WO2025055904A1

WO2025055904A1 - Cd98hc antibody and use thereof

Info

Publication number: WO2025055904A1
Application number: PCT/CN2024/118030
Authority: WO
Inventors: Dong Yang; Weiwei Sun; Jie Xie; Zhiming Cheng; Jing Su
Original assignee: 4b Technologies Beijing Co Ltd; 4b Technologies Suzhou Ltd
Current assignee: 4b Technologies Beijing Co Ltd; 4b Technologies Suzhou Ltd
Priority date: 2023-09-12
Filing date: 2024-09-10
Publication date: 2025-03-20
Anticipated expiration: 2026-03-12

Abstract

Provided is a CD98HC antibody or the antigen-binding fragment thereof, which can be used for delivering a therapeutic entity across blood brain barrier (BBB). Further provided is a fusion protein comprising the CD98HC antibody or the antigen-binding fragment thereof and a therapeutic entity.

Description

CD98HC ANTIBODY AND USE THEREOF

BACKGROUND OF THE INVENTION

The blood-brain barrier (BBB) is a specialized barrier that separates the bloodstream from the brain tissue. It is composed of brain endothelial cells (BECs) , pericytes, and astrocytes. These cells work together to maintain the integrity of the continuous non-fenestrated capillaries through tight junctions. The primary function of the BBB is to regulate the exchange of substances between the blood and the brain, creating a stable and tightly controlled environment necessary for proper brain function. Its presence helps to maintain the homeostasis of the central nervous system (CNS) by preventing the entry of harmful substances, pathogens, and large molecules into the brain. However, the BBB also poses challenges for delivering drugs to the brain, as it restricts the passage of many therapeutic agents. For instance, only a small fraction of circulating antibodies, approximately 0.1%, can cross the intact BBB. Moreover, peptides, antibody-drug conjugates, and proteins face similar limitations in crossing this barrier, substantially compromising the efficacy of treatments for CNS diseases. Some endogenous molecules in circulation can cross the BBB through specific receptors and transporters expressed on the luminal side of brain endothelial cells. These membrane protein pathways offer a promising route for delivering hardly penetrated molecules across the BBB using receptor-mediated transcytosis (RMT) .

CD98 heavy chain (CD98HC) , also known as solute carrier family 3 member 2 (SLC3A2) , is encoded by the SLC3A2 gene. CD98HC interacts with a light subunit from the SLC7 family to form heteromeric amino acid transporters capable of transporting 15 different amino acids across the BBB. CD98HC plays a crucial role in amino acid transport across the plasma membrane and is involved in various physiological and pathological processes, such as immune cell activation, cell adhesion, and tumor progression. CD98HC is highly enriched in brain capillaries and microvessels, located on the luminal and abluminal surfaces of brain endothelial cells, making it a suitable target to mediate hardly penetrated molecules across the BBB.

Taken together, there is an urgent need to develop new methods for the delivery of drugs across BBB.

SUMMARY OF THE INVENTION

The present disclosure provides an antibody or an antigen-binding fragment thereof which can specifically bind to CD98HC. The antibody or the antigen-binding fragment can take the therapeutic entity across the blood-brain barrier. By way of illustration, the applicant has successfully fused a GLP-1 receptor agonist with antibodies specifically targeting CD98HC. This innovative approach has resulted in a notable 20-fold improvement in BBB penetration efficiency compared to Dulaglutide. Similarly, using neurotensin which is a short peptide that could not cross BBB but can induce the hypothermia effect when entered into the brain as an example, the applicant demonstrated that CD98HC-targeting antibodies can efficiently deliver neurotensin into brain and induce a hypothermic effect in mice. Furthermore, using an anti-HEL VHH as an example, the applicant demonstrated that CD98HC-targeting antibodies can effectively deliver large recombinant proteins into the brains of both mice and non-human primates.

In one aspect, the present application provides an antibody or an antigen-binding protein thereof, which is capable of binding to CD98HC, said antigen-binding fragment has one or more characteristics selected from the group consisting of: 1) is capable of binding to CD98HC with a KD of less than about 5×10^-6M; 2) exhibits transcytosis capability; 3) does not interfere with the normal amino acid transportation function of CD98HC; and 4) is capable of mediating therapeutical molecule across the BBB.

In some embodiments, the CD98HC is a human CD98HC.

In some embodiments, the CD98HC comprises the extracellular domain (ECD) of CD98HC.

In some embodiments, the antibody is selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a multi-specific antibody.

In some embodiments, the antigen-binding fragment is selected from the group consisting of: a Fab fragment, a Fab’ fragment, a F (ab) ₂ fragment, a Fv fragment, a VHH and an ScFv.

In some embodiments, the antibody or the antigen-binding fragment thereof is capable of competing with a reference antibody for binding to said CD98HC, wherein said reference antibody comprises light chain CDR1-3 (LCDR1-3) and heavy chain CDR1-3 (HCDR1-3) , said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 80, SEQ ID NO: 15, or SEQ ID NO: 56, said LCDR2 comprises an amino acid sequence as ser forth in SEQ ID NO: 16, SEQ ID NO: 31, or SEQ ID NO: 57, said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50, said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 55.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises at least one CDR of a heavy chain variable region (VH) , wherein said VH comprises an amino acid sequence as set forth in any one of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises at least one CDR of a light chain variable region (VL) , wherein said VL comprises an amino acid sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCDR1, wherein said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 and SEQ ID NO: 27.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCDR1, wherein said HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 27, and SEQ ID NO: 53.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCDR2, wherein said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCDR2, wherein said HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 13, SEQ ID NO: 28, SEQ ID NO: 40, and SEQ ID NO: 54.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an HCDR3, wherein said HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 55.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an LCDR1, wherein said LCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 80, SEQ ID NO: 15, and SEQ ID NO: 56.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an LCDR1, wherein said LCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 42, and SEQ ID NO: 56.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an LCDR2, wherein said LCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 31, and SEQ ID NO: 57.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an LCDR3, wherein the LCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 81 or SEQ ID NO: 50.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises an LCDR3, wherein the LCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 38, or SEQ ID NO: 50.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises HCDRs 1-3 and LCDRs 1-3, wherein said HCDR1-3 and LCDR1-3 comprises the amino acid sequences selected from the group consisting of:

1) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 12, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 13, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 15, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 9;

2) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 29, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 30, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 24;

3) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 40, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 41, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 42, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 38; and

4) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 53, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 54, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 55, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 56, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 57, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) , wherein said VH comprises an amino acid sequence as set forth in any one of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a VH, wherein said VH comprises an amino acid sequence as set forth in SEQ ID NO: 97 or SEQ ID NO: 98.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a light chain variable region (VL) , wherein said VL comprises an amino acid sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 59.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a VL, wherein said VL comprises an amino acid sequence as set forth in SEQ ID NO: 98 or SEQ ID NO: 100.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a VH and a VL, wherein said VH and VL comprises amino acid sequences selected from the group consisting of:

1) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 17, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 18;

2) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 33;

3) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 43, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 44;

4) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 59;

5) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 85, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

6) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 87, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

7) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 88, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

8) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 89, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 90; and

9) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 91, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 92.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region.

In some embodiments, the heavy chain constant region comprises a human IgG constant region.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises a light chain constant region.

In another aspect, the present application provides a fusion protein, comprising the antibody or the antigen-binding fragment thereof of the present application.

In some embodiments, the fusion protein further comprises a therapeutic entity.

In some embodiments, said therapeutic entity is a functional protein.

In some embodiments, said therapeutic entity is an antibody or the antigen-binding fragment thereof.

In some embodiments, the therapeutic entity comprises a GLP-1 protein or a functional fragment thereof.

In some embodiments, the GLP-1 protein or a functional fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 75.

In some embodiments, the therapeutic entity is linked to said antibody or the antigen-binding fragment thereof directly or indirectly.

In some embodiments, the therapeutic entity is linked to said antibody or the antigen-binding fragment thereof via a linker.

In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 76 or SEQ ID NO: 84.

In some embodiments, the fusion protein comprises three polypeptide chain, wherein the first polypeptide chain comprises a light chain of the antibody or the antigen-binding fragment of the present disclosure, the second polypeptide chain comprises a heavy chain of the antibody or the antigen-binding fragment of the present disclosure, the third polypeptide chain comprises the therapeutic entity and an immunoglobulin Fc region.

In some embodiments, the polypeptide chain comprise amino acid sequences respectively selected from the group consisting of:

1) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 63, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 70;

2) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 64, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 71;

3) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 65, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 72; and

4) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 73.

In another aspect, the present application provides a protein conjugate, comprising the antibody or the antigen-binding fragment of the present application, or the fusion protein of the present application.

In another aspect, the present application provides a pharmaceutical molecule, comprising the antibody or the antigen-binding fragment of the present application.

In another aspect, the present application provides an isolated nucleic acid molecule or molecules, encoding for the antibody or the antigen-binding fragment of the present application, or the fusion protein of the present application.

In another aspect, the present application provides a vector or vectors, comprising the isolated nucleic acid molecule or molecules of the present application.

In another aspect, the present application provides a cell, comprising the isolated nucleic acid molecule or molecules or the vector or vectors of the present application.

In another aspect, the present application provides a composition, comprising the antibody or the antigen-binding fragment, the fusion protein, the protein conjugate, the isolated nucleic acid molecule or molecules, the vector or vectors, and/or the cell of the present application, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutically acceptable excipient comprises a buffer.

In another aspect, the present application provides a use of the antibody or the antigen-binding fragment of the present application in taking a therapeutic entity across BBB.

In another aspect, the present application provides a use of the fusion protein, the nucleic acid molecule or molecules, the vector or vectors, the cell, the protein conjugate, the pharmaceutical molecule, or the composition in manufacture of a medicament for preventing and/or treating a disease, wherein said medicament has neuroprotective effects.

In some embodiments, the disease comprises a neurodegenerative disease, cerebrovascular disease, mental disorder, CNS regulating metabolic or endocrine disease.

In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, and/or Amyotrophic Lateral Sclerosis, said cerebrovascular disease comprises stroke, said mental disorder comprises Depression, and/or Schizophrenia, said CNS regulating metabolic or endocrine disease comprises Obesity, and/or Pituitary dwarfism.

In another aspect, the present application provides a method for preventing and/or treating a disease, comprising administering the fusion protein, the pharmaceutical molecule, the nucleic acid molecule or molecules, the vector or vectors, the cell, the protein conjugate, or the composition to a subject in need thereof.

In another aspect, the present application provides the fusion protein, the nucleic acid molecule or molecules, the vector or vectors, the cell, the protein conjugate, the pharmaceutical molecule, or the composition for use in preventing and/or treating a disease.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

Fig. 1 illustrates the binding of recombinant chimeric CD98HC antibodies to cell surface expressed CD98HC.

Fig. 2 illustrates the antibody concentration in the lower chamber of Transwell.

Fig. 3 illustrates the inhibition effect of CD98HC chimeric antibodies on amino acid uptake.

