Multispecific antibodies

Abstract

The present invention relates to a multispecific antibody for transporting a compound across the blood brain barrier (BBB), wherein the antibody binds to at least two target proteins selected from the group consisting of the transferrin receptor (TFRC), CD98 (SLC3A2) and PODXL.

Description

Technical Field

[0001] This invention relates to multispecific antibodies that bind to target proteins expressed on the blood-brain barrier (BBB) ​​and their use for transporting compounds across the BBB. Background Technology

[0002] Brain penetration of drugs for neurological diseases (such as large biologics or small molecule drugs with low brain penetration) is strictly limited by the extensive and impermeable blood-brain barrier (BBB) ​​and other cellular components in the neurovascular unit (NVU). Many strategies to overcome this barrier have been tested, and one strategy utilizes endocytic transport pathways mediated by endogenous receptors (BBB receptors) expressed on the endothelial cells of the brain capillaries. Recombinant proteins, such as monoclonal antibodies or peptides, have been engineered against these receptors to enable receptor-mediated delivery of biologics to the brain. However, strategies to maximize brain uptake while minimizing missorting within brain endothelial cells (BECs) and accumulation in certain organelles within the BECs, particularly those that cause degradation of biologics, remain unexplored.

[0003] Monoclonal antibodies and other biological therapeutic agents hold great therapeutic potential for treating central nervous system (CNS) pathologies. However, their entry into the brain is blocked by the bubonic bifidoid (BBB). Previous studies have shown that a very small percentage (approximately 0.1%) of injected IgG in the bloodstream can penetrate into the CNS compartments (Felgenhauer, Klin. Wschr. 52(1974) 1158-1164). This certainly limits any pharmacological action due to the low concentration of antibodies within the CNS.

[0004] It has been previously found that the percentage of antibodies distributed in the CNS can be improved by utilizing BBB receptors (i.e., transferrin receptor, insulin receptor, etc.) (see, for example, WO 95 / 02421).

[0005] Therefore, there is a need for cross-BBB neurological disease drug delivery systems to efficiently shuttle drugs into the brain.

[0006] Blood-brain barrier shuttles were reported in WO 2014 / 033074.

[0007] Anti-transferrin receptor antibodies and their usage were reported in WO 2014 / 189973. Further reports indicate that targeting the BBB receptor with conventional, high-affinity, specific antibodies typically results in a limited increase in BBB transport. It was later found that, among the anti-BBB antibodies studied, the magnitude of antibody uptake into the CNS and its distribution within the CNS was negatively correlated with its binding affinity to the BBB receptor. For example, low-affinity antibodies against the transferrin receptor (TfR), administered at therapeutic doses, significantly improved BBB transport and CNS retention of anti-TfR antibodies compared to higher-affinity anti-TfR antibodies, and made it easier to achieve therapeutic concentrations in the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). This BBB transport was demonstrated using a bispecific antibody that binds both TfR and the amyloid precursor protein (APP) cleaving enzyme β-secretase (BACE1). A single systemic dose of bispecific anti-TfR / BACE1 antibody engineered with low-affinity antibodies not only induced significant antibody uptake in the brain, but also significantly reduced brain Aβ1-40 levels compared with monospecific anti-BACE1 alone, suggesting that BBB penetration affects the potency of anti-BACE1 (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43; Yu et al., Sci. Transl. Med. 3 (2011) 84ra44).

[0008] A compound delivery system across the BBB is needed to efficiently shuttle compounds into the brain. Summary of the Invention

[0009] In a first aspect, the present invention provides a multispecific antibody for transporting compounds across the blood-brain barrier (BBB), wherein the antibody binds to at least two target proteins selected from the group consisting of: transferrin receptor (TFRC), TfR, CD98 (SLC3A2), and PODXL.

[0010] In one embodiment of the invention, the antibody binds to TfR and CD98.

[0011] In one embodiment of the invention, the antibody binds to TfR and PODXL.

[0012] In one embodiment of the invention, the antibody binds to CD98 and PODXL.

[0013] In one embodiment of the present invention, the antibody is a human antibody.

[0014] In one embodiment of the present invention, the compound is a therapeutic compound.

[0015] In one embodiment of the invention, the antibody is conjugated to a therapeutic compound.

[0016] In one embodiment of the present invention, the target protein is a human protein.

[0017] In one embodiment of the present invention, the antibody includes a first antigen-binding site that binds to TfR and a second antigen-binding site that binds to CD98.

[0018] In one embodiment of the present invention, the antibody comprises a first antigen-binding site that binds to TfR and a second antigen-binding site that binds to PODXL.

[0019] In one embodiment of the present invention, the antibody includes a first antigen-binding site that binds to CD98 and a second antigen-binding site that binds to PODXL.

[0020] In one embodiment of the present invention, the therapeutic compound is a drug for nervous system diseases.

[0021] In one embodiment of the invention, the therapeutic compound forms part of a multispecific antibody.

[0022] In one embodiment of the invention, the therapeutic compound forms one or two antigen-binding sites of a multispecific antibody.

[0023] In one embodiment of the invention, a therapeutic compound that forms one or two antigen-binding sites of a multispecific antibody recognizes a brain antigen.

[0024] In one embodiment of the invention, the brain antigen is selected from the group consisting of: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), TREM2, MS4A, TrkB, and caspase 6.

[0025] In one embodiment of the invention, the antibody has a monovalent binding mode against each target protein.

[0026] In one embodiment of the invention, the TfR binding site has an affinity KD (nM) in the range of 100 to 1000, and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

[0027] In one embodiment of the invention, the TfR binding site has an affinity KD (nM) in the range of 100 to 1000, and the PODXL binding site has an affinity KD (nM) in the range of 0.1 to 100.

[0028] In one embodiment of the invention, the PODXL binding site has an affinity KD (nM) in the range of 0.1 to 100, and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

[0029] In one embodiment of the invention, the TfR binding site has an affinity KD (nM) in the range of 0.01 to 9, and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

[0030] In a second aspect, the present invention provides an antibody for therapeutic purposes, wherein the antibody transports a compound across the blood-brain barrier (BBB), and wherein the antibody binds at least to the target protein PODXL.

[0031] In one embodiment of the present invention, the antibody is a human antibody.

[0032] In one embodiment of the present invention, the compound is a therapeutic compound.

[0033] In one embodiment of the invention, the antibody is conjugated to a therapeutic compound.

[0034] In one embodiment of the present invention, the target protein is a human protein.

[0035] In one embodiment of the present invention, the therapeutic compound is a drug for nervous system diseases.

[0036] In one embodiment of the invention, the antibody is a multispecific antibody and the therapeutic compound forms part of the multispecific antibody.

[0037] In one embodiment of the invention, the therapeutic compound forms one or two antigen-binding sites of a multispecific antibody.

[0038] In one embodiment of the invention, a therapeutic compound that forms an antigen-binding site of a multispecific antibody recognizes a brain antigen.

[0039] In one embodiment of the invention, the brain antigen is selected from the group consisting of: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), TREM2, MS4A, TrkB, and caspase 6.

[0040] In one embodiment of the invention, the antibody has a monovalent binding mode against each target protein. Attached Figure Description

[0041] Figure 1: Overview of the bispecific antibody of the present invention

[0042] Figure 2-4: Dosage-response effect of bispecific antibody on hCMECD3 cells:

[0043] Figure 5: 2D endocytosis transport assay setup

[0044] Figures 6a-k: Endocytotic transport and recycling of the bispecific antibodies of the present invention

[0045] Figures 7a-d: Comparison of maximum endocytic transport of bispecific antibodies against TfR / PODXL, TfR / CD98 and PODXL / CD98 after 5 hours.

[0046] Figure 8: Comparison of maximum endocytic transport of bispecific antibodies against TfR / PODXL / CD98 and PODXL / CD98 after 20 hours.

[0047] Figures 9a–c: Experimental setup for human BBB spheres

[0048] Figure 10: Results of human BBB spheres

[0049] Figures 11a–d: Signal quantification in human BBB spheres. DP47 is a prosthetic conjugate.

[0050] Figure 12: Graphical overview of the endocytic transport rate of the bispecific antibody of the present invention: P1AI8549 (701-PODXL-Low / 1026-TFR-Low) shows increased endocytic transport compared with control P1AI8552 (701-PODXL-Low / Prosthesis), P1AI8553 (1026-TFR-Low / Prosthesis) and control single-arm brain shuttle P1AF3732 (1026-TFR-Low).

[0051] Figure 13: Endocytotic transport and recycling of monospecific PODXL antibody. Detailed Implementation

[0052] definition

[0053] The blood-brain barrier (BBB) ​​refers to the physiological barrier between the peripheral circulation and the brain and spinal cord. It is formed by tight junctions within the cell membranes of brain capillary endothelial cells, creating a tight barrier that restricts the transport of molecules (even very small molecules, such as urea (60 Daltons)) into the brain. The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers within the CNS, collectively referred to as the blood-brain barrier or BBB in this paper. The BBB also includes the blood-CSF barrier (choroid plexus), where the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0054] The mortar-and-pot structure dimer module and its application in antibody engineering are described in Carter P., Ridgway JBB, Presta LG: Immunotechnology, Vol. 2, No. 1, February 1996, pp. 73-73(1).

[0055] The "central nervous system" or "CNS" refers to the complex of neural tissues that control bodily functions and includes the brain and spinal cord.

[0056] "Blood-brain barrier receptors" (referred to as "R / BBB" in this document) are extracellular membrane junction receptor proteins expressed on brain endothelial cells that can transport molecules across the BBB or are used to transport molecules administered exogenously. Examples of R / BBBs in this document include: transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptors (including but not limited to low-density lipoprotein receptor-associated protein 1 (LRP1) and low-density lipoprotein receptor-associated protein 8 (LRP8)), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). The exemplary R / BBB in this document is the transferrin receptor (TfR).

[0057] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art. A preferred method for measuring affinity is surface plasmon resonance (SPR).

