Antibody constructs and conjugates for treatment of disease

Abstract

The disclosure relates to antibodies and antibody-drug conjugates (ADCs) with blood-brain barrier (BBB) and / or cellular penetration and their use in methods of treating neurological disorders, neurodegenerative diseases, and traumatic brain or spinal injuries in a subject.

Description

[0001] GNR-007PC / 115835-5007

[0002] ANTIBODY CONSTRUCTS AND CONJUGATES FOR TREATMENT OF DISEASE FIELD

[0003] The present disclosure relates to the fields of medicine, cell biology, neurology and neurodegenerative disease. More specifically, the disclosure relates to antibodies and antibody-drug conjugates (ADCs) with blood-brain barrier (BBB) and cell penetration (e.g., neurons) properties and their use in methods of treatment, such as for neurological disorders, neurodegenerative diseases, and traumatic brain or spinal injuries.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to and the benefit of U. S. Provisional Application No. 63 / 743,109, filed January 8, 2025, the entire contents of which are hereby incorporated by reference.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing in XML format, which has been submitted electronically and is hereby incorporated by reference in its entirety. The XML Sequence Listing was created on January 8, 2026, named 115835- 5007_Sequence_listing.xml and is approximately 98,304 bytes in size.

[0008] BACKGROUND

[0009] Neurodegenerative diseases and brain or spinal injuries, including without limitation, Alzheimer's disease (AD), Parkinson's disease (PD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (HD), ischemic stroke, and others, are characterized by or result in the loss of neurons. Further, numerous neurological conditions involving mutations in genes that are critical for brain development and / or function are known in the art. While drug delivery is desirable in treating neurological diseases, many therapies can be hindered by limitations in traversing the blood-brain barrier (BBB) and / or cell penetration of neurons. For example, current gene therapies and antisense oligonucleotides targeting the CNS are typically delivered by intrathecal administration to

[0010] DBl / 165335339.4 1GNR-007PC / 115835-5007

[0011] bypass the requirement for transversing the BBB. However, this delivery route is more invasive and painful for the subject than i.v. or subcutaneous routes, for example.

[0012] Antibodies are large, hydrophilic molecules (~50 kDa Fab, ~150 kDa for an antibody), unable to passively diffuse through the tightly packed endothelial cells of the BBB, which are designed to restrict the entry of large molecules. Only a very small percentage (generally 1% or less) of systemically administered antibodies actually cross the BBB, resulting in low concentrations of the therapeutic agent reaching the brain tissue. Antibodies that can cross the BBB may be subject to efflux mechanisms which actively remove larger debris from the brain parenchyma. Due to these challenges, antibody-based agents (as well as other macromolecules) have been largely ineffective at successfully translating into the clinic for neurological indications.

[0013] There remains a need for compositions and methods for targeting therapeutic agents to neurons, for example, for treating neurological diseases.

[0014] SUMMARY OF THE DISCLOSURE

[0015] In accordance with aspects of the present disclosure, there is provided antibodies and antibody-drug conjugates (ADCs) with high penetration of the BBB and / or specific internalization in neurons. In embodiments, the antibody (or ADC) has a complementaritydetermining region (CDR) that targets neurons. For example, in embodiments, the antibody comprises a VH4 heavy chain with a complementarity-determining region (CDR) having at least two codon mutations at positions selected from 3 IB, 32, 40, 56, 57, 60, 81, and 89 with respect to the germline sequence (based on Kabat numbering). In embodiments, the antibody (or ADC) comprises an Fc region having one or more transport mutations that enhance penetration of the BBB, for example and without limitation, by increasing receptor-mediated transcytosis and / or non-classical phagocytosis (NCP). In embodiments, the antibody (or ADC) comprises an Fc region having one or more mutations that enhance internalization by neurons. In embodiments, the one or more mutations that increase penetration of the BBB and / or enhance internalization by neurons are in the CH3 domain.

[0016] In embodiments, the antibody is an IgG monoclonal antibody, and is optionally IgGl or IgG4 isotype.

[0017] DBl / 165335339.4 2GNR-007PC / 115835-5007

[0018] In embodiments, the monoclonal antibody comprises a set of heavy chain CDRs of any one of the heavy chain variable regions represented by the amino acid sequences of SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, and a set of light chain CDRs of any one of the light chain variable regions represented by the amino acid sequences of SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.

[0019] In various embodiments, the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, or a variant thereof as described herein. In embodiments, the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83, or a variant thereof as described herein.

[0020] In some embodiments, the heavy chain comprises a set of heavy chain CDRs of SEQ ID NO: 1 (e.g., amino acid sequences of SEQ ID NOS: 2-4) and the light chain comprises a set of light chain CDRs of SEQ ID NO: 5 (e.g., amino acid sequences of SEQ ID NOS: 6- 8). In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof as described herein, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof as described herein.

[0021] In some embodiments, the one or more mutations that enhance penetration of the BBB comprise a tryptophan at a position corresponding to position 388 of IgGl heavy chain amino acid sequence (according to Kabat numbering), and an aromatic residue (e.g., Phe) at a position corresponding to position 421 of IgGl heavy chain amino acid sequence (according to Kabat numbering). In embodiments, the mutations are at one or more positions corresponding to positions 380, 384, 386, 387, 389, 390, 392, 413-416, 421, 424, and 426 of IgGl heavy chain amino acid sequence according to Kabat numbering. Reference sequences for wild type IgGl heavy constant region (SEQ ID NO: 16) and wild type IgG4 heavy constant region (SEQ ID NO: 17) are provided herein.

[0022] DBl / 165335339.4 3GNR-007PC / 115835-5007

[0023] In some embodiments, the antibody comprises or further comprises additional mutations in the Fc domain. For example, one or more mutations can abrogate FcyR binding, such as mutations corresponding to L234A and L235A, and optionally P329G, with respect to IgGl heavy chain amino acid sequence. In embodiments, one or more mutations can result in increased circulatory half-life and / or serum half-life, such as a YTE mutation (Met252Tyr, Ser254Thr, Thr256Glu) and / or a C6A-66 mutation (Glu294A, Thr307Pro, Asn434Tyr), among others.

[0024] In some embodiments, the one or more mutations that improve BBB penetration or cellular internalization are monovalent, that is, they are present on only one heavy chain of the monoclonal antibody. Such antibodies can be constructed using a knob-in-hole (KIH) approach as is known in the art, e.g., by introducing KIH mutations. For example, KIH mutations are exemplified by a constant region comprising an amino acid sequence selected from SEQ ID NOs: 12, 14, and 18 (knob), and a constant region further comprising an amino acid sequence selected from SEQ ID NOs: 13, 15, and 19 (hole). An exemplary antibody comprises a first heavy chain of SEQ ID NO: 9 and a second heavy chain of SEQ ID NO: 10, and a light chain of SEQ ID NO: 11, including variants as described herein.

[0025] In aspects and embodiments, the present disclosure further provides antibody-drug conjugates (ADCs), where an antibody of this disclosure or an antigen-binding portion thereof is conjugated (either directly or via a linker) to a therapeutic moiety. In these aspects, mutations in the antibody constant region as described herein are optional. In various embodiments, the therapeutic moiety is one or more of a nucleic acid, small molecule, peptide, protein, reactive species, or radionuclide. In some embodiments, the nucleic acid reduces or ablates expression or one or more endogenous neural cell proteins e.g., siRNA, shRNA, or miRNA), or impacts pre-mRNA splicing, for example, by inducing skipping of one or more exons of a target pre-mRNA. In some embodiments, the therapeutic moiety is an enzyme. In some embodiments, the therapeutic moiety is small molecule, such as a cytotoxic agent (e.g., for treatment of malignancy), cytoprotective agent (e.g., for treatment of neurodegenerative disease or neuronal injury), or anticancer agent. In various embodiments, the therapeutic moiety to antibody ratio (“drug-to-antibody ratio,” or DAR) is at least 1, at least 2, at least 3, or at least 4.

[0026] DBl / 165335339.4 4GNR-007PC / 115835-5007

[0027] In various embodiments, the ADC comprises conjugation or association with a therapeutic moiety via a variety of site-specific and stochastic conjugation methods. In embodiments, the conjugation of the therapeutic moiety to the antibody is via a linker molecule. Non-limiting examples of such linker molecules and conjugation chemistries include amide-conjugated linkers, a site- selective linker conjugation (e.g., at one or more native or engineered amino acids, the N-terminus, or C -terminus), a click chemistry- conjugated linker, a pH-sensitive (acid-sensitive) dissociation linker, a redox-sensitive (disulfide) linker, a protease-cleavable (amino acid-based) linker, or an electrostatic release linker (such as protamine, or a protamine-based linker). In some embodiments, the linker is suitable for release of the therapeutic moiety within an endosome or lysosome, such as for example a pH-sensitive linker. In embodiments, the linker allows for release of the therapeutic moiety in the cytosol and / or nucleus.

[0028] In various embodiments, the ADC comprises an endosomal escape element. In embodiments, endosomal escape elements comprises a polypeptide sequence, a small molecule, or a pH-sensitive lipid (e.g., a cationic lipid) that functions under the acidic pH of the endosome / lysosome to facilitate escape of the therapeutic moiety from the endosome after receptor-mediated endocytosis of the ADC. Non-limiting examples of such endosomal escape elements include cationic peptides, amphiphilic peptides, and neutral peptides that promote endosomal escape.

[0029] In aspects, described herein are methods of treating a neurological disorder in a subject by administering the antibodies or ADCs described herein. In various embodiments, the subject has a neurodegenerative disease, a monogenic disease impacting the central nervous system (CNS) (including but not limited to a gain-of-function mutation and / or pathogenic mutation, or haploinsufficiency), traumatic injury, central nervous system (CNS) damage, or peripheral nerve damage. Non-limiting examples of such neurological disorders and diseases include Multiple Sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, lysosomal storage diseases, spinocerebellar ataxia, and ischemic reperfusion injury.

[0030] DBl / 165335339.4 5GNR-007PC / 115835-5007

[0031] Other aspects and embodiments of the present disclosure will be apparent to one of skill in the art in view of the following detailed description, examples, and figures.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts fluorescence microscopy imaging of live cell staining, and demonstrates antibody internalization into SH-SY5Y human neuroblastoma cells.

[0033] FIG. 2 quantifies fluorescence intensity of the live cell staining shown in FIG. 1. FIG. 3 depicts fluorescence microscopy imaging of live cell staining, and showing antibody internalization into SH-SY5Y human neuroblastoma cells at 6 hours post incubation.

[0034] FIG. 4. quantifies fluorescence intensity of the live cell staining shown in FIG. 3. FIG. 5 depicts electrophoresis gel shift assay demonstrating antibody-protamine- siRNA conjugation.

[0035] FIG. 6 depicts quantitative analysis of amyloid precursor protein (APP) expression as assessed by quantitative PCR (qPCR) of U2OS cells treated with antibody-protamine- siRNA conjugates.

[0036] FIGs. 7A-7B depicts western blotting (FIG. 7A) and quantitative analysis of the same (FIG. 7B) confirming the qPCR results of FIG. 6 demonstrating knockdown of APP by antibody-protamine-siRNA conjugates.

[0037] DETAILED DESCRIPTION

[0038] In various aspects and embodiments, the present disclosure provides antibodies and their respective antibody-drug conjugates (ADCs), and methods of using the same for treating or ameliorating genetic or non-genetic neurological disease or neurological injury in a subject in need thereof, including for use in treating neurodegenerative diseases and traumatic brain or spinal injuries for example.

[0039] In some embodiments, the antibodies of this disclosure are based on VH4 antibodies that satisfy the antibody gene signature (AGS) codons described in U. S. Patent 8,394,583, which is hereby incorporated by reference. In some embodiments, the antibodies bind DBl / 165335339.4 6GNR-007PC / 115835-5007

[0040] selectively to neurons or selectively to neurons and astrocytes. Exemplary antibodies are described in US Patent No. 11,518,800 and 11,999,778, which are hereby incorporated by reference in their entireties. Exemplary antibodies were cloned from CSF B cells isolated from patients having clinically isolated syndrome optic neuritis (ON-CIS), clinically isolated syndrome transverse myelitis (TM-CIS) or clinically definite MS, and which contain AGS codons. Such antibodies can be characterized using the EAE mouse model or in vitro cellbased assays to identity / quantify neuroprotective properties. Any antibody with neuronal binding activity and with neuroprotective effects in the EAE model and / or in vitro cell based assays can be modified according to the current disclosure to enhance BBB penetration and cellular internalization. In some embodiments, the antibodies of this disclosure, their target antigens, and the methods of assessing neuroprotective effects (including for the antibody referred to as TGM-010 and its variants), are as described in PCT Publication No. WO 2025 / 014821, which is hereby incorporated by reference.

[0041] In embodiments, the antibodies comprise a VH4 heavy chain with complementarity determining regions (CDRs) having at least two codon mutations at positions selected from 3 IB, 32, 40, 56, 57, 60, 81, and 89 with respect to the germline sequence, for example as described in US 2017 / 0002064, the entire contents of which are hereby incorporated by reference. In various embodiments, the antibody comprises an Fc region having one or more mutations (e.g., in the CH3 region) that enhance penetration of the blood brain barrier (BBB) and / or enhance internalization by neurons, for example and without limitation, by increasing receptor-mediated transcytosis and / or non-classical phagocytosis (NCP). Such mutations are sometimes referred to herein as “transport mutations.” Examples of mechanisms for antibody trafficking across the BBB include transferrin receptor (TfR)-mediated transcytosis, CD98 (also known as CD98 heavy chain (CD98hc) or SLC3A2) receptor- mediated transcytosis, insulin receptor-mediated transcytosis, and transmembrane domain protein 30A (TMEM30A) receptor-mediated transcytosis. In embodiments, the transport mutations enable transferrin receptor (TfR)-mediated transcytosis.

[0042] In embodiments, the antibody described herein is specifically internalized or enriched in neurons.

[0043] DBl / 165335339.4 7GNR-007PC / 115835-5007

[0044] In some embodiments, the antibody is an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), and is optionally IgGl or IgG4 isotype. IgG4 is a poor inducer of Fc-mediated effector functions, and therefore is preferable in some embodiments.

[0045] In various embodiments, the antibody is a monoclonal antibody that comprises a set of heavy chain CDRs of any one of the heavy chain variable regions represented by SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82; and a set of light chain CDRs of any one of the light chain variable regions represented by SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83. In embodiments, the antibody may have one, two, or three amino acid changes within the set of six heavy and light chain CDRs as set forth in the heavy and light chain variable regions described herein. In some embodiments, the antibody comprises a set of heavy chain CDRs from SEQ ID NO: 1 (provided as SEQ ID NOS: 2-4) and a set of light chain CDRs from SEQ ID NO: 5 (provided as SEQ ID NOS: 6-8), optionally with one, two, or three amino acid substitutions (e.g., collectively).

[0046] In various embodiments, the heavy chain variable region (VH) sequence comprises a sequence selected from SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, or a variant thereof; and the light chain variable region comprises a sequence selected from SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83, or a variant thereof. In embodiments, the heavy and light chains are paired as indicated herein by their nomenclature (e.g., antibody name). Variants of any of the foregoing heavy and light chain variable regions will generally have at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to the reference sequence. In embodiments, there are no amino acid modifications or substitutions within the CDRs.

[0047] In some embodiments, the antibody has a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 5 (as included in the antibody known as TGM-010), or a variant thereof.

