Species cross-reactive antibodies capable of crossing the blood-brain barrier (BBB)

Abstract

The present disclosure relates to the field of species cross-reactive antibodies that cross the blood brain barrier (BBB) and related compositions and methods.

Description

SPECIES CROSS-REACTIVE ANTIBODIES CAPABLE OF CROSSING THE BLOODBRAIN BARRIER (BBB)CROSS-REFERENCE TO RELATED APPLICATIONS|0001] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 778,034, filed March 26, 2025, the disclosure of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AG080016 awarded by tire National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The text of the computer readable sequence listing filed herewith, titled “44699-601 SEQUENCE LISTING’’, created March 26, 2026, having a file size of 53.236 bytes, is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The disclosure relates to the field of species cross-reactive antibodies that cross the blood brain barrier (BBB) and related compositions and methods.BACKGROUND

[0005] Antibody-based therapies have shown significant promise for treating a range of diseases, particularly those affecting the central nervous system (CNS). However, the blood-brain barrier (BBB) poses a significant challenge for delivering therapeutic antibodies to the brain. Many current antibody therapies fail to cross the BBB, limiting their effectiveness in treating neurological conditions. Additionally, multiple different antibodies are often needed for pretesting in preclinical animal models and eventually human subjects, further complicating the clinical testing process. These limitations create substantial delays and increased costs in the drug development pipeline, particularly when dealing with complex murine disease models.SUMMARY

[0006] Provided herein are species cross-reactive antibodies that cross the blood brain barrier (BBB) and related compositions and methods.

[0007] In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2. Insome embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-H3 having a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9. SEQ ID NO. 10. SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.

[0008] In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity' to SEQ ID NO. 15, SEQ ID NO. 16. SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19. SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

[0009] In some embodiments of the species cross-rcactivc antibody herein, the at least 70% sequencing identity comprises at least 80% sequence identity. In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 90% sequence identity. In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 95% sequence identity. In some embodiments, the CDR sequences have one, two, three, four, five, six, seven, or eight amino acids substitutions relative to the recited sequences.

[0010] In some embodiments the cross-reactive antibody comprises a heavy chain variable region that includes CDR-H1. CDR-H2. and CDR-H3.

[0011] In some embodiments of the species cross-reactive antibody herein, the heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity' to SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10. SEQ ID NO.11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70%sequence identity to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7. SEQ ID NO. 8, SEQ ID NO. 9, SEQ IDNO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.|0012] In some embodiments the species cross-reactive antibody comprises a light chain variable region that includes CDR-L1, CDR-L2, and CDR-L3.

[0013] In some embodiments of the species cross-reactive antibody herein, the light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20. SEQ ID NO. 21, or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity- to SEQ ID NO. 15. SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

[0014] In some embodiments of the species cross-reactive antibody herein, the said species cross-reactive antibody has a heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity- to SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11. SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO.6. SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 and a light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17. SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18. SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

[0015] In some embodiments of the species cross-reactive antibody herein, the said species cross-reactive antibody has a heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 and a light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO.15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19. SEQ ID NO. 20, SEQ ID NO.21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25.|0016] In some embodiments the species cross-reactive antibody comprises:a) a heavy chain amino acid sequence of SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO.28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO.33. SEQ ID NO. 34 or SEQ ID NO. 35; andb) a light chain amino acid sequence of SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41. SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44 or SEQ ID NO. 45.

[0017] In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising an amino acid sequence selected from SEQ ID NO. 46. SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50. SEQ ID NO. 51. SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54 or SEQ ID NO. 55.

[0018] In some embodiments, the species cross-reactive antibody is a non -targeted antibody.

[0019] In some embodiments, the non-targeted antibody is an IgG antibody.

[0020] In some embodiments, the species cross-reactive antibody comprises one or more single-chain variable fragments (scFvs).

[0021] In some embodiments, the species cross-reactive antibody is functional (e.g., crosses the blood brain barrier and / or binds to one or more target molecules in the brain) in multiple (e.g., 2, 3. 4, 5, etc.) different species.

[0022] In some embodiments, the species cross-reactive antibody is functional (e.g., crosses the blood brain barrier and / or binds to one or more target molecules in the brain) in mammals.

[0023] In some embodiments, the mammal is a human.

[0024] In some embodiments, the species cross-reactive antibody is functional in non-hmnan mammals.

[0025] In some embodiments, the non-human mammal is a mouse.

[0026] In some embodiments, the non-human mammal is a cynomolgus monkey .

[0027] In some embodiments, the species cross-reactive antibody specifically binds to CD98hc.

[0028] In some embodiments, the species cross-reactive antibody is a bispecific antibody.

[0029] In some embodiments, the bispecific antibody binds to a protein on / or in a neuron. In some embodiments, the neuron is associated with a neurological disease and / or condition. In some embodiments, the neurological disease and / or condition is Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS). Huntington’s disease, multiple sclerosis (MS), frontotemporal dementia (FTD), Lewy body dementia (LBD), neuromyelitis optica spectrum disorder (NMOSD), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), stroke, cerebral ischemia, brain microvascular dysfunction, vascular dementia, schizophrenia,intellectual disabilities, autism spectrum disorder (ASD), depression, anxiety disorders, bipolar disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), myasthenia gravis (MG), peripheral neuropathy, glioblastoma (GBM), and other brain tumors such as astrocytomas, meningiomas, and medulloblastomas.[0030| In some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising the species cross-reactive antibody and a pharmaceutically acceptable carrier.[0031| In some embodiments, the presently disclosed subject matter provides the use of a species cross-reactive antibody. For example, in some embodiments, the presently disclosed subject matter provides the use of a species cross-reactive antibody to treat a neurological disease and / or condition or to improve a neurological function.

[0032] In some embodiments, the presently disclosed subject matter provides a method for treating a neurological disease and / or condition or improving a neurological function comprising administering the species cross-reactive antibody to a subject.

[0033] In some embodiments, the neurological disease and / or condition is Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease, multiple sclerosis (MS), frontotemporal dementia (FTD), Lewy body dementia (LBD), neuromyelitis optica spectrum disorder (NMOSD), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), stroke, cerebral ischemia, brain microvascular dysfunction, vascular dementia, schizophrenia, intellectual disabilities, autism spectrum disorder (ASD), depression, anxiety disorders, bipolar disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), myasthenia gravis (MG), peripheral neuropathy, glioblastoma (GBM), and other brain tumors such as astrocytomas, meningiomas, and medulloblastomas.

[0034] In some embodiments, the species cross-reactive antibody is administered intravenously.

[0035] In some embodiments, the species cross-reactive antibody is co-administered with a neuroprotective agent, an anti-inflammatory agent, or a small-molecule therapeutic agent (e.g., for enhanced efficacy).DEFINITIONS

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] The terms “comprise(s).” “include(s),” “having.” “has,” “can.” "contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do notpreclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.[0038| For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4. 6.5. 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0039] The terms “antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g.. which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog. a rat. a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”). single chain antibodies, single domain antibodies. Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti -Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them arc described in Wu, C., ct al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by¬ reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte -binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM. IgD, IgA. and IgY), class (for example, IgGl. IgG2. IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-[analyte] antibody” or merely an “| analyte | antibody”.

[0040] The term “antibody fragment” as used herein refers to a portion of an intact antibody that retains the ability to specifically bind to an antigen (see. generally. Holliger et al.. Nat. Biotech.. 23(9): 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may or may not include the constant heavy chain domains (e.g., CH2. CH3. or CH4. depending on theantibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the 3 complementarity determining regions (CDRs) of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0041] Typically, an immunoglobulin or antibody is a protein that comprises at least one CDR. The CDRs form the “hypervariable region” of an antibody that is responsible for antigen binding. A whole antibody typically consists of 4 polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (X) based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0042] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising 4 framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the [3 sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York. N.Y. (2001)).

[0043] The term "CDR” is used herein to refer to the “complementarity' detennining region” within an antibody variable sequence. There are 3 CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N -terminus of a heavy or light chain, these regions are denoted “CDR1”, "CDR2”, and “CDR3” for each of the variable regions. The term “CDR set” as used herein refers to a group of 3 CDRs that occur in a single variable region that binds the antigen. An antigenbinding site, therefore, may include 6 CDRs comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR. (e.g., a CDR1, CDR2. or CDR3) may be referred to as a “molecular recognition unit.”

[0044] Crystallographic analyses of antigen-antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health. Bethesda. Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the 3 CDRs. These CDRs may be referred to as "Kabat CDRs”. Chothia and coworkers (Chothia and Lesk, J. Mol Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as "LI.” "L2,” and “L3,” or "Hl.” “H2,” and “H3,” where the "L” and the "H” designate the light chain and the heavy chain regions, respectively. These regions may be referred to as "Chothia CDRs.” which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan. FASEB J., 9: 133-139 (1995). and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDR boundary’ definitions may not strictty follow one of the herein systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in view of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may use CDRs defined according to any of these systems, although certain aspects use Kabat- or Chothia-defined CDRs.

[0045] As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds the antigen or epitope with affinity that is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity that is substantially higher” means affinity that is high enough to permit detection of an antigen or epitope that is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 107M ’(e.g., >10'M1. >108M , >109M4, >1010M4, >1011M1, >1012M1, >1013M-1, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

[0046] The term “affinity” refers to the strength of the sum total of noncovalent interactions betw een a single binding site of a molecule (e.g.. an antibody) and its binding partner (e.g.. an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity’ that reflects a1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be represented by the dissociation constant (KD). Affinity may be measured by methods known in the art including those described herein. Specific illustrative and exemplar}' embodiments for measuring binding affinity are described in the following.[0047| The term “heavy chain ammo acid sequence” as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0048] The term “heavy chain variable region” as used herein refers to a region comprising at least three heavy chain CDRs. In some embodiments, the heavy chain variable region includes the three CDRs and at least FR2 and FR3. In some embodiments, the heavy chain variable region includes at least heavy chain CDR1, framew ork (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of a FR1 and / or at least a portion of a FR4.

[0049] The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CHI, CH2, and CH3. Non-function-altering deletions and alterations within the domains are encompassed within the scope of the term “heavy chain constant region,” unless designated otherwise. Nonlimiting exemplary heavy chain constant regions include y, 5, and a, and variants thereof that do not alter the functions of the antibody needed for its intended use. Nonlimiting exemplar}' heavy' chain constant regions also include E and p, and variants thereof that do not alter the functions of the antibody needed for its intended use. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a y constant region is an IgG antibody, an antibody comprising a 5 constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Further, an antibody comprising a p constant region is an IgM antibody, and an antibody comprising an s constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to. IgGl (comprising a yl constant region). IgG2 (comprising a y2 constant region), IgG3 (comprising a y3 constant region), and IgG4 (comprising a y4 constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an al constant region) and IgA2 (comprising an a2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgMl and IgM2.

[0050] The term “light chain amino acid sequence” as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0051] The term “light chain variable region’’ as used herein refers to a region comprising at least three light chain CDRs. In some embodiments, the light chain variable region includes the three CDRs and at least FR2 and FR3. hi some embodiments, the light chain variable region includes at least light chain CDR1, framework (FR) 2. CDR2, FR3, and CDR3. For example, a light chain variable region may comprise light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a light chain variable region also comprises at least a portion of a FR1 and / or at least a portion of a FR4.[0052 | The term “light chain constant region” as used herein refers to a region comprising a light chain constant domain, CL. Nonlimiting exemplary light chain constant regions include X and K, and variants thereof that do not alter the functions of the antibody needed for its intended use.

[0053] The “% sequence identity” of an amino acid sequence refers to the degree of identical amino acids when tw o or more amino acid sequences for comparison are optimally aligned. The identity of an amino acid sequence can be calculated using analysis tools that are commercially available or available through telecommunication lines (internet). For example, the identity may be calculated using the commercially available software GENETYX (Genetyx Corporation) or using default parameters in the homology algorithm BLAST (Basic Local Alignment Search Tool) (littp: / / www. ncbi.nhn.nih.gov / BLAST / ) of the National Centre for Biotechnology7Information (NCBI). As used herein, and throughout, “at least 70%” sequence identity' comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity', or values therein between.

[0054] The terms “sample” and “biological sample” are used in their broadest sense and encompass samples or specimens obtained from any source that contains or may contain eukaryotic cells, including biological and environmental sources. As used herein, the term “sample" when used to refer to biological samples obtained from organisms, includes bodily fluids (e.g., blood or saliva), feces, biopsies, swabs (e.g., buccal swabs), isolated cells, exudates, and the like. The organisms include fungi, plants, animals, and humans. However, these examples are not to be construed as limiting the types of samples or organisms that find use with the present invention. In addition, in order to perform research or study the results related to use of a method or composition of the invention, in some embodiments, a "sample” or “biological sample” comprises fixed cells, treated cells, cell lysates, and the like. Samples also include environmental samples.

[0055] The term “variant” is used herein to describe a peptide or polypeptide that differs in amino acid sequence by' the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity'. Representative examples of “biological activity” include the ability' to bind to another molecule. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid w ith a different amino acid of similar properties (e.g., hy drophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. “Variant” also can be used todescribe a polypeptide or a fragment thereof that has been differentially processed, such as by¬ proteolysis, phosphorylation, or other post-translational modification, yet retains its antigen reactivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0057] FIG 1. shows purification and characterization of CD98hc variants from different species. (A) SDS-PAGE analysis of CD98hc variants following purification via Ni-NTA affinity chromatography and SEC. Lane order: mouse CD98hc. human CD98hc, cyno CD98hc. human CD98hc with a FLAG tag, mouse CD98hc loop 1 mutant, human CD98hc loop 1 mutant, cyno CD98hc loop 1 mutant, mouse CD98hc loop 2 mutant, human CD98hc loop 2 mutant, cyno CD98hc loop 2 mutant.(B-K) SEC profiles of CD98hc variants: (B) human CD98hc, (C) human CD98hc loop 1 mutant, (D) human CD98hc with a FLAG tag, (E) human CD98hc loop 2 mutant, (F) mouse CD98hc, (G) mouse CD98hc loop 1 mutant, (H) mouse CD98hc loop 2 mutant, (I) cyno CD98hc, (J) cyno CD98hc loop 1 mutant, (K) cyno CD98hc loop 2 mutant. Black curves represent samples purified using protein A beads, whereas red curves denote samples subjected to additional purification by SEC.

[0058] FIG 2. shows an overview of the active learning approach for evolving CD98hc antibodies with broad species cross-reactivity. A lead antibody that recognizes human and cyno CD98hc - but fails to recognize mouse CD98hc - was first analyzed using single histidine mutational scanning in HCDR3 to identify permissive sites that retained at least 50% of wild-type binding despite the substitution mutations. This revealed ten permissive sites that were subsequently diversified using soft randomization, the resulting antibody library w as displayed on the surface of yeast, and it was sorted by FACS to enrich for variants that bound human and mouse CD98hc. Next, the enriched libraries were deep sequenced, and a second-generation HCDR3 library with reduced diversity was designed with predicted improvements in species cross reactivity. In parallel, two additional libraries were designed by diversifying sites close to the antigen, one in which HCDR1 / HCDR2 were diversified and the other in w hich LCDR1 / LCDR3 w ere diversified. After enriching the three libraries for binding to both mouse and human CD98hc, the libraries w ere shuffled together, further enriched for species cross-reactivity-, and Sanger sequenced to identify antibody variants with similar affinities for mouse, cyno, and human CD98hc.

