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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480047722.4 (22) Application Date 2024.07.23 (30) Priority Data 23187260.7 2023.07.24 EP 23213859.4 2023.12.04 EP (85) PCT International Application Entering National Phase Date 2026.01.16 (86) PCT International Application Application Data PCT / EP2024 / 070818 2024.07.23 (87) PCT International Application Publication Data WO2025 / 021790 EN 2025.01.30 (71) Applicant: Hofmeister AG, Address: Switzerland (72) Inventors: J. Nivena, T. Serra, T.E. Voss, C.M. Simono (74) Patent Agency: Beijing Zhongzi Law Firm, 11247 Patent Attorneys: Zhang Li, Huang Gesheng (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 39 / 00 (2006.01) C07K 16 / 18 (2006.01) C07K 16 / 40 (2006.01) (54) Invention Title: Multispecific Antibody (57) Abstract: This invention relates to 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), CD98 (SLC3A2) and PODXL. Claims (2 pages), Description (28 pages), Sequence Listing (electronic publication), Drawings (17 pages), CN 121548587 A, 2026.02.17, CN 1 21 54 85 87 A. 1. A multispecific antibody for use in a cross-blood-brain barrier (BBB) ​​transport compound, 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 used according to claim 1, wherein the antibody binds to TfR and CD98. 3. The multispecific antibody used according to claim 1, wherein the antibody binds to TfR and PODXL. 4. The multispecific antibody used according to claim 1, wherein the antibody binds to CD98 and PODXL. 5. The multispecific antibody used according to claims 1 to 4, wherein the antibody is a human antibody. 6. The multispecific antibody used according to claims 1 to 5.7. The multispecific antibody used according to claim 6, wherein the antibody is conjugated to the therapeutic compound. 8. The multispecific antibody used according to claims 1 to 7, wherein the target protein is a human protein. 9. The multispecific antibody used according to claims 1 to 8, wherein the antibody comprises a first antigen-binding site binding to TfR and a second antigen-binding site binding to CD98. 10. The multispecific antibody used according to claims 1 to 8, wherein the antibody comprises a first antigen-binding site binding to TfR and a second antigen-binding site binding to PODXL. 11. The multispecific antibody used according to claims 1 to 8, wherein the antibody comprises a first antigen-binding site binding to CD98 and a second antigen-binding site binding to PODXL. 12. The multispecific antibody used according to claims 6 to 11, wherein the therapeutic compound is a drug for neurological diseases. 13. The multispecific antibody used according to claims 6 to 12, wherein the therapeutic compound forms a portion of the multispecific antibody. 14. The multispecific antibody of claim 13, wherein the therapeutic compound forms one or two antigen-binding sites of the multispecific antibody. 15. The multispecific antibody of claim 13 or 14, wherein the therapeutic compound forming one antigen-binding site of the multispecific antibody recognizes a brain antigen. 16. The multispecific antibody of claim 15, wherein 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. 17. The multispecific antibody used according to claims 1 to 16, wherein the antibody has a monovalent binding mode against each target protein. 18. The multispecific antibody used according to claim 2, wherein the TfR binding site has an affinity KD (nM) in the range of 100 to 1000.Furthermore, the CD98 binding site has an affinity KD (nM) in the range of 0.1 to 100. 19. The multispecific antibody used 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 used 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 used 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. Claims 2 / 2 Page 3 CN 121548587 A Multispecific Antibody Technology Field

[0001] The present 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 Art

[0002] The brain penetration of drugs for neurological diseases (such as, for example, large biotherapeutic drugs 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 is to utilize the endocytic transport pathway mediated by endogenous receptors (blood-brain barrier receptors) expressed on the endothelial cells of brain capillaries. Recombinant proteins such as monoclonal antibodies or peptides have been designed to target these receptors, enabling receptor-mediated delivery of biotherapeutic agents 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 causing biotherapeutic agent degradation, remain unexplored.

[0003] Monoclonal antibodies and other biotherapeutic agents hold great therapeutic potential for treating central nervous system (CNS) pathologies. However, their pathways into the brain are blocked by the 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.Because the concentration of antibodies in the CNS is low.

[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 receptors, insulin receptors, etc.) (see, for example, WO 95 / 02421).

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

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

[0007] Anti-transferrin receptor antibodies and methods of use are reported in WO 2014 / 189973. It has been further reported that targeting BBB receptors with conventional specific high-affinity antibodies usually results in a limited increase in BBB transport. It was later found that, in the anti-BBB antibodies studied, the magnitude of antibody uptake into the CNS and distribution in the CNS was negatively correlated with its binding affinity to the BBB receptor. For example, low-affinity antibodies targeting the transferrin receptor (TfR), administered at therapeutic dose levels, significantly improve BBB transport and CNS retention of anti-TfR antibodies compared to higher-affinity anti-TfR antibodies, and make it easier to achieve therapeutic concentrations in the CNS (Atwal et al., Sci. Transl. Med. 3 (2011) 84ra43). This BBB transport has been demonstrated using bispecific antibodies that bind both TfR and the amyloid precursor protein (APP) cleaving enzyme β-secretase (BACE1). A single systemic dose of a bispecific anti-TfR / BACE1 antibody engineered with a low-affinity antibody not only induced significant antibody uptake in the brain, but also significantly reduced brain Aβ1-40 levels compared to 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 cross-BBB compound delivery system is needed to efficiently shuttle compounds into the brain.

[0009] In a first aspect, the present invention provides a multispecific antibody for transporting compounds across the blood-brain barrier (BBB), as specified in the specification page 1 / 28, CN 121548587 A, 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 invention, the antibody is a human antibody.

[0014] In one embodiment of the 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 invention, the target protein is a human protein.

[0017] In one embodiment of the invention, the antibody comprises 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 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 invention, the antibody comprises 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 neurological diseases.