Fig. 4 illustrates the binding of GLP-1-CD98HC antibody fusion proteins to cell surface expressed CD98HC.

Fig. 5 illustrates the protein concentration of GLP-1-CD98HC antibody fusion proteins in the lower chamber of Transwell.

Fig. 6 illustrates the exact permeability of CD98HC chimeric antibody and GLP-1-CD98HC antibody fusion protein.

Figs. 7A-7C illustrate the GLP-1 receptor activation activity of GLP-1-CD98HC antibody fusion proteins.

Fig. 8 illustrates the concentration of GLP-1-CD98HC antibody fusion proteins and Dulaglutide in brain parenchyma of human CD98HC knock-in mice at 24 hours after administration.

Fig. 9 illustrates the concentration of GLP-1-CD98HC antibody fusion proteins and Dulaglutide in brain microvessel of human CD98HC knock-in mice at 24 hours after administration.

Fig. 10 illustrates the IHC staining result of human IgG at different brain regions of human CD98HC knock-in mice.

Fig. 11 illustrates the neurotensin-CD98HC antibody fusion protein concentration in the brain homogenate of human CD98HC knock-in mice at 72 hours after administration. As shown by fold change over isotype control.

Fig. 12 illustrates the neurotensin-CD98HC antibody fusion protein concentration in the brain parenchyma of the human CD98HC knock-in mice at 24 hours and 72 hours after administration.

Fig. 13 illustrates the neurotensin-CD98HC antibody fusion protein concentration in the brain microvessel of the human CD98HC knock-in mice at 24 hours and 72 hours after administration.

Fig. 14 illustrates the neurotensin-CD98HC antibody fusion protein induced hypothermia effect in human CD98HC knock-in mice.

Fig. 15 illustrates the VHH-CD98HC antibody fusion protein concentration in the brain homogenate of human CD98HC knock-in mice at 72 hours after administration. As shown by fold change over isotype control.

Fig. 16 illustrates the VHH-CD98HC antibody fusion protein concentration in the serum of non-human primates.

Fig. 17 illustrates the VHH-CD98HC antibody fusion protein concentration in the CSF of non-human primates.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term “blood-brain barrier” or “BBB” generally refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., the CNS) which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Daltons) . The blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS and are herein collectively referred to as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.

The term “CD98HC (CD98 heavy chain) ” and “SLC3A2” can be used interchangeably, generally refers to any native CD98HC from any source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats) , etc. The term may include full length CD98HC or the fragment thereof. For example, the term may include the ECD of CD98HC. The term may also include the variants or analogues of CD98HC.

The term “antibody” , as used herein, generally refers to an immunoglobulin or an immunoglobulin-like molecule capable of specifically recognizing or binding to an antigen. An antibody may comprise a light chain (L) and a heavy chain (H) . The light chains of an antibody can be classified as к and λ light chains. The heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of an antibody are defined as IgM, IgD, IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtype) , IgA and IgE, respectively. Each heavy chain may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH) . The heavy chain constant region may comprise three domains (CH1, CH2 and CH3) . Each light chain may comprise a light chain variable region (VL) and a light chain constant region (CL) . The light chain constant region may comprise a CL domain. The VH and VL regions can also be subdivided into regions with high variability known as complementarity determining regions (CDRs) interspersed with more conserved regions known as framework regions (FRs) . Each VH and VL consists of 3 CDRs and 4 FRs arranged from N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding site, respectively. Distribution of amino acids to regions or domains follows the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term “antibody” is not limited by any antibody-producing method. For example, it includes recombinant antibodies, monoclonal antibodies, and other forms of antibodies. In some cases, an antibody of the present disclosure is an isolated antibody.

The term “antigen-binding fragment” , as used herein, generally refers to one or more fragments of a full-length antibody that retain the ability to bind the same antigen to which the antibody binds (e.g., CD98HC) and/or competes against an intact antibody for an antigen-specific binding. Antigen-binding fragment can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, the antigen-binding fragment includes Fab, Fab’ , F (ab’ ) ₂, F(ab) ₂, VHH, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., ScFv) , chimeric antibodies, diabodies, and polypeptides that comprise at least a portion of an antibody that is sufficient to confer specific antigen-binding ability to the polypeptide.

The term “binding specificity” , as used herein, generally refers to an ability of one substance to bind another substance specifically, and not substantially bind to any other substance at random. For example, one protein may bind to another protein specifically due to their specific structures. Binding specificity may be measured by, e.g., cross-competing assays or other binding assays known in the art.

The term “K_D” , as used herein, generally refers to the dissociation constant, a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex falls apart into its component molecules. The dissociation constant is the inverse of the association constant. In the specific case of antibodies (Ab) binding to antigen (Ag) , usually the term affinity constant refers to the association constant.

The term “K_on” , as used herein, generally refers to on rate constant for association of a binding protein (e.g., an antibody or an antigen-binding fragment thereof) to the antigen to form a bound complex (e.g., an antibody/antigen complex) . The term “K_on” also means “association rate constant” , or “ka” , as is used interchangeably herein. This value indicates the binding rate of a binding protein to its target antigen or the rate of complex formation between a binding protein, (e.g., an antibody) and the corresponding antigen.

The term “monoclonal antibody” , as used herein, generally refers to antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have a monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody) . Sometimes, monoclonal antibodies may also be multi specific, such as bispecific or tri specific. It has become an important tool in biochemistry, molecular biology, and medicine. Several monoclonal antibody technologies have been developed recently, such as phage display, single B cell culture, single cell amplification from various B cell populations and single plasma cell interrogation technologies.