[0059] FIG 3. shows generation and sorting of the mutant HCDR3 library for antibody binding to human and mouse CD98hc. (A) Human CD98hc binding results for single histidine HCDR3 mutants of the wild-type (WT) antibody. The antibodies were displayed on yeast as single-chain variable fragments (scFvs), and variants that were tolerant to histidine mutations - those that retained at least 50% of WT binding - are highlighted in red. (B) Crystal structure of the WT antibody with HCDR3 residues that are tolerant to mutations highlighted in red relative to those that are not highlighted in blue. (C) Progressive sorts of the yeast-displayed HCDR3 library for binding to bothmouse and human CD98hc. The library was sorted by FACS using 300 nM mouse and human CD98hc ectodomains, and the percentage of yeast cells collected in each sort is shown in the upper right quadrant of the cytograms. hi (A), the human CD98hc concentration was 100 nM, the normalized data are averages of three independent experiments, and the errors are standard deviations. In (B), the crystal structure is from the Protein Data Bank (7DF1).

[0060] FIG 4. shows the initial sorting campaign of the HCDR3 library. The initial HCDR3 library was enriched through 3 rounds of magnetic-activated cell sorting (MACS) using 300 nM mouse CD98hc, followed by 2 rounds of fluorescence-activated cell sorting (FACS) for either mouse or human CD98hc. In each FACS sort, the library was measured for scFv display on the surface of yeast (x-axis) and antibody binding to CD98hc (y-axis). Cells with high expression and high CD98hc binding were collected, and the rest of the library w as discarded.

[0061] FIG 5. shows the antibody library profiling using deep sequencing identifies optimal HCDR3 residues for the design of a second-generation library against human and mouse CD98hc. (A) Results of selected library clones binding to mouse and human CD98hc (300 nM). Purple clones showed the best binding to both mouse and human CD98hc, and gray clones primarily bound either mouse or human CD98hc or did not bind either protein. (B) Receiver operating characteristic (ROC) curves illustrate the performance of four scoring metrics in characterizing the input library (third-round MACS-enriched HCDR3 library) and the first-round FACS-enriched HCDR3. The metrics evaluated include frequency, amino acid enrichment ratio, position-specific scoring matrices (PSSM), and position-specific enrichment ratio matrices (PSERM). (C) A table summarizing the PSERM scores for the amino acids at each mutated position in the HCDR3 library. Red-filled boxes denote amino acids enriched in the first-round FACS-enriched library, while blue-filled boxes indicate those that were depleted. Amino acids selected for redesigning the HCDR3 library are marked with black circles, and WT amino acids are highlighted with red stars. (D) Distributions of average PSERM scores of sequences sampled from the random library (with 20 amino acids randomly selected at each position, shown in red) and the redesigned library (shown in blue). In (A), the data are averages of three independent experiments, and the errors are standard deviations.

[0062] FIG 6. shows that deep sequencing leads to identification of optimal residues by PSERM scores based on second-round FACS against human and mouse CD98hc. (A) Receiver operating characteristic (ROC) curves demonstrating the performance of four scoring metrics in evaluating the third-round MACS-enriched HCDR3 library and the second-round FACS-enriched HCDR3 library. The metrics assessed include frequency analysis, amino acid enrichment ratio, position-specific scoring matrices (PSSM), and position-specific enrichment ratio matrices (PSERM). (B) A table summarizing the PSERM scores for each amino acid at every mutated position in the HCDR3 library'. Red-filled boxes denote amino acids enriched in the second-round FACS-enriched library, while blue-filled boxes indicate those not enriched. Amino acids selected for redesigning the HCDR3 library are marked withblack circles, and wild-tj pe amino acids are highlighted with red stars. (C) Distributions of average PSERM scores (based on second-round FACS against human and mouse CD98hc) of sequences sampled from the random library (with 20 amino acids randomly selected at each position, shown in red) and the redesigned library (shown in blue).

[0063] FIG 7. shows the affinity maturation via CDR swapping mutagenesis leads to the identification of antibodies with broad CD98hc cross-reactivity. (A) The redesigned HCDR3 library -along with HCDR1 / HCDR2 and LCDR1 / LCDR3 libraries- were enriched for binding to both mouse and human CD98hc (30-100 nM) via FACS. The enriched libraries were next combined using CDR shuffling and further enriched for mouse / human CD98hc binding (3-30 nM). (B-D) Characterization of affinities of selected clones from the CDR-swapped library against (B) mouse. (C) cyno, and (D) human CD98hc ectodomains. The affinities of WT and all the five cross-reactive antibodies displayed on yeast surface were assessed by flow cytometry, with three independent replicates (middle column). Additionally, the binding of the WT antibody and three of the five cross-reactive variants-D4-4. 3.5D19. and 3.5D5-wcrc further evaluated in their IgG format using surface plasmon resonance (SPR), with at least two independent replicates (values presented in parentheses in the rightmost column). The reported KDvalues represent the mean of independent experiments, with errors expressed as standard deviations.

[0064] FIG 8. shows scFvs obtained from the initial and redesigned HCDR3 libraries demonstrate binding to (A) mouse and (B) human CD98hc binding curves of a panel of scFvs on the surface of yeast, including 4D1, 4D5, 5D7, 4F5, and 5F10 from die redesigned HCDR3 library, MH17, MH24, and D3 from the initial HCDR3 library. The results are from three independent replicates.

[0065] FIG 9. shows generation of HCDR1&HCDR2 and LCDR1&LCDR3 libraries for affinity maturation. The crystal structure highlights the binding interface between human CD98hc (shown in grey, with loop 1 and loop 2 highlighted in green) and the WT antibody (VLdomain in light orange and VH domain in light blue) : (A) 8 residues selected for HCDR1&HCDR2 library construction are depicted with their side chains and color-coded. Seven residues (H33, H50, H52, H53. H54, H56. H58) are shown in pink, while H35 is marked in a distinct peach pink. (B) 9 residues selected for LCDR1&LCDR3 library construction are displayed with their side chains and colored in pink (L27d, L27e, L27f L28, L32, L91. L92, L93. L94). The crystal structure is from the Protein Data Bank (7DF1).

[0066] FIG 10. shows complete sorting path of libraries used for CDR swapping mutagenesis.Three distinct libraries were generated and co-selected for binding to mouse and human CD98hc: a redesigned HCDR3 library with optimized mutations, a library with mutations in HCDR1 and HCDR2, and a library with mutations in LCDR1 and LCDR3. The HCDR1&HCDR2 and LCDR1&LCDR3 libraries were first enriched through two rounds of MACS against both mouse and human CD98hc. Output from these libraries, along with the redesigned HCDR3 library, rmderwent two rounds of FACS against mouse and human CD98hc under progressively lower antigen concentrations. The resultingoutputs were subsequently used for CDR swapping mutagenesis. The CDR-swapped library was then subjected to three rounds of FACS co-selection for mouse and human CD98hc binding, leading to the identification of species cross-reactive antibodies.

[0067] FIG 11. shows representative cytograms illustrating the binding of yeast- displayed scFvs to orthologs of CD98hc. The binding of a panel of scFvs displayed on yeast-including CPI, WT, and D4-4 (a cross-reactive antibody)-was assessed against 11.1 nM mouse, human, and cyno CD98hc ectodomains. In each cytogram, the x-axis represents antibody expression on the yeast surface, while the y-axis indicates antibody binding to the corresponding antigen. The cytograms reveal that CPI binds exclusively to mouse CD98hc, WT binds specifically to human and cyno CD98hc, while D4-4 demonstrates cross-reactivity by binding to mouse, human, and cyno CD98hc.

[0068] FIG 12. shows that species cross- reactive antibodies display favorable developability properties. (A) Quantification of mutations within the CDR regions of the five species cross-reactive antibodies. (B) Comparative analysis of predicted T-cell epitopes and humanness scores across clinical-stage antibodies, previously reported CD98hc antibodies. WT antibody, and the cross-reactive CD98hc antibodies (refer to the Methods section for details). (C) Schematic representation of the bispecific antibody (bAb) design, featuring an IgG with knob-into-hole mutations in the Fc region and an anti-CD98hc scFv fused to the C-terminus of one heavy chain. (D) SDS-PAGE analysis of bAbs following a single (Protein A) purification step. (E) Size-exclusion chromatography measurements of % bAb monomer after a single purification step. (F) Apparent melting temperatures (Tm) of bAbs measured using differential scanning fluorimctry (DSF). In (E) and (F), the measurements arc averages of three independent experiments, and the errors are standard deviations

[0069] FIG 13. shows characterization of CPI, WT, and five selected species cross-reactive antibodies. (A) SDS-PAGE analysis and (B) SEC profiles of CPI, WT. and five species cross-reactive antibodies reformatted as IgG. (C) SEC overlay of CPI, WT, and five selected species cross-reactive antibodies reformatted as bAbs.|0070] FIG 14. shows thermal stability characterization of species cross-reactive antibodies reformatted as bAbs using differential scanning fluorimetry (DSF). The representative thermal stability profiles are shown for (A) CPI, (B) WT, (C) D4-4, (D) D4-9, (E) 3.5D19, (F) ADN2-14, and (G) 3.5D5. The data are averages of three independent experiments, and the errors are standard deviations.

[0071] FIG 15. shows species cross-reactive antibodies bind equivalent loops on the surface of mouse, cyno, and human CD98hc. (A) Crystal structure of the WT antibody bound to the human CD98hc ectodomain (pink; PDB: 7DF1). The WT VL (orange) and Vn (dark blue) domains interact with two exposed loops (loops 1 and 2) on CD98hc, highlighted in light green and cyan. (B) Overlay of the cry stal structures of the ectodomains of mouse (PDB: 6I9Q) and human CD98hc (PDB: 2DH2), and AlphaFold3 -pre dieted structure of cyno CD98hc, as well as (C) the overlay of their respective loopregions. (D-E) Analysis of IgG / D4-4 binding to mouse, human, and cyno WT CD98hc ectodomains or equivalent ectodomains with glycine or serine mutations in either (D) loop 1 or (E) loop 2. The experiments are averages of three independent experiments, and the errors are standard deviations.

[0072] FIG 16. shows AlphaFold3 (AF3)-predicted structures of CD98hcs. The most optimized predicted structure of (A) human CD98hc, (B) cyno CD98hc and (C) mouse CD98hc ectodomains are shown. The amino acid coloring represents AlphaFold3's confidence levels, as measured by per-residue predicted local distance difference test (plDDT) scores. Most residues across the three CD98hc structures exhibit high confidence, with plDDT scores above 90. Notably, residues in the antigenic regions (loop 1 and loop 2) consistently show plDDT scores exceeding 70. The figure also includes the inter-residue predicted aligned error (iPTM) score, which evaluates the overall confidence of the protein structure.

[0073] FIG 17. shows a comparison of the crystal structure and AF3-predicted structures of CD98hc. (A) Superimposition of human CD98hc structures, with a backbone root mean square deviation (RMSD) of 0.85 A (considering only C, N, and O atoms of the peptide backbone). (B) Superimposition of mouse CD98hc structures, with a backbone RMSD of 0.54 A. (C) Alignment of loop 1 and loop 2 regions of human CD98hc, showing an RMSD of 1.02 A. (D) Alignment of loop 1 and loop 2 regions of mouse CD98hc, showing an RMSD of 0.38 A. RMSD was calculated by Biopython (Cock et al. 2009) REF(19304878). The crystal structures are derived from the Protein Data Bank (PDB IDs: 6I9Q and 2DH2).

[0074] FIG 18. shows an assessment of recombinant CD98hc variants loading onto ELISA plates.Loading efficiency was evaluated for the following CD98hc variants (left to right): mouse CD98hc, mouse CD98hc loop 1 mutant, mouse CD98hc loop 2 mutant, human CD98hc, human CD98hc loop 1 mutant, human CD98hc loop 2 mutant, cyno CD98hc, cyno CD98hc loop 1 mutant, cyno CD98hc loop 2 mutant, and a blank control.

[0075] FIG 19. shows characterization of bAbs binding to CD98hc variants from different species via ELISA. Analysis of (A) IgG / CPl, (B) IgG / WT, (C) lgG / D4-9, (D) lgG / 3.5D19, (E) IgG / ADN2-14, and (F) IgG / 3.5D5 binding to mouse, human, and cyno CD98hc ectodomains, as well as their respective loop 1 (left) and loop 2 (right) mutants. Loops 1 and 2 were replaced by a glycine-serine linker in the CD98hc mutants. The EC50 values represent the averages of three independent experiments, with errors expressed as standard deviations.

[0076] FIG 20. shows CD98hc bispecific antibodies mediate strong enhancement of brain delivery in vivo. (A) Brain concentrations, (B) blood concentrations, and (C) brain: blood ratios of injected bAbs (3.6 mg / kg) relative to the equivalent mAb at an equivalent dose (3 mg / kg). Concentrations were measured using12’I radiotracing after intravenous administration, and four mice per group were evaluated for each time point.

[0077] FIG 21. shows biodistribution of IgG and three species cross-reactive bispecific antibodies. Equimolar doses (20 nmol / kg) of IgGs (3.0 mg / kg) and bAbs (3.6 mg / kg) were radiolabeled with125I and injected retro-orb itally . Antibody concentrations were measured in various organs and blood at 1 hour. 1 day. and 7 days post-injection for (A) IgG, (B) IgG / D4-4, (C) IgG / 3.5D19, and (D) IgG / 3.5D5. Data represent measurements from n = 4n mice per time point.

[0078] FIG 22. shows functional paratope mapping of an anti-CD98hc species-cross-reactive antibody (D4-4) using mutational analysis. (A-C) Alanine scanning analysis of the functional paratope of D4-4 for binding human and mouse CD98hc. Single CDR substitution mutations were tested at all positions in HCDR3 as well as sites in other CDRs that were mutated relative to the WT antibody. Native alanine residues were substituted with glycine. (A) The relative binding of the D4-4 mutants to human and mouse CD98hc was assessed via yeast surface display (100 nM mouse or human CD98hc ectodomain). CDR sites in which the alanine mutation reduced binding by >50% are indicated with blue circles. (B-C) The D4-4 CDR sites most important for binding (B) human CD98hc and (C) mouse CD98hc, as defined in (A), are highlighted in blue in the predicted structure of D4-4. (D-F) Reversion scanning analysis of the functional paratope of D4-4 for binding human and mouse CD98hc. Single CDR reversion (substitution) mutations were tested at all sites mutated relative to the WT antibody. The single reversion mutations replaced the D4-4 CDR mutations with the WT residue. (D) The relative binding of the D4-4 mutants to human and mouse CD98hc was assessed via yeast surface display (100 nM mouse or human CD98hc ectodomain). CDR sites in which the reversion mutation reduced binding by >50% arc indicated with blue circles. (E-F) The CDR sites most important for binding to (E) human CD98hc and (F) mouse CD98hc, as defined in (D), are highlighted in blue in die predicted structure of D4-4. In (A) and (D), the experiments are averages of three independent experiments, and the errors represent standard deviations. In (B-C) and (E-F), the structural model of D4-4 was generated using ABodyBuilder68.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0079] SEQ ID NO.l represents a heavy chain CDR-H1: SYYMN{0080] SEQ ID NO.2 represents a heavy chain CDR-H1: SYYMH

[0081] SEQ ID NO.3 represents a heavy chain CDR-H2: LINPRSGSTSYAQKFQG

[0082] SEQ ID NO.4 represents a heavy chain CDR-H2: LINPSKGSTSYAQKFQG.

