WO2025054419A1

WO2025054419A1 - Compositions for transport of therapeutic cargos using antibody binders targeting ca-iv

Info

Publication number: WO2025054419A1
Application number: PCT/US2024/045537
Authority: WO
Inventors: Xinhong Chen; Viviana Gradinaru; Timothy F. SHAY; Kate MALECEK; Erin E. SULLIVAN; Xiaozhe DING; Seongmin Jang
Original assignee: California Institute of Technology
Current assignee: California Institute of Technology
Priority date: 2023-09-08
Filing date: 2024-09-06
Publication date: 2025-03-13
Anticipated expiration: 2026-03-08

Abstract

The present invention provides compositions and methods comprising antibodies as shuttles for the BBB-crossing, for example through the receptor carbonic anhydrase IV (CA-IV).

Description

Attorney Docket No.: RECE-005/01WO 40321/31 Compositions for Transport of Therapeutic Cargos Using Antibody Binders Targeting CA-IV Field of the Invention The invention relates to methods and shuttles for crossing the blood brain barrier. Background The blood brain barrier (BBB) presents a fundamental bottleneck to the development of effective research tools and therapeutics for the central nervous system (CNS). This structure, comprising mainly of brain endothelial cells, requires large molecules to be delivered via invasive intracranial injections, technically challenging focused ultrasound, or receptor-mediated transcytosis. The rational design of BBB-crossing large molecules has long been hampered by the imperfect understanding of the mechanisms involved in transcytosis, with only a handful of targets, such as the transferrin receptor, validated for research and therapies. Thus, the identification of BBB-crossing targets, mechanisms, molecules and methods is needed to improve the efficiencies of research tools and therapies for CNS. Summary The present invention provides compositions and methods comprising antibodies as shuttles for the BBB-crossing, for example through the receptor carbonic anhydrase IV (CA-IV). The present invention provides novel human CA-IV interacting antibodies, including monoclonal antibodies, that provide optimal CA-IV interaction strength and efficiency as bispecific mAbs or antibody drug conjugates (ADC). Aspects of the invention provide a conjugate comprising a blood brain barrier (BBB) shuttle. The shuttle comprises an antibody or fragment thereof comprising a binding region which binds to a portion of carbonic anhydrase IV (CA-IV). The conjugate further comprises a therapeutic cargo conjugated to the shuttle. The antibody or fragment thereof may comprise a heavy chain sequence having at least 95% sequence identity with a selected from SEQ ID NO: 91-135. The antibody or fragment thereof may comprise a heavy chain sequence having at least 95% sequence identity with a Attorney Docket No.: RECE-005/01WO 40321/31 selected from SEQ ID NO: 1-45. The antibody or fragment thereof may comprise a light chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 136- 180. The antibody or fragment thereof may comprise a light chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 46-90. The heavy and light chain sequences may be paired as shown in column 1 of each of Table 1, Table 2, and Table 3 below. The antibody or fragment thereof may be a humanized antibody. The antibody or fragment there may comprise one or more framework regions. The one or more framework regions may be derived from immunoglobulin G (IgG). The therapeutic cargo may be conjugated to the shuttle via a linker. Therapeutic cargo may covalently conjugated to the shuttle. The therapeutic cargo may be a biological molecule, for example a nucleic acid (for example, RNA, siRNA, DNA, or an ASO), a protein (for example, an enzyme), a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof.. The therapeutic cargo may be a non-biological molecule, for example a small molecule or a dye. In aspects of the invention, the payload may comprise DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutics, interfering RNA, gene editing cargo, chemotherapeutic agents, toxins, radioisotopes, enzymes, chelators, boron compounds, photoactive agents, dyes, metals, metal alloys, nanoparticles, or other larger synthetic molecules and biologics in vitro and in vivo. When the therapeutics cargo is an antibody or fragment thereof, the shuttle together with the therapeutic cargo may form a bispecific antibody. For example, the shuttle may comprise a heavy and light chain forming one fragment antigen-binding (Fab) arm of the antibody and the therapeutic cargo may form a second Fab arm of the antibody. The bispecific antibody may be IgG-like or non-IgG like. For example, the bispecific antibody may further comprise a Fc region, advantageously the antibody may form a trifunctional antibody. The two arms may be linked from the introduction of “knob into holes” mutations. Alternatively, the two arms of the antibody may together form of a single chain variable fragment (scFv). Advantageously, the shuttle may be a carbonic anhydrase IV (CA-IV) shuttle. Thereby, when provided to a cell expressing CA-IV as a surface protein, binding of the shuttle to the CA- Attorney Docket No.: RECE-005/01WO 40321/31 IV protein mediates transcytosis of the therapeutic cargo across the BBB. Further advantageously, conjugates of the invention may be delivered to the brain or eye of a subject and provide therapeutic effect. The therapeutic cargo may be a therapeutic cargo for the treatment of a disorder affecting the central nervous system. The CA-IV shuttle may be a shuttle for human CA-IV. In aspects of the invention, antibody sequences may also be provided with or without other protein scaffolds in other delivery systems, including viral vectors (e.g., lentivirus, adenovirus, AAVs), non-viral nanoparticles, exosomes, antibodies, antibody-drug conjugates, or proteins. Aspects of the invention further provide methods and uses that comprise delivery of a therapeutic cargo across the BBB of a subject. Methods and uses of the invention (including in the formulation of a medicament), comprise providing to a subject a conjugate of the invention, as described throughout this application, comprising an antibody shuttle of the invention and a therapeutic cargo conjugated to the shuttle. For sequences disclosed throughout this application, it is understood nucleic acid molecules and peptides may comprise one or more substitutions, for example conservative substitutions, that allow sequences to continue to function. Accordingly, sequences may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence disclosed. A conservative substitution refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. For example, conservative substitutions may include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp Attorney Docket No.: RECE-005/01WO 40321/31 or W). Additionally, or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitutions groups include sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. For example, heavy and light chains of antibodies included in the present invention, including at a % sequence identity, are provided in Table 1 and Table 2 below: Table 1: Select heavy chain sequences SEQ Antibody Heavy chain sequence ID