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

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

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

[0024] In one embodiment of the present 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 (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 present invention, the antibody has a monovalent binding mode against each target protein.

[0026] In one embodiment of the present 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 present 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 a target protein PODXL.

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

[0032] In one embodiment of the invention, the compound is a therapeutic compound. Specification 2 / 28 pages 5 CN 121548587 A

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

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

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

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

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

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

[0039] In one embodiment of the present 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 (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 present invention,The antibody has a monovalent binding mode for each target protein. Figure Descriptions

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

[0042] Figures 2-4: Dose-response of the bispecific antibody to hCMECD3 cells:

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

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

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

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

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

[0048] Figure 10: Results of human BBB spheres

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

[0050] Figure 12: Graphical overview of endocytic transport rates of the bispecific antibodies of the present invention: P1AI8549 (701-PODXL-Low / 1026-TFR-Low) showed increased endocytic transport compared to control P1AI8552 (701-PODXL-Low / Pseudo), P1AI8553 (1026-TFR-Low / Pseudo) and control single-arm brain shuttle P1AF3732 (1026-TFR-Low).

[0051] Figure 13: Endocytic transport and recycling of monospecific PODXL antibodies. Detailed Implementation

[0052] Definition

[0053] The "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord. It is formed by tight junctions within the endothelial cell membranes of brain capillaries, constituting 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 herein as the blood-brain barrier or BBB. The BBB also includes the blood-CSF barrier (choroid plexus).The barrier is composed of ependymal cells rather than capillary endothelial cells.

[0054] The mortar-and-pot structure dimer module and its use 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] The “blood-brain barrier receptor” (abbreviated as “R / BBB” herein) is an extracellular membrane-connecting receptor protein expressed on brain endothelial cells that can transport molecules across the BBB or is 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 receptor (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). An 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] A “monovalent binding entity” refers to a molecule capable of specifically binding to R / BBB in a monovalent binding mode. The blood-brain shuttle and / or conjugates of the present invention are characterized by the presence of a single unit of monovalent binding entity, i.e., the blood-brain shuttle and / or conjugates of the present invention comprise a single 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] A “monovalent binding mode” refers to specific binding to R / BBB, wherein the interaction between the monovalent binding entity and R / BBB occurs through a single epitope. Due to the single epitope interaction point,The monovalent binding mode prevents any dimerization / multimerization of R / BBB. The monovalent binding mode prevents changes in the intracellular sorting of R / BBB.

[0060] The term "epitope" 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] A "transferrin receptor" ("TfR") is a transmembrane glycoprotein (molecular weight of about 180,000) involved in iron uptake in vertebrates, consisting of two disulfide-bonded subunits (each with an apparent molecular weight of about 90,000). In one embodiment, the TfR herein is the human TfR, which contains an amino acid sequence such as, for example, 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 an N-terminal intracellular cytoplasmic domain of 75 amino acids, a single transmembrane domain, and a C-terminal extracellular domain of 426 amino acids (Parmacek et al., Nucleic Acids 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 for amino acid transport functions. 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. 118: 889-899, 2005).

[0063] PODXL, also known as podocyte marker protein-like protein-1, PCLP1, or PCX,PODXL is a type I transmembrane protein of the sialomucin family, belonging to CD34. The human PODXL sequence is listed in UNIPOROT accession number O00592. Retaining the basic structure of family member 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 within 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 knot syndrome, Cockayne syndrome, Hallervorden-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), cancer (e.g., CNS cancer and / or brain cancer, including brain metastases caused by cancer in other parts of the body).

[0064] A “neurological disease drug” is a drug or therapeutic agent for treating one or more neurological diseases. The neurological 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, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), ribozymes and small molecules, or active fragments of any of the foregoing. Exemplary neurological disease drugs of the present invention are described herein.And including but not limited to: antibodies, aptamers, proteins, peptides, inhibitory 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, progerin, ApoE, glioma or other CNS cancer markers and neurotrophic factors. Non-limiting examples of drugs for neurological diseases 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)-antibody, 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 having one or more properties that allow direct or indirect detection of its presence and / or location. Examples of such imaging agents include proteins and small molecule compounds incorporating labeled entities that allow detection.

[0066] A “CNS antigen” or “brain target” is an antigen and / or molecule 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 protein β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), 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), and caspase 6. In one embodiment, the antigen is BACE1.

[0067] The term "natural sequence" as used herein refers to a protein that comprises 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" as used herein is used in the broadest sense.And specifically encompasses 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” herein includes portions of a complete antibody that retain the 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 the same epitopes, except for possible variants that may arise during the production of the monoclonal antibody, which are typically present in small amounts. Unlike polyclonal antibody formulations, which typically consist of different antibodies targeting different determinants (epitopes), 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 derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be prepared by the 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 antibody" can also be isolated from phage antibody libraries using techniques described, for example, 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. Monoclonal antibodies as used herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody 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 a corresponding sequence in an antibody 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)). The target 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] The “humanized” form of non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) in which residues in the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, which has the desired specificity, affinity, and function. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not present in receptor antibodies or, as per the specification page 6 / 28, 9 CN 121548587 A. These modifications are intended to further enhance antibody performance. Generally, humanized antibodies will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable regions of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences, except for the FR substitutions mentioned above. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region, which is typically a human immunoglobulin. For further 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" as used herein refers to an antibody comprising an amino acid sequence structure corresponding to the amino acid sequence structure of antibodies obtainable from human B cells, and including an antigen-binding fragment of the human antibody. Such antibodies can be identified or prepared by various techniques, including but not limited to: those capable of being produced without the generation of endogenous immunoglobulins.Production in transgenic animals (e.g., mice) that produce human antibodies after immunization (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); U.S. Patent Nos. 5,565,332 and 5,573,905; generated via in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275); and isolated from human antibodies that produce hybridomas.