[0083] SEQ ID NO.5 represents a heavy chain CDR-H3: GYWDGATGFPSIFFDN

[0084] SEQ ID NO.6 represents a heavy chain CDR-H3: GYWDQVTGFPSIFFDN

[0085] SEQ ID NO.7 represents a heavy chain CDR-H3: GYWDEVTGFPSIFFDI

[0086] SEQ ID NO.8 represents a heavy chain CDR-H3: GYWDELTGFPSIFFDN

[0087] SEQ ID NO.9 represents a heavy chain CDR-H3: GYWDEVTAFPSIFFDN

[0088] SEQ ID NO.10 represents a heavy chain CDR-H3: GYWDGVTGFPSIFFDK

[0089] SEQ ID NO.11 represents a heavy chain CDR-H3: GYWDGATGFPSIFFDD

[0090] SEQ ID NO.12 represents a heavy chain CDR-H3: GYWDGITGFPSIFFDA

[0091] SEQ IDNO.13 represents a heavy chain CDR-H3: GYWDGVTGFPSIFFDT

[0092] SEQ ID NO.14 represents a heavy chain CDR-H3: GYWDQVTGFPSIFFDD

[0093] SEQ ID NO.15 represents a light chain CDR-L1 : KSSQSVLYPYMNKNYLA

[0094] SEQ ID NO.16 represents a light chain CDR-L1: KSSQSVLYSHMNKNYLA

[0095] SEQ ID NO.17 represents a light chain CDR-L1: KSSQSVLFSPNNKNYLA

[0096] SEQ ID NO.18 represents a light chain CDR-L1: KSSQSVLYHPMNKNYLA

[0097] SEQ ID NO.19 represents a light chain CDR-L1: KSSQSVLYSSNNKNYLA

[0098] SEQ ID NO.20 represents a light chain CDR-L1: KSSQSVLYSSMNKNYLA

[0099] SEQ ID NO.21 represents a light chain CDR-L1: KSSQSVLYPWNNKNYLA

[0100] SEQ ID NO.22 represents a light chain CDR-L1 : KSSQSVLYTPMNKNYLA

[0101] SEQ ID NO.23 represents a light chain CDR-L2: WASTRES

[0102] SEQ ID NO.24 represents a light chain CDR-L3: QQYYNVPIT

[0103] SEQ ID NO.25 represents a light chain CDR-L3: QQAYNVPIT

[0104] SEQ ID NO.26 represents a variable heavy (VH) chain amino acid sequence of a D4-4 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSNSTVYMELSSLRSEDTAVYYCARGYWDGATGFPSIFFDNWGQGTM VTVSS

[0105] SEQ ID NO.27 represents a variable heavy (VH) chain amino acid sequence of a D4-9 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDQVTGFPSIFFDNWGQGTM VTVSS

[0106] SEQ ID NO.28 represents a variable heavy (VH) chain amino acid sequence of a 3.5D19 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDNAVYYCARGYWDEVTGFPSIFFDIWGQGTMV TVSS

[0107] SEQ ID NO.29 represents a variable heavy (VH) chain amino acid sequence of a ADN2-14 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDELTGFPSIFFDNWGQGTMV TVSS[0108J SEQ ID NO.30 represents a variable heavy (VH) chain amino acid sequence of a 3.5D5 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDEVTAFPSIFFDNWGQGTM VTVSS

[0109] SEQ ID NO.31 represents a variable heavy (VH) chain amino acid sequence of a 6D10 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGVTGFPSIFFDKWGQGTM VTVSS

[0110] SEQ ID NO.32 represents a variable heavy (VH) chain amino acid sequence of a ADN5 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGATGFPSIFFDDWGQGTM VTVSS

[0111] SEQ ID NO.33 represents a variable heavy (VH) chain amino acid sequence of a 3.5D1 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPSKGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGITGFPSIFFDAWGQGTMV TVSS

[0112] SEQ ID NO.34 represents a variable heavy (VH) chain amino acid sequence of a 3.5D3 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPSGGATA YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGVTGFPSIFFDTWGQGT MVTVSS

[0113] SEQ ID NO.35 represents a variable heavy (VH) chain amino acid sequence of a ADN1-4 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AHKFQGSVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDQVTGFPSIFFDDWGQGTM VTVSS

[0114] SEQ ID NO.36 represents a variable light (VL) chain amino acid sequence of a D4-4 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYPYMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK{0115] SEQ ID NO.37 represents a variable light (VL) chain amino acid sequence of a D4-9 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSHMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQAYNVPITFGQGTRLEIK

[0116] SEQ ID NO.38 represents a variable light (VL) chain ammo acid sequence of a 3.5D19 antibody:DIVMTQSPDSLAVSLGERATINCKSSQSVLFSPNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQAYNVPITFGQGTRLEIK

[0117] SEQ ID NO.39 represents a variable light (VL) chain amino acid sequence of a ADN2-14 antibody:DIVMTQSPDSLAVSLGERATINCKSSQSVLYHPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0118] SEQ ID NO.40 represents a variable light (VL) chain amino acid sequence of a 3.5D5 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0119] SEQ ID NO.41 represents a variable light (VL) chain amino acid sequence of a 6D10 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0120] SEQ ID NO.42 represents a variable light (VL) chain amino acid sequence of a ADN5 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYPYMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0121] SEQ ID NO.43 represents a variable light (VL) chain amino acid sequence of a 3.5D1 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYPWNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0122] SEQ ID NO.44 represents a variable light (VL) chain amino acid sequence of a 3.5D3 antibody: DIVMTQSPDSLAVSLGERATINCKSSQSVLYTPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQAYNVPITFGQGTRLEIK

[0123] SEQ ID NO.45 represents a variable light (VL) chain amino acid sequence of a ADN1-4 antibody:DIVMTQSPDSLAVSLGERATINCKSSQSVLYTPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0124] SEQ ID NO.46 represents heavy and light chain amino acid sequences a D4-4 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSNSTVYMELSSLRSEDTAVYYCARGYWDGATGFPSIFFDNWGQGTM VTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYPYMNKNYLAWYQQKPGQPPKLLIYW ASTRESGVPDRFSGSGSGTDFTLT1SSLQAEDVAVYYCQQYYNVPITFGQGTRLE1K

[0125] SEQ ID NO.47 represents heavy and light chain amino acid sequences of a D4-9 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDQVTGFPSIFFDNWGQGTMVTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYSHMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQAYNVPITFGQGTRLEIK

[0126] SEQ ID NO.48 represents heavy and light chain amino acid sequences of a 3.5D19 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDNAVYYCARGYWDEVTGFPSIFFDIWGQGTMV TVSS

[0127] SEQ ID NO.49 represents heavy and light chain amino acid sequences of a ADN2-14 antibody:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDELTGFPSIFFDNWGQGTMV TVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYHPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0128] SEQ ID NO.50 represents heavy and light chain amino acid sequences of a 3.5D5 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDEVTAFPSIFFDNWGQGTM VTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0129] SEQ ID NO.51 represents heavy and light chain amino acid sequences of a 6D10 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGVTGFPSIFFDKWGQGTM VTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTIS SLQ AED VA V Y YCQQ Y YN VP1TFGQGTRLE1K

[0130] SEQ ID NO.52 represents heavy and light chain amino acid sequences of a ADN5 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGATGFPSIFFDDWGQGTM VTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYPYMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0131] SEQ ID NO.53 represents heavy and light chain amino acid sequences of a 3.5D1 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPSKGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGITGFPSIFFDAWGQGTMV TVSSDIVMTQSPDSLAVSLGERATINCKSSQSVLYPWNNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK

[0132] SEQ ID NO.54 represents heavy and light chain amino acid sequences of a 3.5D3 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPSGGATA YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDGVTGFPSIFFDTWGQGT MVTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYTPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQAYNVPITFGQGTRLEIK

[0133] SEQ ID NO.55 represents heavy and light chain amino acid sequences of a ADN1-4 antibody: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMNWVRQAPGQGLEWMGLINPRSGSTSY AHKFQGSVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYWDQVTGFPSIFFDDWGQGTM VTVSS DIVMTQSPDSLAVSLGERATINCKSSQSVLYTPMNKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNVPITFGQGTRLEIK DETAILED DESCRIPTION

[0134] The present disclosure relates to species cross-reactive antibodies that cross the blood brain barrier (BBB) and related compositions and methods.

[0135] The antibodies provided herein feature species cross-reactivity, making them highly suitable for use as therapeutic antibodies and making pre-clinical studies more efficient, cost-effective, and reliable.

[0136] Exemplary antibodies provided herein are unusual in their ability’ to recognize murine, cyno, and human CD98hc, relative to previously reported antibodies and engineered proteins specific for human CD98hc, including several used for brain shuttling (Hayes et al., Int. J. Cancer 137, 710-720 (2015); WO2013078377; Itoh et al., Cancer Sci. 98, 1696-1700 (2007); Lee et al., Nat. Struct. Mol. Biol. 26, 510-517 (2019); Chew et al., Nat. Commun. 14, 5053 (2023); Edavettal et al., Med 3, 860-882. e815 (2022); WO2021205361: Deuschle etal., Theranostics 10, 2172 (2020); and Tian etal., Nat. Biomed. Eng. 7, 8-23 (2023)). Historically, the identification of human / cy no-cross-reactive antibodies with similar affinities has proven challenging, with antibodies often demonstrating greater than two- to five-fold higher affinity for human CD98hc. Both the lack of binding to the murine ortholog and the difference in cyno / human affinity have proven problematic preclinical development of CD98hc CNS shuttles, as their pharmacokinetics, pharmacodynamics, and therapeutic efficacy should be tested in genetically altered animals. In some cases, this requires complex mouse lines that necessitate humanization of both CD98hc and a parenchymal target or disease gene (e.g., amyloid precursor protein).

[0137] In some embodiments, the antibodies bind to a protein or other factor that facilitates crossing the blood -brain-barrier (BBB). In some embodiments, the antibodies are provided as bispecific or multi-specific antibodies. In some such embodiments, a first specificity is to a protein or other factor that facilitates crossing die BBB (e.g., CD98hc) and a second specificity is to a target in the brain useful for research, diagnostic, or therapeutic purposes. In some embodiments, the antibodies are bound to or otherwise associated with a cargo molecule (e.g., drug) to be delivered to one or more specific cells, tissues, or regions of the brain. In some embodiments, the bispecific antibody binds to a protein on / or in a neuron. In some embodiments, the neuron is associated with a neurological disease and / or condition or is involved in improving a neurological function. In some embodiments, the neurological disease and / or condition or improving a neurological function is Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS). Huntington’s disease, multiple sclerosis (MS), frontotemporal dementia (FTD), Lewy body dementia (LBD), neuromyelitis optica spectrum disorder (NMOSD), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), stroke, cerebral ischemia, brain microvascular dysfunction, vascular dementia, schizophrenia, intellectual disabilities, autism spectrum disorder (ASD), depression, anxiety disorders, bipolar disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), myasthenia gravis (MG), peripheral neuropathy, glioblastoma (GBM), and other brain tumors such as astrocytomas, meningiomas, and medulloblastomas. In some embodiments, the improved neurological function is memory , focus, creativity, sleep, skill performance, enhanced positive sensory perception, and reduced negative sensory perception (e.g., pain, heat, cold, etc.).[0138J It is notable that the bispecific antibodies, with broad CD98hc species reactivity, show similar brain uptake and retention in wild-type mice relative to previous, mouse-specific CD98hc shuttles

[0023] , This includes similar brain levels (~10x higher than unshuttled IgG) and brain-to-blood levels (~100x higher than unshuttled IgG). This is unexpected given that the engineered antibodies possess unique epitopes and lower affinity relative to the CD98hc mouse-specific antibody. More generally, the engineered bispecific antibodies reported here open the door to many future preclinical and clinical studies in mouse models of human disease that do not need to be transgenic for CD98hc, which is significant because it overcomes several practical and intellectual property issues and expedites clinical translation.

[0139] A species cross-reactive antibody, as described herein, refers to an antibody that specifically recognizes and binds to a target antigen across multiple species. Such antibodies are capable of binding epitopes present in different species while maintaining specificity for the intended antigen. Unlike species-specific antibodies, which exhibit binding restricted to a single organism, species cross-reactive antibodies are engineered to recognize structural or sequence-conserved regions of a target molecule that are shared among species. This cross-reactivity facilitates their use in comparative biological studies, preclinical research involving multiple model organisms, and therapeutic applications requiring activity across different species.

[0140] In some embodiments, the species cross-reactive antibody may be specific for 2 or more species. In some embodiments, the species cross-reactive antibody may be specific for 3 or more species. In some embodiments, the species cross-reactive antibody may be specific for 4 or more species. In some embodiments, the species cross-reactive antibody may be specific for 5 or more species. In some embodiments, the species cross-reactive antibody may be specific for 6 or more species. In some embodiments, the species cross-reactive antibody may be specific for 7 or more species. In some embodiments, the species cross-reactive antibody may be specific for 8 or more species. In some embodiments, the species cross-reactive antibody may be specific for 9 or more species. In some embodiments, the species cross-reactive antibody may be specific for 10 or more species.

[0141] Non-limiting exemplary embodiments of the species cross-reactive antibody may include any two or more species, and variants thereof that do not alter the intended binding properties of the antibody. For example, in exemplary embodiments, the species cross-reactive antibody may be specific for human and non-human mammals, such as human and rat. In exemplar} embodiments, the species cross-reactive antibody may be specific for any two or more of a human, a rat, a mouse, a dog, a guinea pig, a hamster, a rabbit, a cat. a pig, a goat, a sheep, a horse, a cow, a macaque, a baboon, a marmoset, a chimpanzee, a chicken, a zebrafish, and a frog.

[0142] In some embodiments, the presently disclosed subject matter provides a species cross-rcactivc antibody comprising a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-H3 having a sequence of SEQ ID NO. 5. SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8. SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 6. SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9. SEQ ID NO. 10. SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.

[0143] In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO. 16. SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21 , or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 15, SEQ ID NO. 16. SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19. SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least70% sequence identity to SEQ ID NO. 23. In some embodiments, the presently disclosed subject matter provides a species cross-reactive antibody comprising a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity' to SEQ ID NO. 24 or SEQ ID NO. 25.[0144| In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 80% sequence identity. In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 90% sequence identity. In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 95% sequence identity. In some embodiments of the species cross-reactive antibody herein, the at least 70% sequencing identity comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity.

[0145] In some embodiments, the CDR sequences have one. two, three, four, five, six. seven, or eight amino acids substitutions relative to the recited sequences. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. One ty pe of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and try ptophan; unnatural amino acids like p-amino phenylalanine, a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur -containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. In some embodiments, a variant may have “non-conservative” changes (e.g., replacement of a gly cine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Variants can be tested in functional assays.