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ Antibody Heavy chain sequence ID NO

Table 2: Select light chain sequences SEQ A tibd Liht hi ID

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ Antibody Light chain sequence ID NO

Attorney Docket No.: RECE-005/01WO 40321/31 Table 3: Select heavy and light chain variable regions SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 36 37 38 39 40 41

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 42 43 44 45 46 47 48

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 49 50 51 52 53 54

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 55 56 57 58 59 60 61

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 62 63 64 65 66 67 68

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 69 70 71 72 73 74 75

Attorney Docket No.: RECE-005/01WO 40321/31 SEQ SEQ Antibody Heavy Chain variable region ID Light Chain variable region ID NO 76 77 78 79 80

Brief Description of the Drawings FIG.1 is an antibody discovery workflow of the invention. FIG.2 is a set of images of fluorescent binding of antibodies of the invention. FIG.3 is a workflow for characterizing antibody binding and internalization. FIG.4A-B are graphs of Surface Plasmon Resonance (SPR) Assay results of antibody CA-IV binding. FIG.5 shows images from immunohistochemistry (IHC) staining following transfection with an antibody of the invention. Attorney Docket No.: RECE-005/01WO 40321/31 FIG.6 shows images from IHC staining following transfection with an antibody of the invention. FIG.7 shows images from fluorescent staining following transfection with antibody mutants of the invention. FIG.8A-B shows images from fluorescent staining following transfection with antibody mutants of the invention under permeabilizing and non-permeabilizing stain conditions, respectively. FIG.9A-D are graphs of Puncta analysis from fluorescent assays from antibody mutants of the invention showing antibody count. FIG.10A-D are graphs of Puncta analysis from fluorescent assays from antibody mutants of the invention showing antibody intensity. Detailed Description The present invention provides compositions and methods comprising antibodies as shuttles for the BBB-crossing, for example through the receptor carbonic anhydrase IV (CA-IV). Receptors for Enhanced Blood-Brain Barrier Crossing Blood-brain barrier (BBB) has emerged as a complex, dynamic, adaptable interface that controls the exchange of substances between the central nervous system (CNS) and the blood, to prevent the uncontrolled leakage of substances from the blood into the brain. The cells that make up the structure of the BBB include mostly brain endothelial cells, which constantly communicate with the other cells of the CNS (e.g., astrocytes, microglia, neurons, mast cells and pericytes, as well as circulating immune cells), adapting their behaviors to serve the needs of the CNS, responding to pathological conditions, and in some cases participating in the onset, maintenance or progression of disease. The complexity of BBB functions explains much of the difficulty in developing drugs that can cross the BBB. Utilizing receptors on the BBB interface can offer a method of crossing BBB. The present invention provides shuttles for receptors on the BBB interface and methods of using the same to enhance BBB crossing and CNS potency, such as increasing the permeability of the BBB and delivering a therapeutic agent across the BBB to a nervous system, specifically carbonic anhydrase IV. Without being bound by any theory, the novel target Attorney Docket No.: RECE-005/01WO 40321/31 receptors disclosed herein may facilitate enhanced BBB receptor-mediated transcytosis across various species, including mammals such as human. In some embodiments, a method of increasing permeability of the BBB comprises providing a shuttle capable of binding to a BBB crossing receptor (e.g., carbonic anhydrase IV), thereby increasing permeability of the BBB (e.g., through transcytosis). In some embodiments, at least one activity of the BBB-crossing receptor (e.g., carbonic anhydrase IV) can be reduced through binding to a small molecule. Accordingly, in some embodiments, a method of increasing permeability of the BBB comprises reducing the activity of carbonic anhydrase IV, thereby increasing permeability of the BBB. In some embodiments, a shuttle binds to one or more of the zinc binding site (e.g., a catalytic pocket) and substrate binding site of the carbonic anhydrase IV. The carbonic anhydrase IV can be a vertebrate carbonic anhydrase IV including non-human primates