The term “chimeric antibody” , as used herein, generally refers to an antibody in which the Variable (V) region of light and heavy chains is of mouse origin, while the Constant (C) region is of human origin. In general, the chimeric antibody may retain the specificity and affinity of the original mouse monoclonal antibody, but Human Anti Mouse Antibody (HAMA) response may be significantly reduced.

The term “humanized antibody” , as used herein, generally refers to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The amino acid sequence of a humanized antibody may be essentially identical to that of a human variant, despite the non-human origin of some of its complementarity determining region (CDR) segments responsible for the ability of the antibody to bind to its target antigen.

The term “fully human antibody” and “human antibody” are used interchangeably herein, and generally refers to an antibody that comprises a human variable region and, most preferably a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) .

The term “Fab fragment” , as used herein, generally refers to a portion (such as an antigen-binding fragment) of an immunoglobulin molecule. A Fab fragment may comprise one light chain and part of a heavy chain with a single antigen-binding site. A Fab fragment may be obtained by papain digestion of an immunoglobulin molecule. For example, a Fab fragment may be composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain may contain the paratope (the antigen-binding site) , comprising a set of the complementarity determining regions, at the amino-terminal end of the immunoglobulin molecule. The enzyme papain may be used to cleave an immunoglobulin molecule into two Fab fragments and one Fc fragment. The enzyme pepsin cleaves below the hinge region, so a F (ab') ₂ fragment and a pFc'fragment is formed. Divalent F(ab) ₂ or F (ab') ₂ fragments have two antigen binding regions that are linked by disulfide bonds. Reduction of F (ab) ₂ or F (ab') ₂ fragments produce 2 monovalent Fab or Fab'fragments, which have a free sulfhydryl group that is useful for conjugation to other molecules.

The term “Fv fragment” , as used herein, generally refers to the smallest fragment made from enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have the antigen-binding site made of the VH and VL regions, but they lack the CH1 and CL regions. The VH and VL chains may be held together in Fv fragments by non-covalent interactions.

The term “scFv” , as used herein, generally refers to a single-chain antibody fragment. An scFv may be a recombinant single chain polypeptide molecule in which VH and VL of an antibody are connected, either directly or via a peptide linker. Single chain antibodies (scFv) generally do not include portions of the Fc region of antibody, although methods are known for adding such regions to known scFv molecules if desired. See Helfrich et al., A rapid and versatile method for harnessing scFv antibody fragments with various biological functions. J Immunol Methods 237: 131-145 (2000) and de Haard et al., Creating and engineering human antibodies for immunotherapy. Advanced Drug Delivery Reviews 31: 5-31 (1998) .

The term “fusion protein” , as used herein, generally refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of a polypeptide fused directly or indirectly (e.g., via a linker) to an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide of a different origin, sequence or structure) .

The term “protein conjugate” , as used herein, generally refers to a conjugate comprising a protein (e.g., an antibody or a functional fragment thereof) conjugated to one or more additional moieties, such as cytotoxic agents, e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) , a label (e.g., a fluorescent label) and/or a radioactive isotope (i.e., a radio-conjugate) .

A number of CDR definitions are in use and are encompassed herein. The Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., ibid. ) . Chothia definition is based on the location of the structural loops (Chothia &Lesk J. (1987) Mol. Biol. 196: 901-917) . The AbM definition is a compromise between the Kabat and the Chothia definitions and is used by Oxford Molecular's AbM antibody modelling software (Martin ACR et al., (1989) PNAS USA 86:9268-9272; Martin ACR et al., (1991) Methods Enzymol. 203: 121-153; Pedersen JT et al., (1992) Immunomethods 1: 126-136; Rees AR et al., (1996) In Sternberg M.J.E. (ed. ) , Protein Structure Prediction. Oxford University Press, Oxford, 141-172) . The contact definition has been recently introduced (MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745) and is based on an analysis of the available complex structures available in the Protein Databank. The definition of the CDR by the international ImMunoGeneTics information (http: //www. imgt. org) is based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONs of all species (the international ImMunoGeneTics information Lefranc MP et al., (1999) Nucleic Acids Res. 27 (1) : 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28 (1) : 219-21; Lefranc MP (2001) Nucleic Acids Res. 29 (1) : 207-9; Lefranc MP (2003) Nucleic Acids Res. 31 (1) : 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol. 29 (3) : 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics &Proteomics, 6 (4) : 253-64) .

The term “isolated nucleic acid molecule or molecules” as used herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from its native environment, or that is artificially synthesized.

The term “vector or vectors” as used herein, generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material elements carried in the vector can be expressed in a host cell by transforming, transducing, or transfecting the host cell with the vector. A vector may contain a variety of elements that control expression, including promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. It is also possible that the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein shells, but not only these substances.

The term “cell” as used herein, generally refers to a cell that may be used to carry the vector or vectors of the present disclosure, or be used to express or produce the antibody, the antigen-binding fragment of the present disclosure. A cell of the present disclosure may be a host cell.

The terms “disease” and “disorder” may be used interchangeably herein, and generally refer to any condition that impairs the normal functioning of the body.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. For example, the subject may be human.

The term “pharmaceutically acceptable excipient” , as used herein, generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration.

The term “about” , as used herein, generally refers to an approximation to a given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, it may refer to a value that is no more than 10%above or below the value being modified by the term.

The terms “polypeptide” or “protein” , as used herein, generally refers to macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass CD98HC binding proteins, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about 5 to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 1 10, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a CD98HC-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and/or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

The term “isolated protein” (such as isolated antibody) , as used herein, generally refers to a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an “isolated protein” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50%of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.