[0146] It was found that the sequence of HCDR3 is more mutable than would have been otherwise expected. Scanning histidine mutagenesis revealed that most of the sites (10 out of 16) could be mutated to a large amino acid with little impact on binding, highlighting that HCDR3 can be extensively changed while maintaining affinity for the original targets (human and cyno CD98hc). Notably, a panel of five antibodies shown below in the Examples have 6-8 mutations out of 16 residues in HCDR3, which corresponds to mutagenesis of 38-50% of the sequence. This level of mutagenesis of HCDR3 is much higher than previous studies aimed at introducing species cross-reactivity into other antibodies [37-40], The five antibodies with broad cross-reactivity lacked histidine mutations in HCDR3, revealing that this process of identifying mutable sites in HCDR3 did not bias the selection toward histidine mutations, experimentally The HCDR3 sites that contributed to binding was experimentally defined so as to onlyallow permissive sites in the libraries, as structure-based predictions of HCDR3 residues important for binding were incorrect at several sites. For example, the WT paratope obtained using the crystallized co-complex (PDB: 7DF1) excludes H95, H100G, H100H, and H101, which would suggest they can be mutated, but it was found that experimentally mutations at these sites reduce binding to human CD98hc by >50%. Conversely, it was found that multiple sites in the paratope (H99, H100, H100B, H100C, H100D, and H100E) - which nominally are expected to be important for binding - are mutable while maintaining >50% binding to human CD98hc. These insights played a role, as eliminating H95, H100G, Hl OOH. and Hl 01 was expected to significantly improve the fraction of functional antibodies in the library, and mutating paratope sites (H100C, H100D, and H100E) led to conserved mutations at both sites in all five antibodies.

[0147] It is also notable that this approach of reinforcing the HCDR3 mutations with mutations in additional CDRs used the opposite approach, namely intentionally mutating CDR sites in the paratope. It was hypothesized that non-HCDR3 mutations would be less likely to disrupt binding, even for those in the antibody paratope. It was identified that there were conserved mutations in all five antibodies in HCDR2 (three conserved mutations), HCDR1 (one conserved mutation), and LCDR3 (one conserved mutation) in addition to several non-conserved mutations in these three CDRs as well as in LCDR1.

[0148] Antibodies may be produced using any method known in the art. In some embodiments, antibodies are produced by recombinant expression in a host organism (e.g., E. coli), followed by purification. As a nonlimiting example, an antibody to a particular antigen (such as CD98hc described herein) may be produced by immunizing an animal (such as a mouse, rat, rabbit, goat, sheep, horse, etc.) with the antigen and isolating antibodies from the serum of the animal and / or immortalizing primary B cells from the animal to produce hybridomas that express the antibodies. Phage display technology' may also be used to produce antibodies that bind to the polymerases described herein. Phage display libraries are commercially available and methods of selecting antibodies from such libraries are known in the art. See, e.g., Vaughan et al., 1996, Nature Biotechnology', 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991. J. Mol. Biol., 222:581.

[0149] The compositions and pharmaceutical compositions thereof may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal. buccal, intrathecal, intracranial, intranasal or topical routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age. health, and weight of the recipient, kind of concurrent treatment, if any. frequency of treatment, and the nature of the effect desired. In some embodiments, the species cross-reactive antibody7is co-administered with a neuroprotective agent, an antiinflammatory agent, or a small-molecule therapeutics for enhanced efficacy.

[0150] In exemplary' embodiments, neuroprotective agents include but are not limited to nootropics, antioxidants, neurotrophic factors, NMDA receptor antagonists, calcium channel blockers, and mitochondrial protectants. In exemplary embodiments, anti-inflammatory agents include but are not limited to non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, cytokine inhibitors, COX-2 inhibitors, and inflammatory modulators, hi exemplary embodiments, small-molecule therapeutics include but are not limited to acetylcholinesterase inhibitors, dopamine agonists, antiepileptic drugs, glutamate antagonists, selective serotonin reuptake inhibitors (SSRIs), cholinergic agents, and tyrosine kinase inhibitors. In exemplary embodiments, supporting agents include but are not limited to vasodilators, gene therapy agents, and gene editing tools.

[0151] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of die present disclosure and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.EXAMPLES

[0152] The present disclosure has multiple aspects, illustrated by the non-limiting examples as described herein.

[0153] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0154] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.Experimental Methods|0155] Cloning of antibody and antigen genes. For yeast display, the variable heavy (VH) and light (VL) domains of CPI [23.44] (GenBank ID: OR253074.1). WT

[0028] (PDB ID: 7DF1), and selected variants were either isolated from yeast plasmids or synthesized as gene fragments (IDT). The VH and VL domains were cloned into pD208 yeast display vectors in the scFv format, which was Aga2-linker-VH-linker-VL for WT and variants thereof and Aga2-linker-VL-linker-VH for CPI. The PCR-amplified fragments and expression vectors were digested with restriction enzymes EcoRI-HF and Xhol (New England Biolabs. Cat. No. R3101L and R0146L. respectively).

[0156] For producing soluble bispecific antibodies, three pTT5 mammalian expression vectors were used that encoded for a phosphorylated tau-targeting IgG with a CD98hc -targeting scFv fused to one of the C -termini of the heavy chains (GenBank ID: OR253074.1). These vectors corresponded to the light chain, heavy chain #1 (hole chain), and heavy chain #2 (knob chain) of human IgGl antibodies. Heavy chain #2 contained knob mutations [S(365)C and T(377)W]

[0045] REF(9661204) and an scFv attached to the C-terminus. Heavy chain #1 contained hole mutations [Y(360)C, T(377)S, L(379)A and Y(418)V],

[0045] The variable regions of various scFvs - including CPI, WT. and evolved variants thereof - were PCR amplified from yeast plasmids and cloned into heavy chain #2. This was performed using restriction enzyme digests with Kpnl-HF and BamHl-HF (New England Biolabs. Cat. No. R3142L and R3136L, respectively).

[0157] For producing CD98hc antigens, gene fragments were synthesized (IDT) and cloned into pTT5 mammalian expression vectors with an N-terminal 6*His tag. Restriction digests for cloning were performed using EcoRI-HF and BamHI-HF (New England Biolabs, Cat. R3101L and R3136L, respectively).

[0158] For cloning each type of protein, D A fragments and vectors were purified using 1% agarose gels and Qiagen PCR Gel Extraction Kits (Qiagen, Cat. No. 28104), ligated using T4 DNA ligase (New England Biolabs, Cat. No. M0202L), and transfonned into competent E. coli DH5a cells. Antibody and antigen sequences were verified by Sanger sequencing.

[0159] Recombinant antigen production. The HEK293-6E cell line (L-11565, National Research Council Canada) was maintained in disposable conical tubes using either Fl 7 medium (Thenno Fisher Scientific. Cat. No. 50591354) or BalanCD HEK293 medium (Fujifilm Irvine Scientific. Cat. No.91165). Cultures were incubated at 37 °C with 5% CO2 and shaken at 250 rpm. To express recombinant CD98hc ectodomains. 15 pg of the corresponding plasmid was mixed with 45 pL of 40 kDa polyethylenimine (1 mg / mL, PEI MAX; Polysciences Inc., Cat. No. 247651) and 3 mL of culture medium for transfection. After a 15-minutes incubation at room temperature, the DNA-PEI complexes were added to 25 mL cultures of HEK293-6E cells at a density of 2x 106cells / mL, and the cultures were returned to the incubator. 24 hours post-transfection. 0.75 mL of 20% (w / w) yeastolate (Thermo Fisher Scientific. Cat. No. B92804) was added to each tube. Cells were harvested 6 days after transfection by centrifugation at 3500 g for 40 min. The supernatant was filtered (Thermo Fisher Scientific, Cat. No.166-0045) for downstream purification.

[0160] For recombinant CD98hc purification. Ni-NTA agarose beads (Qiagen, Cat. No. 30250) were added to supernatants containing recombinant CD98hc and incubated overnight at 4 °C with shaking. Beads were collected in centrifuge columns (Thermo Fisher Scientific, Cat. No. 89898). washed three times with 10 mL PBS, and sequentially incubated with 50 mM imidazole (pH = 7.4) for 15 min. The columns were centrifuged at 1000 g for 2 minutes to remove the flow-through. The beads were then incubated with 500 mM imidazole (pH = 7.4) for 15 min. Following incubation, the columns werecentrifuged again at 1000 g for 2 minutes to collect the eluate, which was immediately buffer-exchanged into PBS (pH = 7.4) using desalting columns (Thermo Fisher Scientific, Cat. No. 89891) and subsequently stored at -80 °C. Protein concentration was determined by NanoDrop absorbance at 280 mn, and purity was evaluated using SEC with the same column described above. CD98hc preparations with purity <90% monomer underwent additional SEC -based purification to ensure high quality. All the samples after 2 step purification were assessed by SDS-PAGE.

[0161] Biotinylation of recombinant CD98hc ectodomains. Filtered PBS (180 pL) was added to 1 mg of pre-aliquoted biotin (Fisher Scientific, Cat. No. A39257) to create a stock concentration of 10 mM. The volume of biotin needed to biotinylate desired proteins at a 20: 1 molar ratio of biotin to protein was calculated, added to the proteins, and incubated for 1 hour at room temperature (approximately 10 pL for 1 mL of a protein at 1 mg / mL). The volume of 1.5 M hydroxylamine at a 10:1 molar ratio of hydroxylamine to biotin was calculated, added to the biotinylated proteins (approximately 1 pL for 1 mL of a protein at 1 mg / mL), and incubated for 1 hour at room temperature to quench the biotin reaction. Proteins were aliquoted and stored at -80 °C until needed.

[0162] Histidine scanning analysis. Histidine scanning mutagenesis was performed on 16 residues in HCDR3 (based on the Kabat numbering system). Mutagenic primers were designed to replace the wildtype codons with histidine codons. Each mutant was cloned following the protocol outlined in the cloning section, and sequence-verified plasmids were subsequently transformed into Saccharomyces cerevisiae EBY100 cells as described below.

[0163] EBY 100 yeast cells were cultured overnight in 5 mL of YPD medium (10 g / L yeast extract, 20 g / L bacto peptone, 20 g / L dextrose) at 30 °C. For each variant, 500 pL of cells were transferred to a 1.5 mL conical tube and centrifuged at 16,000 g for 1 minute. The cell pellets were resuspended in 500 pL of ice-cold plate solution buffer (400 g / L PEG (molecular weight, 3350), 6.6 g / L LiAc, 0.4 mM EDTA and 10 mM Tris HC1, pH = 7.5) and mixed with 10 pL of 100 ng / pL plasmid DNA and 10 pL of 10 mg / mL single-stranded DNA (Sigma- Aldrich, Cat. No. D7656). The mixture was incubated on ice for 15 minutes, followed by a 15 minutes heat shock at 40°C. Afterward, the cells were centrifuged again at 16,000 g for 1 minute. The supernatant was discarded, and the pellets were resuspended in 200 pL of DI water. The cell suspension was plated on dropout plates (15 g / L agar, 20 g / L dextrose, 6.7 g / L yeast nitrogen base (without amino acids), 3.8 g / L tryptophan dropout media) and incubated at 30 °C for 2 days.

[0164] Single colonies were then picked and cultured in 5 mL yeast minimal media (SDCAA (16.75 g / L sodium citrate. 4 g / L citric acid (anhydrous), 6.7 g / L yeast nitrogen base (without amino acids), 5 g / L acid casein peptone (casamino acids), 20 g / L dextrose)) at 30 °C and 225 rpm for 2 days. Cells were transferred from SDCAA to a yeast induction media (SDGCAA. (6.76 g / L sodium phosphate dibasic dihydrate, 8.56 g / L sodium phosphate monobasic monohydrate, 6.7 g / L yeast nitrogen base (without amino acids), 5 g / L acid casein peptone (casamino acids), 20 g / L galactose, 2 g / L dextrose) at a startingOD of 0.5-1.0. Cells were grown in SDGCAA for 48 hours at 20 °C or 24 hours at 30 °C. All media was spiked with 100 pg / mL Ampicillin, 100 pg / mL Kanamycin, 100 pg / mL Streptomycin and 100 units / mL Penicillin prior to cell culture.

[0165] After culturing yeast cells in SDGCAA. the cells were harvested and washed twice with 0.1% PBSB (prepared by combining 100 mL 10 x PBS, 900 mL DI water, and 1 g BSA). The cell suspension was aliquoted into a 96-well plate (Fisher Scientific, Cat. No. 7000609) at a density of 1 x 105cells per well. The cells w ere then incubated in PBSB containing: 1:1000 dilution of anti-myc tag antibody (Cell Signaling Technology. Cat. No. 2276S). 1% milk and 100 nM of biotinylated mouse or human CD98hc protein. The mixture was incubated at room temperature for 3 hours on an orbital shaker at 300 rpm. Following incubation, the plate was washed with cold 0.1% PBSB, and each well was resuspended in 200 pL of 0.1% PBSB containing: 1:1000 dilution of streptavidin Alexa Fluor 647 (Invitrogen, Cat. No. S32357) and 1:200 dilution of anti-mouse Fc antibody conjugated with Alexa Fluor 488 (Invitrogen, Cat. No. Al 1001). The plate was incubated on ice for 4 min. washed with cold 0.1% PBSB, and resuspended in 150 pL of cold 0.1% PBSB per well. Samples were analyzed using a BioRad ZE5 cell analyzer, and the resulting data were processed and visualized using GraphPad Prism software.

[0166] Yeast library construction. For the initial HCDR3 library. 10 sites in HCDR3 of the WT antibody were selected for mutagenesis. The targeted residues (H96. H97, H98, H99, H100, H100B, H100C, H100D, H100E, H102) were subjected to soft random mutagenesis.

[0046] This approach sampled the WT residue with a ~50% probability and the remaining 19 amino acids with a -50% probability (theoretical diversity’ of ~1.0x 1013).

[0167] For die redesigned HCDR3 library’, the same 10 HCDR3 sites were targeted for mutagenesis as in the initial HCDR3 library based on PSERM metrics to introduce specific substitutions. NGS analysis was performed on the sequences from all the libraries, including HCDR3-I (MACS #3 output of HCDR3 library against mouse CD98hc), HCDR3-D1 (FACS #1 output of HCDR3 library against both mouse and human CD98hc), HCDR3-D2 (FACS #2 output of HCDR3 library' against both mouse and human CD98hc), HCDR3-M1 (FACS #1 output of HCDR3 library against mouse CD98hc), and HCDR3-H1 (FACS #1 output of HCDR3 library against human CD98hc). The average PSERM score for the 10 HCDR3 sites yvas calculated across all sequences based on the PSERM metrics derived from the library HCDR3-D1. Additionally, the average PSERM score was determined for the 53 individual clones, and variants with PSERM scores >0.169 were considered to be species cross-reactive. Sequences from all libraries with an average PSERM >0.169 yvere further analyzed to evaluate amino acid distributions at each mutation site. Based on this analysis, mutagenic primers, yvere designed to construct a redesigned HCDR3 library. Degenerate codons were selected to preferentially encode amino acids corresponding to a cumulative probability of -90% at each position, with limited redundancy incorporated at specific sites to minimize the number of primers required, (theoretical library diversity of approximately 3.0* 105). The reconstructed HCDR3 library yvas subsequently characterized by NGS.

[0168] Two affinity maturation libraries were also generated, one for VH (HCDR1 and HCDR2) and the other for VL (LCDR1 and LCDR3). Residue selection for constructing these libraries was guided by the crystal structure of the human CD98hc-WT antibody complex (PDB: 7DF1). Distances were calculated between the Ca atoms of residues in the WT antibody CDRs and the corresponding epitope residues on human CD98hc (loop 1 and 2). LCDR2 was excluded due to its greater distance from the epitope. Residues from the four CDR loops with a minimum distance to the epitope of <8 A were identified as potential mutation sites. In particular, residues with a minimum distance to the epitope of <5 A were selected regardless of the direction of their sidechains, (residues H52, H54, H56, and L92). Residues with a minimum distance to the epitope between 5-8 A were selected if their side chains pointed toward the epitope, as determined by a Cf> (antibody)-epitope distance at least 0.3 A shorter than the corresponding Ca (antibody)-epitope distance.