[0048] In some embodiments, the antibody blocks or competes with the binding of TGM- 010 to neuronal targets according to an in vitro binding assay. Any of the known binding

[0049] DBl / 165335339.4 8GNR-007PC / 115835-5007

[0050] assays and instrumentations can be used to determine whether a candidate antibody and TGM-010 bind to the same epitope or an overlapping epitope. For example, the binding candidate antibody may bind with an affinity of less than or equal to 1 x 10'7M, or less than or equal to 1 x 10'8M, less than or equal to 1 x 10'9M, less than or equal to 1 x IO'10M, less than or equal to 1 x 10'11M, or less than or equal to 1 x 10’12M. Binding affinity as well as binding competition with TGM-010 may be determined by an ELISA, immunofluorescence, immunohistochemistry (ICC), spinning disk confocal microscopy, or surface plasmon resonance (SPR, e g., BIACORE assay), biolayer interferometry (BLI), as well as other techniques known in the art.

[0051] In some embodiments, the antibody Fc region comprises one or more mutations that enhance penetration of the blood brain barrier (BBB), which can increase transferrin receptor-mediated transcytosis. In embodiments, the one or more mutations enhance internalization into neurons. Such mutations are sometimes referred to herein as transport mutations.

[0052] In various embodiments, one or more mutations are in the CH3 region, and comprise a tryptophan at a position corresponding to position 388 of IgGl heavy chain (HC) amino acid sequence. The numbering of mutations according to this disclosure are with regard to the well-known Kabat system. A wild-type IgGl HC constant region amino acid sequence for reference is provided herein as SEQ ID NO: 16. In embodiments, the one or more mutations further comprises the introduction of an aromatic residue at a position corresponding to position 421 of IgGl HC amino acid sequence. In embodiments, the aromatic residue is Phe, Trp, or Tyr. In embodiments, the aromatic residue is Phe. In some embodiments, the one or more mutations further comprises mutation at one or more positions corresponding to positions 380, 384, 386, 387, 389, 390, 392, 413-416, 424, and 426 of IgGl HC amino acid sequence. In some embodiments, the positions are selected from, or comprise, mutations corresponding to positions 384, 386, 387, 389, 390, 413, 415, and 416 of the IgGl HC amino acid sequence. In some embodiments, the mutations are selected from an amino acid residue corresponding to one or more of E380W, E380L, N384L, N384Y, Q386H, Q386T, P387V, P387E, E388W, N389A, N389S, N390V, N390S, K392R, D413P, D413T, K414R, S415E, R416T, R416E, N421W, N421F, S424T, and S426G with respect to the IgGl HC amino acid sequence. In some embodiments, the mutations are selected from,

[0053] DBl / 165335339.4 9GNR-007PC / 115835-5007

[0054] or comprise E380W, N384Y, Q386T, P387E, E388W, N389S, N390S, K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426G.

[0055] In some embodiments, the mutations are selected from or comprise each of E380W, N384Y, Q386T, P387E, E388W, N389S, N390S, K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426G. Exemplary IgGl constant regions with these mutations are shown herein as SEQ ID NOS: 12 and 14. An exemplary IgG4 constant region with these mutations is shown herein as SEQ ID NO: 19. It is understood with respect to the HC constant regions described herein, that one or several amino acids can be deleted from the C-terminus, such as the C-terminal Lys, which is optional.

[0056] In embodiments, the one or more mutations that improve BBB penetration or cellular internalization are monovalent, that is, they are present on only one heavy chain of the monoclonal antibody. Such antibodies can be constructed using a knob-in-hole (KIH) approach as is known in the art, by introducing the known KIH mutations (e g., T366W in the knob chain; and T366S, L368A, and Y407V in the hole chain). In embodiments, the one or more mutations that improve BBB penetration or cellular internalization are constructed in the knob chain, but in embodiments, could be constructed in the hole chain.

[0057] In embodiments, the antibody comprises mutations, or further comprises additional mutations, in the constant region to improve pharmacodynamics (e.g., with or without mutations described above). For example, one or more mutations can abrogate FcyR binding, such as mutations corresponding to L234A and L235A with respect to IgGl sequence. These mutations are sometimes referred to herein a LALA mutations. In embodiments, such mutations may further comprise a P329G mutation, sometimes referred to herein as a LALAPG mutation. In embodiments, one or more mutations can result in increased circulatory half-life and / or serum half-life, such as a YTE mutation (Met252Tyr, Ser254Thr, Thr256Glu) and / or a C6A-66 mutation (Glu294A, Thr307Pro, Asn434Tyr), among others. The mutations in this paragraph are optionally bivalent, that is, present in both heavy chains. It is understood that the mutations in this paragraph are optional.

[0058] In some embodiments, the IgGl heavy chain constant region comprises one chain with an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14 (heavy chain constant region with knob mutation and mutations for increasing BBB penetration and / or cell internalization), and a second chain comprising an amino acid sequence of SEQ ID NO: 13

[0059] DBl / 165335339.4 10GNR-007PC / 115835-5007

[0060] or 15 (heavy chain constant region with hole mutations). In some embodiments, the IgG4 heavy chain constant region comprises one chain with an amino acid sequence of SEQ ID NO: 18 (heavy chain constant region with knob mutation and mutations for increasing BBB penetration and / or cell internalization), and a second chain comprising an amino acid sequence of SEQ ID NO: 19 (IgG4 constant region with hole mutation).

[0061] In embodiments, the antibody may have a first heavy chain comprising SEQ ID NO: 9 (knob) and a second heavy chain comprising SEQ ID NO: 10 (hole). These TGM-010 IgGl constructs include LALPG and YTE mutations, although one or both sets of mutations can be removed (e.g., reverted) in embodiments. In embodiments, the antibody is an IgG4 variant of TGM-010, constructed with the heavy chain variable region of SEQ ID NO: 1, and constant regions of SEQ ID NOS: 18 (knob) and 19 (hole).

[0062] In various embodiments, the one or more mutations for CD98 (CD98hc) or SLC3 A2) receptor-mediated transcytosis are selected from, or comprise E380L, E382N, N384K, N384R, N384H, G385F, G385Y, Q386E, Q386V, P387L, N421E, V422L, V422I, S424A, S426N, M428Y, Y436R, Q438F, S440N, S442A, S442R, and S442H, with respect to a wildtype IgGl Fc domain. The mutations can be monovalent (i.e., occurring within a single heavy chain) or bivalent. One or more of these mutations may improve antibody affinity with a human CD98hc receptor complex (and / or a cynomolgus CD98hc or another homologous protein). It is understood with respect to the HC constant regions described herein, that one or more amino acid mutations in an IgGl Fc domain can be made in equivalent positions in an IgG4 antibody (among other isotypes).

[0063] In some embodiments, the one or more mutations for receptor-mediated transcytosis (e.g., for improved BBB trafficking) includes conjugation or fusion of the Fc domain (e.g., at the C-terminus) with one or more peptides or proteins having binding affinity for a receptor, including for example, TfR, CD98hc, insulin, TMEM30A, etc. The protein domains can include one or more antigen-binding domains (e.g., single chain variable fragments (scFvs)) or protein ligands attached to the Fc domain of an antibody for improved receptor-mediated transcytosis. Exemplary antigen-binding domains that can be fused or conjugated to antibodies herein for improved BBB penetration include the BRAIN SHUTTLE (ROCHE, anti-TfR fusion), or BBB targeting using a variable domain (VHH) of a llama heavy chain antibody (“FC5”) which recognizes a BBB epitope, for example as

[0064] DBl / 165335339.4 11GNR-007PC / 115835-5007

[0065] described in published PCT WO 2007 / 036022, the entire contents of which are incorporated by reference. Fusion of peptides or protein domains to antibodies can be achieved using a variety of linkers known in the art, including without limitation, glycine (G) and serine (S) based linkers.

[0066] In various embodiments, the Fc domain can be engineered to have non-native antigen binding sites which increase binding to one or more receptors present in the BBB (e.g., TfR, CD98hc, insulin receptor, TMEM30A, etc.). Techniques are known to those skilled in the art to engineer receptor specificity into the Fc domain of antibodies. For example, bispecific, tetravalent antibodies can be produced by introducing Fc antigen binding sites using the methods described in U. S. Patent No. 11,827,720, the entire contents of which are incorporated by reference. Fc-mediated engineering can be performing using standard phage display library construction for directed evolution of the Fc domain coupled with phage screening by measuring mutant Fc binding to a target receptor. Methods of measuring receptor binding are described herein.

[0067] In various embodiments, the Fc domain comprises one or more (e.g., from 1 to 5 or from 1 to 3) amino acid substitutions, insertions, or deletions, relative to SEQ ID NOs: 12, 13, 14, 15, 18, or 19. In some embodiments, the one or more amino acid substitutions, insertions, or deletions attenuates Fc-mediated effectors functions, and / or improves pharmacokinetic (PK) and / or pharmacodynamic (PD) properties. Among these properties includes increased half-life (e.g., circulating or serum half-life), improved blood-brain barrier (BBB) penetration, and / or reduced off-target or non-specific uptake. In various embodiments, the antibodies described herein do not appreciably accumulate (relative to neurons in the brain) in cells comprising the BBB, including brain endothelial cells (BECs), pericytes (PCs), capillary basement membrane, and astrocyte end-feet. Further, the constant region can harbor one or more mutations to allow for drug conjugation, as described elsewhere herein.

[0068] In various embodiments, the antibody comprises one or more amino acid substitutions, insertions, or deletions (e.g. from 1 to 3), relative to SEQ ID NO: 11 (TGM- 010 light chain) and / or relative to the light chain framework region.

[0069] In various embodiments, the antibodies have one or more mutations that alter Fc receptor binding (e.g., FcγRI, FcγRII and FcγRIII, as well as to complement component

[0070] DBl / 165335339.4 12GNR-007PC / 115835-5007

[0071] Clq). In some embodiments, the one or more mutations correspond to a LALA or a LALAPG mutation, which reduces IgGl Fc domain binding affinity e.g., to FcγR allowing more efficient antibody recycling). In embodiments, the one or more mutations correspond to C6A-66 mutations e.g., Glu294A, Thr307Pro, and / or Asn434Tyr), or alternatively C6A- 78 mutations (e.g., Thr256Asn, Ala378Val, Ser383Asn, and / or Asn434Tyr). These mutations are part of the C6A collection of mutations that maintain some degree of binding to FcγRIIIa, and may even increase FcRn binding, but with greatly improved serum half-life and with some reduced effector functions (e.g., ADCC)

[0072] In some embodiments, the one or more mutations for increased circulatory half-life and / or serum half-life are introduced at the Fc domain CH2-CH3 interface. For example, the YTE mutation (e.g., Met252Tyr, Ser254Thr, and / orThr256Glu) or LS mutation (e.g., M428L and / or N434S) with respect to the IgGl heavy chain sequence can confer enhanced FcRn binding and increased circulating half-life, which may translate to improved BBB penetration.

[0073] In other aspects, the present disclosure further provides antibody-drug conjugates (ADCs), where an antibody of this disclosure, or an antigen-binding portion thereof, is conjugated (either directly or via a linker) to a therapeutic moiety. In embodiments, the antigen-binding fragment for use with an ADC does not comprise an Fc domain. For example, the antigen-binding fragment can be F(ab')2, Fab, Fab', Fv, or Fd fragment. In some embodiments, the antibody is constructed as a single chain variable fragment (scFv). scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of the antibody, connected with a short linker peptide of ten to about 25 amino acids. These smaller formats can be engineered for increased tissue or cell penetration in some embodiments, as well as for non-parenteral administration routes such as intranasal, which may allow for better CNS penetration. In some embodiments, the antigen-binding portion (e.g., scFv) is multimerized to enhance antigen binding. Other formats that find use are variable heavy domain only antibodies (e.g., nanobodies), which may also be linked in tandem.

[0074] In other embodiments, the antibody for use as an ADC contains an Fc domain, including as already described. In such embodiments, the modifications to the constant region as described herein are optional. That is, TGM-010 and its variants, or other neuronbinding antibodies described herein, can be employed as delivery vehicles without constant

[0075] DBl / 165335339.4 13GNR-007PC / 115835-5007

[0076] region modifications (e g., including as IgGl or IgG4 isotype). In other embodiments, the disclosed modifications to the constant region can enhance delivery to neurons or improve circulating or serum half-life.

[0077] In embodiments, the therapeutic agent is conjugated directly or indirectly to the antibody or antigen binding fragment, or alternatively is encapsulated in a nanoparticle carrier, which can be conjugated to the antibody or antigen-binding fragment.

[0078] In some embodiments, the therapeutic agent is encapsulated by or conjugated to the surface of inorganic particles, liposomes, lipid nanoparticles, or polymeric particles. In some embodiments, the polymeric particles comprise poly(lactic acid)-polyethylene glycol (PLA- PEG) or poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) copolymers. In such embodiments, the antibody described herein as a neuronal targeting agent can be conjugated to PEG as a targeting ligand. Particles can be sized to improve penetration through the BBB, such as a size of less than about 200 nm or a size of less than about 150 nm, or a size less than about 100 nm.

[0079] In other embodiments, the particles are lipid nanoparticles, which can find use for encapsulating nucleic acid agents, for example. In embodiments, lipid nanoparticles comprise a PEG-conjugated lipid. Exemplary PEG lipids are selected from one or more of a PEG-modified phosphatidylethanolamine, a PEG- modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, and a PEG-modified di alkylglycerol. A PEG lipid may be selected from PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-Cholesterol, PEG tocopherol, or a PEG- DSPE lipid.

[0080] In some embodiments, the lipid nanoparticles further comprise a cationic or ionizable lipid, a neutral lipid or phospholipid, and a structural lipid. Exemplary structural lipids can be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherols (e.g, alpha tocopherol). In some embodiments, the structural lipid is cholesterol. In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from the group consisting of cardiolipins, sterol modified lipids (modified with a cholesterol moiety attached at the sn-2 carbon of the glycerol backbone), mixed-acyl glycerophospholipids, and symmetrical acyl glycerophospholipids. Head groups for acyl

[0081] DBl / 165335339.4 14GNR-007PC / 115835-5007

[0082] glycerophospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidylserine. Exemplary phospholipids are selected from 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-gly cero-3 -phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 - phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1-oleoyl — 2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2-dilinolenoyl-sn-gly cero-3 - phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2-didocosahexaenoyl- sn-gly cero-3 -phosphocholine, l,2-dioleoyl-sn-glycero-3 -phosphoethanol amine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- gly cero-3 -phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3 -phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-gly cero-3 -phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0083] In various embodiments, the ADC comprises an overall structure of antibody-linker- therapeutic moiety or antibody-therapeutic moiety, where the therapeutic moiety is associated or conjugated directly or indirectly with the antibody via an amino acid side chain, the N-terminus, and / or the C-terminus of the antibody heavy chain or light chain. The therapeutic moiety may also be referred to as a “payload.” Linkage of the payload to the antibody and / or linker may be covalent, or may be via non-covalent association via electrostatics, ionic interaction, Van der Waals, pi-stacking, etc., or may include a combination of covalent and non-covalent interactions. For example, a linker may form a covalent bond with the antibody and a cationic peptide (e.g., protamine), where the cationic peptide forms non-covalent interactions with a nucleic acid payload. A variety of linker and linker chemistries are useful for payload conjugation and release, including in non-limiting embodiments, amide-based conjugation, site- selective and click chemistry conjugation, protamine-based payload association, pH-sensitive (acid-sensitive) dissociation linkers, redox-sensitive (disulfide) linkers, and electrostatic payload release linkers.