[0058] "Monovalent binding entity" refers to a molecule capable of specifically and monovalently binding to the R / BBB. The blood-brain shuttle and / or conjugates of the present invention are characterized by the presence of a single unit of monovalent binding entity; that is, the blood-brain shuttle and / or conjugates of the present invention comprise one unit of monovalent binding entity. Monovalent binding entities include, but are not limited to, proteins, polypeptides, peptides, and antibody fragments, including Fab, Fab', Fv fragments, and single-chain antibody molecules (such as, for example, single-chain Fab, scFv). Monovalent binding entities can be, for example, scaffold proteins engineered using existing techniques such as phage display or immunoengineering. Monovalent binding entities can also be peptides.

[0059] "Monovalent binding mode" refers to the specific binding to R / BBB, where the interaction between the monovalent binding entity and R / BBB occurs through a single epitope. Due to this single epitope interaction site, the monovalent binding mode prevents any dimerization / multimerization of R / BBB. The monovalent binding mode also prevents changes in the intracellular sorting of R / BBB.

[0060] The term "epitaph" includes any polypeptide determinant capable of specifically binding to an antibody. In some embodiments, the epitope determinant includes chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is an antigenic region that binds to an antibody.

[0061] The transferrin receptor ("TfR") is a transmembrane glycoprotein (molecular weight approximately 180,000) involved in iron uptake in vertebrates, consisting of two disulfide-bonded subunits (each with an apparent molecular weight of approximately 90,000). In one embodiment, the TfR described herein is the human TfR, which contains an amino acid sequence such as that found, for example, in Schneider et al., Nature 311: 675-678 (1984).

[0062] CD98 (also known as CD98 heavy chain; 42F heavy chain; SLC3A2) is a type II transmembrane glycoprotein. The human CD98hc sequence is listed in UNIPROT accession number P08195. This protein comprises a 75-amino acid N-terminal intracellular cytoplasmic domain, a single transmembrane domain, and a 426-amino acid C-terminal extracellular domain (Parmacek et al., NucleicAcids Res. 17: 1915-1931, 1989). CD98 is covalently linked via disulfide bonds to one of several light chains (SLC7A5, 6, 7, 8, 10, or 11), which are L-type amino acid transporters. This interaction is essential for the cell surface expression of the light chains and their amino acid transport function. CD98 is also associated with three subunits of integrin, thereby regulating integrin signaling that controls cell proliferation, survival, migration, and epithelial adhesion / polarity (Cai et al., J. Cell Sci. 1 18:889-899, 2005).

[0063] PODXL, also known as podocyte marker protein-like protein-1, PCLP1, or PCX, is a type I transmembrane protein of the sialomucin family of CD34. The human PODXL sequence is listed in UNIPOROT accession number O00592. Retaining the basic structure of family members CD34 and endosaccharides, PODXL consists of a highly conserved cytoplasmic domain with a C-terminal PDZ-binding region (DTHL motif), a single-channel transmembrane domain, and extensively O-glycosylated and sialylated extracellular domains. As used herein, “nervous system disease” refers to a disease or condition affecting the CNS and / or having an etiology in the CNS. Exemplary CNS diseases or conditions include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or conditions, viral or microbial infections, inflammation, ischemia, neurodegenerative diseases, seizures, behavioral disorders, and lysosomal storage diseases. For the purposes of this application, the CNS will be understood to include the eye, which is normally isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological diseases include, but are not limited to, neurodegenerative diseases (including but not limited to Lewy body disease, post-poliomyelitis syndrome, Shy-Draeger syndrome, oligopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatal substantia nigra degeneration, Tau protein diseases (including but not limited to Alzheimer's disease and supranuclear palsy), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru disease, Gerstmann syndrome, chronic wasting diseases, and fatal familial insomnia), bulbar palsy, motor neuron diseases, and abnormal neurodegenerative diseases (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscin deposition disease, Alexander disease, Tourette syndrome, Menkes kink syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Lafra disease, Rett syndrome, Wilson's disease, Lesch-Niehan syndrome, and Unverricht-Lundborg syndrome). Syndromes), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), and cancer (e.g., CNS cancer and / or brain cancer, including brain metastases caused by cancer in other parts of the body).

[0064] "Nervous system disease drugs" are drugs or therapeutic agents for treating one or more nervous system diseases. The nervous system disease drugs of the present invention include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, aptamers, repressive nucleic acids (i.e., small repressive RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes, and small molecules, or active fragments of any of the foregoing. Exemplary nervous system disease drugs of the present invention are described herein, and include, but are not limited to: antibodies, aptamers, proteins, peptides, repressive nucleic acids, and small molecules, and active fragments of any of the foregoing, which are themselves CNS antigens or target molecules or specifically recognize and / or act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or portions thereof, amyloid β, β-secretase, γ-secretase, tau, α-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophic factors. Non-limiting examples of medications for neurological disorders and their corresponding conditions may be used to treat: brain-derived neurotrophic factor (BDNF), chronic brain injury (neurogenesis); fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor brain cancer; (EGFR)-antibodies, glial cell line-derived neurotrophic factor for Parkinson's disease (GDNF); brain-derived neurotrophic factor (BDNF), amyotrophic lateral sclerosis, depression; lysosomal enzymes, lysosomal storage diseases of the brain; ciliary neurotrophic factor (CNTF), amyotrophic lateral sclerosis; neuromodulatory factor-1, schizophrenia; anti-HER2 antibodies (e.g., trastuzumab), brain metastases from HER2-positive cancers.

[0065] An "imaging agent" is a compound that has one or more properties that allow for the direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds incorporated into labeled entities that allow for detection.

[0066] "CNS antigens" or "brain targets" are antigens and / or molecules expressed in the CNS (including the brain) that can be targeted by antibodies or small molecules. Examples of such antigens and / or molecules include, but are not limited to: β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), and caspase 6. In one embodiment, the antigen is BACE1.

[0067] The term "natural sequence" in this article refers to a protein that contains the amino acid sequence of a naturally occurring protein, including naturally occurring variants of the protein. The terminology used herein includes proteins isolated from or recombinantly produced from their natural sources.

[0068] The term “antibody” is used in the broadest sense in this article and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired biological activity.

[0069] The term "antibody fragment" in this article refers to a portion of a complete antibody that retains its ability to bind to an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bisomatic antibodies; linear antibodies; single-chain antibody molecules, such as, for example, single-chain Fab, scFv, and monospecific antibodies formed from antibody fragments. The "single-chain Fab" format is described, for example, in Hust M. et al., BMC Biotechnol. March 8, 2007; 7:14.

[0070] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitopes, and are typically present in small amounts, except for possible variants that may arise during the production of the monoclonal antibody. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage of being synthesized without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described by Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). Specific examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, and human antibodies, including their antigen-binding fragments. The monoclonal antibodies described herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to the corresponding sequences in antibodies from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of those antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Targeted chimeric antibodies described herein include “primate-derived” antibodies, which contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., such as a baboon, rhesus monkey, or cynomolgus monkey) and a human constant region sequence (US Patent No. 5,693,780).

[0071] "Humanized" nonhuman (e.g., mouse) antibodies are chimeric antibodies containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody), where residues in the hypervariable region of the receptor are replaced by residues from the hypervariable region of a nonhuman species (donor antibody), such as mice, rats, rabbits, or nonhuman primates, possessing the desired specificity, affinity, and function. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, the humanized antibody may contain residues not present in the receptor antibody or donor antibody. These modifications are intended to further refine antibody performance. Generally, the humanized antibody will substantially contain at least one variable domain, typically two, where all or substantially all hypervariable loops correspond to the hypervariable region of the nonhuman immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence, except for the FR substitutions mentioned above. The humanized antibody may also optionally contain at least a portion of the immunoglobulin constant region, which is typically a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992).

[0072] The term "human antibody" in this article refers to an antibody containing an amino acid sequence structure that corresponds to the amino acid sequence structure of antibodies obtainable from human B cells, and includes an antigen-binding fragment of the human antibody. Such antibodies can be identified or prepared using various techniques, including but not limited to: production by transgenic animals (e.g., mice) capable of producing human antibodies after immunization in the absence of endogenous immunoglobulin production (see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807); selection from phage display libraries expressing human antibodies or fragments of human antibodies (see, for example, McCafferty et al., Nature 348:552-553 (1990); Johnson et al., Current Opinion in Structural Biology 3:564-571). (1993); Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991); Griffith et al., EMBO J. 12:725-734 (1993); US Patent Nos. 5,565,332 and 5,573,905; generated via in vitro activated B cells (see US Patents 5,567,610 and 5,229,275); and isolated from human antibodies that produce hybridomas.

[0073] The term "multispecific antibody" as used herein refers to an antibody that has binding specificity to at least two distinct epitopes. Exemplary multispecific antibodies may bind to both R / BBB and brain antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites are also covered (see, for example, US Appln No. US 2002 / 0004587 Al, Miller et al.). Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0074] The antibodies described in this article include “amino acid sequence variants” with altered antigen-binding or biological activity. Examples of such amino acid alterations include antibodies with enhanced affinity for antigens (e.g., “affinity-matured” antibodies), and antibodies with altered Fc regions (if present) (e.g., altered (increased or decreased) antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)) (see, for example, WO 00 / 42072, Presta, L. and WO 99 / 51642, Iduosogie et al.); and / or increased or decreased serum half-life (see, for example, WO00 / 42072, Presta, L.).

[0075] "Affinity-modified variants" have one or more substituted hypervariable regions or framework residues of a parent antibody (e.g., a parental chimeric antibody, a humanized antibody, or a human antibody) that alter (increase or decrease) affinity. In one embodiment, the resulting variant selected for further development has a reduced affinity for the R / BBB according to the invention. A convenient method for generating such substituted variants is phage display. Briefly, several hypervariable sites (e.g., 6 to 7 sites) are mutated to produce all possible amino substitutions at each site. The resulting antibody variant is displayed monovalently from filamentous phage particles as a fusion with the M13 gene III product packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and its target. According to the techniques described in this paper, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the variant set is screened, and antibodies with altered affinities can be selected for further development.