[0169] For the heavy chain (HCDR1 and HCDR2) library, antibody D3, derived from the initial HCDR3 library, served as the starting point. Eight residues in the heavy chain CDR1 and CDR2 regions (H33, H35, H50. H52, H53, H54. H56, H58) were selected for mutagenesis. Degenerate codons were designed using soft-codon mutagenesis. This library had a theoretical diversity of ~2.6 x IO10. For the light chain (LCDR1 and LCDR3) library, antibody D3 also served as the starting point. Nine residues in the light chain CDR1 and CDR3 regions (L27D, L27E, L27F, L28, L32, L91, L92, L93, L94) were targeted for mutagenesis using soft-codon mutagenesis. This library' achieved a theoretical diversity' of —5.1 x 1011. Finally, the three enriched libraries (HCDR1 / HCDR2, LCDR1 / LCDR3, and redesigned HCDR3) were combined using CDR swapping library [30,31],

[0170] To transform each library', EBY100 yeast cells were grown in 5 mL YPD media overnight at 30 °C and 225 rpm. This culture was transferred to a 50 mL culture of YPD media and allowed to grow overnight at 30 °C and 225 rpm. The next day, EBY100 from the 50 mL culture was transferred to 100 mL culture containing YPD such that the initial OD600 of the 100 mL culture was -0.3. This culture was allowed to grow at 30 °C and 225 rpm until the OD600 reached -1.6 (-4-6 hours post inoculation). Once the targeted OD600 was reached, 50 mL EBY 100 per library was aliquoted into conical tubes and centrifuged at 2500 g for 5 minutes at 4 °C. The cells were first resuspended with 25 mL ice-cold sterile DI water, and then with 25 mL ice-cold electroporation buffer (I M sorbitol, 1 inM calcium chloride). After each resuspension step, the cells were centrifuged as described above. Cells were then resuspended in 25 mL conditioning buffer (0.1 M lithium acetate. 10 mM 1.4-dithiothreitol). incubated for 15 minutes at 30 °C and 225 rpm, and centrifuged. Cells were washed with 25 mL of ice-cold electroporation buffer and centrifuged. Cells were then resuspended in the electroporation buffer to a final volume was -400 pL. The ethanol-precipitated DNA was added to the cells and mixed until homogenous. Cells were transferred to pre-chilled 2 mm gap electroporation cuvettes (Fisher Scientific, Cat. No. FBI 02) and electroporated using a Bio-Rad electroporator (Bio-Rad Gene Pulser Xcell) at 2500 V, 200 Q, and 25 pF to transform the library into yeast. The library' was immediately transferredto 8 mL of recovery media (1:1 mixture of YPD and 1 M sorbitol mixture) and incubated for 1 hour at 30 °C and 225 rpm.|0171] The library' was then centrifuged and resuspended in 1 mL of SDCAA. Dilutions of the library', sparming a range from 10-1 to 10-5, were prepped in 250 pL of DI water, plated on tryptophan dropout plates, and allowed to grow for 2 days at 30 °C. The remaining library was added to 200 mL of SDCAA and allowed to grow for 2 days at 30 °C and 225 rpm. After 2 days, the number of colonies on each of the dropout plates was counted to calculate the diversity of the library. The library itself was stored at 4 °C until use.

[0172] Magnetic-activated cell sorting (MACS). The constructed libraries were initially grown in SDCAA medium for 24 hour at 30°C. Subsequently, the cells were transferred to SDGCAA medium at a starting OD of 0.5-1.0 and incubated for 48 hours at 20°C or 24 hours at 30°C. After cultivation, yeast cells were harvested, with 109 cells collected for the first round and 108cells for subsequent rounds of sorting. Cells were centrifuged at 2500 g for 5 minutes, and the supernatant was discarded. They were then washed with 0.1% PBSB using 25 mL for 109cells or 1 mL for 108cells. After centrifugation at 2500 g for 5 min, the washing step was repeated.

[0173] Cells were incubated with 300 nM biotinylated mouse or human CD98hc diluted in 0.1% PBSB containing 1% milk for 3 hours at room temperature with gentle mixing. Following incubation, cells were washed with icc-cold 0.1% PBSB and centrifuged at 2500 g for 5 minutes. Streptavidin microbeads (Miltenyi Biotec, Cat. No. 130-048-101) were prepared, using 750 pL for 109 cells or 200 pL for 108 cells, and mixed with cold 0.1% PBSB. The cells and beads were incubated together for 40 minutes at 4 °C with steady mixing. The cells were washed again with cold 0.1% PBSB, centrifuged at 2500 g for 5 minutes, and kept on ice. LS columns (Miltenyi Biotec, Cat. No. 130-042-401) were mounted in two MACS magnets (hivitrogen, Cat. No. 12321) in series with a collection tray placed underneath. The columns were primed with 5 mL cold 0.1% PBSB. Cells were resuspended in 5 mL cold 0.1% PBSB, passed through a 70 pm filter (Miltenyi Biotec, Cat. No. 130-098-462) and then loaded onto the MACS columns. The columns were washed with 5 mL cold 0.1% PBSB to remove unbound material. Afterward, the columns were removed and filled with 5 mL SDCAA medium. The cells were eluted by using the column plungers, and the eluate was collected into 40 mL SDCAA. To evaluate library diversity, 200 pL of 10 x and 100 x dilutions of the eluted libraries were plated on tryptophan dropout plates. The remaining eluted library' was supplemented with antibiotics as described above and cultured at 30 °C for 48 hours.

[0174] Fluorescence-activated cell sorting (FACS). The library was regrown and cultured as described in the MACS section. Following yeast cell culture, 107 cells per sorting condition were harvested, washed with 1 mL of 0.1% PBSB, and centrifuged at 16,000 g for 1 minute. This washing step was repeated tw ice. The cells were incubated with biotinylated mouse CD98hc and FLAG-tagged human CD98hc ectodomains at equivalent concentrations in 0.1% PBSB containing 1% milk. Theconcentration of CD98hc varied depending on the number of prior sorting rounds, and the incubation volume was adjusted to maintain a molar ratio of CD98hc to scFv of approximately 10:1. Incubation was carried out at room temperature for 3 hours with gentle mixing. Following incubation, cells were washed with ice-cold 0.1% PBSB, centrifuged at 16,000 g for 1 min, and incubated on ice with a 1: 1000 dilution of anti-myc tag antibody (Cell Signaling Technology, Cat. No. 2276S) in 0.1% PBSB for 15 minutes. Cells were washed again with ice-cold 0.1% PBSB, centrifuged at 16,000 g for 1 minutes, and then incubated with 1 mL of a solution containing: 1:1000 dilution of streptavidin Alexa Fluor 647 (Invitrogen, Cat. No. S32357). 1:200 dilution of rabbit anti-FLAG antibody conjugated with Alexa Fluor 568 (Invitrogen. Cat. No. Al 1077), 1:200 dilution of anti-mouse Fc antibody conjugated with Alexa Fluor 488 (Invitrogen. Cat. No. A11001). All reagents were diluted in 0.1% PBSB. Following incubation, the cells were washed again with ice-cold 0.1% PBSB. centrifuged at 16.000 * g for 1 minute, and sorted for simultaneous binding to mouse and human CD98hc using a Sony MA900 cell sorter. The sorted cells were collected in SDCAA medium supplemented with antibiotics as previously described and cultured at 30°C for 48 hours.

[0175] Analysis of single clones from enriched libraries. Individual antibody sequences were selected and constructed following the protocol outlined in the cloning section. Sequence-verified plasmids were then transformed into EBY100 cells, which were cultured in SDCAA and subsequently incubated in SDGCAA. The binding of the mutants to human and mouse CD98hc was assessed via flow cytometry, as detailed in the histidine scanning section, using a CD98hc concentration of 300 nM. The binding signal of each mutant to both mouse and human CD98hc was normalized to the binding signal of the WT antibody to human CD98hc. Mutants exhibiting binding signals greater than 4% for both mouse and human CD98hc were classified as species cross-reactive antibodies, while those below this threshold were categorized as non-species cross-reactive. This dataset was subsequently used to evaluate the predictive performance of four metrics: frequency (dataset: HCDR3-D1; dual FACS #1 output), enrichment ratio (input dataset: HCDR3-I (output of MACS #3), output dataset: HCDR3-D1), PSSM score (dataset: HCDR3-D1), and PSERM score (input dataset: HCDR3-I, output dataset: HCDR3-D1).

[0176] Next-generation sequencing of libraries and data analysis of NGS datasets. Yeast libraries were cultured at 30 °C for 24 hours, and plasmid DNA was extracted using the ZymoPrep Y east Plasmid Miniprep Kit (Zymo Research Corp, Cat. No. D2004). Sequencing primers incorporating Illumina adapter sequences (p5 and p7) and indexing regions (i5 and i7) for sample demultiplexing were designed

[0021] , To enhance sequencing quality, the primers were further modified to include phasing elements [29.47], The HCDR3 region of the scFv library was amplified using the custom primers and Q5 polymerase (New England Biolabs, Cat. No. M0491) in a two-round PCR process. The PCR products were visualized on a 1% agarose gel, purified using the Qiagen PCR Gel Extraction Kit (Qiagen. Cat. No. 28704) and quantified with the Qubit 1 * dsDNA High Sensitivity Assay (Invitrogen. Cat. No. Q22321). The purified libraries were pooled at specific ratios and submitted to the University ofMichigan Advanced Genomics Core for sequencing using the Illumina MiSeq platform with 2 x 300 paired-end reads.

[0177] The data was processed in three main steps. Firstly, the two reads as fastq files are combined with FLASH

[0048] and converted to fasta files. Secondly, with Biopython

[0043] , the DNA sequences were translated into protein sequences. Finally, the residues that are mutated during the library generation process were determined for each sequence and analyzed with four different metrics (frequency, enrichment ratio, PSSM and PSERM), as described previously [29,49], The frequency of the sequences was calculated by dividing the number of observances of each sequence by the total number of sequences within the same library. The enrichment ratio was calculated by taking the log2 of the output library sequence frequencies divided by the input library sequence frequencies. The PSSM values for each residue were calculated based on the observance of each amino acid at a given position (Caa), as described in Equations 1 and 2. A pseudo count (s=l) and a background probability (pa=l / 20) were included in the calculations to eliminate problems related to logarithmic calculations.

[0021] X as Cfta + (1)F■ “ Pa

[0178] Similarly to the enrichment ratio, the PSERM values were calculated using the site-specific frequencies in the input and output libraries, as described in Equation 3:PSERM&aiog:2(3)

[0179] The total PSSM and PSERM scores are the summation of corresponding values for each residue, respectively.

[0180] Assessment of antibody affinities in scFv format using flow cytometry. Individual antibody sequences selected from the enriched libraries were constructed following the protocol described in the cloning section. Sequence-verified plasmids were then transformed into EBY100 cells, which were cultured in SDCAA and subsequently incubated in SDGCAA, as detailed in the histidine scanning section. The affinities of the mutant antibodies for human, cyno, and mouse CD98hc were evaluated by flow cytometry using serial dilutions of CD98hc proteins from each species, as described in the histidine scanning section. The resulting data were processed and visualized using GraphPad Prism software to determine the affinity of each antibody mutant.

[0181] Humanness and T cell epitope predictions. The sequences of clinical-stage antibodies were obtained from tire Therapeutic Antibody Database. The humanness score of each antibody was evaluated by AbNatiV

[0050] in FASTA format. The average of the AbNatiV VH and VL scores was considered as the humanness score for each antibody. The T cell epitope prediction was performed on NetMHCIIpan-4.3

[0051] , The sequences were input in FASTA fonnat. and the peptide length was set to 15 amino acids by default. A total of 27 MHC molecules were selected for the sequences to be predicted against, covering HLA DR, DQ. and DP specificities

[0052] , The prediction results were then used to identify peptides that were in the top 2% in % rank as strong binders, and peptides between the strong and weak thresholds (10%) were considered weak binders. Among all strong and weak binders. 15-mer peptides with the same predicted peptide core and MHC were grouped, and only the strongest binding peptides based on % rank were retained for further analysis. The germlines of each evaluated antibody obtained from ANARCI

[0053] and IM GT

[0054] databases were analyzed using the same process for T cell epitope predictions. Prediction results for each antibody were then compared to its corresponding germlines to identify whether the peptide cores were also germline. The number of predicted T cell epitopes for each antibody was calculated by excluding germline sequences from the total number of unique predicted strong binding core peptides.

[0182] Epitope and paratope analysis of CD98hc antibodies. The paratopes of the antibodies and other engineered protein binders, as well as the corresponding epitopes of human CD98hc and the associated buried surface areas, were derived from the co-cr stal structures of human CD98hc / binder complexes available in the Protein Data Bank. In this context, the epitope is defined as die set of amino acids in human CD98hc that are within 4 A of any atom in the corresponding binder. Similarly, the paratope is defined as the set of amino acids in tire binder that are within 4 A of any atom in human CD98hc. The calculations were performed using Afpdb

[0055] , Residues 374-401of human CD98hc form a solvent-accessible loop region characterized by relative flexibility (Residues 374-378 adopt a short a-helical conformation). Within this region, residues 386-390 are buried within the protein core, displaying low relative solvent accessibility (<15%), with specific values as follows: residue 386 (0.6%), residue 387 (14.9%), residue 388 (1.1%), residue 389 (0.5%), and residue 390 (8.8%). This buried segment effectively divides the loop into tw o sub-regions: loop 1 (residues 374-385) and loop 2 (residues 391-401). Most residues hi these sub-loops exhibit high solvent accessibility (>15%), except for residue 375 (10.3%), residue 378 (0%), and residue 395 (1.2%).

[0183] Antibody production. To produce antibodies. 15 pg of the corresponding plasmids were prepared for transfection. For bispecific antibodies, the mixture included 1.25 pg each of the light chain, heavy chain #1, and heavy chain #2 plasmids, whereas for IgG production, 1.875 pg each of the light and heavy chain plasmids were used. Additionally, 1.125 pL of 10 mg / mL single-stranded DNA (Sigma-Aldrich, Cat. No. D7656) was included. This plasmid and DNA mixhire was combined with 45 pL of 40 kDa PEI and 3 mL of culture medium. The transfection process was performed as describediii the antigen expression section. Cells were harvested 6 days after transfection by centrifugation at 3500 g for 40 minutes. The supernatant was filtered (Thermo Fisher Scientific, Cat. No. 166-0045) for downstream purification.

[0184] For antibody purification, supernatants containing antibodies were incubated overnight with Protein A agarose beads (Thermo Fisher Scientific, Cat. No. PI20334) at 4 °C with shaking. Beads were collected in centrifuge columns (Thermo Fisher Scientific. Cat. No. Pf89898), washed 3 times with 10 mL of PBS, and eluted with 0.1 M glycine buffer (pH = 3.0) after 15 minutes of incubation. The eluted antibodies were neutralized and exchanged into 20 mM sodium acetate buffer (pH = 5.0) using desalting columns (Thermo Fisher Scientific. Cat. No. 89891) and stored at -80 °C. Antibody concentration was determined by absorbance at 280 nm using a NanoDrop spectrophotometer. Purity was assessed by SDS-PAGE and SEC using a Superdex 200 Increase 10 / 300 GL column (Cytiva, Cat. No. 28990944). Antibodies with purity <90% monomer after Protein A purification underwent further fractionation via SEC to achieve the desired purity.