“Antigen binding region” or “antigen-binding fragment” means a protein, or a portion of a protein, that specifically binds a specified antigen (e.g., a paratope) . For example, that portion of an antigen binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen binding protein its specificity and affinity for the antigen is referred to as “antigen binding region” . An antigen binding region typically includes one or more “complementary binding regions” ( “CDRs” ) . A “CDR” is an amino acid sequence that contributes to antigen binding specificity and affinity.

Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics” ) , chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates” ) , and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

The term “compete” , when used in the context of antigen binding proteins (e.g., antigen binding proteins or antibodies) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., CD98HC or a fragment thereof, such as the ECD thereof) . Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA) , solid phase direct or indirect enzyme immunoassay (EIA) , sandwich competition assay (see, e.g., Stahli et al, 1983, Methods in Enzymology 9: 242-253) ; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J. Immunol. 137: 3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press) ; solid phase direct label RIA using I-125 label (see, e.g., Morel et al, 1988, Molec. Immunol. 25: 7-15) ; solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176: 546-552) ; and direct labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82) . Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Usually, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%or 75%or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97%or more.

The term “antigen” , as used herein, generally refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunological functional fragment thereof) . In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that are capable of interacting with different antigen binding proteins, e.g., antibodies.

The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

The term “prevent” does not require the 100%elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) . Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) ) , which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Antibody or an antigen-binding fragment thereof

In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, which binds to CD98HC. The antibody, or the antigen-binding fragment thereof may specifically bind to CD98HC.

In some embodiments, the CD98HC may comprise CD98HC or fragment thereof. For example, the CD98HC may comprise the extracellular domain (ECD) of CD98HC. For example, the ECD of CD98HC may comprise an amino acid sequence as set forth in SEQ ID NO: 1.

The antibody, or the antigen-binding fragment thereof may bind to CD98HC with a K_D of less than about 5.0 ×10^-6M. For example, with a K_D of less than about 3 ×10^-6M, less than about 1 ×10^-6M, less than about 5 ×10^-7M, less than about 4 ×10^-7M, less than about 3 ×10^-7M, less than about 2×10^- ⁷M, less than about 1 ×10^-7M, less than about 5 ×10^-8M, less than about 4 ×10^-8M, less than about 3 ×10^-8M, less than about 2×10^-8M, less than about 1 ×10^-8M, less than about 5 ×10^-9M, less than about 4 ×10^-9M, less than about 3.5 ×10^-9M, less than about 3×10^-9M, less than about 2.8 ×10^-9M, less than about 2.7×10^-9M, less than about 2.5×10^-9M, less than about 2×10^-9M, less than about 1.5×10^-9M, less than about 1×10^-9M, less than about 8×10^-10M, less than about 5×10^-10M, less than about 5×10^-10M, less than about 4.5×10^-10M, less than about 4×10^-10M, less than about 3.5×10^-10M, less than about 3×10^-10M, less than about 2.5×10^-10M, less than about 2×10^-10M, less than about 1.5×10^-10M, less than about 1×10^-10M, less than about 1×10^-11M, less than about 1×10^-12M, or less than about 1×10^-13M, or a K_D value that is even smaller.

In some embodiments, the antibody, or the antigen-binding fragment thereof comprises one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs) . In some embodiments, the antibody, or the antigen-binding fragment thereof comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted into and/or joined to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be, or comprise, the framework of a naturally occurring antibody, or fragment or variant thereof, or can be completely synthetic in nature.

In certain embodiments, the polypeptide structure of the antibody, or the antigen-binding fragment thereof is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics” ) , chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates” ) , and portions or fragments of each, respectively. In some instances, the antibody, or the antigen-binding fragment thereof is an immunological fragment of an antibody (e.g., a Fab, a Fab’ , a F (ab’ ) ₂, or a scFv) .

Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called “complementarity determining regions” or CDRs. The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g., CD98HC) . From N-terminal to C-terminal, naturally occurring VH and VL both typically conform with the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) , or Chothia &Lesk, 1987, J. MoL Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883.

Various VH and VL are provided herein. In some embodiments, each of these variable regions can be attached to a heavy and light chain constant region to form a complete antibody heavy and light chain, respectively. Further, each of the so generated heavy and light chain sequences can be combined to form a complete antibody structure.

Specific examples of some of the variable regions of the light (VL) and heavy (VH) chains of the antibodies are provided and their corresponding amino acid sequences are summarized in Table 1 below.

Table 1

Each of the exemplary variable heavy chains listed in Table 1 can be combined with any of the exemplary variable light chains shown in Table 1 to form an antibody. Table 1 shows exemplary light and heavy chain pairings found in several of the antibodies disclosed herein. In some instances, the antibodies include at least one variable heavy chain and one variable light chain from those listed in Table 1. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody or antigen-binding fragment thereof can include a heavy chain and a light chain, two heavy chains, or two light chains. In some embodiments, the antibody or the antigen-binding fragment thereof comprises (and/or consists of) 1, 2, and/or 3 heavy and/or light CDRs from at least one of the sequences listed in Table 1. In some embodiments, all 6 CDRs (CDRl-3 from the light (LCDR1, LCDR2, LCDR3) and CDR 1-3 from the heavy (HCDR1, HCDR2, and HCDR3) ) are part of the antibody or the antigen-binding fragment thereof. In some embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the antibody or the antigen-binding fragment thereof. In some embodiments, one heavy and one light CDR from the CDRs in the sequences in Table 1 is included in the antibody or the antigen-binding fragment thereof. In some embodiments, additional sections are also included in the antibody or the antigen-binding fragment thereof. Optional light chain variable sequences (including CDR1, CDR2, and CDR3) can be selected from the following: SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92. Optional heavy chain variable sequences (including CDR1, CDR2 and CDR3) can be selected from the following: SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises (and/or consists of) LCDR1 and LCDR3 from at least one of the sequences listed in Table 1.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises (and/or consists of) HCDR1 and HCDR3 from at least one of the sequences listed in Table 1.