[0076] The antibodies described herein may be “glycosylated variants,” thus any carbohydrates (if present) attached to the Fc region will be altered. For example, the antibody described in US Patent Application No. 2003 / 0157108 (Presta, L.) has a mature carbohydrate structure lacking fucose attached to the antibody Fc region. See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Antibodies containing bipartite N-acetylglucosamine (GlcNAc) in the carbohydrates attached to the antibody Fc region are cited in WO 2003 / 011878, Jean-Mairet et al., and US Patent No. 6,602,684, Umana et al. WO 1997 / 30087, Patel et al., also reported antibodies containing at least one galactose residue in the oligosaccharide attached to the antibody Fc region. See also WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) concerning antibodies with modified carbohydrates attached to the antibody's Fc domain. See also US 2005 / 0123546 (Umana et al.) which describes antibodies with modified glycosylation. As used herein, the term "hypervariate region" refers to the amino acid residue in the antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the complementarity-determining region or the CDR (e.g., residues 24-34 (LI), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, and residues 31-35 (HI), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the hypervariable ring (e.g., residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and residues 26-32 (HI), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol). Biol. 196:901-917 (1987)). “Frame” or “FR” residues are those variable domain residues other than the hypervariable region residues defined herein.

[0077] A "full-length antibody" is an antibody that contains an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CHI, CH2, and CH3. The constant domains can be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof.

[0078] "Naked antibody" is an antibody that is not conjugated to a foreign molecule such as a cytotoxic entity, polymer, or radiolabeled substance (as defined herein).

[0079] An antibody "effective function" refers to the biological activity attributed to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and so on. In one embodiment, the antibody described herein is substantially lacking in effector function.

[0080] The term "antibody-dependent cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody in the presence of effector cells. The term "complement-dependent cytotoxicity (CDC)" refers to a process initiated by the binding of complement factor C1q to the Fc moiety of most IgG antibody subclasses. The binding of C1q to the antibody is caused by a protein-protein interaction defined at a so-called binding site. Such Fc moiety binding sites are known in the art. These Fc moiety binding sites are characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (according to Kabat EU index numbers). Antibodies against subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, including C1q and C3 binding, while IgG4 does not activate the complement system and does not bind C1q and / or C3.

[0081] Full-length antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant domains. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known. The term "recombinant antibody" as used herein refers to an antibody (e.g., chimeric, humanized, or human antibody or its antigen-binding fragment) expressed by a recombinant host cell containing a nucleic acid encoding the antibody. Examples of “host cells” used for the production of recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NSO cells), young hamster kidney (BHK), HeLa and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Tobacco (e.g., tobacco); (4) yeast cells, such as yeast cells belonging to the genus Yeast (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells, etc.

[0082] As used herein, “specific binding” or “specific binding to” refers to the selective or preferential binding of an antibody to an antigen. Binding affinity is typically determined using standard assays (such as Scatchard assays) or surface plasmon resonance techniques (such as those using BIACORE®).

[0083] "Antibody that binds to the same epitope as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of that antibody to its antigen by 50% or more in a competitive assay.

[0084] An "imaging agent" is a compound that has one or more properties that allow for the direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds incorporated into labeled entities that allow for detection.

[0085] “Label” is a marker conjugated to the antibody described herein and used for detection or imaging. Examples of such labels include radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the label is a radiolabel for medical imaging, such as TC99M or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.

[0086] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as lysozymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof.

[0087] The "therapeutic effective amount" of a drug (e.g., a pharmaceutical preparation) refers to the amount that effectively achieves the desired therapeutic or preventive outcome at the necessary dosage and time period.

[0088] The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. The Fc region contains the CH2 and CH3 domains of the immunoglobulin. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0089] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0090] As used herein, the term "CH2-CH3 Ig entity" refers to a protein entity derived from the CH2 or CH3 domain of an immunoglobulin. A "CH2-CH3 Ig entity" comprises two "CH2-CH3" polypeptides forming a dimer. The immunoglobulin can be IgG, IgA, IgD, IgE, or IgM. In one embodiment, the CH2-CH3 Ig entity is derived from IgG immunoglobulin and is referred to herein as a "CH2-CH3 IgG entity." The term includes both the native sequence and variant CH2-CH3 domains. In one embodiment, a "CH2-CH3 Ig entity" is derived from the human heavy chain CH2-CH3 IgG domain, which extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the CH2-CH3 domain or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0091] "Conjugate" refers to the fusion protein of the present invention conjugated to one or more heterologous molecules (including but not limited to labels, drugs for neurological diseases, or cytotoxic agents).

[0092] As used herein, "connector" refers to a chemical or single-chain peptide connector that covalently links different entities of the multispecific antibodies and / or fusion proteins and / or conjugates of the present invention. The connector links, for example, a compound to a monovalent binding entity. For example, if the monovalent binding entity comprises a CH2-CH3 Ig entity and an sFab pointing to a blood-brain barrier receptor, the connector links the scFab to the C-terminus of the CH3-CH2 Ig entity. A connector linking a brain effector entity to a monovalent binding entity (the first connector) and a connector linking the scFab to the C-terminus of the CH2-CH3 Ig domain (the second connector) may be the same or different.

[0093] A single-chain peptide linker consisting of one to twenty amino acids linked by peptide bonds can be used. In some embodiments, the amino acids are selected from twenty naturally occurring amino acids. In some other embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is a chemical linker. In some embodiments, the linker is a single-chain peptide having an amino acid sequence of at least 25 amino acids, preferably 32 to 50 amino acids. In one embodiment, the linker is (GxS)n, where G = glycine, S = serine (x = 3, n = 8, 9, or 10, and m = 0, 1, 2, or 3) or (x = 4, and n = 6, 7, or 8, and m = 0, 1, 2, or 3), preferably x = 4, n = 6 or 7, and m = 0, 1, 2, or 3, more preferably x = 4, n = 7, and m = 2. In one embodiment, the connector is (G4S)4 (Seq.Id.No. 17). In another embodiment, the connector is (G4S)6G2 (Seq.Id.No. 13).

[0094] A variety of chemical linkers can be used for conjugation. For example, monovalent binding entities or fusion proteins and compounds can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimino-3-(2-pyridyldithio)propionate (SPDP), succinimino-4-(N-maleiminomethyl)cyclohexane-l-carboxylate (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl diimide adipate hydrochloride), active esters (such as disuccinimino octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The linker can be a "cleavable linker" that facilitates the release of effector progeny upon delivery to the back of the brain. For example, acid-labile, peptidase-sensitive, light-labile, dimethyl, or disulfide-containing adapters can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0095] Covalent conjugation can be performed directly or via a linker. In some embodiments, direct conjugation is performed by constructing a protein fusion (i.e., by fusing two genes encoding a monovalent binding entity targeting R / BBB and an effector entity and expressing them as a single protein). In some embodiments, direct conjugation is performed by forming a covalent bond between a reactive group on one of the two parts of the monovalent binding entity targeting R / BBB and a corresponding group or receptor on the compound. In some embodiments, direct conjugation is performed by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to include a reactive group (as a non-limiting example, a thiol or carboxyl group), which, under appropriate conditions, forms a covalent link with the other molecule to be conjugated. As a non-limiting example, a molecule (i.e., an amino acid) having the desired reactive group (i.e., a cysteine ​​residue) can be introduced into, for example, a monovalent binding entity targeting R / BBB antibodies and a disulfide bond formed with a neurological drug. Methods for covalently conjugating nucleic acids to proteins are also known in the art (i.e., photocrosslinking, see, for example, Zatsepin et al. Russ. Chem. Rev. 74: 77-95 (2005)). Various linkers can also be used for conjugation. For example, the monovalent binding entity and the effector entity can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimide-3-(2-pyridyldithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-l-carboxylate (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and biactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers comprising 1 to 20 amino acids linked by peptide bonds can also be used. In some of these embodiments, the amino acid is selected from 20 naturally occurring amino acids. In some other such embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker can be a “cleavable linker” that facilitates the release of effector entities after delivery to the brain. For example, acid-labile linkers, peptidase-sensitive linkers, light-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0096] A “marker” is a biomarker coupled to the fusion protein described herein and used for detection or imaging. Examples of such marks include radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the mark is a radiolabel for medical imaging, such as TC99M or 1123, or a spin mark for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc. An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0097] "Isolated" antibodies are antibodies that have been separated from components in their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99% by means of, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0098] The term "pharmaceutical formulation" refers to a formulation in which the bioactive ingredient contained therein is in a form in which the activity is effective and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0099] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subjects, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0100] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of the disease in the treated individual, and may be performed for prevention or during the course of clinicopathological progression. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow its progression.