[0084] DBl / 165335339.4 15GNR-007PC / 115835-5007

[0085] In various embodiments, conjugation of therapeutic moi eties and / or linkers can occur via a variety of stochastic and site-specific chemistries. For example, stochastic conjugation can occur via one or more lysine side chains (NH₂, amine-based conjugation) or cysteine side chains (thiol-based conjugation, e.g., maleimide chemistry or thiol-Michael click chemistry) within the antibody or fragment sequence. A variety of site-specific conjugation methods are available, such as via engineering one or more non-native lysines or cysteines into the antibody or fragment for site-specific conjugation. In addition, non-canonical amino acids can be incorporated within the antibody or fragment at defined sites using the appropriate tRNA synthase during protein production, e.g., followed by click chemistry. Site-specific disulfide rebridging can be used (reducing the bridging disulfide bond of the antibody and inserting a drug within the disulfide, relinking the antibody). Enzyme-assisted ligation can occur via reactive side chains (e.g., one well-known example is the N297 mutation in the constant region of an antibody Fc which can be ligated via TGase). Sitespecific glycoconjugation can occur which can then serve as the substrate for linkage, e.g., via click chemistry.

[0086] In various embodiments, the therapeutic moiety is conjugated via a linker. In some embodiments, the linker is a cleavable linker, for example, where the linker is selected to undergo enzymatic or proteolytic degradation (e.g., β-glucuronidase cleavable linker, peptide linker, etc.), or non-enzymatic, hydrolysable linkers (e.g., acid-liable linkers, glutathione reducible linkers, etc.). In some embodiments, the linker is selected from hydrazone, thioether, maleimidocaproyl (me), valine-citrulline (VC), para-amino benzyl alcohol (PABC), maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (mc-VC- PABC), peptide linker (dipeptide, triglycyl peptide linker, tetrapeptide), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC), sulfo-SMCC, cleavable complicated PEG3-, PEG4-, PEG8- and triazole-containing PABC-peptide-mc linker (CL2A), dibenzocyclooctyne (DBCO), DBCO-PEG3-SS-NHS, DBCO-PEG4-hydrazone- NHS, DBCO-PEG4-NHS, mal-PEG4-N-hydroxysuccinimide, N-hydroxysuccinimidyl-4- (2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), mal-cyclobutane-1 (cBu-Cit), butanoic acid linker (4-(40-acetylphenoxy)), β-glucuronide linker, acid-sensitive linker, disulfide linker, and derivatives and analogues thereof. A variety of peptide-based linker sequences

[0087] DBl / 165335339.4 16GNR-007PC / 115835-5007

[0088] are known in the art, such as valine-alanine (va) and phenylalanine-lysine (pl). A variety of chemical moieties used as ADC linkers are known in the art.

[0089] In various embodiments, conjugation of therapeutic moieties and / or linkers occurs via amide-based conjugation. In embodiments, amide-based conjugation includes using one or more lysine residues on the antibody or linker (peptide linker) to serve as a conjugation site. Amide linkages provide chemical stability during in vivo circulation and compatibility with diverse linker and payload classes. Amide-based conjugation to antibodies typically involves a reaction between a carboxylic acid and a primary amine, often utilizing the E- amino group of lysine residues on the antibody. To facilitate this, the therapeutic payload and / or linker may be functionalized with an N-hydroxysuccinimide (NHS) ester, which reacts with lysine amines to form a stable amide bond. While many amide bonds are designed for stability, cleavable amide-based linkers, such as those containing valinecitrulline (Val-Cit) dipeptides, are engineered to facilitate payload release. These linkers are cleaved under the acidic conditions of the lysosome, or by specific proteases like cathepsin B once the conjugate is internalized.

[0090] In various embodiments, conjugation of therapeutic moieties and / or linkers occurs via site-selective and / or click chemistry-based conjugation strategies. In embodiments, site- selective and click chemistry-based conjugation enhance conjugation precision by ensuring a consistent drug-to-antibody ratio (DAR) and homogeneous product. This approach typically involves the genetic or enzymatic introduction of specific conjugation handles, such as unnatural amino acids (e.g., p-acetylphenylalanine), engineered cysteines (e.g., THIOMABs), or azide-functionalized residues, which then react with a complementary alkyne or tetrazine group on the drug-linker via thiol-maleimide coupling, strain-promoted azide-alkyne cycloaddition (SPAAC), or inverse electron-demand Diels-Alder (IEDDA) reactions. In some embodiments, site-selective and click chemistry-based conjugation payload release occurs after internalization into the target cell, where the acidic lysosomal environment (pH ~4.5-5.0) or specific intracellular proteases (e.g., cathepsin B) cleaves the linker. Exemplary linkers include a valine-citrulline dipeptide or an acid-labile hydrazone.

[0091] In various embodiments, the therapeutic moiety is conjugated via pH-sensitive dissociation linkers (e.g., acid-sensitive linkers). In embodiments, these linkers are designed to exploit the natural pH gradient between systemic circulation and intracellular

[0092] DBl / 165335339.4 17GNR-007PC / 115835-5007

[0093] compartments. In some embodiments, such linkers utilize acid-labile functional groups such as hydrazones, acetals, ketals, or carbonates to bridge the antibody and the therapeutic payload. The conjugation often occurs through the s-amino group of lysine residues or via thiol -reactive groups on cysteines (native or engineered), where the linker is attached as an electrophilic handle. Under physiological conditions (approx. pH 7.4), these bonds remain relatively stable, but they can be engineered to undergo rapid hydrolysis in the acidic environment of endosomes (approx. pH 5.5-6.5) or lysosomes (approx. pH 4.0-5.0) following internalization. The pH shift triggers the hydrolysis / cleavage of the bond, thereby releasing the payload within the endosome or lysosome of the targeted cell.

[0094] In various embodiments, the therapeutic moiety is conjugated via a redox-sensitive linker (disulfide linker). In embodiments, redox-sensitive linkers utilize disulfide bonds (S- S) to bridge the antibody and the therapeutic payload, exploiting the significant difference in reduction potential between the extracellular (e.g., blood / plasma) and intracellular environment. These linkers are typically conjugated to the antibody via the sulfhydryl groups of cysteine residues or the s-amino groups of lysines using heterobifunctional crosslinkers, such as N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) or N-succinimidyl 4-(2- pyridyldithio)sulfobutyrate (sulfo-SPDB).

[0095] Crosslinkers described herein are considered part of the linker molecule, and ADCs using a particular linker contain any crosslinker necessary to attach the linker molecule and bridge the linker to the payload, optionally through a spacer molecule, including but not limited to spacers comprising hydrophilic polymers such as polyethylene glycol (PEG).

[0096] Payload release from redox- sensitive linkers often occurs within the cytosol, where higher concentrations of glutathione (GSH, 1-10 mmol / L) and other thiols facilitate the reduction of the disulfide bond. The linker remains relatively stable in the bloodstream, where GSH concentrations are nearly 1,000-fold lower. Redox-sensitive linkers can also incorporate steric hindrance nearby the linkage to prevent premature reduction by circulating thiols, for example, by engineering alkylation, such as methyl groups, adjacent to the disulfide bond of the linker.

[0097] In various embodiments, the therapeutic moiety is conjugated via a protease- cleavable linker. In embodiments, protease-cleavable linkers are engineered to release payloads in response to trafficking to an environment having a specific enzymatic capacity.

[0098] DBl / 165335339.4 18GNR-007PC / 115835-5007

[0099] These linkers typically utilize a peptide sequence or peptidomimetic structure. Exemplary protease-cleavable linkers include the dipeptide valine-citrulline (Val-Cit), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), alanine-phenylalanine-lysine (Ala-Phe-Lys), legumain-sensitive linkers, and tetrapeptide linkers, each of which can be conjugated to an antibody via the sulfhydryl groups of cysteines (often using a maleimide handle) or the s- amino group of lysines. To ensure the payload is released, in various embodiments, the cleavable linker is attached to the payload through a “self-immolative” spacer. In embodiments, the spacer is considered a portion of the linker molecule. Exemplary self- immolative spacers include p-aminobenzyloxycarbonyl (PABC) and its derivative molecules, such as p-aminobenzyl ether (PABE) and p-hydroxybenzyl alcohol (PHOBA). P ABC-based linkers use suitable for non-hydroxyl-containing payloads due to the resulting carbonate bond being prone to premature hydrolysis in plasma. In some embodiments, hydroxyl-containing payloads are conjugated with linkers such as methylene alkoxy carbamate (MAC, cleavable by β-glucuronidase) or glycine-proline (Gly-Pro) diketopiperazine-forming linkers. Additional exemplary linker spacer molecules useful herein include N, N'-dimethylethylene diamine (DMED) or amino benzylamine (ABA), particularly useful for bulky payloads, or hemiacetal or hemiaminal spacers, which are particularly useful for payloads that may be sensitive to extraneous reaction with leftover electron withdrawing groups after release.

[0100] In various embodiments, payload release occurs inside the lysosome after the antibody is internalized via receptor-mediated endocytosis. Here, higher concentrations of lysosomal proteases, such as cysteine cathepsins (cathepsin B, L, S, and K), asparaginyl endopeptidase, and plasmin, cleave the specific protease-cleavable linkage between an amino acid and the spacer molecule. Payload release from protease-cleavable linkers can trigger spontaneous chemical breakdown of the PABC, PABE, or PHOBA group (e.g., 1,6- elimination), releasing the therapeutic molecule.

[0101] In various embodiments, the therapeutic moiety is conjugated via an electrostatic release linker. In some embodiments, the electrostatic release linker represents a non- covalent conjugation strategy where the therapeutic molecule is tethered to the antibody via ionic interactions rather than a covalent bond. This method typically involves a polycationic linker, such as protamine or synthetic poly-arginine peptides, which are either genetically

[0102] DBl / 165335339.4 19GNR-007PC / 115835-5007

[0103] fused or chemically conjugated to the antibody, usually via one or more lysine or cysteine residues. Exemplary linkers useful for electrostatic payload release include protamine and protamine-derived polypeptides, poly-arginine peptides (9-Arg), and cationic polymers such as low-molecular-weight polyethylenimine (PEI) derivatives or poly-L-lysine (PLL). Such linker molecules, in embodiments, are fused to the C-terminus of an antibody chain.

[0104] In some embodiments, the therapeutic payload has a strong negative charge, e.g., such as nucleic acid or through the use of poly-anionic derivatives (e.g., sulfonated or phosphorylated drugs), to facilitate stable electrostatic complexation. These conjugates are stabilized by the high-affinity ionic "zip" between the positive charges of the linker and the negative charges of the payload. Release can be triggered within the acidic and high-ionic- strength environment of the lysosome, where the presence of competitive polyanions (such as heparin or cellular glycosaminoglycans) weaken the electrostatic interactions, causing the drug to dissociate. This "reversible" linkage is particularly advantageous for avoiding chemical modification of the payload, ensuring the payload is released in its native form, and for achieving high drug-to-antibody ratios (DARs).

[0105] In some embodiments, the therapeutic moiety is conjugated via protamine or a protamine-based conjugation. Protamine or a protamine-derived peptide is a cationic peptide that is chemically activated using a heterobifunctional crosslinking reagent. In embodiments, the protamine or protamine-derived peptide is at least about 5 amino acids in length to about 60 amino acids in length, or about 10 to about 50 amino acids in length. Exemplary protamine or protamine-derived peptides are basic amino acid-rich polypeptides, such as peptides having polyarginine-rich repeats, or polyarginine-serine repeats. Non-limiting embodiments of protamine or protamine-based amino acid sequences include human protamine 1 (PRM1) (MARYRCCRSQSRSRYYRQRQRSRRRRRRSCQTRRRAMRCCRPRYRPRCRRH, SEQ ID NO: 94), salmon protamine (MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR, SEQ ID NO: 95), low molecular weight protamine (LMWP) (VSRRRRRRGGRRRR, SEQ ID NO: 96), and F5-P fusion fragment (RSQSRSRYYRQRQRSRRRRRRSSSCQTRRRAMRCCRPRYRPRCRRH, SEQ ID NO: 97). In some embodiments, the ADC comprises an amino acid linker (as described herein) between the protamine or protamine-based peptide sequence and the antibody sequence, for

[0106] DBl / 165335339.4 20GNR-007PC / 115835-5007

[0107] example, at the C-terminus of the antibody heavy chain. Exemplary crosslinkers include maleimide-N-hydroxysuccinimide (NHS) ester reagents, such as sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC). Cationic protamine and protamine-derived peptides are useful for binding anionic payloads, such as nucleic acids (as further described herein). In embodiments, protamine or a protamine-based peptide is contacted with the crosslinker under aqueous conditions, allowing reaction between primary amine groups of protamine and the NHS ester moiety of the crosslinker. In embodiments, protamine-based conjugation utilizes a fusion protein approach where a protamine segment is genetically linked to the C-terminus of an antibody heavy chain or light chain, or chemically conjugated via bifunctional linkers like sulfo-SMCC, which targets the ε-amino group of lysine or sulfhydryl groups of cysteine. The reaction yields an activated protamine intermediate bearing a maleimide functional group capable of subsequent conjugation to thiol-containing biomolecules (such as antibody domains).

[0108] In various embodiments, antibody-protamine conjugates are capable of electrostatically binding nucleic acids (e.g., siRNA, shRNA, ASO molecules) through the cationic portions of protamine. The highly cationic nature of protamine allows it to bind negatively charged therapeutic molecules through electrostatic (ionic) interactions, eliminating the need for complex covalent chemical modifications of the payload itself. Release of the therapeutic molecule from the protamine complex is facilitated by the competitive displacement or dissociation that occurs within the acidic and high-salt environment of the lysosome after receptor-mediated endocytosis. In embodiments, this type of reaction is sufficient to achieve partial substitution with nucleic acid payloads, preserving nucleic acid-binding capacity of the payload. In embodiments, nucleic acid (e.g., siRNA, shRNA, miRNA, ASO) is added to the antibody-protamine conjugate at a defined molar ratio (e.g., antibody conjugate to nucleic acid ratio of 0.1:1 to 1:0.1), for example using an excess of nucleic acid relative to the antibody to form non-covalent complexes.

[0109] In embodiments, protamine conjugation is useful to both to attach the therapeutic moiety to the ADC and to enhance endosomal escape of the therapeutic moiety. Protamine and protamine-derived polypeptides influence endosomal escape of payloads by acting as a cell-penetrating peptide. The strong electrostatic attraction between protamine and the drug backbone protects sensitive payloads (like RNA) from enzymatic degradation by nucleases.

[0110] DBl / 165335339.4 21GNR-007PC / 115835-5007

[0111] Unlike fixed-site chemical conjugation, the drug-to-antibody ratio (DAR) via protamine can be adjusted by varying the concentration / ratio of the payload during the complexation process.

[0112] In various embodiments, the ADC comprises an endosomal escape element. A limitation to delivery of therapeutic molecules via receptor-mediated endocytosis is the that the payload may remain trapped in endosomes, where it may eventually be destroyed. To address this limitation and further improve antibody-delivered therapeutic molecules, different conjugation chemistries, small molecules, fusogenic peptides, cell-penetrating peptides (CCPs), and / or pH-sensitive lipids are used in the ADC to induce “leakage” in the endosome to release the therapeutic moiety into the cytosol. In some embodiments, the endosomal escape element is triggered by acidic pH (<7.0, or pH 4.0-6.5).