[0185] Surface Plasmon Resource (SPR). The affinities of four antibodies (WT, D4-4, 3.5D19, and 3.5D5) for mouse, human, and cyno CD98hc were determined using SPR on a Nicoya Alto instrument (Version 1.2, Nicoya, Canada). Antibodies in IgG format were immobilized onto a 16-channel CMD Carboxyl Cartridge (Nicoya, KC-CBX-CMD-16) via the Carboxyl Surfacing Kit (Nicoya, ALTO-R-CBX-SURF). The assay was conducted using Multi-Cycle Kinetics (MCK) mode, consisting of five cycles, where immobilized antibodies interacted with defined concentrations of CD98hc. Each cycle included specific association and dissociation phases for affinity determination, followed by surface regeneration in accordance with Nicoya’s standard protocols. Surface capture binding and regeneration conditions were as follows: Protein G was diluted to 50 pg / mL in 10 mM sodium acetate (pH 4.5) and covalently linked to EDC / NHS (200 mM)-activated carboxyl sensors for 10 minutes. Unreacted amine groups were subsequently blocked with 1 M ethanolamine (pH 8.5) for 5 minutes. The antibodies were prepared in running buffer (0.05% PBST) at a final concentration of 25 nM and immobilized onto the sensors for 5 minutes via Protein G interaction. CD98hc antigens were prepared using the same running buffer at the following 3-fold serial dilutions: mouse CD98hc: 500, 166.7, 55.6, 18.5, and 6.2 nM, human and cyno CD98hc: 333.3, 111.1, 37.0, 12.3. and 4.1 nM. Binding assays were performed at 25°C by injecting samples over the functionalized sensor surfaces. The association and dissociation phases were set for 6 minutes and 20 minutes, respectively. Sensor regeneration was achieved by removing bound antibodies and antigens using 10 mM Glycine-HCl (pH 2.0) for 1 minute. The regenerated surface was subsequently incubated with 25 nM antibodies for the next cycle. Data analysis was conducted using Nicosystem (Nicoya). Kinetic parameters (ka. kd, and KD) were evaluated using a 1 : 1 Langmuir kinetic binding model. Each binding interaction was measured at least twice independently to ensure reproducibility and accuracy in affinity determination.

[0186] In vitro enzyme linked immuno- adsorbent (ELISA) assay. The binding affinity and epitope of bispecific antibodies were determined by ELISA. For affinity assessment, hydrophilic 96 well plates (Coming, Cat. No. 3915) were coated with recombinant mouse, human, or cyno CD98hc ectodomains at 10 pg / mL and incubated overnight at 4 °C with shaking. For epitope mapping, mouse, human, and cyno CD98hc proteins were engineered with glycine-serine linkers replacing the predicted antibodybinding regions, designated as loop 1 and loop 2 mutants. Both the wild-ty pe and mutant antigens were coated onto ELISA plates at 10 pg / mL under the same conditions. After coating, all plates were washed three times with PBST (0.5% Tween-20 in lx PBS) and blocked with 3% BSA in PBS for 1 hour at room temperature. Following another PBST wash, a dilution series of bispecific antibodies, prepared in 1% BSA in PBS, was added to the wells and incubated for 2 hour at room temperature. For epitope detection, an anti-6 x His tag monoclonal antibody (Thermo Fisher Scientific, Cat. No. 4E3D10H2 / E3) was used to bind the His-tagged antigens. After antibody binding, plates were washed three times with PBST. For bispecific antibody detection. 100 pL of anti-human Fc HRP -conjugated secondary antibody (Thermo Fisher Scientific, Cat. No. A18817) diluted 1:40,000 in PBS was added to the corresponding wells. For detecting the His-tagged antigen, 100 pL of anti-mouse Fc HRP -conjugated secondary antibody (Jackson ImmunoLabs, Cat. No. 115-035-164) diluted 1:30,000 in PBS was used. Plates were incubated for 1 hour at room temperature, followed by another PBST wash. TMB substrate (Thermo Fisher Scientific. Cat. No. ENN301) was added at 100 pL per well, and the plates were incubated in the dark at room temperature for 5 minutes. The reaction was stopped by adding 100 pL of 0.18 M sulfuric acid (Thermo Fisher Scientific, Cat. No. A300-212) to each well. Absorbance at 450 lun was recorded using a plate reader (Molecular Devices Spectramax). Results were analyzed using GraphPad Prism software to determine affinity7and epitope specificity7.

[0187] Melting temperature analysis. The melting temperature (Tm) of bispecific antibodies was evaluated using differential scanning fluorimetry (DSF). Antibodies were diluted to a final concentration of 0.12 mg / mL in 20 mM sodium acetate buffer (pH = 5.0) and mixed with Protein Thermal Shift Dye (Applied Biosystems, Cat. No. 4461146) at a 1 x final concentration. The prepared mixtures were dispensed into a 384-well plate (Corning, Cat. No. 3680), with buffer-only wells serving as blanks. Thermal scanning was performed at the University of Michigan Advanced Genomics Core using a QuantStudio Real-Time PCR System. The temperature was ramped from 25 °C to 98 °C, and measurements were conducted in triplicate. Data analysis was performed using GraphPad Prism software to determine the melting temperatures, providing insights into the thermal stability of the antibody samples.

[0188] Pharmacokinetic analysis. Radiolabeling of antibodies was performed using [125I] Nal (Perkin Elmer) and the Pierce iodination reagent (Thermo Fisher Scientific, Cat. No. 28601). Labeled antibodies underwent purification using Zeba desalting columns to remove unbound iodine, and radiochemical integrity7was evaluated by thin-layer chromatography (TLC) on aluminum silica gel plates (MilliporeSigma, Cat. No. 105554), employing a solvent system of 75% methanol and 25% 1 M sodium acetate (pH = 6.8). The efficiency of radiolabeling routinely exceeded 75%, and only samples containing less than 5% free [125I] were deemed suitable for downstream use. For tracer studies, approximately 1 pg of [125I] -labeled IgG or bAb was combined with an appropriate amount of unlabeled antibody to reach a target radioactivity of 1x106counts per minute (cpm), as confirmed using a Wizard2 2470 gamma counter. The administered doses were standardized to 3 mg / kg (20 nmol / kg) for IgGs and 3.6 mg / kg (20 nmol / kg) for bispecific shuttles. Prior to injection, mice were weighed, and the doses were filtered through 0.2 pm sterile membranes and prepared in sterile PBS to a final volume of 110 pL. Mice were anesthetized with isoflurane (Henry Schein. Cat. No. 66794-017-25), and intravenous injections were performed via the retroorbital plexus. Data was collected at 1 hour, 1 day, and 7 days postadministration, with four mice analyzed per time point. At each time point, mice were re-anesthetized and transcardially perfused with ice-cold PBS. Blood and target organs were harvested, weighed, and subjected to gamma counting for quantitative analysis. Data analysis was performed using GraphPad Prism software.[0189 J Quantification and statistical analysis. The generation of receiver operating characteristic (ROC) cun e and the calculation of areas under the curves (AUC) in Fig.5 and Fig.6 were performed using the scikit-learn package on Python. The distribution of amino acids within the sequences of relevant libraries, the interatomic distances between residues in CDR regions and their respective epitopes, as well as the theoretical clones and corresponding average PSERM scores of the redesigned HCDR3 library (Fig. 5), were generated, analyzed and calculated using custom Python scripts.Abbreviations

[0190] PSERM: Position-Specific Enrichment Ratio Matrix

[0191] PSSM: Position-Specific Scoring Matrix

[0192] PBSB: Phosphate buffer saline with 0.1% BSA

[0193] Cyno: cynomolgus monkey

[0194] NGS: next-generation sequencing

[0195] FACS: Fluorescent- Activated Cell Sorting

[0196] MACS: Magnetic-Activated Cell SortingExample 1: Active learning permits directed evolution of species cross-reactive antibodies targeting CD98hc

[0197] To generate antibodies with broad species cross-reactivity for CD98hc, a previously reported antibody, referred to as wild-type (WT), was identified that bound human and cyno CD98hc but not mouse CD98hc

[0028] , The structure of the WT antibody has been reported in complex with the human CD98hc ectodomain (PDB: 7DF1). To overcome this species restriction, an active-learning strategy was employed to mutate the CDRs of a human antibody specific for human and cyno CD98hc and progressively evolve it toward an ultra-rare region of sequence space that permit broad cross-reactivity1(Fig. 2). First, permissive sites in the longest and most critical CDR, heavy chain CDR3 (HCDR3), were identified using mutational scanning to permit recognition of mouse CD98hc. Thus, the antibody was displayed on the surface of yeast as a single-chain variable fragment (scFv) and evaluated binding to recombinant human CD98hc (Fig. 1) before and after mutating each residue to histidine (Fig. 3A).It was found that most of the CDR sites (10 of 16) tolerated such mutations, as judged by <50% reduction of binding (Fig. 3B).

[0198] Next, a diverse HCDR3 library was generated by encoding mutations at the 10 permissive sites in HCDR3 and performed library selections for binding both human and mouse CD98hc (Fig. 3C).Progressive enrichment for binding to both human and mouse CD98hc occurred over multiple rounds of selection, with deep sequencing revealing the specific residues enriched at each site in HCDR3. Selections were separately performed against only human CD98hc or only mouse CD98hc using the same libraries as controls (Fig. 4). From the input library and three enriched libraries, a total of 53 variants were randomly selected and evaluated for their binding to both mouse and human CD98hc (Fig.5A). While all the variants displayed relatively modest binding, there was a cluster of 20 variants highlighted in purple that displayed the most promising binding to both mouse and human CD98hc. Therefore, different metrics were evaluated for predicting variants with similar species cross-reactivity using the deep sequencing data, including conventional methods - such as enrichment ratio, frequency, and position-specific scoring matrix (PSSM) - and a non-conventional method - namely positionspecific enrichment ratio matrix (PSERM) (Fig.5B)

[0029] ,

[0199] Given that the PSERM score was the best predictor of the species cross-reactive antibodies, this metric was used for redesigning the HCDR3 library (Fig. 5C). Mutations were primarily sampled with positive site-specific enrichment ratios based on the deep sequencing data, while avoiding mutations with strongly negative enrichment ratios. This resulted in sampling as few as one residue per HCDR3 position - such as the non-WT residues Trp at H97 and Gin at H100D - to as many as 16 residues at H99 and H102. The overall diversity of the redesigned library - which was predicted to be more species cross-reactive (Fig. 5D) - was smaller (~105variants) than that of the first-generation library (~1013variants), permitting exhaustive experimental screening of the resulting HCDR3 variants. The library designs were minimally impacted by whether the deep sequencing data was used after FACS sort #1 (Fig.5) or #2 (Fig.6) of the initial random library sorted against both mouse and human CD98hc.

[0200] Therefore, the redesigned library was sorted for binding to both mouse and human CD98hc, but now at lower concentrations (100 nM CD98hc for FACS #1 and 30 nM CD98hc #2) relative to that used for the initial random libraries (300 nM CD98hc; Fig. 7A). Individual clones from the final sort of the redesigned library were Sanger sequenced and evaluated. All randomly selected variants from the redesigned HCDR3 library exhibited significantly higher binding than the top-performing clones from the initial library (Fig. 8), validating the effectiveness of active learning in enriching cross-reactive antibodies. However, their binding to mouse CD98hc did not saturate at high concentrations due to theirmodest affinities, a limitation that was subsequently addressed by introducing mutations in other CDR regions. To improve the affinity', two additional libraries in parallel were generated (Fig. 9), one with diversity' in heavy chain CDRs 1 (HCDR1) and 2 (HCDR2), and another one with diversity in light chain CDRs 1 (LCDR1) and 3 (LCDR3). The diversified sites in these additional libraries were chosen based on those nearest to the epitope. After enriching lire three libraries for binding to both human and mouse CD98hc, the enriched libraries were shuffled together [30. 31], and the resulting library was further enriched for binding to both human and mouse CD98hc (Figs. 7). Individual clones isolated from the final sort of the shuffled library were sequenced and characterized. Five antibody variants from the shuffled library' were identified with affinity to mouse CD98hc - which is unique relative to the WT antibody (Fig. 7) - with equilibrium dissociation constant (KD) values of -10-60 nM. as measured by both flow cytometry and surface plasmon resonance methods (Fig. 7). Notably, the five variants retained their affinity for cyno (Fig. 7) and human (Fig. 7) CD98hc, and they display modest differences in affinity (e.g., <2-3-fold) across the three CD98hc orthologs. The level of humanness and the number of predicted T cell epitopes for the evolved antibodies were also evaluated and the properties were found favorable relative to other anti-CD98hc antibodies and clinical -stage antibodies (Fig. 12). While the level of humanness was modestly reduced (0.91-0.93 relative to 0.95 for WT) and the number of T cell epitopes was modestly increased (2-8 relative to 2 for WT) for the evolved variants, these values were similar to the best antibodies analyzed, including another anti-CD98hc antibody [BBBB578(#4)] and trastuzumab.Example 2: Bispecific antibodies targeting CD98hc possess favorable in vitro and in vivo properties

[0201] To facilitate the use of these species cross-reactive antibodies for brain shuttling applications, they were reformatted as bispecific antibodies in which each single-chain antibody was attached to the C-terminus of one of the tw o heavy' chains of a control (non-targeted) IgG using standard knob-into-hole pairing mutations in die Fc region (Fig. 12A). The resulting 2x1 bispecific antibodies were expressed in mammalian cells (HEK293-6E) and purified using Protein A chromatography. The resulting bispecific antibodies largely displayed one band on an SDS-PAGE gel before reduction and the expected three bands after reduction, including a band above the normal heavy chain due to the attached scFv (Fig. 12B).

[0202] A common problem with bispecific antibodies is that they display modest purities after single-step purification. However, the bispecific versions of the species cross-reactive antibodies displayed >90% monomer after Protein A purification - as observed for their WT equivalent - and better than the previously reported shuttle targeting mouse CD98hc (IgG / CPl ; Figs.12C and Fig.13)

[0023] . In addition, the cross-reactive shuttles also display relatively high stability, as their first unfolding transitions occur >65 °C (Figs. 12D and Fig. 14).

[0203] The evolved antibodies were tested for recognition of equivalent epitopes in mouse, cyno, and human CD98hc (Fig. 15). The WT antibody recognized human CD98hc by contacting two solvent-exposed loops, which was refer to as loops 1 (374-DAAALPGQPMEA-385) and 2 (391-DESSFPDIPGA-401), as shown in the crystal structure of the co-complex (PDB: 7DF1; Fig. 15A).Structural alignment of the predicted (cyno CD98hc) and experimental (mouse and human CD98hc) structures of each CD98hc ortholog (Fig. 15B) revealed that both loops 1 and 2 are structurally similar for human and cyno CD98hc and different for mouse CD98hc (Fig. 15C). While the cyno CD98hc structure was predicted and not experimentally validated, it was confirmed that AlphaFold3 predicted the structures of mouse, cyno, and human CD98hc with relatively high confidence (Fig. 16) and the predicted structures of mouse and human were in close agreement with the experimental structures (Fig.17).