[0101] For neurological disorders, the available neurological medications include analgesics, including but not limited to narcotic / opioid analgesics (i.e., morphine, fentanyl, hydrocodone, meperidine, methadone, hydroxymorphone, pentazocine, propoxyphene, tramadol, codeine, and hydroxycodone), and nonsteroidal anti-inflammatory drugs (NSAIDs) (i.e., ibuprofen, naproxen, diclofenac, diflunisal, etodoxacin, fenprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam, nabumetone, oxapazine, piroxicam, and sulin). (Acids and tometidine), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), anti-migraines (i.e., sumatriptin, amotriptan, fultriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan, and ergotamine), acetaminophen, salicylates (i.e., aspirin, salicylcholine, magnesium salicylate, diflunisal, and disalicylate), anticonvulsants (i.e., carbamazepine, clonazepam, gaba). Pentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomicaine, dimethicaine, propyloxycaine, procaine, novocacaine, prilocaine, tetracaine, articaine, bupivacaine, carticaine, cincocaine, eticaine, levobupivacaine, lidocaine, mepivacaine, piperacaine, prilocaine, ropivacaine, trimethaine, clam toxin, and tetrodotoxin), and cox-2-inhibitors Formulations (i.e., celecoxib, rofecoxib, and vardicoxib). For neuropathic conditions accompanied by vertigo, antivertigo drugs can be selected, including but not limited to meclopramide, diphenhydramine, promethazine, and diazepam. For neuropathic conditions accompanied by nausea, antinausea drugs can be selected, including but not limited to promethazine, chlorpromazine, prochlorperazine, trimethoprim, and metoclopramide. For neurodegenerative diseases, neuropathic drugs containing growth hormone or neurotrophic factors can be selected.Examples include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophic factor-4 / 5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophic factor (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), and interleukin-1. Receptor antagonists (IL-1ra), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophins, platelet-derived growth factor (PDGF), regulatory proteins, neuromodulators, leukosheath embryonic factor, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, netrin, cardiotrophin-1, hedgehog, leukemia inhibitory factor (LIF), metaphase factor, pleiotropic growth factor, bone morphogenetic protein (BMP), netrin, sapogenin, serotonin, and stem cell factor (SCF). For cancer, neurological drugs can be selected as chemotherapy agents. Examples of chemotherapy agents include alkylating agents, such as thiotepa and CYTOXAN®. Cyclophosphamides; alkyl sulfonates, such as busulfan, indomethacin, and piperazine; aproidine derivatives, such as benzotiprine, carboquinone, metoprolol, and urotepiperazine; ethylene imines and methylmelamines, including hexamethylmelamine, tratamido, triethylenephosphamide, triethylenethiophosphate, and trimethylolmelamine; polyacetic acids (especially bratasine and bratasine ketone); δ-9-tetrahydrocannabinol (drocannabinol, MARINOL®); β-lapaquinone; laparol; Colchicine; betulinic acid; camptothecin (including synthetic analogues such as topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolamine, and 9-aminocamptothecin); bryostatin; sponge polyene ketones; CC-1065 (including its synthetic analogues adolexin, carzeolitexin, and bizeolitexin); podophyllotoxin; podophyllic acid; teniposide; cryptophytes (especially cryptophyte 1) And cryptophytin 8); dolalastatin; pyruvic oxychloride (including synthetic analogs KW-2189 and CB1-TM1); arugulatin; pancratistatin; spruein; spongin; nitrogen mustards, such as chlorambucil, naphthalenemus, chophosphatamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trazophos, uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine;Antibiotics, such as enediyne antibiotics (e.g., kazimycin, especially kazimycin γll and kazimycin ωll (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)); enediyne anthracyclines, including enediyne anthracycline antibiotic A; espomycin; and new carcinogen chromophores and related pigment protein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azoserine, bleomycin, cactinomycin, carabicin, erythromycin, carcinomamycin, chromomycin, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN® Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, maceralomycin, mitomycin C, mycophenolate mofetil, nopramine, olivomycin, pepromycin, purulentin, triamcinolone acetonide, rhodopsin, streptomycin, streptozotocin, and tuberculosis-killing agents. Sulfadiazine, ubenmethoxazole, fenestrone, zorobacterium; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, methotrexate, pteroxate, trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine Glycosides, deoxyfluorouridine, enoxabin, fluorouridine; androgens, such as carotestosterone, drotahistone propionate, cyclothiosterol, meandrolone, testrolide; antiadrenergic drugs, such as aminoglutethimide, mitotane, trelostan; folic acid supplements, such as folinic acid; acetoglucan lactone; hydroxyphosphatidylcholine; aminopyruvate; enuramicin; acridine; bestrabucil; bisacodyl; edatrazine; defofamine; colchicine; diacodyl; elfornithine; elifetrazine; epomycin; etogluconate; gallium nitrate; hydroxyurea; lentinan; chlordamine; maytansine, such as metansine and anthraquinone; mitotoxin; mitotoxantrone; mopidazole; nitraacidine; pentostatin; promethazine; pirarubicin; loxoantrone; 2-ethylhydrazine; procarbazine; PSK® Polysaccharide complexes (JHS Natural Products, Eugene, OR); razorylene; lisoxane; cizonan; germanospiramine; cyclohexane ketone; triamine quinone; 2,2',2''-trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, verracurin A, baculosporin A, and serpentin); urethane; vinblastine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; dibromomannitol; dibromoeusol; piperobromane;gacytosine; cytarabine (“Ara-C”); thiotepa; taxanes, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor-free ABRAXANETM, albumin-engineered paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vincristine (VELBAN®); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); anotoxin; edaraxazole; donomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovovin.

[0102] This definition of chemotherapy agents also includes antihormonal agents, which work by regulating, reducing, blocking, or inhibiting the effects of hormones that promote cancer growth, and are typically in the form of systemic or generalized therapy. These may be the hormones themselves. Examples include: anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trivoxifen, raloxifene, LY117018, onanasone, and FARESTON® toremifene; antiprogesterone drugs; estrogen receptor downregulators (ERDs); drugs that suppress or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) antagonists, such as LUPRON® and ELIGARD® leuprorelin acetate, goserelin acetate, and buserelin acetate. And tripterelin; other antiandrogens such as flutamide, nilumethoxazole and bicalutamide; and aromatase inhibitors that inhibit aromatase and thus regulate estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vortexazole, FEMARA® letrozole and ARIMIDEX® anastrozole. Additionally, the definition of such chemotherapeutic agents includes bisphosphonates such as clodronates (e.g., BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronic acid, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® teludronate, or ACTONEL® risedronate; and trisatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccines and gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID® vaccines; LURTOTECAN® topoisomerase 1 inhibitors; and ABARELLX®. rmRH; lapatinib dimethylbenzenesulfonate (a small molecule inhibitor of dual tyrosine kinases ErbB-2 and EGFR, also known as GW572016); and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

[0103] Another class of compounds that can be selected as nervous system drugs for cancer treatment or prevention are anticancer immunoglobulins (including, but not limited to, trastuzumab, bevacizumab, alemtuzumab, cetuximab, gemtuzumab, oxzomicin, tiimomab, panitumumab, and rituximab). In some cases, combinations of antibodies with toxic markers can target and kill desired cells (i.e., cancer cells), including but not limited to radiolabeled tosimomumab.

[0104] For eye diseases or conditions, the following neurological medications can be selected: anti-angiogenic ophthalmic agents (i.e., bevacizumab, ranibizumab, and peganob), ophthalmic glaucoma medications (i.e., carbacholine, epinephrine, dimethoprim, benzodiazepine, brimonidine, bullinazolamide, levobenolol, timolol, betalolol, dazolamide, bimatoprost, carteolol, meteolol, dipiformin, travoprost, and latanoprost), carbonic anhydrase inhibitors (i.e., acetazolamide and acetazolamide), ophthalmic antihistamines (i.e., naphazoline, phenylephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prednisolone, clotiprednisolone, dexamethasone, difluprednisolone, limexolone, fluocinolone acetonide, metronidazole, and triamcinolone acetonide), and ophthalmic anesthetics (i.e., lidocaine, propranolol, etc.). Cacaine and tetracaine), ophthalmic anti-infectives (i.e., levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin / polymyxin B, sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin, sulfamethoxazole, gentamicin, idoxuridine, erythromycin, natamycin, bacitracin, neomycin, ofloxacin, trifluralin, ganciclovir, vidarabine), ophthalmic disinfectants Inflammatory agents (i.e., napalfenamide, ketoprofen, flurbiprofen, sulofen, cyclosporine, triamcinolone, diclofenac, and bromofenac) and ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromoglycine, tetrahydrozoline, pyrimethamine, betahistine, naphazoline, phenylephrine, nedolomem, lodusamide, phenylephrine, emestine, and azelastine). For epileptic seizures, the neurological medications that can be chosen are anticonvulsants or antiepileptic agents, including but not limited to barbiturate anticonvulsants (i.e., primidone, methadone, toluidine, allobarbital, amobarbital, alpratobarbital, phenobarbital, barbital, bromhexine, and phenobarbital), benzodiazepine anticonvulsants (i.e., diazepam, clonazepam, and lorazepam), carbamate anticonvulsants (i.e., felbamate), carbonic anhydrase inhibitor anticonvulsants (i.e., acetazolamide, topiramate, and zonisamide), dibenzodiazepine anticonvulsants (i.e., rufinamide, carbamazepine, and oxcarbazepine), and fatty acid derivative anticonvulsants (i.e., sodium divalproate and valproic acid). ), Gamma-aminobutyric acid analogues (i.e., pregabalin, gabapentin, and vigabatrin), Gamma-aminobutyric acid reuptake inhibitors (i.e., tiagabin), Gamma-aminobutyric acid transaminase inhibitors (i.e., vigabatrin), hydantoin anticonvulsants (i.e., phenytoin, ethylphenytoin, fosphenytoin, and mephenytoin), miscellaneous anticonvulsants (i.e., lacosamide and magnesium sulfate), progestins (i.e., progesterone), oxazolidinediones (i.e., methylethyldione and trimethyldione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximide and methylsuximide), triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants (i.e., phenylacetylurea and phenylbutyrylurea).

[0105] For lysosomal storage disorders, neurological drugs that mimic the activity of impaired enzymes in the disease, either directly or otherwise, can be selected. Exemplary recombinant enzymes for treating lysosomal storage disorders include, but are not limited to, those described, for example, in U.S. Patent Application Publication No. 2005 / 0142141 (i.e., α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosamine glycosidase, N-acetyl-galactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronide glycosidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosamine A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid ceramidinase, asparagylase, palmitoyl-protein thioesterase 1, and tripeptidyl aminopeptidase 1).

[0106] For amyloidosis, selectable neurological medications include, but are not limited to, antibodies or other binding molecules (including, but not limited to, small molecules, peptides, aptamers, or other protein binders) that specifically bind to targets selected from: β-secretase, tau, presenilin, amyloid precursor protein or portions thereof, β-amyloid peptides or oligomers or fibrils thereof, death receptor 6 (DR6), receptor for advanced glycation end products (RAGE), parkin, and huntingtin; cholinesterase inhibitors (i.e., galantamine, donepezil, rivastigmine, and tacrine); NMDA Receptor antagonists (i.e., memantine); monoamine depleting agents (i.e., bubenazine); dihydroergotamine mesylate; anticholinergic anti-Parkinson's drugs (i.e., propiconazole, diphenhydramine, benztropine, piperidone, and benztropine); dopaminergic anti-Parkinson's drugs (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tocapone, and amantadine); bubenazine; anti-inflammatory drugs (including but not limited to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and other compounds listed above); hormones (i.e., estrogens, progesterone, and leuprorelin); vitamins (i.e., folic acid and nicotinamide); danopiformin; high taurine (i.e., 3-aminopropanesulfonic acid; 3 APS); serotonin receptor activity modulators (i.e., zaliroden); interferons and glucocorticoids.

