucleotide via a 3' to 3' bond. An inverted abasic nucleotide at either the terminal 5' or 3' nucleotide can also be referred to as an inverted abasic end cap.

[0292] In some embodiments, one or more of the first 3, 4, or 5 nucleotides at the 5' end and one or more of the last 3, 4, or 5 nucleotides at the 3' end are modified. The modifications can be, for example, 2'-O-Me, 2'-F, inverted abasic nucleotides, phosphorothioate linkages, or other nucleotide modifications known to enhance stability and / or performance.

[0293] In another example, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end can be linked with phosphorothioate linkages.

[0294] In another example, the first three nucleotides of the 5' end and the last three nucleotides of the 3' end can comprise 2'-O-methyl (2'-O-Me) modified nucleotides. In another exa...

Claims

1. A protein-drug conjugate comprising an antigen-binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3) and is conjugated to a molecular cargo.

2. The protein-drug conjugate of claim 1 , wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

3. The protein-drug conjugate comprises a heavy chain variable region (HCVR or V Ｈ ) and / or light chain variable region (LCVR or V Ｌ 3. The protein-drug conjugate of claim 1, comprising:

4. 4. The protein-drug conjugate of claim 3, wherein the antigen-binding protein is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain fragment variable region (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, or a chemically modified derivative thereof.

5. The protein-drug conjugate of claim 3, wherein the antigen-binding protein comprises a fragment antigen-binding region (Fab).

6. The protein-drug conjugate of claim 3, wherein the antigen-binding protein comprises a single-chain fragment variable region (scFv).

7. 7. The protein-drug conjugate of claim 6, wherein the scFv comprises domains arranged from N-terminus to C-terminus in the following orientation: HCVR-LCVR.

8. 7. The protein-drug conjugate of claim 6, wherein the scFv comprises domains arranged from N-terminus to C-terminus in the following orientation: LCVR-HCVR.

9. The protein-drug conjugate of any one of claims 6 to 8, wherein the scFv variable regions are connected by a linker.

10. The protein-drug conjugate of claim 9, wherein the linker is a peptide linker.

11. The peptide linker is -(GGGGS) ｎ - (SEQ ID NO: 321), wherein n is 1 to 10.

12. (i) an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2, 22, 42, 62, 82, 102, 122, 140, 159, 169, 179, 199, or 219 (or a variant thereof); and / or (ii) an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10, 30, 50, 70, 90, 110, 130, 148, 187, 207, or 227 (or a variant thereof).

13. (1) An HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (2) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (3) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (4) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (5) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (6) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (7) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (8) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (9) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (10) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (11) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (12) An HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); or (13) The protein-drug conjugate of any one of claims 1 to 12, comprising an HCVR comprising HCDR1, HCDR2, and HCDR3 of the HCVR comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of the LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).

14. (a) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof); (b) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36 (or a variant thereof); (c) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 (or a variant thereof); (d) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76 (or a variant thereof); (e) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96 (or a variant thereof); (f) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 116 (or a variant thereof); (g) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof); (h) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 146 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (i) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 161 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (j) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 171 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof); (k) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 181 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 191 (or a variant thereof), and an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof); (l) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211 (or a variant thereof), and an LCDR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof); and / or (m) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 221 (or a variant thereof); HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), and an HCVR comprising an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof); LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and 14. The protein-drug conjugate of any one of claims 1 to 13, comprising an LCVR comprising an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 76 (or a variant thereof).

15. (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); or (xiii) The protein-drug conjugate of any one of claims 1 to 14, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof).

16. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 138; (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (l) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (m) the protein-drug conjugate of any one of claims 1 to 3 and 12 to 15, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

231.

17. The protein-drug conjugate of any one of claims 1 to 16, wherein the antigen-binding protein specifically binds to FGFR3b and / or FGFR3c.

18. The protein-drug conjugate of any one of claims 1 to 17, wherein the antigen-binding protein specifically binds to human FGFR3b and / or FGFR3c.