and humans. In some embodiments, the carbonic anhydrase IV is a mouse carbonic anhydrase IV (Car4), a human carbonic anhydrase IV (CA4), or a variant or a homolog thereof. Carbonic Anhydrase IV The present invention provides shuttles for the BBB-crossing suing the receptor carbonic anhydrase IV, capable of facilitating the delivery of a pharmaceutical agent across the BBB (CA- IV shuttles). Carbonic anhydrase IV is an isozyme that belongs to the carbonic anhydrase family, a family of zinc metalloenzymes, which catalyzes the reversible reaction of hydration of CO₂ (H₂O+CO₂⇄HCO₃ −+H+), allowing the enzyme to regulate intra- and extra-cellular concentrations of CO₂, H+, and HCO₃ −. The carbonic anhydrases participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. The carbonic anhydrases show extensive diversity in tissue distribution and in their subcellular localization. There are at least seven genetically distinct isozymes of mammalian carbonic anhydrase, designated I-VII, each of which catalyzes the reversible hydration of carbon dioxide by a zinc- hydroxide mechanism. Physiological functions that are regulated by carbonic anhydrase comprise, for example, removal of HCO₃− in lung by respiration, reutilization of HCO₃− in kidney, production of aqueous humor in eyes, cerebrospinal fluids in brain, gastric juice production in stomach, pancreatic juice, and bone resorption by osteoclasts. Carbonic anhydrase Attorney Docket No.: RECE-005/01WO 40321/31 family members also play important roles in metabolic processes that include ureagenesis, gluconeogenesis, and lipogenesis. Different from other carbonic anhydrases that are either soluble or attached to the plasma membrane by a membrane-spanning domain, carbonic anhydrase IV is a glycosylphosphatidyl- inositol-anchored membrane isozyme. Carbonic anhydrase IV is broadly conserved across vertebrates and has similar CNS expression profiles in humans, with a recent single cell analysis of human brain vasculature confirming CA-IV's expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which is associated with neural discharge and can influence neuronal function through ion-gated channels. In some embodiments, the carbonic anhydrase IV disclosed herein is a human carbonic anhydrase IV (CA-IV). CA-IV is known to localize on the luminal surface of brain endothelial cells throughout the cortex and cerebellum where it enzymatically modulates carbon dioxide- bicarbonate balance. Human CA-IV has been previously characterized as a 35-kDa protein with a “high activity” in CO2 hydration and a higher activity than other isozymes in catalyzing the dehydration of HCO3 −. In general, human CA-IV contains an 18-amino acid signal sequence at the N-terminal of the protein for endoplasmic reticulum (ER) translocation and a 260-amino acid “CA domain” containing active site amino acid residues that shows 30-36% homology with cytoplasmic CAs. At the C-terminal, an additional 27 amino acid residues containing the hydrophobic sequences of 21 amino acids sufficient to span the membrane are preceded by the 6- amino acid signal sequence for GPI-anchoring. The amino acid residue, Ser 266, was identified as the site for the attachment of the GPI anchor. The removal of C-terminal hydrophobic domain found in the CA-IV precursor has important impact on GPI-anchoring, cell surface expression, and realization of the enzyme activity. Based on amino acid sequences deduced from the nucleotide sequence, human CA-IV contains no classical consensus sites (Asn-Xxx-Ser/Thr) for N-glycosylation. Human CA-IV also contains no oligosaccharide chains, while other mammalian carbonic anhydrase IV (e.g. mouse carbonic anhydrases IV (Car4)) are glycoproteins with one to several oligosaccharide side chains. In some embodiments, the carbonic anhydrase IV disclosed herein is a mouse carbonic anhydrase IV. CA-IV has recently been found to be among the mouse proteins most strongly positively correlated with plasma-protein uptake in the brain (slightly stronger than the often- targeted transferrin receptor). This property is useful for identifying receptors for enhanced BBB Attorney Docket No.: RECE-005/01WO 40321/31 crossing. CA-IV is also expressed in the GI tract, kidney, and lung, as well as taste receptor cells where it allows the sensing of carbonation. Mouse CA-IVand human CA-IV are highly homologous, containing the same amino acids at positions crucial for enzyme activity (e.g., histidine residue 64 (His 64)), with several differences including, for example, that mouse CA-IV is an N-linked glycoprotein and the