[0107] For viral or microbial diseases, selectable neurological medications include, but are not limited to, antiviral compounds (including, but not limited to, amantadine antivirals (i.e., rimantadine and amantadine), antiviral interferons (i.e., pegylated interferon α-2b), chemokine receptor antagonists (i.e., maraviro), integrase chain transfer inhibitors (i.e., retegvir), neuraminidase inhibitors (i.e., oseltamivir and zanamivir), non-nucleoside reverse transcriptase inhibitors (i.e., efavirenz, ectrevirine, deraviridine, and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine, and norinosine), protease inhibitors (i.e., darunavir, atazanavir, fosanavir, telanavir, ritonavir, nelfinavir, ampravir, indinavir, and saquinavir), and purine nucleosides (i.e., Valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir) and miscellaneous antiviral agents (i.e., entfuvirtide, foscarnet, palilizumab, and fomivirex), antibiotics (including but not limited to aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temoxicillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bamoxicillin) and other antiviral agents (i.e., entfuvirtide, foscarnet, palilizumab, and fomivirex), and antibiotics (including but not limited to aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temoxicillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bamoxicillin)). (Cypercillin), cephalosporins (i.e., cefazolin, cefalexin, cefotaxime, cefamandole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cefradine, cefanocarbazide, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), carbapenem / penem (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem), monoamides (i.e., amtram, tegafur, and nocachlor A) β-lactamase inhibitors (such as clavulanic acid, triazobactam, and subaktam), aminoglucosides (such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, topromycin, and paromomycin), anesarcomycin (such as gerdemycin and atrazine), carbapenems (such as chloramphenicol), glycopeptides (such as teicoplanin and vancomycin), macrolides (such as azithromycin, clarithromycin, erythromycin, and erythromycin). Benzyl, roxithromycin, acetosylmycin, telithromycin, and spectinomycin), monoamides (i.e., aztreonam), quinolones (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, limofloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, gapfloxacin, sparfloxacin, and temaxa), sulfonamides (i.e., sulfamidone, azosulfonamide, acesulfame, sulfadiazine, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfasalazine, sulfisoxazole, methoxyoctidine, methoxyoctidine, and methamidoxaazole), tetracyclines (i.e.,Tetracyclines, demeclocycline, doxycycline, minocycline, and oxotetracycline; anti-inflammatory or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunorubicin, daunorubicin, epirubicin, idarubicin, priloin, mitomycin, pentostatin, and pentorubicin); miscellaneous antimicrobial compounds (i.e., bacitracin, colistin, and polymyxin B); and antifungal agents (i.e., metronidazole, nizopyrazinamide, tebufenozide, etc.). (nitroazole, chloroquine, diiodoquinoline and paromomycin) and antiparasitic agents (including but not limited to quinine, chloroquine, amodiaquine, pyrimethamine, sulfadoxine, chloroguanidine, mefloquine, atovaquinone, primaquine, artemisinin, halofanthracene, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, dimethoprim, ivermectin, rifampin, amphotericin B, melarisol, efornithine and albendazole). For ischemia, selectable neurological medications include, but are not limited to, thrombolytic agents (i.e., urokinase, alteplase, reteplase, and tenecteplase), platelet aggregation inhibitors (i.e., aspirin, cilostazol, clopidogrel, prasugrel, and dipyridamole), statins (i.e., lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin, and pitavastatin), and compounds that improve blood flow or vascular elasticity, including, for example, blood pressure medications.

[0108] For behavioral disorders, neurological medications may be selected from behavior-modifying compounds, including but not limited to atypical antipsychotics (i.e., risperidone, olanzapine, aripiprazole, quetiapine, palapiridone, asenapine, clozapine, irapiridone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thixotropium (i.e., sulfathitropium), miscellaneous antipsychotics (i.e., pimozide, lithium, indole, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, etapiprolam, paroxetine, fluoxetine, and sertraline), and serotonin-norrepinephrine inhibitors. Adenosine reuptake inhibitors (i.e., duloxetine, venlafaxine, divenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, tramipramine, imipramine, protriptyline, and desipramine), tetracyclic antidepressants (i.e., mirtazapine and maprotiline), phenylpiperazine antidepressants (i.e., trazodone and nefazodone), monoamine oxidase inhibitors (i.e., isocarboxazid, phenelzine, selegiline, and tranylcypromine), benzodiazepines (i.e., alprazolam, estazolam, flurazepam, clonazepam, lorazepam, and diazepam), norepinephrine-dopamine reuptake inhibitors (i.e., buprofen), CNS Stimulants (i.e., phentermine, diethylamine, methamphetamine, dextroamphetamine, amphetamine, methylphenidate, dextroamphetamine, lidextroamphetamine, modafinil, pimolin, benztrozine, sennatamethasone, benztrozine, amorfenib, diethylamine, caffeine, atoxetine, doxapram, and morindole), anxiolytics / sedatives / hypnotics (including but not limited to barbiturates (i.e., secobarbital, phenobarbital, and toluidine), benzodiazepines (as described above), and miscellaneous anxiolytics / sedatives / hypnotics (i.e., diphenhydramine, sodium hydroxybutyrate, zaleplon, hydroxyzine, chlorhydrate, zolpidem, buspirone, doxepin, levozoprothiolane, ramelton, meprobamate, and ethylchlorvinox)), secretins (see, for example, Ratliff-Schaub et al., Autism 9: 256-265 (2005)), opioid peptides (see, for example, Cowen et al. J. Neurochem. 89:273-285 (2004)), and neuropeptides (see, for example, Hethwa et al. Am. J. Physiol. 289: E301-305 (2005)).

[0109] For CNS inflammation, one can choose neurological medications that address the inflammation itself (i.e., nonsteroidal anti-inflammatory drugs, such as ibuprofen or naproxen) or neurological medications that treat the underlying cause of the inflammation (i.e., antiviral or anticancer agents).

[0110] In another embodiment, the compound is a complete or full-length antibody. Complete antibodies can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of complete antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known. In one embodiment, complete antibodies lack effector function.

[0111] Techniques for generating antibodies are known, and examples are provided in the definitions section above this document. In one embodiment, the antibody is chimeric, humanized, or a human antibody or its antigen-binding fragment.

[0112] The uptake of the target multispecific antibody and / or conjugate, as well as other biological activities of the multispecific antibody and / or conjugate, can be assessed by means of examples disclosed or known methods. The concentration within the CNS parenchymal space can also be measured using, for example, microdialysis or capillary depletion methods combined with ELISA or radiometric measurements of labeled multispecific antibodies and / or conjugates.

[0113] pharmaceutical preparations

[0114] The therapeutic formulations of antibodies or conjugates used according to the present invention are prepared as lyophilized formulations or aqueous solutions for storage by mixing with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the docking receptor at the doses and concentrations used, including buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) peptides; proteins Proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0115] The formulations described herein may also contain more than one active compound (as needed), optionally those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depend, for example, on the amount of multispecific antibodies and / or conjugates present in the formulation and the clinical parameters of the subject. Exemplary such drugs are discussed below.

[0116] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, cohesive drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or coarse drop emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980).

[0117] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0118] The formulation intended for in vivo administration must be sterile. This can be easily achieved through filtration using a sterile filter membrane. In one embodiment, the formulation is isotonic.

[0119] The multispecific antibodies or conjugates of the present invention can be used in a variety of in vivo methods. For example, the present invention provides a method for transporting a therapeutic compound across the BBB, the method comprising exposing a multispecific antibody and / or conjugate to the BBB such that the multispecific antibody transports the therapeutic compound conjugated thereto across the BBB. In another example, the present invention provides a method for transporting a neurological disease drug across the BBB, the method comprising exposing a conjugate to the BBB such that the multispecific antibody transports the neurological disease drug conjugated thereto across the BBB. In one embodiment, the BBB here is in mammals (e.g., humans), such as mammals suffering from neurological diseases, including but not limited to: Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddell syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, traumatic brain injury, etc.

[0120] In one embodiment, the neurological disease is selected from: neuropathy, amyloidosis, cancer (e.g., involving the CNS or brain), eye disease or condition, viral or microbial infection, inflammation (e.g., inflammation of the CNS or brain), ischemia, neurodegenerative disease, seizures, behavioral disorders, lysosomal storage diseases, etc.

[0121] Neuropathic disorders are diseases or abnormalities of the nervous system characterized by inappropriate or uncontrolled nerve signaling or lack thereof, and include, but are not limited to, chronic pain (including nociceptive pain), pain caused by damage to body tissues, including cancer-related pain, neuropathic pain (pain caused by abnormalities in nerves, spinal cord, or brain), and psychogenic pain (which is wholly or mostly related to a mental disorder), headaches, migraines, neuropathy, and symptoms and syndromes that often accompany such neuropathic disorders, such as dizziness or nausea.

[0122] Amyloidosis is a group of diseases and conditions associated with extracellular protein deposition in the CNS, including but not limited to: secondary amyloidosis, age-related amyloidosis, Alzheimer's disease (AD), mild cognitive impairment (MCI), Lewy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); Guam Parkinson's-dementia complex, cerebral amyloid angiopathy, Huntington's disease, progressive supranuclear palsy, multiple sclerosis; Creutzfeldt-Jakob disease, Parkinson's disease, transmitted spongiform encephalopathy, HIV-related dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), and eye diseases involving β-amyloid protein deposition (i.e., macular degeneration, drusen-related optic neuropathy, and cataracts).

[0123] CNS cancers are characterized by the abnormal proliferation of one or more CNS cells (i.e., nerve cells), and include, but are not limited to, gliomas, glioblastomas multiforme, meningiomas, astrocytomas, acoustic neuromas, chondromas, oligodendrogliomas, medulloblastomas, gangliogliomas, schwannomas, neurofibromas, neuroblastomas, and extradural, intramedullary, or intradural tumors.

[0124] Viral or microbial infections of the central nervous system (CNS) include, but are not limited to, infections caused by viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., Escherichia coli, Staphylococcus aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., and Mycobacterium tuberculosis) and other microorganisms such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., Toxoplasma gondii) or amoebas, which can lead to CNS infection. Pathophysiology, including but not limited to meningitis, encephalitis, myelitis, vasculitis, and abscess, can be acute or chronic. CNS inflammation is inflammation caused by damage to the CNS, which can be physical injury (i.e., due to an accident, surgery, brain trauma, spinal cord injury, or concussion) or injury caused by or related to one or more other CNS diseases or conditions (i.e., abscess, cancer, viral or microbial infection).