[0113] In various embodiments, the endosomal escape element comprises a polypeptide that facilitates therapeutic moiety escape from the endosome. In embodiments, the endosomal escape element comprises a peptide of at least about 5 amino acids in length to about 60 amino acids in length, or about 5 to about 50 amino acids in length, or about 10 to about 30 amino acids in length.

[0114] In embodiments, the endosomal escape element is fused or conjugated to the antibody. In embodiments, the endosomal escape element is fused at the N-terminus or C- terminus of one or both of the antibody heavy chains. In embodiments, the endosomal escape element is fused at the N-terminus or C-terminus of one or both of the antibody light chains. In embodiments, the endosomal escape element is fused at the C-terminus of one or both antibody heavy chains. In some embodiments, the endosomal escape element is crosslinked with the therapeutic moiety. For example, in non-limiting embodiments, the therapeutic moiety is a nucleic acid payload (e.g., siRNA, shRNA, mRNA, ASO, DNA) crosslinked with a peptide or small molecule that acts as an endosomal escape element to assist the nucleic acid in escaping the endosome.

[0115] Exemplary peptide-based endosomal escape elements include CCPs, fusogenic peptides, and viral peptides, including without limitation arginine-rich CCPs such as polyarginine (RRRRRRRRR, R9, SEQ ID NO: 85), peptides derived from HIV-1 Tat (GRKKRRQRRRPPQY, SEQ ID NO: 86), pH-sensitive viral fusion peptides derived from influenza virus hemagglutinin HA2 (GLFEAIAGFIENGWEGMIDGGGYC, SEQ ID NO:

[0116] DBl / 165335339.4 22GNR-007PC / 115835-5007

[0117] 87) or influenza IFN-7 (GLFEAIEGFIENGWEGMIDGWYG, SEQ ID NO: 88), amphipathic peptides with alternating hydrophobic and hydrophilic residues, often adopting helical structure, such as GALA (WEAALAEALAEALAEHLAEALAEALEALAA, SEQ ID NO: 89) or an amphipathic peptide derived from the antimicrobial peptide Aurein 1.2 (GLFDIIKKIAESF, SEQ ID NO: 90), histidine-rich peptides that accumulate protonation in acidic environments such as polyhistidine (HHHHHHHHHH, H10, SEQ ID NO: 91), and insect-based polypeptides such as third helix of the Drosophila Antennapedia homeodomain containing protein penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO: 92) or Apis melifera melittin (GIGAVLKVLTTGLPALISWIKRKRQQ, SEQ ID NO: 93). In some embodiments, protamine and protamine-derived peptides, e.g., SEQ ID NOs: 94-97, or polypeptides having 1 to 10 amino acid substitutions, deletions, or insertions thereto, are useful as endosomal escape elements. In such embodiments, fusion or conjugation of these polypeptide sequences is useful for nucleic acid payload conjugation and escape from the endosome after internalization.

[0118] In various embodiments, the endosomal escape element comprises a pH-sensitive (acid-sensitive) lipid. In embodiments, acid-sensitive lipids include ionizable lipids which undergo a physicochemical change when pH is shifted from physiological (pH ~7.4) to the pH experienced in the endosome / lysosome (pH ~4.0-6.5), or are and nonlamellar lipids which naturally favor the curved membrane structure of the endosome. Such lipids may remain neutrally charged at pH 7.4 (reducing toxicity) but become protonated in the acidic endosome, creating electrostatic interactions with the negatively charged endosomal membrane and causing a transition that ruptures the membrane. Exemplary ionizable lipids for conjugation to the ADC for this purpose include DLin-MC3-DMA (dilinoleyl-methyl-4- dimethylaminobutyrate), SM-102 (8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester), ALC-0315 ([(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)), and DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl) — dioxolane). Exemplary nonlamellar lipids for conjugation to the ADC for this purpose include l,2-dioleoyl-3-dimethylammonium- propane (DODAP) and glycerol monooleate (GMO). In embodiments, lipids are conjugated to the ADC using maleimide functionalization, such as with DPSE-PEG-maleimide for

[0119] DBl / 165335339.4 23GNR-007PC / 115835-5007

[0120] conjugation at a thiol (cysteine), using NHS-ester lipids for conjugation to primary amines of lysines, or using DBCO or azide lipids for click chemistry conjugation (SPAAC).

[0121] In embodiments, polypeptide fusions to the ADC are fused via amino acid linkers. For example, in embodiments, the endosomal escape element is a polypeptide that is fused to the antibody via a flexible or rigid amino acid linker. Exemplary amino acid linkers for fusion of polypeptides herein comprise, are predominately, or consist of, glycine and / or serine residues, including repeated motifs and units thereof (e.g., 1 to 10 repeats). In embodiments, the linker comprises one or more repeated units / motifs comprising predominately glycine (G) and serine (S) residues. Amino acid linkers, in embodiments, are from about 1 to about 50 amino acids in length, such as from about 4 to about 20 amino acids in length.

[0122] In some embodiments, the endosomal escape element is incorporated by conjugation of the therapeutic moiety and / or drug linker. In some embodiments, the endosomal escape element is a part of the linker portion of the conjugate that bridges the antibody and therapeutic moiety. In embodiments, the endosomal escape element is a fusion polypeptide or a conjugated polypeptide (e.g., using one or more chemistries as described herein to conjugate the polypeptide, for example, to a lysine or cysteine).

[0123] In embodiments, the endosomal escape element is located adjacent to where the nucleic acid therapeutic moiety (e.g., siRNA, shRNA, or ASO) is conjugated / associated with the ADC. For example, in embodiment, an endosomal escape element peptide is fused to a protamine polypeptide, or another electrostatic release linker.

[0124] In some embodiments, the endosomal escape element comprises a small molecule that facilitates therapeutic moiety escape from the endosome. In embodiments, small molecule endosomal escape elements are pH-triggered in that they work as “proton sponges” in acidic environments. Such small molecules disrupt the endosomal membrane by inducing osmotic swelling from the uptake of protons. In embodiments, small molecule endosomal escape elements are conjugated to the antibody, either at a location distal from the therapeutic moiety (e.g., conjugated at a different lysine, cystine, or other residue), or fused / conjugated adjacent to the therapeutic moiety, or contacting the therapeutic moiety (conjugated to the therapeutic moiety).

[0125] DBl / 165335339.4 24GNR-007PC / 115835-5007

[0126] Exemplary small molecule-based endosomal escape elements include chloroquine ((RS)-N'-(7-chloroquinolin-4-yl)-N, N-di ethylpentane- 1,4-diamine), which is a well-studied antimalarial that buffers endosomal pH and enhances gene transfer; NP3, which is a Niemann-Pick type Cl (NPC1) inhibitor (l-[4-chloro-2,6-di(propan-2-yl)phenyl]-3-[4-(2- hydroxypropan-2-yl)furan-2-yl] sulfonylurea) that similarly enhances gene silencing potency of RNA systems via endosomal escape; cationic amphiphilic drugs (CADs) that function as membrane destabilizers such as siramesine (l'-{4-[l-(4-Fluorophenyl)-lH- indol-3-yl]butyl}-3H-spiro[2 -benzofuran- l,4'-piperi dine]), which is a σ-2 receptor agonist or the a tricyclic antidepressant, amitriptyline (3-(10,l l-dihydro-5H- dibenzo[a,d]cyclohepten-5-ylidene)-N, N-dimethylpropan-l -amine).

[0127] In embodiments, endosomal escape elements are useful for delivery of nucleic acids, therapeutic enzymes, and genome-editing proteins.

[0128] In various embodiments, the therapeutic moiety is one or more of a nucleic acid, small molecule, peptide, protein, reactive species, or radionuclide.

[0129] In some embodiments, the nucleic acid comprises DNA and / or RNA nucleotides. In some embodiments, the DNA is single stranded (ssDNA), double stranded (dsDNA), linear, or circular DNA, such as an episome, vector, minicircle, or plasmid DNA. In embodiments, the DNA encodes a protein-coding sequence, an expression cassette, or a gene. In embodiments, the nucleic acid is an aptamer, e.g., which may function as a binding molecule. In some embodiments, the RNA is an mRNA, or a small RNA, including without limitation, one or more of a siRNA, shRNA, or miRNA. In embodiments, the nucleic acid is an antisense oligonucleotide (ASO). In some embodiments, the nucleic acid reduces or ablates expression or one or more endogenous neural cell proteins, and such nucleic acids include siRNA or gapmer ASO (which includes a central region of DNA nucleotides to recruit RNaseH). In embodiments, the nucleic acid is an ASO that targets (e.g., blocks or targets for destruction) a miRNA (antagomir) or long non-coding RNA (LncRNA). In other embodiments, the nucleic acid is an ASO (e.g., RNA, PNA, or morpholino ASO) that impacts splicing of a target pre-mRNA, either to enhance or block splicing or to induce skipping of one or more exons of a target pre-mRNA.

[0130] In embodiments, oligonucleotides agents can be modified to enhance their stability and / or enhance biological activity by modification with nuclease resistant groups, for

[0131] DBl / 165335339.4 25GNR-007PC / 115835-5007

[0132] example, 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl groups. Inclusion of locked nucleic acids (LNA) can also increase binding affinity to the target. Exemplary internucleotide linkage modifications include, but are not limited to, phosphorothioate, chiral phosphorothioate, phosphorodithioate, peptide nucleic acid (PNA), morpholino, and thiomorpholino, among others.

[0133] In embodiments, the modifications include locked nucleic acids (LNA) or other bridged nucleotides such as cEt, and / or 2'-O-(2 -Methoxyethyl) (abbreviated as 2’ MOE) or 2'-0Me modifications, whereby at least part or all of the sequence is modified at the 2' position of each nucleotide. Locked nucleic acid (LNA) or “locked nucleotides” are described, for example, in U. S. Patent Nos. 6,268,490; 6,316,198; 6,403,566; 6,770,748; 6,998,484; 6,670,461; and 7,034,133, all of which are hereby incorporated by reference in their entireties. LNAs are modified nucleotides that contain a bridge between the 2' and 4' carbons of the sugar moiety resulting in a “locked” conformation, and / or bicyclic structure. Other suitable locked nucleotides that can be incorporated in the oligonucleotides of this disclosure include those described in U. S. Pat. Nos. 6,403,566 and 6,833,361, both of which are hereby incorporated by reference in their entireties. In exemplary embodiments, the locked nucleotides are independently selected from a 2' to 4' methylene bridge and a constrained ethyl (cEt) bridge (see, US Patent Nos. 7,399,845 and 7,569,686, which are hereby incorporated by reference in their entireties).

[0134] In embodiments, the nucleic acid agent is an ASO and is from 8 to about 50 nucleotides in length, such as 12 to about 25 nucleotides in length.

[0135] In embodiments, the nucleic acid is an siRNA to induce RNA interference. RNA interference (RNAi) is a sequence-specific RNA degradation process to knockdown, or silence, theoretically any gene containing the homologous sequence. In naturally occurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNase III / helicase protein (Dicer) into small interfering RNA (siRNA) molecules, which are dsRNAs of 19-27 nucleotides (nt) with 2-nt overhangs at the 3' ends. Afterwards, the siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC to guide the complex towards a cognate RNA that has a sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, resulting in truncation and inactivation of the targeted RNA.

[0136] DBl / 165335339.4 26GNR-007PC / 115835-5007

[0137] In various embodiments, the siRNA comprises an antisense strand and a sense strand, where the antisense strand comprises a nucleotide sequence that is at least 12, 14, 16, 18, 20, 21 or 22 nucleotides in length, and preferably at least 16 nucleotides in length (e.g., 19-21 nucleotides in length), and which targets (e.g., is complementary to) an RNA for destruction. Sense strands (i.e., passenger strands) for inducing RNAi can be designed as known in the art or as described below. Exemplary sense strands comprise a sequence complementary to the antisense strand, and may have 16, 17, 18, 19, 20, or 21 nucleotides. 3’ nucleotide overhangs (e.g., dTdT) are optional for antisense and sense strands.

[0138] In embodiments, the siRNA comprises a chemical modification, including any of the well-known chemical modifications for siRNA. In embodiments, the chemical modifications increase stability, reduce endonuclease degradation, reduce immunogenicity, and / or reduce Toll-like receptor recognition. In embodiments, the chemical modification is a nucleobase modification, a backbone modification, and / or a sugar modification. In embodiments, the siRNA may have one or more backbone modification(s) selected from phosphorothioate, phosphorodithioate, methylphosphonate, and methoxypropylphosphonate. For example, such modifications may be placed at and / or near the 3 ' end of the antisense strand and / or the sense strand. In embodiments, the siRNA comprises one or more sugar modifications, such as those selected from 2’-methoxy (2’-0me), 2’-O-methoxyethyl (2’-0-M0E), 2’-fluoro (2’-F), 2’-arabino-fluoro (2’-Ara-F), constrained ethyl (cEt), bridged nucleic acid (BNA) and locked nucleic acid (LNA).

[0139] In embodiments, the siRNA comprises a sense and antisense strain, each having a length of about 12 to about 40 nucleotides. In embodiments, the siRNA comprises two substantially complementary RNA strands with a duplex length of about 12 to about 40 base pairs (such as from 16 to 24 base pairs). In embodiments, the siRNA comprises a sense strand overhang and an antisense strand overhang at the 3’ ends. The overhangs may be RNA overhangs or may be deoxythymidine (dTdT) overhangs. In embodiments, the siRNA is an asymmetric siRNA (asiRNA) having a blunt end corresponding to the 5’ end of the antisense strand. Other siRNA formats, including but not limited to short-hairpin RNAs (shRNAs), that may be used are described in US 2008 / 0188430, which is hereby incorporated by reference.

[0140] DBl / 165335339.4 27GNR-007PC / 115835-5007

[0141] In embodiments, the nucleic acid (e.g., siRNA or ASO) may reduce the expression of an endogenous neural cell proteins, which can include disease-relevant proteins (e.., subject to aggregation / inclusion, mutated proteins associated with monogenic diseases or malignancies, proteins related to gene expression regulation, etc.). Non-limiting examples of endogenous neural cell proteins for targeting include alpha-synuclein (SNCA), amyloid precursor protein (APP), elongation factor 1 -alpha 1 (EEF1 Al), elongation factor 1 -alpha 2 (EEF1A2), histone H3.3A (H3-3A), Huntingtin (HTT), superoxide dismutase 1 (SOD1) (e.g., QUALSODY), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), and leucine-rich repeat kinase 2 (LRRK2), and isoforms and orthologs thereof. Various other genes whose mutation can cause haploinsufficiency resulting in impairments of brain development or function are known in the art. In some embodiments, the targeted gene has a mutation resulting in a lysosomal storage disorder.

[0142] In some embodiments, the therapeutic moiety is an enzyme, peptide, or protein, for example, where enzyme delivery for replacement therapy is needed. Generally, any therapeutic agent whose safety and / or efficacy can be improved by targeted delivery to neurons can be employed in the various embodiments.