[0073] The term “multispecific antibody” herein refers to an antibody that has binding specificity to at least two distinct epitopes. Exemplary multispecific antibodies may bind both R / BBB and brain antigens. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Also covered are engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites (see, for example,US Appln No. US 2002 / 0004587 A1, Miller et al. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0074] Antibodies described herein include “amino acid sequence variants” with altered antigen-binding or biological activity. Examples of such amino acid alterations include antibodies with enhanced affinity to antigens (e.g., “affinity-matured” antibodies), and antibodies with altered Fc regions (if present) (e.g., with 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 gene III product of M13 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 technique described on page 7 / 28 of this specification, 10 CN 121548587 A, 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”, so any carbohydrates (if present) attached to the Fc region are altered. For example, the antibody described in US Patent Application No. US 2003 / 0157108 (Presta, L.) has a mature carbohydrate structure.The fucose attached to the Fc region of the antibody is missing. See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). WO 2003 / 011878, Jean-Mairet et al., and US Patent No. 6,602,684, Umana et al., cite antibodies containing bipartite N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody. WO 1997 / 30087, Patel et al., also report antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody. See also WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) concerning antibodies with altered carbohydrates attached to the Fc domain of the antibody. 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 residues in the antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the complementarity-determining region (CDR) or CDR (e.g., residues 24–34 (L1), 50–56 (L2), and 89–97 (L3) in the light chain variable domain, and residues 31–35 (H1), 50–65 (H2), and 95–102 (H3) in the heavy chain variable domain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the hypervariable ring (e.g., residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) in the light chain variable domain, and residues 26–32 (H1), 53–55 (H2), and 96–101 (H3) in the heavy chain variable domain; Chothia and Lesk J.). Mol. Biol. 196:901-917 (1987)). The “framework” or “FR” residues are those variable domain residues other than the hypervariable region residues defined herein.

[0077] A “full-length antibody” is an antibody containing 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 may be native sequence constant domains (e.g.,Human natural sequence constant domain) or amino acid sequence variants thereof.

[0078] A “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 Fc region of the antibody (the Fc region of the natural sequence or the Fc region of the amino acid sequence variant). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), etc. In one embodiment, the antibody 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 portion of most IgG antibody subclasses. The binding of C1q to the antibody is caused by a protein-protein interaction defined at the so-called binding site. Such Fc partial binding sites are known in the prior art. Such Fc partial binding sites are characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (according to the Kabat EU index number). Antibodies of 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 some 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 classes of antibodies are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations 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,For example, plants belonging to the genus *Nicotiana* (e.g., tobacco); (4) yeast cells, such as yeast cells belonging to the genus *Saccharomyces* (e.g., *Saccharomyces cerevisiae*) or the genus *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” means that an antibody selectively or preferentially binds to an antigen. Binding affinity is typically determined using standard assays (such as Scatchard assays) or surface plasmon resonance techniques (e.g., using BIACORE®).

[0083] An antibody “binding to the same epitope” as a reference antibody means 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 that blocks the binding of the antibody to its antigen by 50% or more in a competitive assay.

[0084] An “imaging agent” is a compound having one or more properties that allow 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 detection.

[0085] A “marker” is a biomarker conjugated to the antibody described herein and used for detection or imaging. Examples of such markers include: radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, a marker 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 enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof.

[0087] A “therapeuticly effective amount” of an agent (e.g., a pharmaceutical preparation) means the amount that effectively achieves the desired therapeutic or preventative outcome at the necessary dose and time period.

[0088] The term “Fc region” herein is used to define the C-terminal region of the immunoglobulin heavy chain.The C-terminal region contains at least a portion of the constant region. The Fc region contains the CH2 and CH3 domains of the immunoglobulin. The term includes the native sequence Fc region and the variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from 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 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 variable domain residues other than the hypervariable region (HVR) residues. The variable domain FR is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. Specification 9 / 28 pages 12 CN 121548587 A

[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”. This term encompasses both the native sequence and variants of the CH2-CH3 domain. In one embodiment, the “CH2-CH3 Ig entity” is derived from the human heavy chain CH2-CH3 IgG domain, which extends from Cys226 or Pro230 to the carboxyl 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 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.

[0091] A “conjugate” is a 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, a “connector” refers to a chemical or single-chain peptide connector that covalently links different entities of the multispecific antibody and / or fusion protein and / or conjugate of the present invention. A 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 that links a brain effector entity to a monovalent binding entity (a first connector) and a connector that links the scFab to the C-terminus of the CH2-CH3 Ig domain (a 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 in length, preferably 32 to 50 amino acids in length. 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 one embodiment, the connector is (G4S)6G2 (Seq.Id.No. 13).

[0094] Various chemical connectors can be used for fusion. 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-1-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 posterior 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); US Patent No. 5,208,020).

[0095] Covalent conjugation can be performed directly or via adapters. In some embodiments, direct conjugation is performed by constructing a protein fusion (i.e., by fusing the genes of 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 acceptor on the compound. In some embodiments, direct conjugation is achieved by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to include a reactive group (as a non-limiting example, the reactive group may be 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 pointing to an R / BBB antibody 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,Monovalent binding entities and effector entities can be conjugated using a variety of bifunctional protein conjugates such as N-succinimide-3-(2-pyridyldithio)propionate (SPDP), succinimide-4-(N-maleimidemethyl)cyclohexane-1-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), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated 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 such 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 the effector progenitor upon 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 “tag” is a marker coupled to the fusion protein herein and used for detection or imaging. Examples of such tags include: radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the label is a radioactive label for medical imaging, such as TC99M or 1123, 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. The “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] The “isolated” antibody is an antibody that has been isolated from components of its natural environment. In some embodiments, antibody purification to a purity greater than 95% or 99% is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). A review of methods for assessing antibody purity is provided.See, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0098] The term “pharmaceutical formulation” means a formulation in which the active ingredient is permitted to be contained and is in a biologically effective form, and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0099] “Pharmaceuticalally acceptable carrier” means a component of a pharmaceutical formulation that is non-toxic to a subject, 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 individual being treated, and may be performed for prevention or during the course of clinicopathology. The expected effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or reducing the severity of the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of the disease or slow its progression.