19. The protein-drug conjugate of any one of claims 1 to 18, wherein the antigen-binding protein specifically binds to FGFR3b.

20. The protein-drug conjugate of any one of claims 17 to 19, wherein the FGFR3b is a monomeric and / or dimeric FGFR3b.

21. The protein-drug conjugate of any one of claims 17 to 20, wherein the FGFR3b is a monomeric FGFR3b.

22. The protein-drug conjugate of any one of claims 17 to 20, wherein the FGFR3b is a dimeric FGFR3b.

23. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 40; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 60; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 78, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 80; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 118, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 120; (g) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 136, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 138; (h) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (i) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (j) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (k) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (l) the protein-drug conjugate of claim 22, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

231.

24. The protein-drug conjugate of claim 17 or 18, wherein the FGFR3c is a monomeric and / or dimeric FGFR3c.

25. 25. The protein-drug conjugate of claim 24, wherein the FGFR3c is a monomeric FGFR3c.

26. (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 100; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 155, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 157; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 195, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 197; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 215, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 217; and / or (f) the protein-drug conjugate of claim 25, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 229, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

231.

27. The protein-drug conjugate of any one of claims 1 to 26, wherein the antigen-binding protein specifically binds to both FGFR3b and FGFR3c.

28. The antigen-binding protein is about 1 x 10 －７ K of M Ｄ 28. The protein-drug conjugate of any one of claims 18 to 23 and 27, which binds to human FGFR3b with affinity equal to or higher than 100 ng / mL.

29. The antigen-binding protein is about 1 x 10 －８ K of M Ｄ 29. The protein-drug conjugate of claim 28, which binds to human FGFR3b with an affinity of 100 or higher.

30. The antigen-binding protein is about 1 x 10 －７ K of M Ｄ The protein-drug conjugate of any one of claims 18 and 24 to 27, which binds to human FGFR3c with affinity equal to or higher than 100 ng / mL.

31. The antigen-binding protein is about 1 x 10 －８ K of M Ｄ 31. The protein-drug conjugate of claim 30, which binds to human FGFR3c with an affinity of 100 or higher.

32. 21. The protein-drug conjugate of any one of claims 1 to 20, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

20.

33. The protein-drug conjugate of any one of claims 1 to 11, wherein the antigen-binding protein binds to the same epitope on FGFR3 as an antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1-1.

34. 12. The protein-drug conjugate of any one of claims 1 to 11, wherein the antigen-binding protein competes for binding to FGFR3 with an antibody comprising an HCVR / LCVR amino acid sequence pair set forth in Table 1-1.

35. 1. A protein-drug conjugate comprising an antigen-binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3), said antigen-binding protein being conjugated to a molecular cargo and comprising an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising: a. an epitope comprising the sequence GPTVWVK (SEQ ID NO: 378), and / or an epitope comprising the sequence TQR; b. an epitope comprising the sequence ADVR (SEQ ID NO: 376) and / or an epitope comprising the sequence IGVAEK (SEQ ID NO: 377); c. an epitope comprising the sequence HCKVY (SEQ ID NO: 379), and / or an epitope comprising the sequence KSWISE (SEQ ID NO: 380), and / or an epitope comprising the sequence ADVR (SEQ ID NO: 376); d. an epitope contained within or overlapping with the sequence GPTVWVK (SEQ ID NO: 378) and / or an epitope contained within or overlapping with the sequence TQR; e. an epitope contained within or overlapping with the sequence ADVR (SEQ ID NO: 376), and / or an epitope contained within or overlapping with the sequence IGVAEK (SEQ ID NO: 377), and f. A protein-drug conjugate that binds to one or more epitopes of FGFR3 selected from an epitope contained within or overlapping with the sequence HCKVY (SEQ ID NO:379), and / or an epitope contained within or overlapping with the sequence KSWISE (SEQ ID NO:380), and / or an epitope contained within or overlapping with the sequence ADVR (SEQ ID NO:376).