In some embodiments, the antibody or the antigen-binding fragment thereof comprises (and/or consists of) LCDR1, LCDR3, HCDR1 and HCDR3 from at least one of the sequences listed in Table 1.

Examples of the CDRs of the antibodies shown in Table 1 are listed below (determined according to the IMGT method) in Table 2.

Table 2 (IMGT)

As described herein, the CD98HC antibody or the antigen-binding fragment thereof can comprise a humanized antibody and/or part thereof. An important practical application of such a strategy is the “humanization” of the mouse humoral immune system. In certain embodiments, a humanized antibody is substantially non-immunogenic in humans. In certain embodiments, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived.

In certain embodiments, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity are identified.

In certain embodiments, modification of an antibody by methods known in the art is typically designed to achieve increased binding affinity for a target and/or to reduce immunogenicity of the antibody in the recipient. In certain embodiments, humanized antibodies are modified to eliminate glycosylation sites in order to increase affinity of the antibody for its cognate antigen. See, e.g., Co et al., MoI. Immunol., 30: 1361-1367 (1993) . In certain embodiments, techniques such as “reshaping” , “hyperchimerization” or “veneering/resurfacing” are used to produce humanized antibodies. See, e.g., Vaswami et al., Annals of Allergy, Asthma, &Immunol. 81: 105 (1998) ; Roguska et al, Prot. Engineer., 9:895-904 (1996) ; and U.S. Patent No. 6,072,035. In certain such embodiments, such techniques typically reduce antibody immunogenicity by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibodies.

In certain instances, humanizing antibodies results in a loss of antigen binding capacity. In certain embodiments, humanized antibodies are “back mutated” . In certain such embodiments, the humanized antibody is mutated to include one or more of the amino acid residues found in the donor antibody. See, e.g., Saldanha et ai, MoI Immunol 36: 709-19 (1999) .

In certain embodiments the complementarity determining regions (CDRs) of the light and VH of an antibody to CD98HC can be grafted to framework regions (FRs) from the same, or another, species. In certain embodiments, the CDRs of the VH and VL of an antibody to CD98HC can be grafted to consensus human FRs. To create consensus human FRs, in certain embodiments, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. In certain embodiments, the FRs of an antibody heavy chain or light chain are replaced with the FRs from a different heavy chain or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody are not replaced, while the rest of the FR amino acids are replaced. Rare amino acids are specific amino acids that are in positions in which they are not usually found in FRs. In certain embodiments, the grafted variable regions from an antibody can be used with a constant region that is different from the constant region of an antibody. In certain embodiments, the grafted variable regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in U.S. Patent Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101, and in Jones et al, Nature, 321: 522-525 (1986) ; Riechmann et al, Nature, 332: 323-327 (1988) ; Verhoeyen et al, Science, 239: 1534-1536 (1988) , Winter, FEBS Letts., 430: 92-94 (1998) , which are hereby incorporated by reference for any purpose.

As described herein, an antibody or its antigen-binding fragment that binds to CD98HC can comprise a human (i.e., fully human) antibody and/or part thereof. In certain embodiments, nucleotide sequences encoding, and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions are provided. In certain embodiments, sequences corresponding to complementarity determining regions (CDRs) , specifically from CDR1 through CDR3, are provided. According to certain embodiments, a hybridoma cell line expressing such an immunoglobulin molecule is provided. According to certain embodiments, a hybridoma cell line expressing such a monoclonal antibody is provided.

One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in WO 98/24893, U.S. Patent No. 5,545,807, EP 546073, and EP 546073.

In certain embodiments, one can use constant regions from species other than human along with the human variable region (s) .

The ability to clone and reconstruct megabase sized human loci in yeast artificial chromosomes (YACs) and to introduce them into the mouse germline provides an approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents could provide insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.

Humanized antibodies are those antibodies that, while initially starting off containing antibody amino acid sequences that are not human, have had at least some of these nonhuman antibody amino acid sequences replaced with human antibody sequences. This is in contrast with human antibodies, in which the antibody is encoded (or capable of being encoded) by genes possessed by a human.

Other antibodies that are provided are variants of the antibody or the antigen-binding fragment thereof listed above formed by combination or subparts of the variable heavy and variable light chains shown in Table 1 and comprise variable light and/or variable heavy chains that each have at least 50%, 50%-60%, 60%-70%, 70%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-97%, 97%-99%, or above 99%identity to the amino acid sequences of the sequences in Table 1 (either the entire sequence or a subpart of the sequence, e.g., one or more CDR) . In some instances, such antibodies include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two identical light chains and two identical heavy chains (or subparts thereof) . In some embodiments, sequence comparison can be used in order to identify sections of the antibodies that can be modified by observing those variations that impact binding and those variations that do not appear to impact binding. For example, by comparing similar sequences, one can identify those sections (e.g., particular amino acids) that can be modified and how they can be modified while still retaining (or improving) the functionality of the antibody or the antigen-binding fragment thereof. In some embodiments, variants of the antibody include consensus groups and sequences between alternatives, as described above. The CDRs shown in Table 2 are defined based upon the IMGT method (based on sequence variability, see, e.g., Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242, Kabat et al., (1991) ) .