CO2 hydration rate catalyzed by mouse CA-IV is much lower than human CA-IV. Without being bound by any theory, the lower enzyme activity of mouse CA-IV may be associated with the replacement of Gly 63 in human CA-IV with Gln 63, among several other amino acid replacements. Another difference between mouse CA-IV and human CA-IV is the Val-131-Asp-136 segment (130's segment) that forms an α-helix in mouse CA-IV and an extended loop in human CA-IV. In some embodiments, a carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB crossing can be any carbonic anhydrase IV, such as a mouse CA-IV, a human CA-IV, or a homology or a variant thereof. Carbonic anhydrase IV homologs and/or variants can be derived from a vertebrate species including, but not limited to, mouse, rat, human, bovine, rabbit, monkey, pig, horse, rainbow trout, chimpanzee, squirrel, chicken, goat, and sheep. Carbonic anhydrase IV homologs from various species can be found in public databases identifiable to a person skilled in the art, including for example UniProt, NCBI, and Swiss-Prot. In some embodiments, a small molecule can interact with a carbonic anhydrase IV disclosed herein (e.g., mouse Car4, human CA4 or a homology or a variant thereof), thereby increasing permeability of the BBB (e.g., through transcytosis). In some embodiments, the increase in the permeability of the BBB is achieved by altering (e.g., increasing or decreasing) the carbonic anhydrase IV activity, such as reducing its activity. In some embodiments, the alteration of carbonic anhydrase IV activity is achieved by a shuttle interacting to one or more active sites of the carbonic anhydrase IV including the zinc binding site and the hydrophobic substrate binding pocket. For example, the shuttle can interact with the zinc binding site, the hydrophobic substrate binding pocket, or both. The zinc binding site in carbonic anhydrase IV has a conserved structure dominated by a β-sheet super-structure with a metal binding site formed by at least three His residues. Without being bound by any theory, it is believed that the zinc binding site is on one face of the β-sheet at the bottom of a 15-Å-deep, conical active site cleft in which zinc is liganded by three His residues and hydroxide ion with tetrahedral geometry. The hydrophobic substrate binding pocket Attorney Docket No.: RECE-005/01WO 40321/31 is adjacent to zinc-bound hydroxide, formed in large part by bulky residues such as Val at its base and Val, Trp and Leu at its neck. This pocket is highly conserved among all active isozymes on the basis of phylogenetic comparisons. Without being bound by any theory, it is believed that the hydrophobic pocket has a minimum width and depth for efficient catalysis, and linear free energy relationships indicate that the volume of the amino acid residue at the base of the pocket and the hydrophobicity of residues at the neck of the pocket are critical for activity. Both the zinc binding site and the hydrophobic substrate binding pocket are highly conserved among carbonic anhydrase isozymes. Antibodies An antibody refers to any antigen-binding molecule comprising at least one binding region, e.g. complementarity determining regions (CDRs), that specifically binds to or interacts with a particular antigen. Antibodies may be conjugated to a therapeutic cargo to form an antibody drug conjugate (ADC). The antibody moiety of an ADC dictates its plasma circulation duration, immunogenicity, immune functions, and target specificity. Typical ADCs are predominantly based on immunoglobulin G (IgG), particularly IgG1. IgG1 offers a long serum half-life and strong Fc- mediated immune functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. Selection of a suitable target antigen has also proven to be instrumental in modulating the specificity and processing of an ADC. An ideal target should exclusively, or preferentially, be expressed at high levels on the surface of tumor cells and not on normal cells. The term antibody includes intact antibodies comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof. Each heavy chain typically comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining Attorney Docket No.: RECE-005/01WO 40321/31 regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antigen-binding portion, antigen-binding fragment, or antibody-fragment of an antibody refers to any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from intact antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from commercial sources, DNA libraries, or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. The