36. the antibody or antigen-binding fragment thereof a. an epitope consisting of the sequence GPTVWVK (SEQ ID NO: 378), and / or an epitope consisting of the sequence TQR; b. an epitope consisting of the sequence ADVR (SEQ ID NO: 376), and / or an epitope consisting of the sequence IGVAEK (SEQ ID NO: 377), and c. The protein-drug conjugate of claim 35, which binds to one or more epitopes of FGFR3 selected from the epitope consisting of the sequence HCKVY (SEQ ID NO: 379), and / or the epitope consisting of the sequence KSWISE (SEQ ID NO: 380), and / or the epitope consisting of the sequence ADVR (SEQ ID NO: 376).

37. 1. A protein-drug conjugate comprising an antigen-binding protein that specifically binds to fibroblast growth factor receptor 3 (FGFR3), said antigen-binding protein being conjugated to a molecular cargo and comprising an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising: a. an epitope comprising the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363), and / or an epitope comprising the sequence YSCRQRLTQRVL (SEQ ID NO: 364); b. an epitope comprising the sequence LLAVPAAN (SEQ ID NO: 365), and / or an epitope comprising the sequence VLERSPHRPILQAG (SEQ ID NO: 366), and / or an epitope comprising the sequence YVTVLKSWISE (SEQ ID NO: 367), and / or an epitope comprising the sequence ADVRLR (SEQ ID NO: 368), and / or an epitope comprising the sequence LCRATNFIGVAEKAFW (SEQ ID NO: 369); c. an epitope comprising the sequence GQQEQLVFGSGDAVE (SEQ ID NO: 370), and / or an epitope comprising the sequence VLVGPQRL (SEQ ID NO: 371); d. an epitope comprising the sequence VLERSPHRPILQAG (SEQ ID NO: 372), and / or an epitope comprising the sequence HCKVYSDAQP (SEQ ID NO: 373), and / or an epitope comprising the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO: 374), and / or an epitope comprising the sequence LCRATNFIGVAEKAF (SEQ ID NO: 375); e. an epitope contained within or overlapping with the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363), and / or an epitope contained within or overlapping with the sequence YSCRQRLTQRVL (SEQ ID NO: 364); f. an epitope contained within or overlapping with the sequence LLAVPAAN (SEQ ID NO:365), and / or an epitope contained within or overlapping with the sequence VLERSPHRPILQAG (SEQ ID NO:366), and / or an epitope contained within or overlapping with the sequence YVTVLKSWISE (SEQ ID NO:367), and / or an epitope contained within or overlapping with the sequence ADVRLR (SEQ ID NO:368), and / or an epitope contained within or overlapping with the sequence LCRATNFIGVAEKAFW (SEQ ID NO:369); g. an epitope contained within or overlapping with the sequence GQQEQLVFGSGDAVE (SEQ ID NO: 370), and / or an epitope contained within or overlapping with the sequence VLVGPQRL (SEQ ID NO: 371), and h. A protein-drug conjugate that binds to one or more epitopes of FGFR3 selected from an epitope contained within or overlapping with the sequence VLERSPHRPILQAG (SEQ ID NO:372), and / or an epitope contained within or overlapping with the sequence HCKVYSDAQP (SEQ ID NO:373), and / or an epitope contained within or overlapping with the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO:374), and / or an epitope contained within or overlapping with the sequence LCRATNFIGVAEKAF (SEQ ID NO:375).