[0143] In various embodiments, the therapeutic moiety is a small molecule that is a cytotoxic agent or anticancer agent, for example, where the ADC is used for targeting cancer cells or tissue within the CNS (e.g., neuronal tumor). In some embodiments, the cytotoxic agent or anticancer agent comprises a microtubule or tubulin inhibitor, DNA damaging agent, DNA intercalating agent, DNA synthesis inhibitor, immunomodulator, alkylating agent, antimetabolite, or antitumor antibiotic. In some embodiments, the cytotoxic agent or anticancer agent is selected from a class of vinca alkaloids, anthracy clines, benzoansamacrolides (maytansinoids), pyrrolobenzodiazepines (PBDs), topoisomerase 1 inhibitors, auristatins, tubulysins, calicheamicins, duocarmycins, exatecans, pyrrolobenzodiazepines, antifolates, TLR agonists, and STING agonists. In some embodiments, the cytotoxic agent or anticancer agent is selected from calicheamicin, N- acetyl-y-calicheamicin, monomethyl auristatin E (MMAE), monomethyl auristatin-F (MMAF), derivative of may tansine 1 (DM1), derivative of maytansine 4 (DM4), exatecan derivatives (Dxd, DX-8951), SN-38 (irinotecan), methotrexate, thioguanine, 5-fluorouracil,

[0144] DBl / 165335339.4 28GNR-007PC / 115835-5007

[0145] cytosine arabinoside, cisplatin, doxorubicin, daunorubicin, actinomycin D, camptothecin derivatives, duocarmycin, and derivatives and analogues thereof.

[0146] In other embodiments, the therapeutic moiety is a radionuclide, for example, where the ADC is used for concentrating radiation at the site of cancer cells or tissue within the CNS (e.g., neuronal tumor). Non-limiting examples of radionuclides used for targeted anticancer therapy include Iodine-131 (1-131), Lutetium-177 (Lu-177), Yttrium-90 (Y-90), and Radium-223 (Ra-223). In some embodiments, ADCs herein are useful for trafficking cytotoxic agents, anticancer agents, and radionuclides across the BBB to neurons and neuronal tissue, e.g., for the treatment of cancer e.g., solid tumors, metastasis, and circulating tumor cells).

[0147] In various embodiments, the therapeutic moiety is a small molecule, which is some embodiments is a cytoprotective agent, for example, comprises a neuroprotective agent or an agent for a neurological disease, for example, where the ADC is used for targeting cells or tissue within the CNS implicated in neurological disease. In embodiments, the therapeutic agent comprises one or more of an acetylcholinesterase or cholinesterase inhibitor (tacrine, rivastigmine, galantamine), donepezil, NMDA receptor antagonist or glutamate blocker (memantine), masitinib, donepezil, carbamazepine, lamotrigine, levetiracetam, topiramate, valproic acid, phenytoin, diazepam, clonazepam, pregabalin, gabapentin, ketamine, antiviral medications, including derivatives and analogues of the foregoing.

[0148] In some embodiments, the therapeutic moiety is a reactive species comprises nitric oxide (NO donor) or reactive oxygen species (ROS)-generating molecule, for example, for inducing intracellular oxidative damage.

[0149] In some embodiments, the drug-to-antibody ratio (DAR) is controlled based on the use of stochastic or site-specific conjugation techniques, and dependent upon the purification method used to discriminate between different levels of conjugation. In some embodiments, the DAR ranges from between about 1 to about 10, or from about 1 to about 5, depending upon the nature of the therapeutic moiety and the chemistry used for conjugation. In some embodiments, DAR is at least 1, at least 2, at least 3, or at least 4.

[0150] In various embodiments, the antibodies and ADCs disclosed herein include one or more modifications to further alter their physicochemical properties, such as the surface charge, hydrodynamic radius, solubility, and / or hydrophilicity / hydrophobicity, as well as to

[0151] DBl / 165335339.4 29GNR-007PC / 115835-5007

[0152] modify the PK and PD properties, such as renal clearance, half-life, tissue sinking, BBB penetration, and the like. In some embodiments, the modifications include post-translation modifications (PTMs), including without limitation, glycosylation, phosphorylation, alkylation, deamidation, isomerization, oxidation, acetylation, methylation, sumoylation, ubiquitination, lipidation, and ADP ribosylation (ADPr). In some embodiments, the modifications include PEGylation using one or more PEG sizes from about 700 Da to about 40 kDa. In some embodiments, the modifications include fusion or conjugation to a peptide or one or more domains of a protein.

[0153] In some aspects, the disclosure provides pharmaceutical compositions comprising an effective amount of an antibody or ADC of this disclosure, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in accordance with this disclosure are known in the art. In some embodiments, the pharmaceutically acceptable carrier is a physiological solution, e.g., saline or other pharmacologically acceptable solvent or a buffered solution, and may optionally comprise a surfactant. Pharmaceutically acceptable carriers include water, saline, and glycerol. In some embodiments, the formulation may comprise fixed oils, polyethylene glycol, propylene glycol or other solvents.

[0154] The pharmaceutical compositions of the disclosure may be conveniently presented in unit dose forms containing a predetermined amount of the active agent of the disclosure per dose. Auto-injectors such as an “injection pen” are spring-loaded syringes designed to deliver a dose of a particular drug. By design, injection pens are easy to use and are intended for self-administration by patients, or administration by untrained personnel. Injection pens are designed to overcome the hesitation associated with self-administration of the needlebased drug delivery device. The injection pen keeps the needle tip shielded prior to injection and also has a passive safety mechanism to prevent accidental firing (injection). Injection depth can be adjustable or fixed and a function for needle shield removal may be incorporated. By pressing a button, the syringe needle is automatically inserted into the subcutaneous tissue and the drug is delivered. Once the injection is completed some injection pens have a visual or audible indication to confirm that the full dose has been delivered.

[0155] In some embodiments, the injection pen contains from one to ten unit doses or from one to five unit doses. In some embodiments, the unit doses are no more than about 1.5 mL

[0156] DBl / 165335339.4 30GNR-007PC / 115835-5007

[0157] or about 1 mL in volume (whether or not contained or delivered by an injection pen). The injection pen may contain at least four unit doses (e.g., at least about 5, at least about 8, or at least about 10 unit doses).

[0158] In an aspect, the disclosure provides methods of treating a neurological disorder, including genetic and non-genetic disorders. The method comprises administering an effective amount of an antibody or ADC of this disclosure.

[0159] In embodiments, the subject in need has a neurodegenerative disease, a traumatic brain or spinal injury, central nervous system (CNS) damage, or peripheral nerve damage. Exemplary neurological disorders comprise Multiple Sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, Spinal muscular atrophy, and ischemic reperfusion injury (e g., after ischemic or hemorrhagic stroke). In embodiments, the subject receives an effective amount of an antibody described herein, including but not limited to TGM-010 or its variants. In embodiments, the subject receives an ADC having a therapeutic agent that is neuroprotective, or which targets expression of a protein involved in the pathology.

[0160] In embodiments, the subject has a gene mutation resulting in haploinsufficiency, and resulting in impairments of brain development or function. Gene expression in embodiments can be enhanced by an ASO that targets a small RNA or LncRNA that regulates gene expression, or by an ASO that enhances pre-mRNA splicing or otherwise impacts splicing in a manner that increases gene expression. In embodiments, the subject has a gain-of- function or pathogenic mutation, which can be targeted by an exon-skipping approach (inducing skipping of a mutated exon) or ASO that targets the mRNA containing the pathogenic mutation for degradation (e.g., gapmer ASO or siRNA). For example, the subject may have primary erythromelalgia due to a mutation in SCN9A gene, and which can be targeted by an siRNA or gapmer. In embodiments, the subject has spinal muscular atrophy (SMA) for which an ASO approach can improve expression of SMN2 mRNA (as known in the art).

[0161] In some embodiments, the targeted gene has a mutation resulting in a lysosomal storage disorder. In such embodiments, the subject may receive an ADC delivering an ASO

[0162] DBl / 165335339.4 31GNR-007PC / 115835-5007

[0163] or siRNA therapy targeting the relevant pathogenic mutation (which are known in the art) as described.

[0164] In embodiments, the subject has a condition involving expression of a pathogenic protein (whether carrying mutations or not) that may form pathogenic deposits, such as Alzheimer’s disease (e.g., β-amyloid), Huntington’s disease (e.g., mutant huntingtin), Parkinson’s disease (e.g., a-synuclein), ALS (e.g., SOD-1), spinocerebellar ataxia targeting ataxin-1 (SCA1), ataxin-2 (SCA2), or ataxin-7 (SCA7), and Familial amyloidotic polyneuropathy (FAP) (mutated ATTR).

[0165] In embodiments, the subject has a malignancy of the CNS such as a neuronal tumor, which can be targeted using an ADC carrying a cytotoxic or anticancer moiety.

[0166] In various embodiments, the antibody or ADC is administered parenterally. For example, these agents may be administered by subcutaneous administration, intravenous administration, intracerebroventricular administration, intraparenchymal administration, intraarterial administration, intraventricular administration, or intrathecal administration. In some embodiments, compositions are administered via stereotactic injection. In embodiments, the compositions described herein are administered intravenously or subcutaneously. In some embodiments, the agents are administered by continuous infusion. In other embodiments, the agents are administered by a non-parenteral route, such as intranasally. Intranasal delivery provides a practical, non-invasive method of bypassing the blood-brain barrier (BBB) to deliver therapeutic agents to the brain and spinal cord.

[0167] In some embodiments, the antibody or ADC is administered at a determined frequency, for example, to reduce or slow neuronal loss over time in some embodiments. For example, the agent may be administered about daily, about 1-3 times weekly, about weekly, about twice monthly, about monthly (e.g., every 28 days), about once every two months, about once every three months, about quarterly or about semi-annually. In some embodiments, the agent is administered upon symptoms or attack (e.g., relapse) of a neurodegenerative disease, and / or during the recovery from the attack, to minimize neuronal loss. In various embodiments, the agent is administered by continuous intravenous (i.v.) infusion or by bolus administration.

[0168] In some embodiments, the agent (such as an antibody of this disclosure) is administered in a regimen initiated after an acute event, such as stroke, traumatic brain injury

[0169] DBl / 165335339.4 32GNR-007PC / 115835-5007

[0170] (TBI), or spinal injury. In some embodiments, a first administration is within about 24 hours of the acute injury, and within about 12 hours, within about 6 hours, or within about 3 hours. In some embodiments, for example, the regimen may comprise from 1 to about 20 bolus administrations after an acute event. The regimen may have a duration of one week, one month, or a plurality of months (e.g., from 1 to 12 months, or from 1 to 6 months, or from 1 to 4 months). The regimen can involve administrations at varying frequency, such as about daily, about 1-3 times weekly, about weekly, about twice monthly (e.g., every 28 days), or about monthly. In various embodiments, the agent is administered by continuous i.v. infusion for a period of time following the acute injury.

[0171] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0172] As used herein, the term “about” means ±10% of an associated numerical value. Embodiments of the disclosure are now described with reference to the following examples.

[0173] DBl / 165335339.4 33GNR-007PC / 115835-5007

[0174] EXAMPLES

[0175] Example 1: TGM-010 live cell internalization

[0176] TGM-010 (also referred to as AJL10) is a recombinant human IgG antibody that binds to neurons. TGM-010 includes the variable heavy and light chains cloned from a single CSF B cell isolated from a patient having clinically isolated syndrome optic neuritis (ON- cis). TGM-010 is a VH4 antibody that satisfies the antibody gene signature (AGS) codons described in U. S. Patent 8,394,583, which is hereby incorporated by reference. See also, US 2017 / 0002064, US Patent No. 11,518,800, and US 11,999,778, which are hereby incorporated by reference in their entireties.

[0177] A Sy5y human neuroblastoma cell line was used to evaluate the internalization capabilities of TGM-010 and certain variants in vitro. Sy5y cells were cultured to confluency, incubated with either a negative control antibody (JRR-067), an isotype control antibody (IgG), or TGM-010, TGM-010 F(ab’)2 fragment, and its variants. The cells were then fixed, stained, and imaged using fluorescence confocal microscopy at various time points. The control antibodies, TGM-010, and its variants are summarized in Tables 1 and 2.

[0178] Table 1: Description of antibodies used in live cell penetration testing in FIGs. 1-2,

[0179] Isotype Mutation Description

[0180] JRR-067 IgGl Non-specific control

[0181] TGM-010 IgGl Comparator antibody

[0182] Variant 1 IgGl E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0183] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0184] Met252Tyr, Ser254Thr, Thr256Glu

[0185] Variant 2 IgGl E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0186] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0187] Variant 3 IgG4

[0188] Variant 4 IgG4 E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0189] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0190] Variant 5 IgGl Met252Tyr, Ser254Thr, Thr256Glu

[0191] Variant 6 IgGl Glu294A, Thr307Pro, Asn434Tyr

[0192]

[0193] Isotype Ctl IgGl Human IgG isotype control

[0194] DBl / 165335339.4 34GNR-007PC / 115835-5007

[0195] TGM-010 N / A F(ab')2fragment of TGM-010

[0196]

[0197] Fab2

[0198] Table 2: Description of antibodies used in live cell penetration testing in FIGs. 3-4,

[0199] Isotype Mutation Description

[0200] JRR-067 IgGl Non-specific control

[0201] TGM-010 IgGl Comparator antibody

[0202] Variant 1 N / A F(ab')2fragment of TGM-010

[0203] Variant 2 IgGl E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0204] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0205] Met252Tyr, Ser254Thr, Thr256Glu

[0206] Variant 3 IgGl E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0207] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0208] Variant 4 IgG4

[0209] Variant 5 IgG4 E380W, N384Y, Q386T, P387E, E388W, N389S, N390S,

[0210] K392R, D413T, K414R, S415E, R416E, N421F, S424T, and S426

[0211] Variant 6 IgGl Met252Tyr, Ser254Thr, Thr256Glu

[0212]

[0213] Variant 7 IgGl Glu294A, Thr307Pro, Asn434Tyr

[0214] SH-SY5Y cells were seeded onto chamber slides and cultured under starvation conditions in serum-free culture medium at 37°C for 2 hours. Cells were then incubated with 50 pg / ml recombinant antibodies (rAb) in a blocking buffer at 4°C for 30 minutes. Cells were washed in phosphate buffered saline (PBS), then fixed in 4% paraformaldehyde (PF A) for 15 minutes at 37°C. For trafficking assays, cells were incubated at 37°C for 15 minutes or 6 hours to allow endocytosis, washed in phosphate buffered saline (PBS), and fixed in 4% PFA for 15 minutes at 37°C. Staining was performed using a monoclonal anti-a-tubulin antibody (Tubulin) as a marker to delineate neuronal cell bodies, 4',6-diamidino-2- phenylindole (DAPI) blue staining to visualize nuclei, and anti-human IgG secondary antibodies to visualize TGM-010 and its variants (mAb). The variant antibodies (Nos. 1 to 6) each have Fc domain mutations, as described herein, hypothesized to improve blood -brain barrier (BBB) penetration and / or neuronal cell intracellular trafficking.

[0215] DBl / 165335339.4 35GNR-007PC / 115835-5007

[0216] As shown in FIG. 1, imaging of live cell staining of TGM-010 (e.g., variable regions of SEQ ID NOs: 1 and 5 grafted onto a wild-type IgGl) and its Fc variants each showed some degree of internalization. As summarized in FIG. 2, variants 1, 2, and 4 showed statistically significant improvements in live cell internalization over the controls and TGM- 010 as evidenced by fluorescence intensity in comparison to the control antibodies (scale bars = 20 pm). As shown in FIG. 3, fluorescence intensity at 6 hours post-incubation demonstrated that variants 1, 2, and 4 show robust live cell internalization that outpaces the controls and TGM-010. Quantification of the fluorescence intensity at 6 hours (e.g., in FIG.

[0217] 4), shows statistically significant improvements from variants #1 and #2 relative to TGM- 010.

[0218] These data demonstrated, inter alia, that the Fc variants stimulated antibody internalization into neurons at an increased total amount and rate.