variable region or variable domain of an antibody refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of complementarity determining regions (CDRs; i.e., CDR-1, CDR-2, and CDR-3), and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. The amino acid positions assigned to CDRs and FRs may be defined according to the Kabat numbering system. Complementarity Determining Regions (CDRs) are regions within antibody variable sequences. Typically, there are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. Certain sub-portions within CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions are Attorney Docket No.: RECE-005/01WO 40321/31 referred to as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. The term framework regions (hereinafter FR) refer to those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. Common structural features among the variable regions of antibodies, or functional fragments thereof, are well known in the art. The DNA sequence encoding a particular antibody can generally be found following well known methods Fc regions of antibodies refer to the C-terminal region of an antibody heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an antibody heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. A humanized antibody refers to an antibody or a variant, derivative, analog or fragment thereof, which immunospecifically binds to an antigen of interest, and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Humanized forms of non-human (for example, murine) antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Monoclonal antibodies refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence Attorney Docket No.: RECE-005/01WO 40321/31 from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal-antibody preparation is directed against a single epitope on an antigen. A chimeric antibody refers to an antibody that has a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. An epitope or target of an antibody refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Binding affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g., an epitope). The affinity of a binding molecule for its binding partner can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In Attorney Docket No.: RECE-005/01WO 40321/31 one embodiment, the “KD” or “KD value” may be measured by assays known in the art, for example by a binding assay. The KD may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen. The term “kon” refers to the on rate constant for association of an antibody to the antigen to form the antibody/antigen complex, as is known in the art. The term “koff” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex, as is known in the art. The on-rate, rate of association, association rate, or “kon,” and the off-rate, rate of dissociation, dissociation rate, or “koff” may also be determined with the same surface plasmon resonance or biolayer interferometry techniques. In aspects of the invention, antibody sequences may be provided with or without other protein scaffolds in other delivery systems, including viral vectors (e.g., lentivirus, adenovirus, AAVs), non-viral nanoparticles, exosomes, antibodies, antibody-drug conjugates, or proteins. Payload delivery across the BBB Disclosed herein include methods and delivery systems for delivering a payload (e.g., a therapeutic agent) to a nervous system. The method comprises providing a small molecule capable of interacting with a carbonic anhydrase IV or a derivative thereof. The small molecule can be part of a delivery system and the delivery system can comprise a payload to be delivered to a nervous system. The method can further comprise administering the delivery system to the subject. In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a nanoparticle selected from lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide or protein-based particles, lipid- polymer particles, nanolipoprotein particles, and combinations thereof. For example, the payload may include an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) molecule, or a combination of two or more of the agents. Attorney Docket No.: RECE-005/01WO 40321/31 In aspects of the invention, the payload may comprise DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutics, interfering RNA, gene editing cargo, chemotherapeutic agents, toxins, radioisotopes, enzymes, chelators, boron compounds, photoactive agents, dyes, metals, metal alloys, nanoparticles, or other larger synthetic molecules and biologics in vitro and in vivo. The payload can include a neuroactive