38. the antibody or antigen-binding fragment thereof a. an epitope consisting of the sequence SCPPPGGGPMGPTVWVKDGTGLVPSER (SEQ ID NO: 363), and / or an epitope consisting of the sequence YSCRQRLTQRVL (SEQ ID NO: 364); b. an epitope consisting of the sequence LLAVPAAN (SEQ ID NO: 365), and / or an epitope consisting of the sequence VLERSPHRPILQAG (SEQ ID NO: 366), and / or an epitope consisting of the sequence YVTVLKSWISE (SEQ ID NO: 367), and / or an epitope consisting of the sequence ADVRLR (SEQ ID NO: 368), and / or an epitope consisting of the sequence LCRATNFIGVAEKAFW (SEQ ID NO: 369); c. an epitope consisting of the sequence GQQEQLVFGSGDAVE (SEQ ID NO: 370), and / or an epitope consisting of the sequence VLVGPQRL (SEQ ID NO: 371), and d. The protein-drug conjugate of claim 37, which binds to one or more epitopes of FGFR3 selected from the epitope consisting of the sequence VLERSPHRPILQAG (SEQ ID NO: 372), and / or the epitope consisting of the sequence HCKVYSDAQP (SEQ ID NO: 373), and / or the epitope consisting of the sequence YVTVLKSWISESVEADVRLR (SEQ ID NO: 374), and / or the epitope consisting of the sequence LCRATNFIGVAEKAF (SEQ ID NO: 375).

39. 39. The protein-drug conjugate of any one of claims 35 to 38, wherein the antigen-binding protein is selected from a humanized antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain fragment variable region (scFv), a bis-scFv, an (scFv)2, a diabody, a bivalent antibody, a one-arm antibody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a single heavy chain antibody, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof.

40. the molecular cargo is (i) the HCVR of said antigen binding protein; (ii) the LCVR of said antigen-binding protein; (iii) the heavy chain of said antigen-binding protein, and / or (iv) a light chain of the antigen-binding protein.

41. 41. The protein-drug conjugate of any one of claims 1 to 40, wherein said molecular cargo is conjugated to said antigen-binding protein via a glutamine and / or lysine residue.

42. The glutamine residue is (i) introduced at the N-terminus and / or C-terminus of the heavy chain of said antigen-binding protein; (ii) introduced at the N-terminus and / or C-terminus of the light chain of said antigen-binding protein; (iii) is naturally present in the CH2 domain or CH3 domain of the antigen-binding protein; (iv) introduced into said antigen-binding protein by modifying one or more amino acids; and / or (v) the protein-drug conjugate of claim 41, which is Q295 or is mutated from N297 to Q297 (N297Q).

43. 43. The protein-drug conjugate of claim 41 or 42, wherein the antigen-binding protein comprises a glutamine-containing tag, and the molecular cargo is conjugated to the antigen-binding protein via a glutamine residue of the glutamine-containing tag.

44. The glutamine-containing tag is LLQGG (SEQ ID NO: 290), LLQG (SEQ ID NO: 291), LSLSQG (SEQ ID NO: 292), GGGLLQGG (SEQ ID NO: 293), GLLQG (SEQ ID NO: 294), LLQ, GSPLAQSHGG (SEQ ID NO: 295), GLLQGGG (SEQ ID NO: 296), GLLQGG (SEQ ID NO: 297), GLLQ (SEQ ID NO: 298), LLQLLQGA (SEQ ID NO: 299), LLQGA (SEQ ID NO: 300), LLQYQGA (SEQ ID NO: 301), LLQGSG (SEQ ID NO: 302), LLQYQG (SEQ ID NO: 303), LLQLLQG (SEQ ID NO: 304), SLLQG (SEQ ID NO: 305), LLQLQ (SEQ ID NO: 306), LLQLLQ (SEQ ID NO: 307), and LLQGR (SEQ ID NO: 308).

45. The protein-drug conjugate of any one of claims 1 to 44, wherein the antigen-binding protein and the molecular cargo are conjugated via a linker.

46. 46. ​​The protein-drug conjugate of any one of claims 1 to 45, wherein the molecular cargo comprises a polynucleotide molecule, a polypeptide molecule, a carrier, a viral particle, a viral capsid protein, or a small molecule.