[0219] Example 2: TGM-010-siRNA conjugation

[0220] Site-specific TGM-010 conjugation with small interfering RNA (siRNA) payloads was tested with protamine-mediated conjugation. Protamine peptide at 2.7 mM was contacted with a crosslinking agent (4.5 mM) under aqueous conditions, allowing reaction between primary amine groups of the protamine peptide and the NHS ester moiety of the crosslinker (final 2.7 mM concentration). This reaction yielded an activated protamine intermediate bearing a maleimide functional group capable of subsequent conjugation to thiols of the antibody (sulfo-SMCC-protamine). The reaction was conducted at ambient temperature for 2 hrs. sufficient to achieve partial substitution, thereby preserving nucleic acid-binding capacity. The resulting activated protamine was purified to remove unreacted crosslinker using desalting techniques (desalting spin column).

[0221] TGM-10 needs was buffer-exchanged into a physiologically compatible buffer using a desalting spin column and subjected to a mild reducing reagent to expose reactive thiol groups. The activated protamine (540 pM) was then combined with TGM10 (23 pM, 1:23.5 molar ratio) under conditions that allow maleimide-thiol coupling, forming a stable antibody-protamine conjugate at 4°C overnight. Following conjugation, the antibody- protamine conjugate was purified using a desalting spin column and stored at 4°C for protein stability.

[0222] DBl / 165335339.4 36GNR-007PC / 115835-5007

[0223] siRNA (ThermoScientific Cat# 106121 and Cat# AM4635) at 66 pM was coupled with 33 pM TGM-10-sulfo-SMCC (TSP) at 1000 rpm for 2 hr. at 25°C. Each coupling reaction was performed before treating cells with the TSP-siRNA complex. As shown in FIG. 5, electrophoresis gel shift assay confirmed antibody-protamine-siRNA conjugation. Free siRNA (siCtrl, non-specific siRNA sequence and siAPP, anti-APP siRNA) traversed at normal rates to the bottom of the gel without conjugation, whereas antibody-conjugated siRNAs did not migrate through the gel (at top).

[0224] The antibody-protamine-siRNA complexes were used for delivery of anti-APP siRNA to U2OS cells (osteosarcoma cell line). U2OS cells were treated with either the TSP- siRNA complexes (0 to 3.3 pM of TSP-si APP or TSP-siCtrl complexes) for two consecutive days (24 hrs. per each treatment) or transfected with either siRNA alone, siCtrl or siAPP (50 nm), and then analyzed with validated primers for APP and GAPDH expression. Transfection reagent (Lipo., Lipofectamine® RNAiMAX®) was used as a control for siRNA delivery. To test knockdown efficiency, gene silencing was assessed by measuring target mRNA (qRT-PCR). Each qPCR reaction was performed in four replicates and all samples showed a standard deviation within 0.3 ( / / =2). As shown in FIG. 7, TGM-10 conjugated siRNA achieved a robust, statistically significant internalization and APP knockdown at the mRNA level with all concentrations tested. Protein levels were analyzed by western blotting to confirm the qPCR results using validated anti-APP and anti-GAPDH antibodies. Gel electrophoresis band intensities were quantified using pixelate density with ImageJ software and graphed as fold change (FIG. 7B). As shown in FIGs. 7A-7B, western blotting confirmed that TGM-10 conjugated siRNA achieved robust APP knockdown at the protein level.

[0225] These data demonstrated, inter alia, that the conjugates enable targeted, receptor- mediated intracellular delivery of nucleic acid payloads without covalent modification of the siRNA. These data also demonstrated, inter alia, that upon binding to the target receptor, the antibody-conjugate complexes were internalized via receptor-mediated endocytosis which enabled intracellular delivery of siRNA and subsequent gene silencing, with knockdown confirmed at the mRNA and protein levels.

[0226] Example 3: TGM-OlO-siRNA endosomal escape via fusogenic peptides

[0227] DBl / 165335339.4 37GNR-007PC / 115835-5007

[0228] A limitation to nucleic acid delivery via receptor-mediated endocytosis is that nucleic acid payloads, such as siRNAs, shRNAs, and ASOs, remain trapped in endosomes. To address this limitation and further improve antibody-delivered nucleic acid-mediated intracellular knockdown, different conjugation chemistries and / or fusogenic peptides are used to induce “leakage” in the endosome to release nucleic acid into the cytosol. Table 3 below provides an illustrative listing of TGM-10 conjugates fortesting endosomal escape of siRNA.

[0229] Table 3: Description of TGM-10 conjugates useful for live cell endosomal escape testing. DBCO = dibenzocyclooctyne, SS = disulfide, NHS = N-hydroxysuccinimide, Cy5 = cyanine 5 (fluorescent dye), PEG3 = 3x oxyethylene, PEG4 = 4x oxyethylene.

[0230] Illustrative TGM-10 conjugate

[0231] 1 TGM- 10-DBCO-PEG3 -S S-NHS-3 ’ siRNA-5 ’ -Cy 5

[0232] 2 TGM- 10-DBCO-PEG4-hydrazone-NHS-3 ’ siRNA-5 ’ -Cy 5

[0233] 3 TGM- 10-DB CO-PEG4-NHS-3 ’ siRNA-5 ’ -Cy 5

[0234] 4 TGM-10-DBCO-PEG4-hydrazone-NHS-3’siRNA-5’-IFN7 peptide-Cy5 5 TGM- 10-DBCO-PEG4-hydrazone-NHS-3 ’ ASO-5 ’ -Cy 5

[0235] 6 TGM-10-luciferase

[0236]

[0237] 7 TGM- 10-Sulfo-SMCC-Protamine-siRNA

[0238] The use of different conjugation chemistries, and fusogenic peptides in particular, are evaluated for endosomal escape using fluorescence microscopy (and fluorescent dyes) to assess subcellular localization within the endosome and escape into the cytosol. ADCs herein are suitable for conjugation with fluorophore, fluorescence proteins, and other luminescent, fluorescent, or colorimetric probes and dyes. ADCs herein may include fluorophore-labelled therapeutic moieties, such as fluorophore-labelled oligonucleotides. For example, Cy5 is an optional fluorescent probe used to, e.g., visualize subcellular and tissue trafficking and for manufacturing purposes. The use of Cy5 in the conjugates shown in Table 3 is optional. Nucleic acid payload escape is further measured by qPCR and western blotting to evaluate gene silencing, e.g., in comparison to nucleic acid conjugates lacking endosomal escape molecules.

[0239] DBl / 165335339.4 38GNR-007PC / 115835-5007

[0240] SEQUENCES SEQ ID NO: 1 - TGM10 Heavy Chain Variable Amino Acid Sequence (CDRs in underline) QVQLQESGPGLVKPSETLSLTCSVSGGAVSNYYWSWIRQSAGKGLEWLGRI YINGTTYYNPSLRSR VSMSVDTSKGQFSLRLTSVTAADTAIYYCARWGALLGDYYYGLDVWGQGTTVTVSS SEQ ID NO: 2 - TGM10 HC CDR1

[0241] GGAVSNYY SEQ ID NO: 3 - TGM10 HC CDR2

[0242] IYINGTT SEQ ID NO: 4 - TGM HC CDR3

[0243] ARWGALLGDYYYGLDV SEQ ID NO: 5 - TGM10 Light Chain Variable Amino Acid Sequence (CDRs in underline) DIVMTQSPLSLPVTPGEPASISCRSTQSLLHSNEYIYLDWYVQKPGQSPQLLIFLASNRASGVPDR FSGSASGTDFTLKISRVEAEDVGVYYCMQALEAPWTFGQGTRLEIK SEQ ID NO: 6 - TGM10 LC CDR1

[0244] QSLLHSNEYIY SEQ ID NO: 7 - TGM10 LC CDR2

[0245] LAS SEQ ID NO: 8 - TGM10 LC CDR3

[0246] MQALEAPWT SEQ ID NO: 9 - TGM-010 Variant - - Knob IgGl HC (CDRs in bold, amino acids subject to mutation underlined and in bold) QVQLQESGPGLVKPSETLSLTCSVSGGAVSNYYWSWIRQSAGKGLEWLGRI YINGTTYYNPSLRSR VSMSVDTSKGQFSLRLTSVTAADTAIYYCARWGALLGDYYYGLDVWGQGTTVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVWWESYGTEWSSY RTTPPVLDSDGSFFLYSKLTVTREEWQQGFVFTCGVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 - TGM-010 Variant - - Hole IgGl HC (CDRs in bold, amino acids subject to mutation underlined and in bold) QVQLQESGPGLVKPSETLSLTCSVSGGAVSNYYWSWIRQSAGKGLEWLGRI YINGTTYYNPSLRSR VSMSVDTSKGQFSLRLTSVTAADTAIYYCARWGALLGDYYYGLDVWGQGTTVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 - TGM-10 LC (CDRs in bold)

[0247] DB1 / 165335339.4 39GNR-007PC / 115835-5007

[0248] DIVMTQSPLSLPVTPGEPASISCRSTQSLLHSNEYIYLDWYVQKPGQSPQLLIFLASNRASGVPDR FSGSASGTDFTLKISRVEAEDVGVYYCMQALEAPWTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSS P VTKS FNRGE C SEQ ID NO: 12 - HC IgGl constant region variant. Knob 1 (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVWW E SYGTEWSS YRTTPPVLDS DGS F FL YS KL TVTREE WQQGFVFTCGVMHE AL HNHYTQKS LS LS PGK SEQ ID NO: 13 - HC IgGl constant region variant, Hole 1 (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW E SNGQ PENNYKTTPPVLDS DGS F FLVSKLTVDKSRWQQGNVFS CSVMHE AL HNHYTQKS LS LS PGK SEQ ID NO: 14 - HC IgGl constant region variant, Knob 2 (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVWW ESYGTEWSSYRTTPPVLDSDGSFFLYSKLTVTREEWQQGFVFTCGVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 15 - HC IgGl constant region variant, Hole 2 (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16 - IgGl constant region (wild type) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW E SNGQ PENNYKTTPPVLDS DGS F FL YS KLTVDKSRWQQGNVFS CSVMHE AL HNHYTQKS LS LS PGK SEQ ID NO: 17 - IgG4 HC constant region (wild type) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0249] DBl / 165335339.4 40GNR-007PC / 115835-5007

[0250] SEQ ID NO: 18 - IgG4 HC constant region variant - - Knob (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVWWESY GTEWSSYRTTPPVLDSDGSFFLYSRLTVTREEWQEGFVFTCGVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 19 - IgG4 HC constant region variant - - Hole (amino acids subj ect to mutation underlined and in bold) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 20 - AJL01 Heavy TGVHSQVQLQQWGAGLLKPSETLSLTCAVYGGSFNEFYWSWIRQPARKGLEWIGEISHSGRANYNP SLKSRVTLSVDRSKNQFSLNLSPVAAADTAVYYCARREIWTVRGRRAFDIWGQGTMVTVSSAS SEQ ID NO: 21 - AJL01 Light

[0251] TGVHS E I VLTQS PGTLS LS PGERAALS CRAS QS L I GS FLAWYQQKPGQAPRLL I YHTSNRASG I PD RFSGGGFGTDFTLTI SRLE PEDFAVYYCQQYDS SP I TFGQGTRLE IKRT SEQ ID NO: 22 - AJL02 Heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGHYWSWIRQSPGKGLEWIGNVYYSGSTYY TPSLDSRLTISLDTSKNQFSLRLSNVTVADTAVYYCARGRNWEGEFDPWGQGTLVTVSSAS SEQ ID NO: 23 - AJL02 Light TGVHSDIQMTQSPSSLSASLGDRVTITCRASQGISSSVNWFQQKPGKAPELLI YAASTLQSGVPSR FSGSGSGTDFTLTVSSLQPEDFATYYCQQSYSPPRTFGQGTKLEIKRT SEQ ID NO: 24 - AJL03 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGASISSSRSYWGWIRQPPGKGLEWIGSMYQSGSTYY SPSLKSRVTISMDTSKNQFSLNLTSVTAADTAVYFCARHSNPGTANKLRLGEFSPWGQGTLVTVSS AS SEQ ID NO: 25 - AJL03 Light TGVHSDIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWFQQQPGKAPKLLIYDASKLQMGVPSR FSGSASGTDFTFTISSLQPEDIGTYYCQQYYNLPYTFGQGTKLEIKRT SEQ ID NO: 26 - AJL04 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCTVSGDSIRSNFWTWIRQSPGRGLEWIGYFSYSGGINYSP SLKSRVTISVDTSKNQFSLKLTSVTAADAAVYYCARDPNGDYEVNWWGQGTLVTVSSAS SEQ ID NO: 27 - AJL04 Light

[0252] TGVHSDIQMTQSPSSLSASVGDRVTITCRASQDII IYLAWFQQRPGKAPRSLI YSASTLQSGVPSK FSGSGSGTYFTLTISSLQPEDSATYYCQQYKSYPITFGQGTRLEIKRT SEQ ID NO: 28 - AJL05 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSIRNSNYYWDWIRQPPGKGLEWIGSGYYSGSAYY HSSLKSRVSISVDTSKNQFSLNLTSVTAADTAFYYCARRSYYYASGSHDYWGQGTLVTVSSAS DBl / 165335339.4 41GNR-007PC / 115835-5007

[0253] SEQ ID NO: 29 - AJL05 Light TGVHSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLAWFQQKPGKAPKSLI YGASSLQSGVPSK FSGSGSGTDFTLTISGLQPEDFATYHCQQYRSFPITFGQGTRLEIKRT SEQ ID NO: 30 - AJL06 Heavy TGVHSQLQLQESGSGLVKPSQTLSLTCAVSGGSINTGNYFWSWIRQPPGKGLEWIGYMFRSTSTYY NPSLKGRVTISGGTSLSQFSLRLDSVTAADTAI YYCARGRYYCGVNCHPFDSWGQGTLVTVSSAS SEQ ID NO: 31 - AJL06 Light

[0254] TGVHSDIQMTQSPSSLSASVGDRVI ITCQASQDISIYLNWYQVKPGKAPKLLIYDASNLQAGVPSR FSGSGSGTDFSFTISSLQPEDVAAYYCHQYDSLPSFGQGTKLEIKRT SEQ ID NO: 32 - AJL07 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCNVSGGSINNYYWSWIRQPPGKGLEWIGYIYYNGNINYNP S LKSRVT I S RDMS KNQFSLNLRS VTAADTAVYYCG I G YS AVAAGTVD YWGHGTLVTVS S AS SEQ ID NO: 33 - AJL07 Light TGVHSAIQLTQSPSSLSASVGDRVTITCRASQGISSGLAWYQQEPGKAPKLLI YDASTLESGVPSR FSGSGSAIDFTLTISSLQPEDFATYYCQQFNTFPYTFGQGTKLEIKRT SEQ ID NO: 34 - AJL08 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCAVSGFSITSGYYWGWIRQPPGKGLEWIGSIYHTGTTYYN PSLKSRVTISVDTSKNQFSLNLNSVTAADTAFYYCARDPLFPGRNLLSVFDNWGQGTLVTVSSAS SEQ ID NO: 35 - AJL08 Light TGVHSDIQMTQSPSTLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPRLLIYKASSLESGVPSR FSGSGSGTEFTLTISGLQPDDFANYSCQQYNIYPFTFGQGTKLEIKRT SEQ ID NO: 36 - AJL09 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCSVSGGSISSYYWGWIRQSPGKGLECIGYIYFSGSTSYNP SLKSRVTISVDTAKNQISLNLTSVTAADTAVYFCARVWGSSWYANWFDPWGQGTLVTVSSAS SEQ ID NO: 37 - AJL09 Light TGVHSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQHKPGKAPKLLI FDASTLAAGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYVLTFGQGTRLEIKRT SEQ ID NO: 38 - AJL10 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCSVSGGAVSNYYWSWIRQSAGKGLEWLGRIYINGTTYYNP SLRSRVSMSVDTSKGQFSLRLTSVTAADTAI YYCARWGALLGDYYYGLDVWGQGTTVTVSSAS SEQ ID NO: 39 - AJL10 Light TGVHGDIVMTQSPLSLPVTPGEPASISCRSTQSLLHSNEYIYLDWYVQKPGQSPQLLIFLASNRAS GVPDRFSGSASGTDFTLKISRVEAEDVGVYYCMQALEAPWTFGQGTRLEIKRT SEQ ID NO: 40 - AJL11 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCSVSGGSITSTSYYWGWIRQSPGKGLEWIGSVYYSGNTFY NASLKSRVTISIDTSKYQFSLMLRSVTAADTAVYYCARRHDWFWFDPWGQGTLVTVSSAS SEQ ID NO: 41 - AJL11 Light