polypeptide, for example, a neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for a receptor expressed by a CNS cell, polypeptides involved in lysosomal storage disease or any combination thereof. In another example, the payload can include an IL-1 receptor antagonist (IL-1Ra), dalargin, an interferon-β, Glial-derived neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-4/5, neurotrophin (NT)-3, a neurturin, neuregulin, a netrin, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), a semaphorin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-cx, TGF-B, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), heregulin, artemin, persephin, interleukins, granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, erythropoietin (EPO), bone morphogenetic proteins (BMPs), netrins, saposins, any fragment thereof, or any combination thereof. Aspects of the invention also provide for delivery of the conjugate to a subject in order to transport a therapeutic agent across the BBB. In aspects of the invention, delivery of the therapeutic payload may be for the treatment of a disease, disorder, or injury of the CNS. In aspects of the invention, the therapeutic agent may be released from the conjugate following entry into the CNS. In certain aspects, the disease, disorder, or injury of the CNS can be, without limitation, multiple sclerosis (MS), amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, stroke, neuropathic pain, neurodegeneration, neuroinflammation, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease), Wallerian Degeneration, optic neuritis, transverse myelitis, post radiation injury, neurologic complications of chemotherapy, acute ischemic optic Attorney Docket No.: RECE-005/01WO 40321/31 neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, primary tumors, secondary metastases, or any combination thereof. Experimental examples Antibody Screen In-house immunization in mice with human receptor proteins, including human CA-IV, was performed. At the end of the immunization schedule, a microfluidic instrument (trade name BERKELEY LIGHTS BEACON) was utilized for the isolation, culture, characterization, and recovery of single cells, to isolate individual plasma B cells from human CA-IV immunized mice into individuals nanopens. FIG.1 is an antibody discovery workflow of the invention. As shown, animals were immunized with the human receptor proteins. At the end of the immunization schedule, the spleen was collected from the animal and splenocytes were extracted. After two rounds of CD138 positive selection, plasma B cells were concentrated and loaded onto BEACON to perform multiplexed antigen specificity assays. Cells of interest were exported, and BCR sequences was recovered. After assembly into an expression vector, the identified antibody pairs were moved into production. IgG capture beads and fluorescently labeled human CA-IV antigen were then used to assay for reactive antibodies. A main channel was filled with IgG capture beads, a labeled secondary anti-IgG antibody, and soluble fluorescently labeled antigens. Secreted antibodies were detected as the local concentration of the bound anti-IgG and labeled antigen fluorophores increased when their diffusion in the main channel was limited by secreted antibody binding. FIG.2 is a set of images of fluorescent binding of antibodies of the invention. As shown, in the BEACON machine, IgG capture beads were employed and combined with fluorescently tagged human CA-IV antigen to test for responsive antibodies. The primary channel was loaded with IgG capture beads, a marked secondary anti-IgG antibody, and soluble fluorescent antigens. As secreted antibodies were present, there was observed increase in the local concentration of the attached anti-IgG and fluorescent antigen due to restricted diffusion in Attorney Docket No.: RECE-005/01WO 40321/31 the main channel caused by the binding of the secreted antibody. This phenomenon appears as a distinct “bloom” of signal originating from a single nanopen. 19 IgG secreted cells were identified with strong human CA-IV reactivity. The cells of interest were recovered for sequencing studies. Though the processes of RNA purification and DNA strand synthesis, B cell receptor (BCR) regions from cDNA were then amplified. VH- and VL-specific PCR reactions for GGA inserts were performed and 21 VH and VL pairs for antibodies were identified that could bind to human CA-IV. Each of the pairs is provided in Table 1 and Table 2, reproduced below: Table 1: Select heavy chain sequences SEQ Antibody Heavy chain sequence ID O