[0101] For neurological conditions, selectable neurological drugs are 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, oxaprazin, piroxicam, succinate). (e.g., lindac 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, salicylate choline, magnesium salicylate, diflunisal, and disalicylate), anticonvulsants (i.e., carbamazepine, clonazepam, ... Gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomecaine, dimethicaine, propyloxycaine, procaine, novocacaine, propylmecaine, tetracaine, articaine, bupivacaine, carticaine, cincocaine, eticaine, levobupivacaine, lidocaine, mepivacaine, piperacaine, prilocaine, ropivacaine, trimethaine, clam toxin, and tetrodotoxin),And cox-2-inhibitors (i.e., celecoxib, rofecoxib, and vardicoxib). For neurological conditions accompanied by vertigo, antivertigo medications can be selected, including but not limited to meclopramide, diphenhydramine, promethazine, and diazepam. For neurological conditions accompanied by nausea, antinausea medications can be selected, including but not limited to promethazine, chlorpromazine, prochlorperazine, trimebenamide, and metoclopramide. For neurodegenerative diseases, neurotrophic drugs such as 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), interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurotrophins, platelet-derived growth factor (PDGF), regulatory proteins, neuromodulators, leptospirin, persephin, interleukins, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, etc. Netrin, Cardiac Nutrient-1, Hedgehog, Leukemia Inhibitor Factor (LIF), Mid-term Factor, Pleurotrophic Growth Factor, Bone Morphogenetic Protein (BMP), Netrin, Saponin, Signal Phenylexin, and Stem Cell Factor (SCF). For cancer, these are neurological drugs that can be used as chemotherapy agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, inprossurfan, and piperossurfan; acridine derivatives such as benzotipane, carboquinone, metopepa, and urotepa; ethylene imines and methylmelamine derivatives, including hexamethylmelamine, tratamiamine, triethylenephosphamide, triethylenethiophosphate, and trihydroxymethylmelamine; polyacetic acids (especially bratasin and bratasinone); δ-9-tetrahydrocannabinol (drocannabinol, MARINOL®); β-lapaquinone; laparol; colchicine; betulinic acid; camptothecin (including synthetic analogues such as topotecan (HYCAMTIN®) and CPT-11 (irinotecan,CAMPTOSAR®, acetylcamptothecin, scopolamine and 9-aminocamptothecin); bryostatin; sponge polyene ketones; CC-1065 (including its synthetic analogues adolaxine, calcein and pyrazin); podophyllotoxin; podophyllic acid; teniposide; cryptophytes (especially cryptophyte 1 and cryptophyte 8); dolalastatin; pyroxine (including the synthetic analogue KW-2189) And CB1-TM1); arugula; pancratistatin; scutellarin; spongin; nitrogen mustards, such as chlorambucil, naphthalenemus, chophosphatamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, benzyl mustard cholesterol, prednimustine, trazophosphatide, uracil mustard; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine; antibiotics, such as enediynes. (Instructions 12 / 28 pages 15 CN 121548587 A) Antibiotics (e.g., kazimycin, especially kazimycin γ-II and kazimycin ω-II (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186) (1994)); enediyne anthracycline antibiotics, including enediyne anthracycline antibiotic A; esporamycin; and new carcinogen chromophores and related pigment proteins (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, mycophenolic acid, nopramine, olivomycin, pepromycin, purulin, triamcinolone acetonide, rhodorubicin, streptomycin, streptozotocin, tuberculin, ubenmex, fenestrated statin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-F... U); folic acid analogs, such as folate, methotrexate, pteroxate, trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine; androgens, such as calotestosterone, drotalonone propionate, cyclothionol, meandrolone, testrolide; antiadrenergic drugs, such as aminoglutethimide, mitotane, trelostan; folic acid supplements,Such as leucovorin; glucuronolactone; hydroxylphosphamide glycoside; aminopyruvate; enuramicin; acridine; bestra bucil; bisaccharin; edatrazine; defofamine; colchicine; diaccharin; elfornithine; elifonitrile; epomycin; etanertin; gallium nitrate; hydroxyurea; lentinan; chlordamine; maytansine, such as metansine and anthraquinone; mitoxantrone; mitoxantrone; mopiperol; nitroacin; pentostatin; promethazine; pirarubicin; loxoantrone; 2-ethylhydrazine; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razorylene; lisoxantrone; cizonan; germanium spiroamine; Alternaria spp. ketoacid; triamine; 2, 2',2''-Trichlorotriethylamine; trichothecene compounds (especially T-2 toxin, verracurin A, baculosporin A, and serpentin); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; dibromomannitol; dibromoeusine; piperobromethane; gacytosine; cytarabine (“Ara-C”); thiotepa; taxanes, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), 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; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); naringoside; edaraxal; 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. This is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX.This is an abbreviation for the treatment regimen of oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovovin.