47. The protein-drug conjugate of claim 46, wherein the molecular cargo comprises a polynucleotide molecule.

48. 48. The protein-drug conjugate of claim 47, wherein the polynucleotide molecule is an interfering nucleic acid molecule, a guide RNA, a ribozyme, an aptamer, a mixmer, a multimer, or an mRNA.

49. 49. The protein-drug conjugate of claim 48, wherein the interfering nucleic acid molecule is an siRNA, shRNA, miRNA, an antisense oligonucleotide, or a gapmer.

50. 50. The protein-drug conjugate of claim 48 or 49, wherein the interfering nucleic acid molecule is an siRNA.

51. 51. The protein-drug conjugate of claim 50, wherein the siRNA comprises a sense strand 21 nucleotides in length.

52. 52. The protein-drug conjugate of claim 50 or 51, wherein the siRNA comprises an antisense strand 23 nucleotides in length.

53. The protein-drug conjugate of any one of claims 50 to 52, wherein the siRNA comprises two phosphorothioate linkages in the first and second internucleoside linkages at the 5' end of the sense strand.

54. The protein-drug conjugate of any one of claims 50 to 53, wherein the siRNA comprises two phosphorothioate linkages in the first and second internucleoside linkages at the 3' end and / or 5' end of the antisense strand.

55. 50. The protein-drug conjugate of claim 48 or 49, wherein the interfering nucleic acid molecule is an antisense oligonucleotide.

56. 49. The protein-drug conjugate of claim 48, wherein the polynucleotide molecule is a guide RNA.

57. 57. The protein-drug conjugate of any one of claims 48 to 56, wherein said polynucleotide molecule targets a gene or gene product associated with a neurological disease and / or disorder.

58. 58. The protein-drug conjugate of claim 57, wherein the gene is APOE4, GFAP, MECP2, AQP4, or STAT3.

59. The protein-drug conjugate of any one of claims 48 to 58, wherein the polynucleotide molecule comprises one or more modified nucleotides.

60. 47. The protein-drug conjugate of claim 46, wherein the molecular cargo comprises a polypeptide molecule.

61. 61. The protein-drug conjugate of claim 60, wherein the polypeptide molecule is an enzyme, a neuroprotective molecule, or an antigen-binding protein that binds to a target other than FGFR3.

62. 62. The protein-drug conjugate of claim 60 or 61, wherein the polypeptide molecule is associated with a neurological disease and / or disorder.

63. 63. The protein-drug conjugate of claim 62, wherein the polypeptide molecule is a protective ApoE isoform or variant, ATPase 13A2 (encoded by ATP13A2), sulfatase modifying factor 1 (encoded by SUMF1), fragile X messenger ribonucleoprotein (FMRP) (encoded by FMR1), or glutamate transporter 1 (encoded by GLT1).

64. 64. The protein-drug conjugate of claim 63, wherein the protective ApoE isoform or variant is ApoE2, ApoE Christchurch, or ApoE Jacksonville.

65. 63. The protein-drug conjugate of claim 62, wherein the polypeptide molecule is a neurotrophic factor, an antibody or antibody fragment, an antibody receptor fusion protein, or a cytokine signaling inhibitor.

66. 66. The protein-drug conjugate of claim 65, wherein the neurotrophic factor is ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), or insulin-like growth factor 1 (IGF).

67. 66. The protein-drug conjugate of claim 65, wherein the antibody receptor fusion protein is an anti-amyloid beta Gas6 fusion protein.

68. 66. The protein-drug conjugate of claim 65, wherein the suppressor of cytokine signaling is suppressor of cytokine signaling 3 (Socs3).

69. 69. The protein-drug conjugate of any one of claims 1 to 68, wherein said molecular cargo is conjugated to said antigen-binding protein at a drug-to-antibody ratio (DAR) of at least 1 to at least 10.