In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 90%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91. In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 95%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91. In certain embodiments, the antibody or its antigen-binding fragment comprises a heavy chain comprising a variable region comprising an amino acid sequence at least 99%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.

In some embodiments, the antibody or its antigen-binding fragment comprises a sequence that is at least 90%, 90-95%, and/or 95-99%identical to one or more CDRs from the CDRs in at least one of sequences of SEQ ID NO: 77, SEQ ID NO: 27, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 55. In some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99%identical to the above sequences) is present.

In certain embodiments, the antibody or its antigen-binding fragment comprises a light chain comprising a variable region comprising an amino acid sequence at least 90%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92. In certain embodiments, the antibody or its antigen-binding fragment comprises a light chain comprising a variable region comprising an amino acid sequence at least 95%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92. In certain embodiments, an antigen-binding fragment thereof comprises a light chain comprising a variable region comprising an amino acid sequence at least 99%identical to an amino acid sequence selected from at least one of the sequences of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92.

In some embodiments, the antibody or its antigen-binding fragment comprises a sequence that is at least 90%, 90-95%, and/or 95-99%identical to one or more CDRs from the CDRs in at least one of sequences of SEQ ID NO: 80, SEQ ID NO: 15, SEQ ID NO: 56, SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 57, SEQ ID NO: 81, and SEQ ID NO: 50. In some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99%identical to the above sequences) is present.

In light of the present disclosure, a skilled artisan will be able to determine suitable variants of the antibody or the antigen-binding fragment thereof as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar antibodies. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

In certain embodiments, antigen-binding fragment thereof variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

According to certain embodiments, amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physicochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain (s) forming intermolecular contacts) . In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence or disrupt other types of secondary structure that characterizes the parent sequence) . Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984) ) ; Introduction to Protein Structure (C. Branden &J. Tooze, eds., Garland Publishing, New York, N. Y. (1991) ) ; and Thornton et al, Nature, 354: 105 (1991) , which are each incorporated herein by reference.

In some embodiments, the variants are variants of the nucleic acid sequences of the antibody or the antigen-binding fragment thereof disclosed herein. One of skill in the art will appreciate that the above discussion can be used for identifying, evaluating, and/creating antibodies and protein variants and also for nucleic acid sequences that can encode for those protein variants. Thus, nucleic acid sequences encoding for those protein variants (as well as nucleic acid sequences that encode for the antibody or the antigen-binding fragment thereof in Table 1, but are different from those explicitly disclosed herein) are contemplated.

In certain embodiments, the antibody, or the antigen-binding fragment thereof are produced by immunization with an antigen (e.g., CD98HC) . In certain embodiments, antibodies can be produced by immunization with full-length CD98HC, a soluble form of CD98HC, the extracellular domain alone, a splice variant form of CD98HC, or a fragment thereof. In certain embodiments, the antibodies of the present disclosure can be polyclonal or monoclonal, and/or can be recombinant antibodies. In certain embodiments, antibodies of the present disclosure are human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (see, for example, PCT Application No. WO 93/12227) .

In certain embodiments, certain strategies can be employed to manipulate inherent properties of an antibody, such as the affinity of an antibody for its target. Such strategies include, but are not limited to, the use of site-specific or random mutagenesis of the polynucleotide molecule encoding an antibody to generate an antibody variant. In certain embodiments, such generation is followed by screening for antibody variants that exhibit the desired change, e.g., increased or decreased affinity.

In certain embodiments, the amino acid residues targeted in mutagenic strategies are those in the CDRs. In certain embodiments, amino acids in the framework regions of the variable domains are targeted. In certain embodiments, such framework regions have been shown to contribute to the target binding properties of certain antibodies. See, e.g., Hudson, Curr. Opin. Biotech., 9: 395-402 (1999) and references therein.

As will be appreciated, antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference) . The transformation procedure used depends upon the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2) , human epithelial kidney 293 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with constitutive CD98HC binding properties.

In certain embodiments, the antibody, or the antigen-binding fragment thereof comprises an immunoglobulin molecule of at least one of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype. In certain embodiments, the antibody, or the antigen-binding fragment thereof comprises a human kappa light chain and/or a human heavy chain. In certain embodiments, the heavy chain is of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, the antibody, or the antigen-binding fragment thereof has been cloned for expression in mammalian cells. In certain embodiments, the antibody, or the antigen-binding fragment thereof comprises a constant region other than any of the constant regions of the IgGl, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype.

Epitopes to which anti-CD98HC antibodies bind are provided. In some embodiments, epitopes that are bound by the presently disclosed antibodies are particularly useful. In some embodiments, the antibody, or the antigen-binding fragment thereof that binds to any of the epitopes that are bound by the antibodies described herein are useful. In some embodiments, the epitopes bound by any of the antibodies listed in Table 1 are especially useful. In some embodiments, the epitope is on the extracellular domain of CD98HC.

In some embodiments, the antibody, or the antigen-binding fragment thereof disclosed herein bind specifically to CD98HC ECD. In some embodiments, the domain (s) /region (s) containing residues that are in contact with or are buried by an antibody can be identified by mutating specific residues in CD98HC (e.g., a wild-type antigen) and determining whether the antigen-binding fragment thereof can bind the mutated or variant CD98HC protein. By making a number of individual mutations, residues that play a direct role in binding or that are in sufficiently close proximity to the antibody such that a mutation can affect binding between the antigen-binding fragment thereof and antigen can be identified. From a knowledge of these amino acids, the domain (s) or region (s) of the antigen that contain residues in contact with the antigen-binding fragment thereof or covered by the antibody can be elucidated. Such a domain can include the binding epitope of an antigen-binding fragment thereof. One specific example of this general approach utilizes an arginine/glutamic acid scanning protocol (see, e.g., Nanevicz, T., et ai, 1995, J. Biol. Chem., 270: 37, 21619-21625 and Zupnick, A., et al, 2006, J.Biol. Chem., 28_L: 29, 20464-20473) .