[0125] As used in this article, CNS ischemia refers to a group of conditions or causes associated with abnormal blood flow or vascular behavior in the brain, including but not limited to focal cerebral ischemia, global cerebral ischemia, stroke (i.e., subarachnoid hemorrhage and intracerebral hemorrhage), and aneurysm.

[0126] Neurodegenerative diseases are a group of diseases and conditions associated with the loss of function or death of nerve cells in the CNS, and include, but are not limited to, adrenoleukodystrophy, Alexander disease, Alpert disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, Baden disease, Cockayne syndrome, basal cortical degeneration, degeneration caused by or related to amyloidosis, Friedreich ataxia, frontotemporal degeneration, Kennedy disease, multiple system atrophy, multiple sclerosis, primary lateral sclerosis, progressive supranuclear palsy, spinal muscular atrophy, transverse myelitis, Rifsum disease, and spinocerebellar ataxia.

[0127] Epilepsy disorders and conditions involving the CNS involve inappropriate and / or abnormal conduction in the CNS, and include, but are not limited to, epilepsy (i.e., absence seizures, atonic seizures, benign motor epilepsy, childhood absence seizures, clonic seizures, complex partial seizures, frontal lobe seizures, febrile seizures, infantile spasms, juvenile myoclonic seizures, juvenile absence epilepsy, Rengork syndrome, Ranke syndrome, Clawfield syndrome, Otahara syndrome, Wechsler syndrome, myoclonic seizures, mitochondrial diseases, progressive myoclonic epilepsy, psychogenic seizures, reflex seizures, Rasmussen syndrome, simple partial seizures, secondary generalized seizures, temporal lobe seizures). Seizures include: clonic seizures, tonic seizures, psychomotor seizures, limbic seizures, partial seizures, generalized seizures, status epilepticus, abdominal seizures, akinetic seizures, vegetative seizures, massive bilateral myoclonus, menstrual seizures, fall-related seizures, emotional seizures, focal seizures, laughing seizures, Jacksonian walking, Lafra disease, motor seizures, multifocal seizures, nocturnal seizures, photosensitive seizures, pseudoseizures, sensory seizures, microseizures, jungle seizures, withdrawal seizures, and visual reflex seizures.

[0128] Behavioral disorders are CNS conditions characterized by abnormal behavior in the affected part of the subject, and include, but are not limited to, sleep disorders (i.e., insomnia, deep sleep, night terrors, circadian rhythm sleep disorders, and narcolepsy), mood disorders (i.e., depression, suicidal depression, anxiety, chronic affective disorders, phobias, panic attacks, obsessive-compulsive disorder, attention deficit hyperactivity disorder (ADHD), attention deficit disorder (ADD), chronic fatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolar disorder), eating disorders (i.e., anorexia or bulimia), major mental illnesses, developmental behavioral disorders (i.e., autism, Rett syndrome, Asperger's syndrome), personality disorders, and mental disorders (i.e., schizophrenia, delusional disorder, etc.).

[0129] Enzymatic storage disorders are metabolic disorders that are associated with or have CNS-specific symptoms in certain cases; these disorders include, but are not limited to: Tay-Sachs disease, Gaucher disease, Fabry disease, mucopolysaccharidosis (types I, II, III, IV, V, VI, and VII), glycogen storage diseases, GM1 ganglioside storage disorders, metachromatic leukoencephalopathy, Fabry disease, Carnafan leukodystrophy and neuronal ceroid lipofuscinosis type 1 and 2, Niemann-Pick disease, Pompe disease, and Clapey disease.

[0130] The multispecific antibodies and / or conjugates of the present invention can be used in therapy, alone or in combination with other agents. For example, the multispecific antibodies and / or conjugates of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a therapeutic agent that is effective in treating the same or different neurological disease as the neurological disease treated by the multispecific antibodies and / or conjugates of the present invention. Exemplary additional therapeutic agents include, but are not limited to: various neurological drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (such as memantine), amyloid-β peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors (statins), ampaquinone, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid-β peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid-β peptide antibodies. In some embodiments, at least one additional therapeutic agent is selected because of its ability to reduce one or more side effects of neurological drugs.

[0131] The aforementioned combination therapies encompass both combined administration (where two or more therapeutic agents are included in the same or separate formulation) and single administration. In the case of single administration, the administration of the multispecific antibodies and / or conjugates of the present invention may be performed before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants. The multispecific antibodies and / or conjugates of the present invention may also be used in combination with other interventional therapies, such as, but not limited to, radiotherapy, behavioral therapy, or other therapies known in the art and suitable for treating or preventing neurological diseases. The multispecific antibodies and / or conjugates of the present invention (and any additional therapeutic agents) may be administered by any suitable manner, including parenteral, intrapulmonary, and intranasal administration, and, if desired, for local treatment or intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0132] Administration can be carried out via any suitable route, such as by injection, including intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. This article considers various dosing schedules, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0133] The multispecific antibodies and / or conjugates of the present invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a practicing physician. The multispecific antibodies and / or conjugates of the present invention are not mandatory, but may optionally be formulated in conjunction with one or more agents currently used for the prevention or treatment of the disease in question. The effective amount of such other agents depends on the amount of multispecific antibodies and / or conjugates present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically / clinically to be appropriate.

[0134] For the prevention or treatment of disease, the appropriate dose of the multispecific antibody and / or conjugate of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of multispecific antibody and / or conjugate, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the multispecific antibody and / or conjugate, and the discretion of the attending physician. The multispecific antibody and / or conjugate may be administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, a multispecific antibody and / or conjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be an initial candidate dose for administration to the patient, whether, for example, by single or multiple administrations alone, or by continuous polymeric administration. Depending on the factors mentioned above, a typical daily dose may range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, treatment will generally continue until the desired suppression of disease symptoms is achieved, depending on the condition. An exemplary dose of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses, or for example about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be available. Progression of this therapy can be easily monitored using routine techniques and assays.

[0135] Table 1: Amino acid sequences of bispecific antibodies

[0136] Target 1 is recognized by light chain A and heavy chain K.

[0137] Target 2 is recognized by light chain B and heavy chain H.

[0138]

[0139] Table 2: Bispecific antibodies with target clone affinity

[0140]

[0141] Table 3: Antibody affinity based on SPR measurement

[0142] PODXL and CD98-705 affinity classification based on flow cytometry binding assay.

[0143]

[0144] Table 4: Detailed information on PODXL antibodies

[0145]

[0146] Table 5: Detailed information on CD98 antibodies

[0147]

[0148] Table 6: Monospecific control antibodies

[0149]

[0150] Example

[0151] Dose-response of bispecific antibodies to hCMECD3 cells:

[0152] Figure 2a shows the binding assay results of the bispecific antibodies P1AI8556 (PODXL-698-high / TfR-1026-low), P1AI8559 (PODXL-698-high / pseudo), P1AI8549 (PODXL-701-low / TfR-1026-low), P1AI8553 (pseudo / TfR-1026-low), and P1AI8552 (PODXL-701-low / pseudo) on hCMECD3 cells. The binding assay showed that P1AI8556 (698-PODXL-high / TFR-low) bound to hCMECD3 cells the most. Compared with the single-specific binding controls P1AI8559 (698-PODXL-high / prototype) and P1AI8553 (1026-TfR-low / prototype), the combination of TFR-low and 698-PODXL-high increased binding properties.

[0153] Figure 2b shows the binding assay results of the bispecific antibodies P1AI8557 (PODXL-698-high / TfR-Duta 3-high), P1AI8550 (PODXL-701-low / TfR-Duta 3-low), and P1AI8547 (CD98-705-low / TfR-Duta 3-high) on hCMECD3 cells. The binding assay showed that P1AI8557 bound to hCMECD3 cells most strongly. Compared with the two monospecific binding controls P1AI8559 (698-PODXL / prototype), the combination of PODXL-698-high / TfR-Duta 3-high enhanced binding properties.

[0154] Figure 2c shows the binding assay results of the bispecific antibodies P1AI8558 (PODXL-698-high / TfR-Duta 1-medium), P1AI8559 (PODXL-698-high / pseudo), P1AI8555 (pseudo / TfR-Duta 1-medium), P1AI8551 (PODXL-701-low / TfR-Duta 1-medium), P1AI8552 (PODXL-701-low / pseudo), P1AG3698 (PODXL-698-high-single-arm), and P1AG3701 (PODXL-701-low-single-arm) on hCMECD3 cells. The binding assay showed that P1AG3698, P1AI8559, and P1AI8558 bound to hCMECD3 cells most strongly. Compared to the monospecific TfR conjugate control P1AI8555 (prosthetic / Duta-1-medium), the combination PODXL-698-high / TfR-Duta-1-medium showed increased binding properties, but did not show increased binding properties compared to the PODXL conjugate controls P1AI8558 (PODXL-698-high / TfR-Duta-1-medium) and P1AG3698 (PODXL-698-high-single-arm). Compared to both monospecific controls P1AI8552 (PODXL-701-low / prosthetic) and P1AI8555 (prosthetic / TfR-Duta-1-medium), the combination PODXL-701-low / TfR-Duta-1-medium showed increased binding affinity.

[0155] Figure 2d shows the binding assay results of the bispecific antibodies P1AK1324 (TfR-Duta 3-high / CD98-703-high) and P1AK1327 (pseudo / CD98-703-high) on hCMECD3 cells. The binding assay showed that P1AK1327 (pseudo / CD98-703-high) bound to hCMECD3 cells the strongest. Compared with the monospecific control P1AK1327 (pseudo / CD98-703-high), the combination of TfR-Duta 3-high / CD98-703-high (P1AK1324) showed reduced binding affinity.