[0255] TGVHS E I VLTQS PGTLS LS PGERVTLS CRAS QS VS S S YLAWYQQKPGQAPRLL I YDS S S RATG I PD RFSGSGSGTDFTLTISRLDPEDFAVYYCQQYGSSPSTFGQGTKLEIKRT DBl / 165335339.4 42GNR-007PC / 115835-5007

[0256] SEQ ID NO: 42 - AJL12 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSVTSSDYYWAWIRQPPGKGPEWIGSISNSGNTYY SPSLKSRVSISGDTSKKQFSLNLSSVTDADTAVYYCTRHGHYVSGGLGPWGQGTLVTVSSAS SEQ ID NO: 43 - AJL12 Light

[0257] TGVHS E I VLTQS PGTLS LS PGERATLS CRAS QS VGS Y YLAWYQQKPGQAPRLL I EGAS S RATGTPD RFSGSGSGTDFTLTISKLEPEDFALYYCQQYGPSPWTFGQGTKVEIKRT SEQ ID NO: 44 - AJL13 Heavy TGVHSQVQLQESGPGLVKPSETLSLTCNVSGGSINNYYWSWIRQPPGKGLEWIGYIYYNGNINYNP SLKSRVTISRDMSKNQFSLNLRSVTAADTAVYYCGIGYSAVAAGTVDYWGQGTLVTVSSAS SEQ ID NO: 45 - AJL13 Light TGVHGDWMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLNWFQQRPGQSPRRLIYTVSKRGS GVPDRFSGSGSGTDFTLKISRVEAEDVGDYYCMQGTHWPWTSGQGTRLEIKRT SEQ ID NO: 46 - AJL14 Heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSLSSVNYYWNWIRQPAGKGLEWMGRIYASGYTTY NPSFQSRVTISLDPSKNQISLKVTSLTAADTAIYYCARHDLGHCSSTSCYLSWFDAWGQGTTVTVS SAS SEQ ID NO: 47 - AJL14 Light TGVHSDIVMTQSPDSLAVSLGERATINCRSSQTIFFSPNNNNHLAWYQQKPGQPPRLLI YWASTRE SGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQYYSLPYTFGQGTKLEIKRT SEQ ID NO: 48 - AJL15 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSITSRNNYWGWIRQSPGKGLEWIGSLYYTGSDYY NPSLKSRVTISVDTSKNQFSLRLSSVTAADTAVYYCVRVNVDDFWSGLGGAWFDPWGQGTLVTVSS AS SEQ ID NO: 49 - AJL15 Light TGVHGDIVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTHLYWYLQKPGQSPQSLIYEVSKRFS GVPDRFTGSGSGTDFTLKISRVEAEDVGLYYCMQSAQLPYTFGQGTKLEIKRT SEQ ID NO: 50 - AJL16 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSPYYWGWIRQPPGKGLEWIGSIYYSGHTYY NPSLKSRVTISVDTSKNQFSLRLTSVTAADTSVYYCAKQTDDYGDYASRGWFDPWGQGTLVTVSSA S SEQ ID NO: 51 - AJL16 Light TGVHSDIQMTQSPSSLSASVGDRVTFTCQASHDISNYLNWYQQKPGKVPELLI YDASNLKTGVPSR FSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGQGTRLEIKRT SEQ ID NO: 52 - AJL18 Heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGDSISSDKYYWTWIRQLPGKGLEWIGYISYSGTTYY NPSLKSRVSISVDTSGNQFSLRLSSVTAADTARYYCARVYYDVLSAYYNMGSWVDPWGQGTLVTVS SAS SEQ ID NO: 53 - AJL18 Light TGVHSDIQMTQSPSSLPASVGDRVTITCRASQSISSYLNWYQQKPGRAPKLLIYAASSLQSGVSSR FSGSGSGTDFTLTISSLQPEDSATYYCQQSYSTPWTFGQGTKVEIKRT DBl / 165335339.4 43GNR-007PC / 115835-5007

[0258] SEQ ID NO: 54 - AJL19 Heavy TGVHSQVQLQQWGAGLLKPPETLSLTCAVFGGSLSGYYWSWIRQPPGKGPEWIAEINHSGDANYNP SLKSRVTISVDTSKNQFSLKMSSVTVADTALYYCATQGSRLTTFAFDVWGQGTMVTVSSAS SEQ ID NO: 55 - AJL19 Light TGVHSEIVLTQSPGTLSLSPGERVTLSCRTSQSVSSDSLAWYQQKPGQTPRLLIYHTSTRAAGIPD RFSGTGSGTDFTLTIARLEPEDFAVYYCQHYGRSSLFTFGQGTKLEIKRT SEQ ID NO: 56 - AJL20 Heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSISGSSFYWGWVRQPPGRGLEWIGTIYYRGTTYY TPSLKSRVTISVDTSKNQFSLRLNSVTAADTAI YYCASLPHYDFWSGSVFFDYWGQGTLVTVSSAS SEQ ID NO: 57 - AJL20 Light TGVHSDIQMTQSPSSLSASVGDRVTITCRASQGIANYLAWYQQKPGNIPKLLIYAASTLQSGVPSR FSGSGSGTDFALTI S CLQPEDVATYYCQKYNSAPLTFGGGTKVE I KRT SEQ ID NO: 58 - WR01 heavy TGVHSQVQLQESGPGLVKPSETLSLTCTVSGGSIDTYYWTWIRQPPGKGLEWIGYIYSTGSPKYKP SLKSRWMSVDTSTNEFALRLSSVTAADTAVYYCARSSGFYVEHLEKWGQGTTVTVSSAS SEQ ID NO: 59 - WR01 light

[0259] TGVHSEIVLTQSPATLSLS PGERATLS CRASQTVSSSYLDWFQQKPGQAPRLLIYGASSRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQFATSPYTFGQGTKLEIKRT SEQ ID NO: 60 - WR02 heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYHWSWIRQPPGKGLEWIGNINYNGGAYH NPSLTNRVIMSVDTSKNHFSLKLTSVTAADTAVYYCARESQWLRYGAFGMDVWGQGTTVTVSSAS SEQ ID NO: 61 - WR02 light

[0260] TGVHS E I VLTQS PGTLS LS PGERATLS CRAS QS VS S S HLVWYQQKAGQAPRLVI YGANRRASGTPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIKRT SEQ ID NO: 62 - WR03 heavy TGVHSQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSYWWSWVRQPPGKGLEWVGEIYHSGGANYS PSLKSRVTISVDKSKNQFSLNLISVTAADTAVYFCARSRMLVGADGGGAFDIWGQGTMVTVSSAS SEQ ID NO: 63 - WR03 light

[0261] TGVHS E I VLTQS PGTLS LS PGERATLS CG S QS VS S S YL WYQQKPGLAPRLL I YDAS S RATG I PD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPQTFGQGTKVEIKRT SEQ ID NO: 64 - WR04 heavy TGVHSQLQLQQSGPGLVKPSETLSLTCTVSGGSISSGGSYWGWIRQAPGKGLEWIGSMYYSGSTFY NPSVKSRVTISVDRSKEQFSLNLNAVTAADTAVYYCVRHRRSEPSDSWGQGTLVTVSSAS SEQ ID NO: 65 - WR04 light

[0262] TGVHS E I VMTQS PATLS VS PGERATLS CRAS QS VS SNLAWYQQKPGQAPRLL I YGTS TRATG I PAR FSGSGSGTEFTLTISSLQSEDSAVYFCQQYNNWPLYTFGQGTKVEIKRT SEQ ID NO: 66 - WR05 heavy TGVHSQVQLQESGPGLVKPSETLSLTCSVSGGSVSSNGHFWSWIRLPPGKGLEWIGYVYNTGTSGY SPSLKSRVTISVDTSKNQFSLTLRSVTAADTAI YYCARGLTGNYPSHWGQGTLVTVSSAS DBl / 165335339.4 44GNR-007PC / 115835-5007

[0263] SEQ ID NO: 67 - WR05 light TGVHSDIQMTQSPSSVSASVGDRVTITCRASRGVSTWLAWYQQKPGEAPKLLI YASSRLEGGVPAR FSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPLTFGGGTKVEIKRT SEQ ID NO: 68 - WR06 heavy TGVHSQLQLQESGPGLVKPSETLSLTCTVSGGSVSSSAYWWAWIRQPPGGGLEWIGHIYYFGNKYY KSSLESRVTISLDASQNQFSLKLTSVTAADTALYYCARVDTALAFDFWGQGTMVTVSSAS SEQ ID NO: 69 - WR06 light TGVHSDIQMTQSPSSLSASVGDRVTITCRASQYVSSSLNWYQQKPGKAPTLLI YLASNLRSGVPSR FSGSESGTDFTLTINSLQPEDVATYFCQQSYSLPRTFGPGTKVDIKRT SEQ ID NO: 70 - WR07 heavy TGVHSQLQLQESGPGLVKPSETLSLTCSVSGGSVSSTTYYWGWIRQSPGKGLEWIGSIYHSGKTYY NPSLKSRVTISVDTSKNQFSLNLSSVTAADTAVYYCARENSHHYDSSGYYLGGFDYWGQGTLVTVS SAS SEQ ID NO: 71 - WR07 light

[0264] TGVHS E I VLTQS PGTLS LS PGERATLS CRAS QS VS S S YLAWYQQKPGQAPRLL I YGAS S RATG I PD RFSGSGSGTDFTLTISRLEPEDFAVYFCQHYGSPSTFGQGTKVEIKRT SEQ ID NO: 72 - WR08 heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYWSWIRQHPGKGLDCIGYIHYTGTTYYN PSLKSRLTISVDTSKNQFSLNLTSVTAADTAVYYCAREEYTTSSVDYWGRGTLVTVSSAS SEQ ID NO: 73 - WR08 light TGVHGDWMTQSPLSLPVTLGQPASISCRSSESLVSVDGNTYLNWFHQRPGQSPRRLIYKVSNRDS GVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQATHWLRTFGQGTRLEIKRT SEQ ID NO: 74 - WR09 heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYWSWIRQHPGKGLEWIGYIHYTGTTYYN PSLKSRVTISVDTSKNQFSLNLTSVTAADTAVYYCAREEYTTSSVDYWGRGTLVTVSSAS SEQ ID NO: 75 - WR09 light

[0265] TGVHS E I VLTQS PGTLS LS PGERATLS CRAS QRVS SG FLAWYQQKPGQAPRLL I YGAS S RATG I PD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYESSPSPYNFGQGTKLEIKRT SEQ ID NO: 76 - WR10 heavy TGVHSQVQLQESGPGLVKPSGTLSLTCAVSGGSISNNKWWNWVRQSPGKGLEWIGEIYHSGGTNYN PSLKSRVTISVDKSKNLFSLKLSSVTAADTAVYYCASATTMVRGLSLYYYGLDVWGPGTTVTVSSA S SEQ ID NO: 77 - WR10 light

[0266] TGVHS E I VLTQS PGTLS LS PGERAALS CRAS QS L I GS FLAWYQQKPGQAPRLL I YHTSNRASG I PD RFSGGGFGTDFTLTI SRLE PEDFAVYYCQQYDS SP I TFGQGTRLE IKRT SEQ ID NO: 78 - WR11 heavy TGVHSQVQLQESGPGLVKPSGTLSLTCAVSGGSISNNKWWNWVRQSPGKGLEWIGEIYHSGGTNYN PSLKSRVTISVDKSKNLFSLKLSSVTAADTAVYYCASATTMVRGLSLYYYGLDVWGQGTTVTVSSA S

[0267] DBl / 165335339.4 45GNR-007PC / 115835-5007

[0268] SEQ ID NO: 79 - WR11 light

[0269] TGVHS E I VLTQS PGTLS LS PGERATLS CRAS QS VS S FLAWYQQKPGQAPRLL I YDASNRATG I PAR FSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPPSLTFGGGTKVEIKRT SEQ ID NO: 80 - WR12 heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGDSVSSNDHYWSWIRQPPGQGLEWIGYISHGGTTYY NPSLKSRVTMSIDTSTNQFSLRVTSVRAADMAVYFCARAPAPITTFGMVTPVPYFHSWGQGTLVTV SSAS SEQ ID NO: 81 - WR12 light TGVHGDIVMTQSPLSLPVSPGEPASISCRSSQSLLHSNGYNYLSWYLQKPGQSPQLLIFSSSIRAS GVPDRFSGSGSGTDFTLTINRVEAEDVGVYYCMQALQTPLTFGGGTKLEIKRT SEQ ID NO: 82 - WR13 heavy TGVHSQVQLQESGPGLVKPSQTLSLTCTVSGDSVSSNDHYWSWIRQPPGQGLEWIGYISHGGTTYY NPSLKSRVTMSIDTSTNQFSLRVTSVRAADMAVYFCARAPAPITTFGMVTPVPYFHSWGQGTLVTV SSAS SEQ ID NO: 83 - WR13 light TGVHGDIVMTQSPLSLPVSPGESASISCRSSQSLLHSNGYNYLSWYLQKPGQSPQLLIFSSSIRAS GVPDRFSGSGSGTDFTLTINRVEAEDVGVYYCMQALQTPLTFGQGTKLEIKRT SEQ ID NO: 84 - cathepsin L-specific tetrapeptide linker

[0270] GGFG SEQ ID NO: 85 - polyarginine CCP

[0271] RRRRRRRRR SEQ ID NO: 85 - HIV-1 Tat peptide

[0272] GRKKRRQRRRPPQY SEQ ID NO: 87 - influenza virus hemagglutinin HA2 GLFEAIAGFIENGWEGMIDGGGYC SEQ ID NO: 88 - influenza virus IFN-7

[0273] GL FEAI EGF I ENGWEGM I DGWYG SEQ ID NO: 89 - GALA peptide

[0274] WEAALAEALAEALAEHLAEALAEALEALAA SEQ ID NO: 90 - Aurein 1.2 peptide

[0275] GLFDI IKKIAESF SEQ ID NO: 91 - polyhistidine peptide

[0276] HHHHHHHHHH SEQ ID NO: 92 - Drosophila Antennapedia penetratin peptide RQIKIWFQNRRMKWKK SEQ ID NO: 93 - Apis melifera melittin peptide

[0277] G I GAVLKVLTTGL PAL I S W I KRKRQQ

[0278] DBl / 165335339.4 46GNR-007PC / 115835-5007

[0279] SEQ ID NO: 94 - Human protamine 1 (PRM1) MA YRCCRSQSRSRYYRQRQRSRRRRRRSCQTRRRAMRCCRPRYRPRCRRH SEQ ID NO: 95 - Salmine (salmon protamine)

[0280] M PRRRRS S S RPVRRRRR PR VS RRRRRRGGRRRR SEQ ID NO: 96 - Low molecular weight protamine (LMWP) VSRRRRRRGGRRRR SEQ ID NO: 97 - F5-P Fusion Fragment RSQSRSRYYRQRQRSRRRRRRSSSCQTRRRAMRCCRPRYRPRCRRH

[0281] DB1 / 165335339.4 47

Claims

GNR-007PC / 115835-5007CLAIMS1. An antibody comprising a heavy chain and a light chain, wherein the heavy chain is a VH4 heavy chain with complementarity determining regions (CDRs) having at least two codon mutations at positions selected from 31B, 32, 40, 56, 57, 60, 81, and 89 with respect to the germline sequence, and wherein the antibody comprises an Fc region having one or more transport mutations that enhance penetration of the blood brain barrier (BBB) and / or enhance internalization of neurons.