[0102] This definition of chemotherapeutic agents also includes anti-hormonal agents, which act to regulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are usually in the form of systemic or generalized therapy. They may be the hormones themselves. Examples include: anti-estrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trivoxifen, raloxifene, LY 117018, onanasone, and FARESTON® toremifene; anti-progesterone drugs; estrogen receptor downregulators (ERDs); and 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® vortexilazole, 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® tiludronate, 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, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELLX® rmRH; lapatinib dimethylbenzenesulfonate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

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

[0104] For eye diseases or conditions, the following neurological medications can be selected: anti-angiogenic ophthalmic agents (i.e., bevacizumab, ranibizumab, and peganob), and ophthalmic glaucoma medications (i.e., carbachol, epinephrine, dimethoprim, adaloride, brimonidine, bullinazolamide, levobunolol, timolol, betalolol, dazolide, bimatoprost, carteolol, meteolol, dipiformin, travoprost, and latanoprost). Carbonic anhydrase inhibitors (i.e., acetazolamide and acetazolamide), ophthalmic antihistamines (i.e., naphazoline, norepinephrine, and tetrahydrozoline), ophthalmic lubricants, ophthalmic steroids (i.e., fluorometholone, prednisolone, clotiprednisolone, dexamethasone, difluprednisolone, limexolone, fluocinolone acetonide, methaqualone, and triamcinolone acetonide), ophthalmic anesthetics (i.e., lidocaine, promecaine, 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 anti-inflammatory agents (i.e., napafenamide, ketoprofen, flurbiprofen, sulofen, cyclosporine, triamcinolone, diclofenac, and bromofenac), and ophthalmic antihistamines or decongestants (i.e., ketotifen, olopatadine, epinastine, naphazoline, cromoglycine, tetrahydrozoline, pyrimisulfan, betasine, naphazoline, phenylephrine, nedolomethil, lodusamide, phenylephrine, emesine, and azelastine). For epileptic seizures, the neurological medications that can be chosen are anticonvulsants or antiepileptic drugs, including but not limited to barbiturate anticonvulsants (i.e., primidone, methadone, toluidine, allobarbital, amobarbital, alpratobarbital, phenobarbital, barbital, bromhexine, and phenobarbital), and 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., rufenamide, carbamazepine, and oxcarbazepine), fatty acid derivative anticonvulsants (i.e., sodium valproate and valproic acid), Gamma-aminobutyric acid analogs (i.e., pregabalin, gabapentin, and vigabatrin), Gamma-aminobutyric acid reuptake inhibitors (i.e., tiagabine), 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), progesterone package insert 14 / 28 pages 17 CN 121548587 A Anticonvulsants include oxazolidinediones (i.e., progesterone), oxazolidinediones (i.e., methylethyldione and trimethyldione), pyrrolidine anticonvulsants (i.e., levetiracetam), succinimide anticonvulsants (i.e., ethosuximide and mesuximide), 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 the treatment of lysosomal storage diseases 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 drugs 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 a target selected from the following: β-secretase, tau, presenilin, amyloid precursor protein or a portion thereof, β-amyloid peptide 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 depletion agents (i.e., buphenazine); dihydroergotamine mesylate; anticholinergic anti-Parkinson's agents (i.e., propiconazole, diphenhydramine, trihexyphenidyl, benzalkonium chloride, biperiden and trihexyphenidyl); dopaminergic anti-Parkinson's agents (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, progesters, and leuprolide); vitamins (i.e., folic acid and nicotinamide); danopiformin; high taurine (i.e., 3-aminopropanesulfonic acid; 3APS); serotonin receptor activity modulators (i.e., zaliroden); interferon 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 antiviral agents (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, etravirine, deraviridine, and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine, and norinosine), and protease inhibitors (i.e., darunavir, atazanavir, fosanavir, telanavir, ritonavir, nelfinavir, ampravir, indinavir, and salbutavir). Quinavir), purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir), and miscellaneous antiviral agents (i.e., entfuvirtide, phosphonoformate, palilizumab, and fomivirex), and antibiotics (including but not limited to aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temoxicillin, azlocillin, carbenicillin)). Ticarcillin, Meropenem, Piperacillin, and Bambucil), cephalosporins (i.e., cefazolin, cefalexin, cefotaxime, cefamandole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cefadroxil, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), and carbapenem / penem (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem). Monoamide antibiotics (i.e., amtremone, tegafur, nocardiin A, and nicotinic acid-β-lactam), β-lactamase inhibitors in combination with another β-lactam antibiotic (i.e., clavulanic acid, triazobactam, and subaktam), aminoglucosides (i.e., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, topromycin, and paromomycin), product information 15 / 28 pages 18 CN 121548587 A sarsaparilla (i.e.,Geldermycin and atrazine), carbapenems (i.e., chloramphenicol), glycopeptides (i.e., teicoplanin and vancomycin), macrolides (i.e., azithromycin, clarithromycin, erythromycin, roxithromycin, acetomycin, telithromycin, and spectinomycin), monoamides (i.e., aztreonam), quinolones (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, limoxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, gapfloxacin, sparfloxacin, and temaxacin), sulfonamides (i.e., sulfamidone, azosulfonamide, acesulfame potassium, sulfadiazine, sulfadiazine, sulfasal ... Sulfisoxazole, methoxyoctidine, methoxyoctidine and methoxymethoxazole), tetracyclines (i.e. tetracycline, demecycline, doxycycline, minocycline and oxtetracycline), anti-inflammatory or cytotoxic antibiotics (i.e. doxorubicin, mitoxantrone, bleomycin, daunorubicin, daunorubicin, epirubicin, idarubicin, priloin, mitomycin, pentostatin and pentorubicin) And miscellaneous antimicrobial compounds (i.e., bacitracin, colistin, and polymyxin B), antifungal agents (i.e., metronidazole, nitrozonide, tinidazole, 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, neurological medications that can be selected 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 drugs may be selected from behavior-modifying compounds, including but not limited to atypical antipsychotics (i.e., risperidone, olanzapine, aripiprazole, quetiapine, palapiridone, asenapine, clozapine, ipraridone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thixothralose (i.e., sulfathione), miscellaneous antipsychotics (i.e., pimozide, lithium, indole, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, etapaplan, paroxetine, fluoxetine, and sertraline), serotonin-norepinephrine reuptake inhibitors (i.e., duloxetine, venlafaxine, divenlafaxine), tricyclic antidepressants (i.e.,Doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, tramipram, 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, succinylphenamine, 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, neurological drugs that address the inflammation itself (i.e., nonsteroidal anti-inflammatory agents, such as ibuprofen or naproxen) or neurological drugs that treat the underlying cause of the inflammation (i.e., antiviral agents or anticancer agents) can be selected.