[0204] Recombinant versions of the CD98hc ectodomains were generated, incorporating either 6 (loop 1) or 7 (loop 2) substitution mutations, and tested to determine whether the evolved antibodies required both loops for binding (Fig. 15E-F). Notably, either mutating the equivalent loop 1 (Fig. 15E) or 2 (Fig. 15F) eliminated binding for the IgG / D4-4 bispecific antibody for human, cyno, and mouse CD98hc despite loading similar amounts of WT and mutant versions of CD98hc ectodomains (Fig.18).Similar results were obtained for the other four bispecific antibodies (Fig. 19), and the ELISA EC50 values for these antibodies. The first-generation CD98hc shuttle exclusively relied on loop 2 of mouse CD98hc for binding (Fig. 19).

[0205] Finally, the pharmacokinetics of IV-administered bispecific antibodies were evaluated in WT mice to evaluate their delivery to the brain and other organs (Figs. 20 and Figs.21). Notably, a dose of 3.6 mg / kg (3 mg / kg IgG equivalent) resulted in maximal brain concentrations of 3.0-3.6 nM after 1 day, and these elevated brain levels were maintained at 1.7-2.7 nM for 1 week (Figs. 20A). As expected, engaging CD98hc resulted in relatively fast clearance from blood relative to the corresponding control IgG (Figs. 20B). This resulted 30-50x (1 day) and 66-140x (1 week) higher brain-to-blood ratios for the shuttled IgGs than the control IgG (Figs.20C).Example 3: Anti-CD98hc antibody affinity characterization on yeast[0206| Five species cross-reactive variants were expressed and identified — D4-4, D4-9, 3.5D19. ADN2-14, and 3.5D5 — as single chain Fvs (scFvs) on the surface of yeast. To validate the binding of these variants, binding assays were performed by incubating recombinant mouse, human, and cynomolgus monkey (cyno) CD98hc ectodomain with the yeast-displayed scFvs. It was observed that all five of these antibodies bound mouse, human, and cyno CD98hc ectodomain. Furthermore, each variant demonstrated similar affinity to each species of CD98hc. Notably, D4-4 and D4-9 showed higher affinities, with binding constants around 12-34 nM, while 3.5D19 and 3.5D5 exhibited relatively lower affinities, ranging from 24-70 nM.Example 4: CD98hc IgG and Control IgG / CD98hc bispecific antibody production and characterization

[0207] Having identified several cross-reactive variants using yeast surface display, five lead candidates were produced in both the IgG and CD98hc-bispecifc antibody shuttle (bAb) formats. These bAb shuttles comprise an intact phosphorylated human tau-targeted IgG, with a cross-reactive CD98hc-targeted scFv on the C-terminus of one of the heavy chains. The IgGs and bAb shuttles were able to be successfully produced at 20-50 mg / L and 10-20 mg / L in HEK cell culture respectively. Additionally, both the IgG and bAb formats of the selected cross-reactive variants achieved > 90% monomeric purity. Both IgG and bAb formats, as characterized by SDS-PAGE and SEC, demonstrated favorable biophysical properties.

[0208] To characterize affinity, an ELISA was performed on a dilution series of mouse, human, and cyno CD98hc ectodomains. To characterize the epitope, mouse, human, and cyno CD98hc ectodomains were designed and expressed with the predicted epitope replaced with a glycine-serine linker (referred to as CD98hc loop knockouts). Then an ELISA on a dilution series of the CD98hc loop knockouts were performed. It was observed that EC50s between 1-10 nM for mouse, human, and cyno CD98hc for the five bAbs. Additionally, similar affinities towards the relative affinities for each of the bAbs matched their yeast display ed-scFv counterparts were shown. Likewise, it was noted that a 10-fold increase in affinity in the bAb format compared to the scFv format for all five variants, despite both formats being monovalent for CD98hc. This can be attributed to the format of the binding assay. Furthermore, it was observed a ncar-complctc reduction of binding of each of the variants to the CD98hc loop knockouts. It was also shown that the wild type bAb loses binding to the human and cyno CD98hc loop knockouts, demonstrating that throughout the engineering process, die epitope of the original antibody was maintained.Example 5: Control IgG / CD98hc pharmacokinetic analysis

[0209] To understand the pharmacokinetic profile of the species cross-reactive antibodies, quantitative radiotracing was preformed. Bispecific antibodies pTau-D4-4, pTau-3.5D19 and pTau-3.5D5 were radiolabeled with125I and injected WT mice IV (retro-orbital) at 3.6 mg / kg (20 nmol / kg). Mice were sacrificed at different time points (1 horn and 1 day), and various organs including the brain, as well as blood were collected.

[0210] It was observed that there was significant brain uptake and retention for all three bAbs. Antibody concentrations in the brain were 2-3 nM at 1 hour post-injection, increasing to over 3 nM after 24 hours. When analyzing the distribution of the bAb shuttles in blood and other organs over time, it was found that antibody levels rapidly decreased as they were cleared from circulation and most organs, except for the kidney and brain. It was anticipated that only the brain will maintain the antibody levels over an extended period (e.g., 7 days), given the strong binding of the shuttles to CD98hc antigens at the blood-brain barrier, which was expected to prolong their retention in the brain.Example 6: Functional Paratope Mapping of Species Cross-Reactive Anti-CD98hc Antibodies

[0211] To understand the molecular basis for species cross-reactivity, functional paratope mapping was performed on the D4-4 antibody using alanine scanning and reversion mutagenesis (see FIG. 22).

[0212] Single alanine substitution mutations were introduced at all positions in HCDR3 as well as sites in other CDRs that were mutated relative to the WT antibody. Native alanine residues were substituted with glycine. The relative binding of the D4-4 mutants to human and mouse CD98hc was assessed via yeast surface display using 100 nM mouse or human CD98hc ectodomain.

[0213] The alanine scanning analysis revealed that the D4-4 antibody relies primarily on HCDR3 residues for antigen binding. Of 13 CDR sites identified as important for binding (defined as those reducing binding by >50% upon mutation to alanine), 12 were located in HCDR3. Notably, D4-4 showed differential dependence on specific HCDR3 residues for binding to mouse versus human CD98hc. Alanine mutations at 11 of 16 HCDR3 sites reduced D4-4 binding to mouse CD98hc by >50%, while only 3 HCDR3 sites showed equivalent importance for binding to human CD98hc.

[0214] Only three CDR residues were identified as important for D4-4 binding to both human and mouse CD98hc: Tyr-96 and Trp-97 in HCDR3, and Leu-50 in HCDR2. The identification of Leu-50 in HCDR2 as a key residue for dual-species binding is particularly significant, as this position corresponds to one of the conserved mutations formd across all five species cross-reactive antibodies (D4-4, D4-9, 3.5D19, ADN2-14, and 3.5D5).

[0215] The key HCDR3 residues for D4-4 binding to human CD98hc were identified as H96-97 and H100B, representing a compact binding interface. In contrast, the key HCDR3 residues for mouse CD98hc binding spanned a broader region including H96-99 and H100C-101, indicating that species cross-reactivity involves engagement of additional paratope residues to accommodate the structural differences between human and mouse CD98hc.

[0216] To identify which mutations acquired during directed evolution were important for species cross-reactivity, reversion mutagenesis was performed. Single CDR reversion mutations were introduced at all sites mutated relative to the WT antibody, replacing each D4-4 CDR mutation with the corresponding WT residue.

[0217] The reversion scanning analysis identified six key CDR sites that, when reverted to WT sequence, led to >50% reduction in D4-4 binding to mouse CD98hc. In contrast, only two reversion mutations had similar effects on D4-4 binding to human CD98hc, demonstrating that the acquired mutations are more important for mouse CD98hc recognition than for maintaining affinity’ to human CD98hc.

[0218] One reversion mutation in HCDR3 (Ser-HIOOE to Pro) eliminated binding to both human and mouse CD98hc. Without wishing to be bound by theory, this is believed to result from the incompatibility of two consecutive proline residues in the HCDR3 loop, as tire adjacent residue at position H100D was mutated to proline in D4-4.

[0219] Significantly, a proline mutation in LCDR1 (Pro-L27E) was identified as important for D4-4 binding to mouse CD98hc but dispensable for human CD98hc binding. This finding highlights the importance of light chain CDR mutations in facilitating species cross-reactivity and explains why the directed evolution strategy of combining mutations from multiple CDR libraries (HCDR3, HCDR1 / HCDR2, and LCDR1 / LCDR3) was important to achieve broad cross-reactivity.

[0220] The paratope analysis demonstrates that the species cross-reactive antibodies achieve dual recognition through an expanded binding interface that engages both conserved and species-specific epitope residues on CD98hc. The HCDR3 mutations facilitate recognition of the structurally distinct loop regions on mouse CD98hc, while mutations in HCDR2 and LCDR1 provide additional contacts necessary for high-affinity binding across species.

[0221] Structural models of D4-4 were generated using ABodyBuilder to visualize the spatial distribution of key paratope residues. The models revealed that the key CDR sites for binding human CD98hc (H96-97, H100B, H50) form a relatively compact cluster at the center of the antigen-binding site. In contrast, the key sites for mouse CD98hc binding extend beyond this central cluster to include peripheral HCDR3 residues (H100C-101) and residues in HCDR1 (H35) and LCDR1 (L27E), indicating that species cross-reactivity involves engagement of a broader paratope surface.

[0222] These findings demonstrate that the active learning approach employed in the directed evolution of species cross-reactive antibodies successfully identified mutations that expand the functional paratope to accommodate the structural differences betw een human and mouse CD98hc orthologs while maintaining high-affinity binding to both targets.Example 7: Pharmacokinetic Analysis of Species Cross-Reactive Antibodies in Humanized CD98hc Mice

[0223] To validate the brain delivery properties of the species cross-reactive antibodies in a model expressing human CD98hc, pharmacokinetic studies were conducted in transgenic mice in which the extracellular domain of murine CD98hc was replaced with that of human CD98hc (humanized CD98hc mice).

[0224] Prior to pharmacokinetic analysis, CD98hc expression levels on brain endothelial cells were quantified in both wild-type and humanized CD98hc mice. Mouse brains were harvested following transcardial perfusion with ice-cold phosphate-buffered saline to remove circulating blood cells. Brain tissue was processed using enzymatic dissociation, and cells were stained with anti-mouse CD45. antimouse CD31, and species-appropriate anti-CD98hc antibodies conjugated to Alexa Fluor 647. Endothelial cells were identified by CD31 positivity, and receptor density was quantified using calibration beads.

[0225] The humanized CD98hc mice expressed approximately 270,000 CD98hc molecules per brain endothelial cell, which was substantially higher than the approximately 40,000 CD98hc molecules per cell observed in wild-type mice. This elevated expression level in humanized mice is consistent withthe replacement of murine CD98hc with the human ortholog and provides a suitable model for evaluating antibodies targeting human CD98hc.|0226] To evaluate the brain delivery of the species cross-reactive D4-4 shuttle in humanized CD98hc mice, radiolabeled bispecific antibodies were administered intravenously at a dose of 60 imiol / kg (10.8 mg / kg for IgG / scFv shuttles or 9 mg / kg for IgG controls). Brain and blood concentrations were measured at 1 day and 1 week post-injection using125I radiotracing. For comparison, the previously reported human / cyno-specific antibody transport vehicle TV6.8 was evaluated in parallel experiments. To provide an appropriate control for the IgG / D4-4 shuttle, the TV6.8 construct was reformatted to include the same control IgG Fab domain used in lgG / D4-4, replacing the anti-dinitrophenyl Fab domain used in the original report of TV6.8.

[0227] The species cross-reactive IgG / D4-4 shuttle demonstrated brain uptake and retention in humanized CD98hc mice that was comparable to the human / cyno-specific TV6.8 shuttle. At 1 day postinjection, IgG / D4-4 achieved brain concentrations that were similar to those observed for TV6.8. Brain levels for both shuttles remained elevated at 1 week post-injection, demonstrating sustained retention consistent with CD98hc-mediated transcytosis and brain parenchymal accumulation.

[0228] The human / cyno-specific WT bispecific antibody (IgG / WT), which binds human and cynomolgus CD98hc but not mouse CD98hc, showed enhanced brain uptake in humanized CD98hc mice compared to its negligible uptake in wild-type mice. This result confirms that the humanized CD98hc mouse model faithfully recapitulates CD98hc -mediated brain deliver}' for antibodies targeting human CD98hc.

[0229] The control IgG lacking a CD98hc-targeting moiety showed minimal brain uptake in humanized CD98hc mice, consistent with the limited ability of unshuttled antibodies to cross the bloodbrain barrier. This control confirms that the enhanced brain deliver}' observed for IgG / D4-4 and TV6.8 is attributable to CD98hc-mediated transcytosis rather than non-specific mechanisms.

[0230] Blood concentrations of IgG / D4-4 and TV6.8 were similar at both the 1 day and 1 week timepoints, indicating comparable systemic pharmacokinetics for the two shuttles. Both CD98hc-targeting bispecific antibodies showed accelerated clearance from blood compared to the control IgG, consistent with CD98hc-mediated uptake into tissues expressing the target receptor.

[0231] The similar blood pharmacokinetics of IgG / D4-4 and TV6.8 suggest that differences in epitope or affinity between these two shuttles do not substantially impact systemic disposition in the humanized CD98hc mouse model. This finding supports the utility of IgG / D4-4 as a translational tool for evaluating CD98hc-mediated brain delivery in preclinical models.

[0232] Biodistribution analysis revealed that IgG / D4-4 and TV6.8 showed similar accumulation in peripheral organs at both 1 day and 1 week post-injection. Both shuttles demonstrated elevated uptake in the kidney and spleen compared to the control IgG, consistent with CD98hc expression in these tissues. The similar peripheral biodistribution profiles of IgG / D4-4 and TV6.8 further support theconclusion that the species cross-reactive D4-4 antibody engages human CD98hc with comparable efficiency to previously reported human / cyno-specific shuttles.|0233] Liver and lung concentrations were similar across all antibodies tested, indicating that CD98hc-mediated uptake does not substantially impact distribution to these organs. Heart concentrations were also comparable across groups.

[0234] To contextualize the brain delivery performance of the species cross-reactive antibodies, results from humanized CD98hc mice were compared to those obtained in wild-type mice.

[0235] In wild-type mice, the species cross-reactive shuttles (IgG / D4-4, IgG / 3.5D19, and IgG / 3.5D5) achieved peak brain concentrations of 3.1±0.19 nM, 3.1±0.17 nM, and 3.6±0.41 nM. respectively, at 1 day post-injection at a dose of 20 nmol / kg. These brain concentrations were sustained at 1 week, with levels of 1.8±0.1 nM for IgG / D4-4, 2.7±0.1 nM for IgG / 3.5D19, and 1.9±0.1 nM for IgG / 3.5D5. The brain-to-blood ratios for shuttled antibodies were 31-47 fold higher than control IgG at 1 day and 66-144 fold higher at 1 week.

[0236] At a higher dose of 60 nmol / kg in wild-type mice, IgG / 3.5D5 achieved the highest brain concentrations among the tested shuttles, with 6.9±0.3 nM at 1 day and 4.5±0.7 nM at 1 week. The human / cyno-specific IgG / WT shuttle showed minimal brain uptake in wild-type mice, with brain concentrations comparable to the control IgG, confirming its lack of mouse CD98hc cross-reactivity.