2. The antibody of claim 1, wherein the heavy chain comprises a set of heavy chain CDRs of any one of the heavy chain variable regions of SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

3. The antibody of claim 2, wherein the heavy chain comprises a variable region sequence selected from SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, or a variant thereof.

4. The antibody of any one of claims 1 to 3, wherein the light chain comprises a set of light chain CDRs of any one of the light chain variable regions of SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.

5. The antibody of claim 4, wherein the light chain comprises a variable region sequence selected from SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83, or a variant thereof.

6. The antibody of claim I, wherein the heavy chain comprises a set of heavy chain CDRs defined by SEQ ID NOS: 2 to 4.

7. The antibody of claim 6, the heavy chain comprises a variable region sequence of SEQ ID NO: 1.

8. The antibody of claim 6 or 7, wherein the light chain comprises a set of light chain CDRs defined by SEQ ID NOS: 6 to 8.DBl / 165335339.4 48GNR-007PC / 115835-50079. The antibody of claim 8, wherein the light chain comprises a variable region sequence of SEQ ID NO: 5.

10. The antibody of any one of claims 1 to 9, wherein the antibody is an IgG isotype.

11. The antibody of claim 10, wherein the antibody is IgGl or IgG4 isotype.

12. The antibody of any one of claims 1 to 11, wherein the one or more transport mutations that enhance penetration of the blood brain barrier (BBB) increase receptor-mediated transcytosis.

13. The antibody of claim 12, wherein the one or more mutations comprises a tryptophan at a position corresponding to position 388 of IgGl amino acid sequence, and an aromatic residue at a position corresponding to position 421 of IgGl amino acid sequence.

14. The antibody of claim 13, wherein the one or more amino acid mutations comprises mutation at one or more positions corresponding to positions 380, 384, 386, 387, 389, 390, 392, 413-416, 424, and 426 of IgGl amino acid sequence.

15. The antibody of claim 14, wherein the one or more amino acid mutations comprises mutation at positions corresponding to positions 384, 386, 387, 389, 390, 413, 415, and 416 of IgGl amino acid sequence.

16. The antibody of any one of claims 13 to 15, wherein the one or more amino acid mutations comprises an amino acid residue corresponding to one or more of E380W, E380L, N384L, N384Y, Q386H, Q386T, P387V, P387E, E388W, N389A, N389S, N390V, N390S, K392R, D413P, D413T, K414R, S415E, R416T, R416E, N421W, N421F, S424T, and S426G with respect to IgGl amino acid sequence.

17. The antibody of any one of claims 12 to 16, wherein the one or more mutations are monovalent.

18. The antibody of claim 17, wherein the antibody comprises one or more knob-in-hole (KIH) mutations.

19. The antibody of any one of claims 1 to 18, wherein the antibody further comprises one or more mutations that abrogate FcyR binding.DBl / 165335339.4 49GNR-007PC / 115835-500720. The antibody of claim 19, wherein the antibody comprises a mutation corresponding to L234A and L235A with respect to an IgGl amino acid sequence, and optionally P329G.

21. The antibody of any one of claims 1 to 20, wherein the antibody comprises one or more mutations that increase circulatory half-life.

22. The antibody of claim 21, wherein the one or more mutations that increase serum half-life comprises a YTE mutation.

23. The antibody of any one of claims 1 to 22, having a first heavy chain with a constant region comprising an amino acid sequence selected from SEQ ID NOs: 12, 14, and 18.

24. The antibody of claim 23, having a second heavy chain with a constant region comprising an amino acid sequence selected from SEQ ID NOs: 13, 15, and 19.

25. The antibody of any one of claims 1 to 24, wherein antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, wherein the LALAPG and YTE mutations are optional; and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the LALPG and YTE mutations are optional.

26. The antibody of claim 25, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 11.

27. The antibody of any one of claims 1 to 26, wherein the antibody is conjugated to a therapeutic moiety.

28. The antibody of claim 27, wherein the therapeutic moiety is conjugated to the antibody through a linker, which is optionally a cleavable or hydrolysable linker.

29. The antibody of claim 27 or 28, wherein the therapeutic moiety is one or more of a nucleic acid, small molecule, peptide, protein, reactive species, or radionuclide.

30. The antibody of claim 29, wherein the nucleic acid is an RNA, and which is optionally a siRNA, shRNA, or miRNA.

31. The antibody of claim 30, wherein the therapeutic moiety is an antisense nucleic acid.DBl / 165335339.4 50GNR-007PC / 115835-500732. The antibody of claim 30 or 31, wherein the nucleic acid reduces or ablates expression or one or more endogenous neural cell proteins, or induces skipping of one or more exons of a target pre-mRNA.

33. The antibody of claim 29, wherein the therapeutic moiety is an enzyme.

34. The antibody of claim 29, wherein the small molecule is a cytotoxic agent, cytoprotective agent, or anticancer agent.

35. The antibody of claim 34, wherein the cytoprotective agent comprises a neuroprotective agent or neurological disease therapeutic.

36. The antibody of claim 29, wherein the reactive species comprises nitric oxide (NO donor) or reactive oxygen species (ROS)-generating molecule.

37. The antibody of any one of claims 27 to 36, wherein the therapeutic moiety to antibody ratio (DAR) is at least 1, at least 2, at least 3, or at least 4.

38. An antibody-drug conjugate, comprising:a. an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH is a VH4 heavy chain with complementarity determining regions (CDRs) having at least two codon mutations at positions selected from 3 IB, 32, 40, 56, 57, 60, 81, and 89 with respect to the germline sequence, or an antigen-binding portion thereof; and b. a therapeutic moiety conjugated to the antibody, optionally through a linker.

39. The antibody-drug conjugate of claim 38, wherein the heavy chain comprises a set of heavy chain CDRs of any one of SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82.

40. The antibody-drug conjugate of claim 39, wherein the heavy chain comprises a variable region sequence selected from SEQ ID NOs: 1, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82, or a variant thereof.

41. The antibody-drug conjugate of any one of claims 38 to 40, wherein the light chain comprises a set of light chain CDRs of any one of the light chain variable regions of DBl / 165335339.4 51GNR-007PC / 115835-5007SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.

42. The antibody-drug conjugate of claim 41, wherein the light chain comprises a variable region sequence selected from SEQ ID NOs: 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83, or a variant thereof.

43. The antibody-drug conjugate of claim 41, wherein the heavy chain comprises a set of heavy chain CDRs defined by SEQ ID NOS: 2 to 4.

44. The antibody-drug conjugate of claim 43, wherein the heavy chain comprises a variable region sequence of SEQ ID NO: 1.

45. The antibody-drug conjugate of claim 43 or 44, wherein the light chain comprises a set of light chain CDRs defined by SEQ ID NOS: 6 to 8.

46. The antibody-drug conjugate of claim 45, wherein the light chain comprises a variable region sequence of SEQ ID NO: 5.

47. The antibody-drug conjugate of any one of claims 38 to 46, wherein the antibody is a Fab fragment.

48. The antibody-drug conjugate of any one of claims 38 to 47, wherein the antibody is an IgG isotype.

49. The antibody-drug conjugate of claim 48, wherein the antibody is IgGl or IgG4 isotype.

50. The antibody-drug conjugate of claim 48 or 49, wherein the Fc region comprises one or more transport mutations that enhance penetration of the blood brain barrier (BBB), which optionally increase receptor-mediated transcytosis and / or internalization by neurons.

51. The antibody-drug conjugate of claim 50, wherein the one or more mutations comprises a tryptophan at a position corresponding to position 388 of IgGl amino acid sequence and an aromatic residue at a position corresponding to position 421 of IgGl amino acid sequence.DBl / 165335339.4 52GNR-007PC / 115835-500752. The antibody-drug conjugate of claim 51, wherein the one or more mutations comprise mutation at one or more positions corresponding to positions 380, 384, 386, 387, 389, 390, 392, 413-416, 424, and 426 of an IgGl amino acid sequence.

53. The antibody-drug conjugate of claim 52, wherein the one or more mutations comprise mutation at one or more positions corresponding to positions 384, 386, 387, 389, 390, 413, 415, and 416 of an IgGl amino acid sequence.

54. The antibody-drug conjugate of any one of claims 50 to 53, wherein the one or more mutations comprises an amino acid residue corresponding to one or more of E380W, E380L, N384L, N384Y, Q386H, Q386T, P387V, P387E, E388W, N389A, N389S, N390V, N390S, K392R, D413P, D413T, K414R, S415E, R416T, R416E, N421W, N421F, S424T, and S426G with respect to an IgGl amino acid sequence.

55. The antibody-drug conjugate of any one of claims 50 to 54, wherein the one or more mutations are monovalent.

56. The antibody-drug conjugate of claim 55, wherein the antibody comprises one or more knob-in hole (K1H) mutations.

57. The antibody-drug conjugate of any one of claims 50 to 56, wherein antibody further comprises one or more mutations that abrogate FcyR binding.

58. The antibody-drug conjugate of claim 57, wherein the antibody comprises a mutation corresponding to L234A and L235A with respect to IgGl sequence, and optionally P329G.

59. The antibody-drug conjugate of any one of claims 50 to 58, wherein the antibody comprises one or more mutations that increase circulatory half-life.

60. The antibody-drug conjugate of claim 59, wherein the one or more mutations that increase serum half-life comprises a YTE mutation.

61. The antibody-drug conjugate of claim 50, having a first heavy chain with a constant region comprising an amino acid sequence selected from SEQ ID NOs: 12, 14, and 18.

62. The antibody-drug conjugate of claim 61, having a second heavy chain with aDBl / 165335339.4 53GNR-007PC / 115835-5007constant region comprising an amino acid sequence selected from SEQ ID NOs: 13, 15, and 19.

63. The antibody-drug conjugate of claim 50, wherein antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, wherein the LALAPG and YTE mutations are optional; and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the LALPG and YTE mutations are optional.

64. The antibody-drug conjugate of claim 63, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 11.

65. The antibody-drug conjugate of any one of claims 38 to 64, wherein the therapeutic moiety is conjugated to the antibody through a linker, which is optionally a cleavable or hydrolysable linker.

66. The antibody-drug conjugate of claim 65, wherein the linker comprises an amide- conjugated linker, a site-selective linker, a click chemistry-conjugated linker, a pH- sensitive dissociation linker, a redox-sensitive linker, a protease-cleavable linker, or an electrostatic release linker.

67. The antibody-drug conjugate of claim 65 or 66, wherein the linker is suitable for release of the therapeutic moiety under acidic conditions, optionally under conditions within the endosome or lysosome.

68. The antibody-drug conjugate of any one of claims 65-67, wherein the linker is suitable for conjugation or association with a nucleic acid therapeutic moiety.

69. The antibody-drug conjugate of any one of claims 65-68, wherein the linker is an electrostatic release linker comprising protamine, or a protamine-derived peptide.

70. The antibody-drug conjugate of any one of claims 38 to 69, wherein the therapeutic moiety is one or more of a nucleic acid, small molecule, peptide, protein, reactive species, or radionuclide.

71. The antibody-drug conjugate of claim 70, wherein the therapeutic moiety is an RNA, and which is optionally a siRNA, shRNA, or miRNA.DBl / 165335339.4 54GNR-007PC / 115835-500772. The antibody-drug conjugate of claim 70, wherein the therapeutic moiety is an antisense nucleic acid.

73. The antibody-drug conjugate of claim 71 or 72, wherein the nucleic acid reduces or ablates expression or one or more endogenous neural cell proteins, or induces skipping of one or more exons of a target pre-mRNA.

74. The antibody-drug conjugate of claim 70, wherein the therapeutic moiety is an enzyme.

75. The antibody-drug conjugate of claim 70, wherein the small molecule is a cytotoxic agent, cytoprotective agent, or anticancer agent.

76. The antibody-drug conjugate of claim 75, wherein the cytoprotective agent comprises a neuroprotective agent or neurological disease therapeutic.

77. The antibody-drug conjugate of claim 70, wherein the reactive species comprises nitric oxide (NO donor) or reactive oxygen species (ROS)-generating molecule.

78. The antibody-drug conjugate of any one of claims 38 to 77, wherein the therapeutic moiety to antibody ratio (DAR) is at least 1, at least 2, at least 3, or at least 4.

79. The antibody-drug conjugate of any one of claims 38 to 78, wherein the antibodydrug conjugate further comprises an endosomal escape element.

80. The antibody-drug conjugate of claim 79, wherein the endosomal escape element comprises a polypeptide, a small molecule, or a pH-sensitive lipid.

81. The antibody-drug conjugate of any one of claims 38-80, wherein the antibody-drug conjugate further comprises an amino acid linker, optionally between an antibody domain and an endosomal escape element and / or between an antibody domain the therapeutic moiety, and optionally wherein the amino acid linker is fused at the C- terminus of one or more antibody heavy chain and / or light chain.

82. A method for treating a neurological disorder, comprising administering an effective amount of the antibody of any one of claims 1 to 37 or an effective amount of the antibody-drug conjugate of any one of claims 38 to 81, to a subject in need.

83. The method of claim 82, wherein the neurological disorder is a neurodegenerativeDBl / 165335339.4 55GNR-007PC / 115835-5007disease, a monogenic disease impacting the central nervous system (CNS), traumatic injury, central nervous system (CNS) damage, or peripheral nerve damage.

84. The method of claim 83, wherein the neurological disorder is Multiple Sclerosis (MS), clinically isolated syndrome (CIS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Progressive supranuclear palsy, Friedreich ataxia, Lewy body disease, spinocerebellar ataxia, or Spinal muscular atrophy.

85. The method of claim 83, wherein the neurological disorder is a lysosomal storage disease.

86. The method of claim 83, wherein the neurological disorder comprises ischemic reperfusion injury.

87. The method of any one of claims 83 to 86, wherein the antibody or antibody-drug conjugate is administered parenterally.

88. The method of claim 87, wherein the administration is by subcutaneous, intravenous, intracerebroventricular, intraparenchymal, intraarterial, intraventricular, or intrathecal routes.

89. The method of any one of claims 82 to 88, wherein the antibody or antibody-drug conjugate is administered a plurality of times at a frequency of about daily, about twice weekly, about weekly, about twice monthly, about monthly, about once every two months, or about once every three months.

90. The method of claim 89, wherein the is administered upon symptoms or attack of the neurodegenerative disease, or during recovery from the attack.DBl / 165335339.4 56

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