[0110] In another embodiment, the compound is a complete or full-length antibody. Complete antibodies can be classified into different categories according to the amino acid sequence of their heavy chain constant domain, page 16 / 28 of the specification, 19 CN 121548587 A. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some 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 classes of antibodies are called 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, intact antibodies lack effector function.

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

[0112] The target multispecific antibody and / or conjugate can be assayed to evaluate the uptake of the multispecific antibody and / or conjugate and other biological activities of the multispecific antibody and / or conjugate for systemic administration, as disclosed in the examples or in a known manner. The concentration in the CNS physical space can also be measured using, for example, microdialysis or capillary depletion combined with ELISA or radiometric measurements of labeled multispecific antibodies and / or conjugates.

[0113] Pharmaceutical Formulation

[0114] The therapeutic formulation of the antibody or conjugate used according to the invention is prepared as a lyophilized formulation or an aqueous solution for storage by mixing with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Edited (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the dosage and concentration 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). (1 residue) polypeptides; 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 active compounds having 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 technology or interfacial polymerization, and is encapsulated in a colloidal drug delivery system (e.g.,In liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in 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 a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, in the form of a molded article, such as a membrane or microcapsule. 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 copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Instructions for Use, Pages 17 / 28, 20 CN 121548587 A

[0118] The formulation to be used for in vivo administration must be sterile. This can be easily achieved by filtration through 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 a multispecific antibody transports the neurological disease drug conjugated thereto across the BBB. In one embodiment, the BBB here refers to mammals (e.g., humans), such as mammals suffering from neurological disorders, 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 disorder is selected from: neuropathy, amyloidosis, cancer (e.g., involving the CNS or brain), eye diseases or conditions, viral or microbial infections, inflammation (e.g., inflammation of the CNS or brain), ischemia, neurodegenerative diseases, seizures, behavioral disorders, lysosomal storage diseases, etc.

[0121] A neurological disorder is a disease or abnormality of the nervous system characterized by inappropriate or uncontrolled neural signaling or lack of signaling, and includes, but is 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 entirely or mostly related to a mental disorder), headaches, migraines, neuropathy, and symptoms and syndromes that often accompany such neuropathy, 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-associated dementia, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), and eye diseases involving β-amyloid protein deposition (i.e., macular degeneration, drusen-associated 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 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, thereby causing CNS infection. Pathophysiology, including but not limited to meningitis, encephalitis, myelitis, vasculitis, and abscess,It 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, 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 herein, CNS ischemia refers to a group of conditions or their causes associated with abnormal blood flow or vascular behavior in the brain, and includes, but is 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 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 of 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, clonic seizures, complex partial seizures, frontal lobe seizures, febrile seizures, infantile spasms, juvenile myoclonic seizures, juvenile absence epilepsy, Rengork syndrome, Ranke syndrome, Zachary 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, clonic seizures, tonic seizures, psychomotor seizures, limbic seizures, partial seizures, generalized seizures, status epilepticus, abdominal seizures, akinetic seizures, vegetative seizures, etc.). Numerous bilateral myoclonus, menstrual epilepsy, fall epilepsy, emotional epilepsy, focal epilepsy, laughing epilepsy, Jacksonian walking, Lafra disease, motor epilepsy, multifocal epilepsy, nocturnal epilepsy, photosensitive epilepsy, pseudoepileptic seizures, sensory epilepsy, microepileptic seizures, jungle epilepsy, withdrawal epilepsy, and visual reflex epilepsy.

[0128] Behavioral disorders are CNS conditions characterized by abnormal behavior in parts of the affected 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 and 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 some cases; such 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, GMI ganglioside storage disorders, metachromatic leukoencephalopathy, Fabry disease, Carnafan leukodystrophy and neuronal cerebrolipofuscinosis type 1 and 2, Niemann-Pick disease, Pompe disease, and Krabby disease.

[0130] The multispecific antibodies and / or conjugates of the present invention can be used alone or in combination with other agents in a therapeutic manner. 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 diseases as the neurological diseases for which the multispecific antibodies and / or conjugates of the present invention are used for treatment. 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-secretion enzyme 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 above-described 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 the treatment or prevention of 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 may be performed by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse 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 the practicing physician. The multispecific antibodies and / or conjugates of this invention are not essential, but are optionally 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 preventive 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 can range from about 1 μg / kg to 100 mg / kg or more. For repeated dosing 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 range for antibodies is 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 the 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 dosage regimens may be available. Progression of this therapy can be easily monitored using routine techniques and assays. Instructions for Use 20 / 28 pages 23 CN 121548587 A

[0135] Table 1: Bispecific antibody amino acid sequences

[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 Instructions for Use 21 / 28 pages 24 CN 121548587 A

[0140]

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

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

[0143]

[0144] Table 4: PODXL Antibody Details

[0145] Specification 22 / 28 pages 25 CN 121548587 A

[0146] Table 5: CD98 Antibody Details

[0147]

[0148] Table 6: Monospecific Control Antibody

[0149]