[0156] Figure 3 shows the binding assay results of the bispecific antibodies P1AI8549 (PODXL-701-low / TfR-1026-low), P1AI8553 (pseudo / TfR), and P1AI8552 (PODXL-701-low / pseudo) on hCMECD3 cells. For the low-binding antibodies, P1AI8553 (1026-TFR-low / pseudo) shows the lowest binding to hCMECD3 cells. When this Tfr-conjugate is combined with the 701-PODXL conjugate, this increases the binding strength: P1AI8549 (701-PODXL-low / 1026-TFR-low). However, P1AI8552 (701-PODXL-low / pseudo) shows a higher binding strength as a monospecific conjugate than the bispecific P1AI8549 (PODXL-701-low / TfR-1026-low).

[0157] Figure 4 shows the binding assay results of the bispecific antibodies P1AI8559 (PODXL-698-high / pseudo), P1AG3701 (PODXL-701-low / non-binding), P1AG3698 (PODXL-698-high / non-binding), P1AI8552 (PODXL-701-low / pseudo), P1AG3705 (CD98-705-low), P1AI4681, and P1AI3732 on hCMEDCD3. The single-arm version of 698-PODXL-high (P1AG3698) behaves similarly to the two-arm version, where 698-PODXL-high is combined with the non-binding antibody (P1AI8559 (PODXL-698-high / pseudo)). Therefore, it can be concluded that the observed effect is not due to the antibody format (single-arm vs. dual-arm IgG).

[0158] Results of endocytosis transport assay of bispecific antibodies

[0159] Figures 6a and 6b An overview of the endocytosis transport assay results for the following bispecific antibodies and control antibodies is shown: P1AI8549 (PODXL-701-Low / TfR-1026-Low), P1AI8552 (PODXL-701 / Prototype), P1AI8553 (Prototype / TfR-1026-Low), P1AI8556 (PODXL-698-High / TfR-1026-Low), P1AI8559 (PODXL-698-High / Prototype), P1AK1323 (PODXL-698-High / CD98-703-High), P1AK1326 (PODXL-701-Low / CD98-703-High), P1AK1327 (Prototype / CD98-703-High), and P1AK1325 (TfR-1026-Low / CD98-703-High). Experimental setup: hCMECD3; loading amount: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0160] Figure 6c shows the endocytic transport assay results of the selected bispecific antibodies: P1AI8549 (PODXL-701-low / TfR-1026-low), P1AI8556 (PODXL-698-high / TfR-1026-low), P1AK1323 (PODXL-698-high / CD98-703-high), and P1AK1325 (TfR-1026-low / CD98-703-high). Experimental setup: hCMECD3; loading amount: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0161] Figure 6d shows the endocytic transport assay results of the selected bispecific antibodies: P1AI8552 (PODXL-701 / prototype), P1AI8553 (prototype / TfR-1026-low), P1AI8559 (PODXL-698-high / prototype), and P1AK1327 (prototype / CD98-703-high). Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0162] Figure 6e shows the endocytosis and transport assay results of the bispecific antibody P1AI8549 (PODXL-701-low / Tfr-1026-low) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate: 2.67 µg / ml.

[0163] Figure 6f shows the endocytosis transport assay results of the bispecific antibody P1AI8550 (PODXL-701-low / TfR-1026-low) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0164] Figure 6g shows the endocytosis and transport assay results of the bispecific antibody P1AI8551 (CD98-705-low / TfR-duta 1-medium) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0165] Figure 6h shows the endocytosis and transport assay results of the bispecific antibody P1AI8547 (PODXL-701-low / Tfr-Duta 3-high) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate: 2.67 µg / ml.

[0166] Figure 6i shows the endocytosis and transport assay results of the bispecific antibody P1AI8557 (PODXL-698-high / Tfr-Duta 3-high) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate: 2.67 µg / ml.

[0167] Figure 6j shows the endocytosis and transport assay results of the bispecific antibody P1AI8558 (PODXL-698-high / TfR-Duta 1-medium) + control. Experimental setup: hCMECD3; loading: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0168] Figure 6k shows the endocytosis and transport assay results of the bispecific antibody PIAK1324 (TfR-Duta 3–high / CD98–703-high) + control. Experimental setup: hCMECD3; loading amount: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0169] For endocytic transport, similar effects to binding properties were observed. P1AI8556 (PODXL-698-high / TfR-1026-low) showed increased endocytic transport compared to the following two single-specific controls: P1AI8553 (1026-TFR-low / pseudo) and P1AI8559 (698-PODXL-high / pseudo).

[0170] The same situation was observed with P1AK1323 (PODXL-698-high / CD98-703-high), where P1AK1327 (prosthetic / CD98-703-high) represents a monospecific 703-CD98 control, and P1AI8559 (698-PODXL-high / prosthetic) represents another control.

[0171] P1AK1326 (PODXL-701-low / CD98-703-high) showed increased endocytic transport compared to the control P1AI8552 (701-PODXL-low / prototype).

[0172] P1AK1325 (1026-TFR-Low / CD98-703-High) showed increased endocytosis and transport compared to the control P1AI8553 (1026-TFR-Low / Prototype).

[0173] P1AI8549 (701-PODXL-Low / 1026-TFR-Low) showed increased transgenic cells compared to the control P1AI8552 (701-PODXL-Low / Prosthesis).

[0174] Figure 7 shows the endocytotic transport assay results of the bispecific antibodies targeting TfR / PODXL / CD98 and PODXL / CD98 5 h after transport. After 5 h of intracellular transport, P1AK1325 (1026-TFR-Low / CD98-703-High) showed the highest level of endocytotic transport, followed by P1AK1323 (698-PODXL-High / 703-CD98-High). Furthermore, P1AK1326 (701-PODX-Low / 703-CD98-High), P1AI8559 (698-PODXL-High / Prosthesis), and P1AI8556 (698-PODXL-High / 1026-TFR)-Low also showed high levels of endocytotic transport.

[0175] Figure 8 shows the endocytic transport assay results of the bispecific antibodies targeting TfR / PODXL / CD98 and PODXL / CD98 20 h after transport. After 20 h of intracellular transport, P1AK1323 (698-PODXL-high / 703-CD98-high) showed the highest endocytic transport, followed by P1AK1325 (1026-TFR-low / CD98-703-high).

[0176] In addition, P1AK1326 (701-PODXL-low / 703-CD98-high), P1AI8559 (698-PODXL-high / prosthetic), and P1AI8556 (698-PODXL-high / 1026-TFR-low) also showed high levels of endocytic transport.

[0177] Figure 13 shows the endocytosis transport assay results of the monospecific antibody targeting PODXL. P1AG3700 showed the highest endocytosis transport. Experimental setup: hCMECD3; loading volume: 20.1 µg / ml.

[0178] Materials and methods

[0179] Antibody production, purification, and analysis:

[0180] Expi293 cells were transiently transfected at a production scale of 1.0 L, with the ratio of protein-encoding plasmids being 1:1:1:1. The proteins were then analyzed by affinity chromatography.

[0181] Purification was performed using MabSelect SuRe, ion exchange chromatography (POROS XS), and size exclusion chromatography. For quality control, the antibody was analyzed for endotoxin levels (<0.5 EU / mg (kinetic LAL)), monomer content >96% (aSEC, 1x after freezing / thawing), and purity (capillary gel electrophoresis).

[0182] Combined with measurements:

[0183] For FACS binding assays, hCMECD3 cells were incubated for 1 h with antibodies at concentrations of 1000 nM, 500 nM, 250 nM, 125 nM, 63 nM, 31 nM, 16 nM, 8 nM, 4 nM, 2 nM, and 1 nM. After washing the cells with PBS (calcium-free, magnesium-free, sterile DPBS; PANBiotech; P04-36500) + 5% FBS (FBS Premium, γ-irradiated, 0.2µm sterile filtered fetal bovine serum originating from Australia; PANBiotech; P30-2006), secondary antibody (R-phycoerythrin AffiniPure F(ab')2 fragment donkey anti-human IgG (H+L); Jackson Immuno Research; 709-116-149) (1:100 dilution) was added. After culturing for 1 hour, and washing the cells with PBS (calcium-free, magnesium-free, sterile DPBS; PANBiotech; P04-36500) + 5% FBS (FBS Premium, γ-irradiated, 0.2µm sterile filtered fetal bovine serum originating from Australia; PANBiotech; P30-2006), the PE intensity of the cells was analyzed by flow cytometry (BD FACSCelesta cell analyzer). The results were analyzed using FlowJo and Graphpad Prism software.

[0184] Endocytosis transport assay:

[0185] For endocytosis transport assays, 12-well cell culture inserts (cellQART; 9310402) were coated for 1 h with 400 µl collagen (Corning; 354236) and 200 µl fibronectin (Gibco; 33016-015) at a concentration of 50 µg / ml. The inserts were washed twice with DPBS, CaCl2, and CaMg2 (Gibco; 14040-091) and stored at 4°C for up to one week until cell seeding. Before seeding, plates were filled with preheated cell culture medium (EBM-2 basal medium; Lonza; CC-3156 and EGM-2 MV SingleQuots; Lonza; CC-4147), with 1.6 ml on the basal side and 0.4 ml on the top. HCMECD3 cells were thawed and seeded at 8000 cells / filter in the tip portion of a transwell insert and cultured at 37°C and 5% CO2 for 12 days, with the medium changed every 2–3 days. On day 12, fresh cell culture medium (basolateral) was added, and the tip medium was exchanged with a previously prepared antibody dilution (21.0 µg / ml), followed by incubation at 37°C and 5% CO2 for 1 h to allow intracellular uptake. After washing the basolateral and tip portions five times with DPBS, CaCl2, CaMg2+, and 2.5% fetal bovine serum (Lonza; CC-4102B), fresh cell culture medium was added, and the cells were incubated at 37°C and 5% CO2 for 0 h, 1 h, 2 h, 4 h, 5 h, and 20 h. At each time point, samples of the tip and basolateral supernatant were collected, and cell lysates were prepared, with two replicates for each. For cell lysates, the entire cell culture medium was removed and cells were lysed in RIPA buffer (Sigma Aldrich; R0278). Samples were stored at -80°C and IgG levels were measured via huIgG ELISA and analyzed using Excel and XLfit.