[0237] In humanized CD98hc mice at 60 nmol / kg, the species cross-reactive IgG / D4-4 shuttle achieved brain concentrations comparable to the human / cyno-specific TV6.8 shuttle, demonstrating that the engineered cross-reactivity does not compromise brain delivery efficiency when targeting human CD98hc.Example 8: Assessment of Species Cross-Reactive Anti-CD98hc Antibodies

[0238] To evaluate the suitability of the species cross-reactive antibodies for therapeutic development, comprehensive developability assessments were performed including analysis of self-association propensity', non-specific binding, thermal stability, and aggregation.

[0239] Antibody self-association was evaluated using charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS), a technique that measures the propensity of antibodies to form reversible selfassociations at low concentrations.

[0240] Gold nanoparticles were coated with a mixture of anti-human capture antibody and poly lysine at a 90: 10 weight ratio. After overnight incubation, the gold conjugates were mixed with dilute antibody solutions (11.1 pg / mL) and incubated for 4 horns at room temperature. Absorbance spectra were acquired from 450-650 imr, and the plasmon wavelength was determined by identif ing the maximum absorbance value. Results were normalized using a calibration panel of antibodies with known selfassociation properties.

[0241] In the bispecific antibody format, all species cross-reactive antibodies (IgG / D4-4, IgG / D4-9, IgG / 3.5D19, IgG / ADN2-14, and IgG / 3.5D5) showed similar levels of self-association with nostatistically significant differences betw een them (p-value >0.05, t-test). The self-association scores for the species cross-reactive bispecific antibodies w ere comparable to those of the WT bispecific antibody (IgG / WT) and the mouse-specific CPI bispecific antibody (IgG / CPl), indicating that the mutations introduced during directed evolution did not adversely impact self-association behavior.[0242| In the IgG format, the WT and species cross-reactive IgGs exhibited significantly lower selfassociation than the control IgG and CPI IgG (p-value <0.05, t-test). This finding indicates that the species cross -reactive antibodies possess favorable self-association properties.

[0243] Non-specific binding to membrane proteins was evaluated using the Polyspecificity Particle (PSP) assay, which measures antibody binding to a soluble membrane protein preparation derived from CHO cells.

[0244] The soluble membrane protein (SMP) fraction was prepared from CHO cells by sequential homogenization, sonication, and ultracentrifugation. The resulting SMP preparation was biotinylated for detection purposes. Antibodies were immobilized on Protein A magnetic beads and incubated with biotinylated SMP. Bound SMP was detected using streptavidin conjugated to Alexa Fluor 647, and fluorescence intensity was measured by How cytometry.

[0245] Binding signals were normalized betw een emibetuzumab (a clinical-stage antibody with high non-specific binding) and elotuzmnab (a clinical-stage antibody with low' non-specific binding) to generate a polyspecificity score ranging from 0 (low non-specific binding) to 1 (high non-specific binding). Each antibody was evaluated in nine independent replicates.

[0246] The WT IgG showed low non-specific binding with a poly specificity score that was not significantly different from two of the species cross-reactive antibodies, D4-4 and 3.5D5 (p-value >0.05, t-test). This finding indicates that D4-4 and 3.5D5 maintain the favorable non-specific binding profile of the parental WT antibody despite the mutagenesis performed during directed evolution.

[0247] Three of the species cross-reactive antibodies (D4-9, 3.5D19. and ADN2-14) showed statistically higher levels of non-specific binding compared to the WT and CPI IgGs (p-value <0.05, t-test). However, the polyspecificity scores for these antibodies remained within the range observed for clinical-stage antibodies.

[0248] Thermal stability w as evaluated using differential scanning fluorimetry (DSF), w hich measures protein unfolding as a function of temperature by monitoring binding of a fluorescent dye to hydrophobic regions exposed during denaturation.

[0249] Bispecific antibodies were diluted to 0.12 mg / mL in 20 mM sodium acetate buffer (pH 5.0) and mixed w ith Protein Thermal Shift Dye at 1xfinal concentration. Thermal scanning was performed using a QuantStudio Real-Time PCR System with temperature ramping from 25°C to 98°C. Measurements were conducted in triplicate for each antibody.

[0250] All species cross-reactive bispecific antibodies demonstrated first unfolding transitions (Tml) above 65°C, indicating favorable thermal stability. The specific melting temperatures were as follows:IgG / D4-4 showed Tml of 67.1±0.3°C and Tm2 of 73.1±0.2°C; IgG / D4-9 showed Tml of 66.3±0.1°C; IgG / 3.5D19 showed Tml of 66.1±0.3°C; IgG / ADN2-14 showed Tml of 66.5±0.1°C and Tm2 of 73.6±0.03°C; and IgG / 3.5D5 showed Tml of 66.7±0.04°C and Tm2 of 73.1±0.1°C.

[0251] For comparison, the WT bispecific antibody (IgG / WT) showed Tml of 66.6±0.04°C and Tm2 of 74.6±0.1°C, while the mouse-specific CPI bispecific antibody (IgG / CPl) showed Tml of 64.6±0.1°C with no detectable Tm2.[0252| The presence of two distinct unfolding transitions in several of the bispecific antibodies (IgG / WT. IgG / D4-4, IgG / ADN2-14, and IgG / 3.5D5) is consistent with independent unfolding of the IgG and scFv domains. The Tm2 values of 73-75°C for these antibodies indicate that both domains possess favorable thermal stability.

[0253] Aggregation propensity was evaluated by size-exclusion chromatography (SEC) following single-step Protein A purification. The percentage of monomeric protein after purification is an important developability parameter because antibodies with high aggregation propensity often require complex purification schemes and may exhibit poor storage stability.

[0254] Bispecific antibodies were expressed in HEK293-6E cells and purified using Protein A agarose chromatography. Purified antibodies were analyzed by SEC using a Superdex 200 Increase 10 / 300 GL column, and the percentage of monomer was calculated from the integrated peak areas.

[0255] All species cross-reactive bispecific antibodies demonstrated greater than 90% monomer after single-step Protein A purification. The specific monomer percentages were as follows: IgG / D4-4 showed 92.2±1.8% monomer; IgG / D4-9 showed 95.3± 1.7% monomer; IgG / 3.5D19 showed 93.7±1.0% monomer; IgG / ADN2-14 showed 92.9±1.1% monomer; and IgG / 3.5D5 showed 92.0±2.5% monomer.

[0256] For comparison, the WT bispecific antibody (IgG / WT) showed 92.1±2.3% monomer, while the mouse -specific CPI bispecific antibody (IgG / CPl) showed only 81.0±2.0% monomer. The species cross-reactive antibodies thus demonstrated aggregation propensity comparable to the parental WT antibody and substantially lower than the mouse-specific CPI antibody.

[0257] Correct assembly of the bispecific antibodies was confirmed by SDS-PAGE analysis under reducing and non-reducing conditions. Under non-reducing conditions, the bispecific antibodies migrated predominantly as a single band with molecular weight consistent with the expected heterodimeric assembly of one standard heavy chain, one heavy chain with C-terminal scFv fusion, and two light chains.

[0258] Under reducing conditions, three bands were observed corresponding to the light chain, standard heavy chain, and heavy' chain-scFv fusion. The molecular weights of all bands were consistent with theoretical predictions.

[0259] Potential immunogenicity of the species cross-reactive antibodies was assessed by computational prediction of T cell epitopes and evaluation of humanness scores.

[0260] T cell epitope prediction was performed using NetMHCIIpan-4.3 with peptide length set to 15 amino acids. Sequences were evaluated against 27 MHC class II molecules covering HLA DR, DQ, and DP specificities representative of the general population. Peptides ranking in the top 2% by predicted binding affinity were classified as strong binders, and peptides between 2% and 10% were classified as weak binders. Predicted epitopes matching gennline sequences were excluded from the analysis, as these are expected to be tolerized and unlikely to elicit T cell responses.[02611 The number of predicted non-germline T cell epitopes for the species cross-reactive antibodies ranged from 4 to 8. compared to 2 for the parental WT antibody. While the species cross-reactive antibodies showed modestly increased predicted T cell epitopes relative to WT, the values remained comparable to clinical-stage antibodies including trastuzumab and other approved therapeutics.

[0262] Humanness scores were calculated using AbNatiV, which evaluates antibody sequences for similarity to human germline and mature antibody repertoires. The humanness scores for the species cross-reactive antibodies ranged from 0.91 to 0.93. compared to 0.95 for the parental WT antibody.

[0263] The comprehensive developabilitv assessment demonstrates that tire species cross-reactive anti-CD98hc antibodies possess favorable biophysical and biochemical properties suitable for therapeutic development.REFERENCES

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Claims

CLAIMSWhat is claimed is:

1. A species cross-reactive antibody comprising a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2.

2. A species cross-reactive antibody comprising a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4.

3. A species cross-reactive antibody comprising a CDR-H3 having a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.

11. SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.

4. A species cross-reactive antibody comprising a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO.

16. SEQ ID NO.

17. SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22.

5. A species cross-reactive antibody comprising a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23.

6. A species cross-reactive antibody comprising a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

7. The species cross -re active antibody of any of claims, 1-6, wherein said at least 70% sequencing identity comprises at least 80% sequence identity.

8. The species cross -re active antibody of any of claims, 1-6, wherein said at least 70% sequencing identity comprises at least 90% sequence identity.

9. The species cross-reactive antibody of any of claims. 1-6, wherein said at least 70% sequencing identity comprises at least 95% sequence identity.

10. The species cross-reactive antibody of any of claims 1-6, comprising a heavy chain variable region that includes CDR-H1, CDR-H2, and CDR-H3.

11. The species cross-reactive antibody 10. wherein the heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11, SEQ ID NO.

12. SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO. 8, SEQ ID NO.

9. SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.

12. The species cross-reactive antibody of any of claims 1-6, comprising a light chain variable region that includes CDR-L1. CDR-L2, and CDR-L3.

13. The species cross-reactive antibody of claim 12, wherein the light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO.

18. SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO.

18. SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO. 23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

14. The species cross-reactive antibody of any of claims 1-13, wherein said species cross-reactive antibody has a heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO.

9. SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO.

8. SEQ ID NO.

9. SEQ ID NO.

10. SEQ ID NO.

11. SEQ ID NO.

12. SEQ ID NO.

13. or SEQ ID NO.14 and a light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO.15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO.

20. SEQ ID NO.

21. or SEQ ID NO. 22 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO.

18. SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO.23 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25 or an amino acid sequence with at least 70% sequence identity to SEQ ID NO. 24 or SEQ ID NO. 25.

15. The species cross-reactive antibody of any of claims 1-14, wherein said species cross-reactive antibody has a heavy chain variable region comprises a CDR-H1 having an amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2; a CDR-H2 having a sequence of SEQ ID NO. 3 or SEQ ID NO. 4; a CDR-H3 comprising a sequence of SEQ ID NO. 5, SEQ ID NO.

6. SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.

13. or SEQ ID NO. 14 and a light chain variable region comprises a CDR-L1 having a sequence of SEQ ID NO.

15. SEQ ID NO.

16. SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22; a CDR-L2 having a sequence of SEQ ID NO. 23; and a CDR-L3 having a sequence of SEQ ID NO. 24 or SEQ ID NO. 25.

16. The species cross-reactive antibody of claim 15. comprising:a) a heavy chain amino acid sequence of SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO.28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.

32. SEQ ID NO.33, SEQ ID NO. 34 or SEQ ID NO. 35; andb) a light chain ammo acid sequence of SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO.38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO.

43. SEQ ID NO. 44 or SEQ ID NO. 45.

17. A species cross-reactive antibody comprising an amino acid sequence selected from SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54 or SEQ ID NO. 55.

18. The species cross-reactive antibody of any one of claims 1-17, wherein the species cross- reactive antibody is a non-targeted antibody.

19. The species cross-reactive antibody of claim 18, wherein said non-targeted antibody is an IgG antibody.

20. The species cross-reactive antibody of claim 19, wherein the species cross-reactive antibody comprises a single-chain variable fragment (scFv) at the C-tenninus of the heavy chain.

21. The species cross-reactive antibody of any one of claims 1-17. wherein the species cross- reactive antibody is cross-reactive across three or more species.

22. The species cross-reactive antibody of claim 21, wherein the species cross-reactive antibody is functional in mammals.

23. The species cross-reactive antibody of claim 22, wherein the mammal is a human.

24. The species cross-reactive antibody of claim 21, wherein the antibody is functional in nonhuman mammals.

25. The species cross-reactive antibody of claim 24, wherein the non-human mammal is a mouse.

26. The species cross-reactive antibody of claim 25, wherein the non-human mammal is a cynomolgus monkey.

1. The species cross-reactive antibody of any one of claims 1-26, wherein species cross-reactive antibody specifically binds to CD98hc.

28. The species cross-reactive antibody of claims 27, wherein species cross-reactive antibody is a bispecific antibody.

29. The species cross-reactive antibody of claims 28. wherein the bispecific antibody binds to a protein on / or in a neuron.

30. The species cross-reactive antibody of claims 29, wherein the neuron is associated with a neurological disease and / or condition.

31. The species cross-reactive antibody of claims 30, wherein the neurological disease and / or condition is Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease, multiple sclerosis (MS), frontotemporal dementia (FTD), Lewy body dementia (LBD), neuromyelitis optica spectrum disorder (NMOSD), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), stroke, cerebral ischemia, brain microvascular dysfunction, vascular dementia, schizophrenia, intellectual disabilities, autism spectrum disorder (ASD), depression, anxiety disorders, bipolar disorder, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), myasthenia gravis (MG), peripheral neuropathy, glioblastoma (GBM), and other brain tumors such as astrocytomas, meningiomas, and medulloblastomas.

32. The species cross-reactive antibody of any one of claims 1-31 wherein species cross-reactive antibody is configured to cross the blood brain barrier (BBB).

33. A pharmaceutical composition comprising the species cross-reactive antibody of any of claims 1 -32 and a pharmaceutically acceptable carrier.

34. Use of a species cross-reactive antibody or pharmaceutical composition of any of claims 1-33.

35. Use of a species cross-reactive antibody or pharmaceutical composition of any of claims 1-33 to treat a neurological disease and / or condition or improve a neurological function.

36. A method for treating a neurological disease and / or condition or improving a neurological function comprising administering the species cross-reactive antibody or pharmaceutical composition of any of claims 1 -33 to a subject.

37. The method of claim 36, wherein the neurological disease and / or condition is Alzheimer’s disease (AD), Parkinson’s disease (PD). amyotrophic lateral sclerosis (ALS). Huntington’sdisease, multiple sclerosis (MS), frontotemporal dementia (FTD), Lewy body dementia (LBD), neuromyelitis optica spectrum disorder (NMOSD), chronic inflammatory demyelinating polyneuropathy (CIDP). Guillain-Barre syndrome (GBS), stroke, cerebral ischemia, brain micro vascular dysfunction, vascular dementia, schizophrenia, intellectual disabilities, autism spectrum disorder (ASD), depression, anxiety disorders, bipolar disorder, obsessive- compulsive disorder (OCD), post-traumatic stress disorder (PTSD), myasthenia gravis (MG), peripheral neuropathy, glioblastoma (GBM), and other brain tumors such as astrocytomas, meningiomas, and medulloblastomas.

38. The method of claim 37, wherein the species cross-reactive antibody is administered intravenously.

39. The method of claim 37, wherein the species cross-reactive antibody is co-administered with a neuroprotective agent, an anti-inflammatory agent, or a small-molecule therapeutic agent for enhanced efficacy.

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