70. 70. The protein-drug conjugate of claim 69, wherein said molecular cargo is conjugated to said antigen binding protein with a DAR of 1, 2, 3, or 4.

71. 71. The protein-drug conjugate of claim 69 or 70, wherein said molecular cargo is conjugated to said antigen binding protein with a DAR of 2.

72. 71. The protein-drug conjugate of claim 69 or 70, wherein said molecular cargo is conjugated to said antigen binding protein with a DAR of 4.

73. 73. A protein-drug conjugate according to any one of claims 1 to 72 for use in the treatment or prevention of a neurological disease or disorder.

74. The protein-drug conjugate of any one of claims 57, 62, and 73, wherein the neurological disease or disorder is a neurodegenerative disease, a neurodevelopmental disease, a physical injury, a neuropsychiatric disease, or brain cancer.

75. 75. The protein-drug conjugate of claim 74, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), or prion disease (transmissible spongiform encephalopathy).

76. 75. The protein-drug conjugate of claim 74, wherein the neurodevelopmental disease is Alexander disease, multiple sulfatase deficiency, autism, epilepsy, Rett syndrome, or fragile X.

77. 75. The protein-drug conjugate of claim 74, wherein the physical injury is a traumatic brain injury, a spinal cord injury, a stroke, or cerebral edema.

78. 75. The protein-drug conjugate of claim 74, wherein the neuropsychiatric disease or disorder is major depressive disorder, anxiety disorder, or bipolar disorder.

79. 75. The protein-drug conjugate of claim 74, wherein the brain cancer is glioma.

80. 80. The protein-drug conjugate of claim 79, wherein the glioma is an astrocytoma.

81. 47. The protein-drug conjugate of claim 46, wherein the molecular cargo comprises a carrier.

82. 82. The protein-drug conjugate of claim 81, wherein the carrier is a lipid-based carrier.

83. 83. The protein-drug conjugate of claim 82, wherein the lipid-based carrier is a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex.

84. 84. The protein-drug conjugate of claim 83, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

85. The protein-drug conjugate of claim 84, wherein the LNP further comprises a polynucleotide molecule and / or a polypeptide molecule.

86. The protein-drug conjugate of claim 84 or 85, wherein the LNP comprises one or more components of a gene editing system.

87. The LNP is (a) a Cas nuclease or a nucleic acid encoding said Cas nuclease, and / or 87. The protein-drug conjugate of claim 86, comprising (b) a guide RNA or one or more DNAs encoding the guide RNA.

88. 88. The protein-drug conjugate of claim 87, wherein said Cas nuclease is a Cas9 protein.

89. 89. The protein-drug conjugate of claim 88, wherein the Cas9 protein is derived from a Streptococcus pyogenes Cas9 protein, a Staphylococcus aureus Cas9 protein, a Campylobacter jejuni Cas9 protein, a Streptococcus thermophilus Cas9 protein, or a Neisseria meningitidis Cas9 protein.

90. 90. The protein-drug conjugate of any one of claims 87 to 89, wherein the nucleic acid encoding the Cas nuclease is codon-optimized for expression in a mammalian cell.

91. 91. The protein-drug conjugate of claim 90, wherein the nucleic acid encoding the Cas nuclease is codon-optimized for expression in a human cell.

92. 92. The protein-drug conjugate of any one of claims 87 to 91, wherein the nucleic acid encoding the Cas nuclease is mRNA.

93. The protein-drug conjugate of any one of claims 48, 56, and 87-92, wherein the guide RNA is a single guide RNA (sgRNA).

94. 87. The protein-drug conjugate of claim 86, wherein the LNP comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).

95. The protein-drug conjugate of any one of claims 84 to 94, wherein the LNP comprises a cationic lipid, a neutral lipid, a helper lipid, a stealth lipid, or any combination thereof.

96. 96. The protein-drug conjugate of claim 95, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC).

97. 96. The protein-drug conjugate of claim 95, wherein the helper lipid is cholesterol.