[0186] huIgG ELISA

[0187] To measure the amount of IgG in the supernatant and cell lysate of the endocytosis transport assay, the samples were measured by ELISA. For this purpose, 384-well maxisorp plates (Thermo Scientific; 460372) were coated overnight at 4°C with 30 µl of AffiniPure goat anti-human IgG Fcγ-specific coating antibody (BIOZOL; 109-005-098) at a concentration of 1 µg / ml. On the day of the ELISA, the coating antibody was removed and replaced with 100 µl of Ca2+ and Mg2+-free PBS (PANBiotech; P04-991500) + 0.5% BSA (Thermo Scientific; 37525) + 0.05% Tween 20 (anatrace; T1003). Standard curves for the corresponding antibodies were prepared at concentrations of 0 ng / ml, 0.0004 ng / ml, 0.0011 ng / ml, 0.004 ng / ml, 0.011 ng / ml, 0.03 ng / ml, 0.1 ng / ml, 0.3 ng / ml, 0.9 ng / ml, 2.77 ng / ml, 8.33 ng / ml, and 25 ng / ml in EBM-2 basal medium (Lonza; CC-3156) or RIPA buffer (Sigma Aldrich; R0278). Furthermore, for both supernatant and cell lysate, starting with undiluted samples, 1:3 dilutions up to 1:729 were prepared for each sample in EBM-2 basal medium (Lonza; CC-3156) or RIPA buffer (Sigma Aldrich; R0278). Coated and blocked 384-well plates diluted 1:10 in Millipore H2O were washed four times with 10x PBST buffer (Serva; 42597.01). The standard curve and diluted samples were transferred to 384-well plates and incubated at room temperature with shaking at 450 rpm for 2 h. After washing four times with PBST, 30 µl of 50 ng / ml biotin-SP AffiniPure F(ab')2 fragment goat anti-human IgG (BIOZOL; 109-066-097) was added, and the plates were incubated at room temperature with shaking at 450 rpm for 1 h. After washing six times with PBST, 30 µl of 10 ng / ml streptavidin poly-hrp40 conjugate (Fitzgerald; 65R-S104HRP) was added, and the plates were incubated at room temperature with shaking at 450 rpm in the dark for 20 min.After washing six times with PBST, 100 µl of SuperSignal ELISA Pico chemiluminescent substrate (Thermo; 37069) was added, and the mixture was incubated at room temperature in the dark for 5 min. The luminescence was measured using a Tecan Infinite F200 (TECAN) without oscillation, with attenuation at OD1 and an integration time of 100 ms. Calculations were performed using Excel and XLfit.

[0188] Sphere measurement:

[0189] References:

[0190] Simonneau et al., Fluids Barriers CNS (2021)

[0191] Kassianidou and Simonneau et al., Bio Protoc (2022)

[0192] Fabrication of U-shaped microporous arrays based on hydrogels

[0193] For the generation of high-throughput BBB spheres, microcavities with a diameter of 600 μm and a depth of 720 μm printed in polyethylene glycol (PEG) hydrogel are typically used (GRI3D® 96-well plate, SunBioscience).

[0194] Cultivation conditions

[0195] Primary human astrocytes (HA, ScienCell Research Laboratories) were grown in astrocyte growth medium (AGM) supplemented with 2% FBS, 1% astrocyte growth supplement (AGS, ScienCell Research Laboratories), and 1% penicillin / streptomycin. Human brain microvascular pericytes (HBVP, ScienCell Research Laboratories) were cultured in pericyte growth medium (PGM) supplemented with 2% FBS, 1% pericyte growth supplement (PGS, ScienCell Research Laboratories), and 1% penicillin / streptomycin. Human brain microvascular endothelial cells (hCMEC / D3, Merck) were maintained in culture using EGM-2 (Lonza) endothelial-based medium supplemented with hydrocortisone, GA-1000, 2% FBS, hEGF, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, and heparin (EGM-2 SingleQuots Supplements, Lonza). For HBVP and HA, cells were grown in T-75 flasks coated with 2 μg / cm² of polylysine. The growth medium was changed every two days. For experimental use, cells were grown to 90% confluence before passage; HA and HBVP were maintained between p1 and p4 passages, and hCMEC / D3 cells were used for 10 passages. For BBB spheroidization, VEGF-free EGM-2 medium, hereinafter referred to as spheroid medium (SM), was used.

[0196] BBB Sphere Generation

[0197] HA and HBVP were isolated using 0.05% trypsin / EDTA (ThermoFisher Scientific), and hCMEC / D3 was isolated using 0.25% trypsin / EDTA, and then resuspended in warm SM. Cells were resuspended at a suitable target concentration of 1000 cells per well at a 1:1:1 ratio (i.e., a total of 3000 cells per well) at a seeding volume of 60 μL per well. After 20 min, 150 μL of culture medium was added. Cells were grown in a humidified incubator at 37°C and 5% CO2 for 48 h (with culture medium refreshed after 24 h) to allow for multicellular spheroid self-assembly.

[0198] Assay for endocytosis transport using BBB spherical array

[0199] Forty-eight hours after assembly, the BBB sphere array was incubated with a test antibody targeting the human transferrin receptor and a non-targeting human IgG as a control in SM at 37°C and 5% CO2 for 4 hours. After incubation, the BBB spheres were thoroughly washed six times with preheated SM in an incubator for 5 minutes each time, and then fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 20 minutes. The samples were thoroughly washed with DPBS and permeabilized at RT with 0.6% Triton-X + 10% donkey serum in DPBS and blocked for 1 hour.

[0200] Immunofluorescence staining of BBB spheroids

[0201] The BBB spheres were washed from the plate and transferred to 1.5 mL protein LoBind tubes using wash buffer (DPBS containing 0.1% Triton-X). After 2 to 3 min, when the spheres settled to the bottom of the tube, the supernatant was removed. Then, anti-human FcY (H+L) secondary antibody (Jackson ImmunoResearch 709-545-098; 488 fluorescently labeled) and DAPI (1 μg / mL, Sigma-Aldrich) were added to the spheres, and the mixture was incubated in the dark on a rotary shaker at RT for 1 h. Finally, the sample was washed again with wash buffer, transferred to a coverslip, and mounted with Fluoromount (Electron Microscopy Science).

[0202] Imaging of a BBB sphere

[0203] 3D imaging of the sphere was performed using a Leica Microsystems Thunder imager with a 20 × 0.55 Ph2 dry objective. Images were acquired using 2x2 bins in 16-bit format. Two distinct channels were set up – one for DAPI (UV laser) and one matched to the fluorophore of the secondary antibody used, in this case AlexaFluor488 (488 laser). A z-stacking pattern with a total coverage depth of 8.5 μm was used, employing 8 steps with the core placed at the center (1.21 μm step size).

[0204] At least 10 spheres were obtained for each experiment and each condition. Then, Leica's Instant Computational Clearing (ICC) algorithm was applied to the image.

[0205] Data analysis of endocytic transport assay

[0206] The code for the analysis is available here: https: / / github.com / phagozyt / Fiji / blob / fb365d7c1275a6b013dc32b982504dfef9653d42 / MIP75ROI.

[0207] In short, upon receiving user input, the macro opens a single z-stack. The macro converts the multi-channel z-stack into a multi-channel maximum projection image. It then splits the multi-channel maximum projection image into individual channel images and creates a mask using the DAPI maximum projection image via thresholding. The macro then converts the mask into a target region (ROI) based on its size and shape. The ROI is then reduced to 75% to cover only the core of the sphere, excluding measurements from the sphere's endothelial surface. The reduced ROI is superimposed on the target channels (488 channels), and the relevant measurements are calculated. Fluorescence intensity per µm² is reported by dividing the original integrated density by the area (µm²).

Claims

1. A multispecific antibody for use in transporting a compound across the blood brain barrier (BBB), wherein the antibody binds to at least two target proteins selected from the group consisting of transferrin receptor (TFRC, TfR), CD98 (SLC3A2), and PODXL.

2. The multispecific antibody for use according to claim 1, wherein the antibody binds to TfR and CD98.

3. The multispecific antibody for use according to claim 1, wherein the antibody binds to TfR and PODXL.

4. The multispecific antibody for use according to claim 1, wherein the antibody binds to CD98 and PODXL.

5. The multispecific antibody for use according to claims 1 to 4, wherein the antibody is a human antibody.

6. The multispecific antibody for use according to claims 1 to 5, wherein the compound is a therapeutic compound.

7. The multispecific antibody for use according to claim 6, wherein the antibody is conjugated to the therapeutic compound.

8. The multispecific antibody for use according to claims 1 to 7, wherein the target proteins are human proteins.

9. The multispecific antibody for use according to claims 1 to 8, wherein the antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to CD98.

10. The multispecific antibody for use according to claims 1 to 8, wherein the antibody comprises a first antigen binding site that binds to TfR and a second antigen binding site that binds to PODXL.

11. The multispecific antibody for use according to claims 1 to 8, wherein the antibody comprises a first antigen binding site that binds to CD98 and a second antigen binding site that binds to PODXL.

12. The multispecific antibody for use according to claims 6 to 11, wherein the therapeutic compound is a nervous system disease drug.

13. The multispecific antibody for use according to claims 6 to 12, wherein the therapeutic compound forms a part of the multispecific antibody.

14. The multispecific antibody for use according to claim 13, wherein the therapeutic compound forms one or both antigen binding sites of the multispecific antibody.

15. The multispecific antibody for use according to claims 13 or 14, wherein the therapeutic compound forming an antigen binding site of the multispecific antibody recognizes a brain antigen.

16. The multispecific antibody for use according to claim 15, wherein the brain antigen is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), TREM2, MS4A, TrkB, and caspase 6.

17. The multispecific antibody for use according to claims 1 to 16, wherein the antibody has a monovalent binding mode for each target protein.

18. The multispecific antibody for use according to claim 2, wherein the TfR binding site has an affinity KD (nM) in the range of 100 to 1000 and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

19. The multispecific antibody for use according to claim 3, wherein the TfR binding site has an affinity KD (nM) in the range of 100 to 1000 and the PODXL binding site has an affinity KD (nM) in the range of 0.1 to 100.

20. The multispecific antibody for use according to claim 4, wherein the PODXL binding site has an affinity KD (nM) in the range of 0.1 to 100 and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

21. The multispecific antibody for use according to claim 2, wherein the TfR binding site has an affinity KD (nM) in the range of 0.01 to 9 and the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100.

Images