Table 2: Select light chain sequences SEQ Antibody Light chain sequence ID

Antibody Validation The pairs were assembled into expression vectors and produced using Expi293 cells. Recombinant protein production was successful for all 21 CA-IV antibodies, termed CAAs. A subset of the CAAs were selected to be subject to a biochemical binding assay as well as a high- throughput in vitro cell internalization assay. FIG.3 is a workflow for characterizing antibody binding and internalization. As shown, a Surface Plasmon Resonance (SPR) Assay was used to evaluate the direct binding between antibodies and CA-IV. For the in vitro cell assay, cells were transfected with a plasmid to overexpress human CA-IV. Antibodies were then applied to the cells and a detergent Attorney Docket No.: RECE-005/01WO 40321/31 was used to differentiate internalization from binding. The assay and imaging were conducted in a high-throughput 96 well plate manner. FIG.4A-B are graphs of SPR results of antibody CA-IV binding. As shown, SPR of human or mouse CA-IV binding to CAA7 captured on a protein A chip show clear binding with human CA-IV while showing no binding with mouse CA-IV. The SPR results confirmed CAA binding to human CA-IV. Binding and internationalization assessment For an in vitro cell assay, a pipeline was developed to differentiate between binding and internalization. CAA18 and CAA24b Assessment of human CA-IV enhanced binding and internalization for CAA18 and CAA24b was conducted. HEK293 cells were transfected with pUC18 or human CA-IV plasmid for 48 hours, followed by incubation with an individual antibody. After a 24-hour incubation, the antibodies signal was examined by immunohistochemistry (IHC) staining with or without a detergent. FIG.5 shows images from IHC staining following transfection with an antibody of the invention. The enhanced signal in detergent conditions showed human CA-IV-enhanced internalization for CAA18 and CAA24b. CAA7 Assessment of human CA-IV enhanced binding and internalization for CAA7 was conducted. HeLa cells were transfected with pUC18 or CA-IV plasmid for 48 hours, followed by incubation with an individual antibody. After a 24-hour incubation, the antibody signal was examined by IHC staining with or without detergent. FIG.6 shows images from IHC staining following transfection with an antibody of the invention. Attorney Docket No.: RECE-005/01WO 40321/31 The enhanced signal in detergent conditions showed huCA4-enhanced binding and internalization for CAA7. Altogether, the pipeline was used to confirm that CAA7, CAA18, and CAA24b showed human CA-IV-dependent binding and internalization. CAA residue analysis To further explore the key residues involved in the binding of CAA antibodies with CA- IV, 24 CAA7 antibody mutants were generated, labeled CAA701-CAA724. An in vitro cell assay was conducted to analyze each of the CAA7 antibody mutants. Alanine mutants were labeled with pHrodo red and incubated on HeLa cells expressing human CA-IV at 0.2 µM for 6 hours at 37C. FIG.7 shows images from fluorescent staining following transfection with antibody mutants of the invention. Alanine mutants were further analyzed in membrane permeabilizing and non- permeabilizing stain conditions after incubation at 0.4 µM for 1 hour at 37C. FIG.8A-B shows images from fluorescent staining following transfection with antibody mutants of the invention under permeabilizing and non-permeabilizing stain conditions, respectively. FIG.9A-D are graphs of Puncta analysis from fluorescent assays from antibody mutants of the invention showing CAA count. Puncta within the cells transfected with CA-IV were counted using MetaXPress software. Log transformed average puncta intensity per cell across CAA7 mutants was anaylzed. Asterisks indicate significant results of a one-way Welch ANOVA with Dunnett’s T3 multiple comparisons test where ****p<0.0001 (p>0.0001 not shown). Data collected at 3 hours (W(13.00, 528.8) = [14.92], p = 0.05) and 6 hours (W(13.00, 548.8) = [30.10], p = 0.05). (B) Difference of count group mean between CAA7 mutants and CAA7. Attorney Docket No.: RECE-005/01WO 40321/31 FIG.10A-D are graphs of Puncta analysis from fluorescent assays from antibody mutants of the invention showing CAA intensity. As shown, it was discovered that the mutant CAA713 and CAA714 had lower binding and internalization among CAA7 antibody mutants, indicating the importance of the residue mutated for the interaction between CAA7 antibodies and CA-IV. The other single amino acid alanine scanning mutants CAA712, CAA715-CAA724 show comparable binding and internalization as CAA7, indicating those residues mutated were more tolerant to change in CAA7-CA-IV interactions. Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Equivalents Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Attorney Docket No.: RECE-005/01WO 40321/31 Claims 1. A conjugate comprising: a shuttle comprising an antibody or fragment thereof comprising a binding region which binds to a portion of carbonic anhydrase IV (CA-IV); and a therapeutic cargo conjugated to the shuttle. 2. The conjugate of claim 1, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 91- 135. 3. The conjugate of claim 2, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1-45 4. The conjugate of claim 1, wherein the antibody or fragment thereof comprises a light chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 136- 180. 5. The conjugate of claim 4, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 46- 90 6. The conjugate of claim 1, wherein the antibody or fragment thereof comprises one or more framework regions. 7. The conjugate of claim 6, wherein the one or more framework regions are derived from immunoglobulin G (IgG). 8. The conjugate of claim 1, wherein the therapeutic cargo is conjugated to the shuttle via a linker. Attorney Docket No.: RECE-005/01WO 40321/31 9. The conjugate of claim 1, wherein the therapeutic cargo is covalently conjugated to the shuttle. 10. The conjugate of claim 1, wherein the therapeutic cargo is a biological molecule. 11. The conjugate of claim 10, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof. 12. The conjugate of claim 11, wherein the therapeutic cargo is an antibody fragment, wherein the shuttle and therapeutic cargo together form a bispecific antibody. 13. The conjugate of claim 1, wherein the conjugate is characterized by delivery of the therapeutic cargo across the blood brain barrier (BBB). 14. The conjugate of claim 13, wherein when provided to a cell expressing CA-IV as a surface protein, binding of the shuttle to the CA-IV protein mediates transcytosis of the therapeutic cargo across the BBB. 15. The conjugate of claim 14, wherein when provided to a cell present in the human brain or human eye expressing CA-IV as a surface protein, binding of the shuttle to the CA-IV protein mediates transcytosis of the therapeutic cargo across the BBB. 16. A method of delivering a therapeutic cargo across the BBB of a subject, the method comprising: providing to a subject a conjugate comprising: a shuttle comprising an antibody or fragment thereof comprising a binding region which binds to a portion of carbonic anhydrase IV (CA-IV); and a therapeutic cargo conjugated to the shuttle, Attorney Docket No.: RECE-005/01WO 40321/31 17. The method of claim 16, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 91- 135. 18. The conjugate of claim 17, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 1-45 19. The conjugate of claim 16, wherein the antibody or fragment thereof comprises a light chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 136- 180. 20. The conjugate of claim 19, wherein the antibody or fragment thereof comprises a heavy chain sequence having at least 95% sequence identity with a sequence selected from SEQ ID NO: 46-90 21. The method of claim 16, wherein the antibody or fragment thereof comprises one or more framework regions. 22. The method of claim 21, wherein the one or more framework regions are derived from immunoglobulin G (IgG). 23. The method of claim 18, wherein the therapeutic cargo is conjugated to the shuttle via a linker. 24. The method of claim 18, wherein the therapeutic cargo is covalently conjugated to the shuttle. 25. The method of claim 18, wherein the therapeutic cargo is a biological molecule. Attorney Docket No.: RECE-005/01WO 40321/31 26. The method of claim 25, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof. 27. The method of claim 26, wherein the therapeutic cargo is an antibody fragment, wherein the shuttle and therapeutic cargo together form a bispecific antibody. 28. The method of claim 16, wherein the method results in delivery of the therapeutic cargo across the blood brain barrier (BBB). 29. The method of claim 28, wherein the delivery comprises transcytosis of the therapeutic cargo across the BBB of the subject. 30. The method of claim 29, wherein the method comprises providing the conjugate to a cell present in the human brain or human eye of the subject.