[0150] Examples

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

[0152] Figure 2a shows 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- Binding assays of P1AI8556 (698-PODXL-701-low / prototype) and P1AI8552 (PODXL-701-low / prototype) on hCMECD3 cells were performed. The binding assays showed that P1AI8556 (698-PODXL-high / TFR-low) bound to hCMECD3 cells most strongly. 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. The binding assay showed that P1AI8557 bound to hCMECD3 cells the most. Compared with the two monospecific binding controls P1AI8559 (698-PODXL / prototype), the combination of PODXL-698-high / TfR-Duta 3-high increased 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 single-specific TfR conjugate control P1AI8555 (prosthesis / Duta-1-medium), the combination PODXL-698-high / TfR-Duta-1-medium showed increased binding properties, but compared to the PODXL conjugate controls P1AI8558 (PODXL-698-high / TfR-Duta-1-medium) and P1AG3698 (PODXL-698-high-single-arm), this combination did not show increased binding properties. Compared to the single-specific controls P1AI8552 (PODXL-701-low / prosthesis, page 23 / 28, CN 121548587 A) and P1AI8555 (prosthesis / TfR-Duta-1-medium),The combination of PODXL-701-low / TfR-Duta 1- increases 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. 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) decreased 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. 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: P1AI8549 (701-PODXL-low / 1026-TFR-low). However, P1AI8552 (701-PODXL-low / pseudo) showed 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 bispecific antibodies P1AI8559 (PODXL-698-high / pseudo), P1AG3701 (PODXL-701-low / none), P1AG3698 (PODXL-698-high / none), P1AI8552 (PODXL-701-low / pseudo), P1AG3705 (CD98-705-low), P1AI4681 and P1AI3732 on hCMEDCD3. The single-arm version of the 698-PODXL-High (P1AG3698) behaves similarly to the two-arm version, where the 698-PODXL-High is combined with a non-conjugate (P1AI8559 (PODXL-698-High / prosthesis)). Therefore, we can conclude that...The effects shown are not due to the antibody format (single-arm vs. double-arm IgG).

[0158] Endocytotic transport assay results of bispecific antibodies

[0159] Figures 6a and 6b show an overview of the endocytotic transport assay results of the following bispecific antibodies and control antibodies: P1AI8549 (PODXL-701-low / TfR-1026-low), P1AI8552 (PODXL-701 / pseudo), P1AI8553 (pseudo / TfR-1026-low), P1AI8556 (PODXL-698-high / TfR-1026-low), P1AI8559 (PODXL-698-high / pseudo), 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: 20.1 µg / ml, conjugate 2.67 µg / ml.

[0160] Figure 6c shows the endocytosis transport assay results of the selected bispecific antibodies, namely: 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 endocytosis transport assay results of the selected bispecific antibodies, namely: P1AI8552 (PODXL-701 / pseudo), P1AI8553 (pseudo / TfR-1026-low), P1AI8559 (PODXL-698-high / pseudo), and P1AK1327 (pseudo / CD98-703-high). Experimental setup: hCMECD3; loading amount: 20.1 µg / ml, conjugate 2.67 µg / ml.

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

[0164] Figure 6g shows the endocytosis transport assay results of the bispecific antibody P1AI8551 (CD98-705-low / TfR-duta 1-medium) + control. Experimental setup: hCMECD3; loading amount: 20.1 µg / ml, conjugate concentration 2.67 µg / ml. Instructions for Use, pages 24 / 28, CN 121548587 A

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

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

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

[0168] Figure 6k shows the endocytosis 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 endocytosis transport, similar effects to binding properties were observed, with P1AI8556 (PODXL-698-high / TfR-1026-low) showing increased endocytosis transport compared to the following two monospecific 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 single-specific 703-CD98 control.And P1AI8559 (698-PODXL- High / Prosthesis) represents another control.

[0171] P1AK1326 (PODXL-701- Low / CD98-703- High) showed increased endocytosis compared to the control P1AI8552 (701-PODXL- Low / Prosthesis).

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

[0173] P1AI8549 (701-PODXL- Low / 1026-TFR- Low) showed increased transcellularity 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 TfR / 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). In addition, P1AK1326 (701-PODXL-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 endocytosis transport assay results of the bispecific antibodies targeting TfR / PODXL TfR / CD98 and PODXL / CD98 20 h after transport. After 20 h of intracellular transport, P1AK1323 (698-PODXL-high / 703-CD98-high) showed the highest level of endocytosis transport, followed by P1AK1325 (1026-TFR-low / CD98-703-high).

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

[0177] Figure 13 shows the endocytosis transport assay results of the monospecific antibody targeting PODXL. P1AG3700 showed the highest endocytic 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.The ratio of plasmids encoding the protein was 1:1:1:1. The protein was purified by affinity chromatography

[0181] (MabSelect SuRe), ion exchange chromatography (POROS XS), and size exclusion chromatography. For quality control, the endotoxin level (<0.5 EU / mg (kinetic LAL)), monomer content >96% (aSEC, 1x after freezing / thawing), and purity (capillary gel electrophoresis) of the antibody were analyzed. Specification 25 / 28 pages 28 CN 121548587 A

[0182] Binding assay:

[0183] For the FACS binding assay, hCMECD3 cells were incubated 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 for 1 h. Cells were washed with PBS (calcium-free, magnesium-free, sterile DPBS; PANBiotech; P04-36500) + 5% FBS (FBS Premium, fetal bovine serum from Australia, irradiated with γ and sterile filtered at 0.2µm; PANBiotech; P30-2006), and then the 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, fetal bovine serum from Australia, irradiated with γ and sterile filtered at 0.2 μm; 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 and transport assay:

[0185] For the endocytosis and transport assay, 12-well cell culture inserts (cellQART; 9310402) were coated with 400 µl collagen (Corning; 354236) and 200 µl fibronectin (Gibco; 33016-015) at a concentration of 50 µg / ml for 1 h. After washing the insert twice with DPBS, CaCl2, and CaMg2 (Gibco; 14040-091), it was stored at 4°C for up to one week until cell seeding. Before seeding cells,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 8000 cells / filter were seeded at the top of the transwell insert and cultured at 37°C and 5% CO2 for 12 days, changing the medium every 2–3 days. On day 12, fresh cell culture medium (basal side) was added, and the top medium was exchanged with a previously prepared antibody dilution (21.0 µg / ml), and incubated at 37°C and 5% CO2 for 1 h to allow intracellular uptake. After washing the basal and apical sides 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, apical and basal supernatant samples were collected, and cell lysates were prepared, with each sample being repeated twice. For the cell lysates, the entire cell culture medium was removed, and the cells were lysed in RIPA buffer (Sigma Aldrich; R0278). The samples were stored at -80°C, and the IgG levels were measured by huIgG ELISA and analyzed using Excel and XLfit.