98. 96. The protein-drug conjugate of claim 95, wherein the stealth lipid is PEG2k-DMG.

99. A pharmaceutical composition comprising the protein-drug conjugate of any one of claims 1 to 98 and a pharmaceutically acceptable carrier.

100. A composition or kit comprising the protein-drug conjugate of any one of claims 1 to 98 or the pharmaceutical composition of claim 99, and a further therapeutic agent.

101. 99. A complex comprising the protein-drug conjugate of any one of claims 1 to 98 bound to fibroblast growth factor receptor 3 (FGFR3).

102. 99. A method for making a protein-drug conjugate according to any one of claims 1 to 98, comprising: (a) contacting said antigen binding protein with said molecular cargo under conditions favorable for conjugation of said antigen binding protein to said molecular cargo; (b) optionally isolating the protein-drug conjugate produced in step (a).

103. 69. A method for making a protein-drug conjugate according to any one of claims 60 to 68, wherein the molecular cargo comprises a polypeptide molecule; (a) culturing a host cell comprising a polynucleotide encoding said protein-drug conjugate under conditions that allow expression of said protein-drug conjugate; (b) optionally isolating the protein-drug conjugate produced in step (a).

104. 104. A protein-drug conjugate produced by or obtainable by the method of claim 102 or 103.

105. A container or injection device comprising the protein-drug conjugate of any one of claims 1 to 98 and 104.

106. 105. A method for administering to a subject the protein-drug conjugate of any one of claims 1 to 98 and 104, comprising introducing said protein-drug conjugate into the body of said subject.

107. 104. A method for delivering a molecular cargo to a tissue or cell type expressing FGFR3 in a subject, the method comprising administering to said subject a protein-drug conjugate according to any one of claims 1 to 98 and 104 or a pharmaceutical composition according to claim 99.

108. 108. The method of claim 107, wherein the tissue is the brain, spinal cord, or eye.

109. 108. The method of claim 107, wherein the cell type is an astrocyte or an astrocyte-derived tumor cell.

110. 106. A method for treating or preventing a neurological disease or disorder in a subject in need thereof, comprising administering to said subject an effective amount of a protein-drug conjugate of any one of claims 1 to 98 and 104.

111. 111. The method of claim 110, wherein the neurological disease or disorder is associated with astrocytes.

112. 112. The method of claim 110 or 111, wherein the neurological disease or disorder is a neurodegenerative disease, a neurodevelopmental disease, a physical injury, a neuropsychiatric disease, or brain cancer.

113. 113. The method of claim 112, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or prion disease (transmissible spongiform encephalopathy).

114. 113. The method of claim 112, wherein the neurodevelopmental disease is Alexander disease, multiple sulfatase deficiency, autism, epilepsy, Rett syndrome, or fragile X.

115. 113. The method of claim 112, wherein the physical injury is a traumatic brain injury, spinal cord injury, stroke, or cerebral edema.

116. 113. The method of claim 112, wherein the neuropsychiatric disease or disorder is major depressive disorder, anxiety disorder, or bipolar disorder.

117. 113. The method of claim 112, wherein the brain cancer is glioma.

118. 118. The method of claim 117, wherein the glioma is an astrocytoma.

119. 119. The method of any one of claims 110 to 118, further comprising administering an additional treatment to the subject.

120. 120. The method of any one of claims 106-119, wherein the protein-drug conjugate is administered internally to the subject via intrathecal, intracisternal, intraventricular, or intraparenchymal administration to the central nervous system.

121. 120. The method of any one of claims 106 to 119, wherein the protein-drug conjugate is administered internally to the subject via intravitreal or intraocular administration to the eye.

122. 120. The method of any one of claims 106 to 119, wherein the protein-drug conjugate is administered into the subject's body via systemic administration.

123. 123. The method of claim 122, wherein the protein-drug conjugate is administered to the subject via intranasal administration.

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