[0186] huIgG ELISA

[0187] To measure the IgG levels in the supernatant and cell lysates of the endocytosis transport assay, the samples were measured by ELISA. Therefore, 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 ELISA,Remove the coating antibody and replace it with 100 µl of Ca2+ and Mg2+-free PBS (PAN Biotech; P04-991500) + 0.5% BSA (Thermo Scientific; 37525) + 0.05% Tween 20 (anatrace; T1003). Prepare standard curves for the corresponding antibodies 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). In addition, for both supernatant and cell lysate, starting with undiluted samples, 1:3 dilution series were prepared for each sample up to a 1:729 dilution 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 biotin-SP AffiniPure F(ab')2 fragment goat anti-human IgG (BIOZOL; 109-066-097) at a concentration of 50 ng / ml was added, and the mixture was incubated at room temperature with shaking at 450 rpm for 1 h. After washing six times with PBST, 30 µl of streptavidin poly-hrp40 conjugate (Fitzgerald; 65R-S104HRP) at a concentration of 10 ng / ml was added, and the mixture was 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 shaking, with the decay measured at OD1.The integration time is 100 ms. Calculations were performed using Excel and XLfit.

[0188] Sphere determination:

[0189] References:

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

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

[0192] Fabrication of U-bottom 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) hydrogels are typically used (GRI3D® 96-well plates, SunBioscience).

[0194] Culture Conditions

[0195] Primary human astrocytes (HA, ScienCell Research Laboratories) were grown in astrocyte growth medium (AGM) consisting of astrocyte culture medium (AM, ScienCell Research Laboratories) 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 pericytic growth medium (PGM) consisting of pericytic culture medium (PM, ScienCell Research Laboratories) supplemented with 2% FBS, 1% pericytic 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 passages p1 and p4.hCMEC / D3 cells were used for 10 passages. For BBB spheroid formation, VEGF-free EGM-2 medium was used, which is hereinafter referred to as spheroid medium (SM).

[0196] BBB Spheroid Formation

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

[0198] . Endocytosis and transport assays using BBB spheroid arrays

[0199] After 48 h of assembly, the BBB spheroid arrays were incubated for 4 h at 37°C and 5% CO2 in SM with a test antibody targeting the human transferrin receptor and non-targeting human IgG as a control. After incubation, the BBB spheroids were thoroughly washed 6 times for 5 min each time in a preheated SM in an incubator and then fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 20 min. The samples were thoroughly washed with DPBS and permeabilized and blocked at RT with 0.6% Triton-X + 10% donkey serum in DPBS for 1 h.

[0200] Immunofluorescence staining of BBB spheres

[0201] BBB spheres were washed from the plate with washing buffer (DPBS containing 0.1% Triton-X) and transferred to 1.5-mL protein LoBind tubes. 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 samples were incubated in the dark on a rotary shaker at RT for 1 h. Finally, the samples were washed again with washing buffer and transferred to coverslips.The slides were mounted using Fluoromount (Electron Microscopy Science).

[0202] Imaging of BBB Spheres

[0203] Spheres were imaged using a Leica Microsystems, Thunder Imager 3D assay with a 20 × 0.55 Ph2 dry objective. Images were acquired using 2x2 bins in 16-bit format. Two different channels were set up – one for DAPI (UV laser) and one matched to the fluorophore of the secondary antibody used, in this case Alexa Fluor 488 (488 laser). A z-stacking 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. Leica’s Instant Computational Clearing (ICC) algorithm was then applied to the images.

[0205] Data Analysis of Endocytotic Transport Assay

[0206] The code used 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 single-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 and exclude measurements from the endothelial surface of the sphere. The reduced ROI is superimposed on the target channels (488 channels).The relevant measurement results are calculated. The fluorescence intensity per µm² is reported by dividing the original integrated density by the area (µm²). Instruction Manual 28 / 28 Page 31 CN 121548587 A Figure 1 Figure 2a Instruction Manual Figure 1 / 17 Page 32 CN 121548587 A Figure 2b Figure 2c Instruction Manual Figure 2 / 17 Page 33 CN 121548587 A Figure 2d Figure 3 Instruction Manual Figure 3 / 17 Page 34 CN 121548587 A Figure 4 Figure 5 Instruction Manual Figure 4 / 17 Page 35 CN 121548587 A Figure 6a Figure 6b Figure 6c Instruction Manual Figure 5 / 17 Page 36 CN 121548587 A Figure 6d Figure 6e Figure 6f Instruction Manual Figure 6 / 17 Page 37 CN 121548587 A Figure 6g Figure 6h Figure 6i Instruction Manual Figure 7 / 17 Page 38 CN 121548587 A Figure 6j Figure 6k Figure 7a Instruction Manual Figure 8 / 17 Page 39 CN Figure 7b of the instruction manual, page 40 of 121548587 A; Figure 7c of the instruction manual, page 41 of 10 of 17; Figure 7d of the instruction manual, page 42 of 11 of 17; Figure 8 of the instruction manual, page 43 of 121548587 A; Figure 9c of the instruction manual, page 44 of 13 of 17; Figure 11a of the instruction manual, page 45 of 11c of the instruction manual, page 46 of 15 of 17; Figure 11d of the instruction manual, page 47 of 16 of 17; Figure 13 of the instruction manual, page 47. Instruction manual, Figure 17 / 17, page 48, CN 121548587 A,

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.

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