In some cases, the antibody or the antigen-binding fragment thereof may capable of competing with a reference antibody for binding to the CD98HC, wherein the reference antibody comprises light chain CDR1-3 (LCDR1-3) and heavy chain CDR1-3 (HCDR1-3) , the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 80, SEQ ID NO: 15, or SEQ ID NO: 56, the LCDR2 comprises an amino acid sequence as ser forth in SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 31 or SEQ ID NO: 57, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50, the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 55.

In some cases, the antibody or the antigen-binding fragment thereof may capable of competing with a reference antibody for binding to the CD98HC, wherein the reference antibody comprises VH and VL, the VH comprises the amino acid sequence as set forth in SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 91, and the VL comprises the amino acid sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, or SEQ ID NO: 92.

In some cases, the antibody or the antigen-binding fragment thereof may comprise HCDR3, and the HCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 55.

In some cases, the antibody or the antigen-binding fragment thereof may comprise HCDR2, and the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79. For example, the HCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 13, SEQ ID NO: 28, SEQ ID NO: 40, and SEQ ID NO: 54.

In some cases, the antibody or the antigen-binding fragment thereof may comprise HCDR1, and the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27. For example, the HCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 27, and SEQ ID NO: 53.

In some cases, the antibody or the antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79, and the HCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 55. For example, the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 12, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 13, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 14. For example, the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 28, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 29. For example, the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 27, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 40, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 41. For example, the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 53, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 54, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 55.

In some cases, the antibody or the antigen-binding fragment thereof may comprise LCDR3, and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50. For example, the LCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 38, or SEQ ID NO: 50.

In some cases, the antibody or the antigen-binding fragment thereof may comprise LCDR2, and the LCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 31, and SEQ ID NO: 57.

In some cases, the antibody or the antigen-binding fragment thereof may comprise LCDR1, and the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 80, SEQ ID NO: 15, and SEQ ID NO: 56. For example, the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 42, and SEQ ID NO: 56.

In some cases, the antibody or the antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 80, SEQ ID NO: 15, and SEQ ID NO: 56, the LCDR2 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 31, and SEQ ID NO: 57, and the LCDR3 may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 81 or SEQ ID NO: 50. For example, the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 15, the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 16, and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 9. For example, the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 30, the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 31, and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 24. For example, the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 42, the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 31, and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 38. For example, the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO: 56, the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO: 57, and the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO: 50.

In some cases the antibody or the antigen-binding fragment thereof may comprise LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, and the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 80, SEQ ID NO: 15, or SEQ ID NO: 56, the LCDR2 comprises an amino acid sequence as ser forth in SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 31 or SEQ ID NO: 57, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50, the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 55.

In some cases, the antibody or the antigen-binding fragment thereof may comprise LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, and the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 may comprise the amino acid sequence selected from the group consisting of:

1) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 12, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 13, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 15, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16, the said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 9;

2) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 29, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 30, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 24;

3) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 40, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 41, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 42, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 38;

4) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 53, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 54, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 55, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 56, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 57, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.

In some cases, the antibody or the antigen-binding fragment may comprise VH and VL, the VH may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, and SEQ ID NO: 58, the VL may comprises an amino acid sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, and SEQ ID NO: 59. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 17, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 43, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 44. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 58, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 59. For example, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 85, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 87, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 86. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 88, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 86. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 89, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 90. For example, the VH comprise an amino acid sequence as set forth in SEQ ID NO: 91, and the VL comprise an amino acid sequence as set forth in SEQ ID NO: 92.

In the present application, the antibody, or the antigen-binding fragment thereof may comprise a light chain constant region. The light chain constant region may comprise a human Igκ constant region or a human Igλ constant region. In some embodiments, the light chain constant region may comprise a human Igκ constant region. In some embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 69.

In the present application, the antibody, or the antigen-binding fragment thereof may comprise a heavy chain constant region. The heavy chain constant region may comprise a human IgG constant region (such as a human IgG1, IgG2, or IgG4 constant region) . In some embodiments, the heavy chain constant region comprises a human IgG4 constant region. In some embodiments, the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

The antibody or antigen-binding fragment may also encompass a homologue or a variant thereof having substantially the same function/property thereto. In some cases, the homologue or variant may have an amino acid sequence different from that of the antibody or antigen-binding fragment of the present disclosure by at least one amino acid. For example, the homologue or variant may be a polypeptide different from the antibody or the antigen-binding fragment thereof by an addition, deletion or substitution of one or more amino acid, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids. In some cases, the homologue or variant may be a polypeptide having a sequence identity of at least 80%with the antibody or antigen-binding fragment thereof. For example, the homologue or variant may be a polypeptide having a sequence identity of 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or higher) to the antibody or antigen-binding fragment thereof.

The term “percent (%) sequence identity, ” as used in the context of polypeptide sequences identified herein, generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical with the amino acid residues or nucleotides of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid/nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide/polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide/polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Fusion protein

In another aspect, the present application provides a fusion protein, comprising the antibody or the antigen-binding fragment thereof of the present disclosure.

In the present application, the fusion protein may comprise the antibody or the antigen-binding fragment thereof of the present disclosure, and a therapeutic entity.

In the present application, the antibody or the antigen-binding fragment thereof may take the therapeutic entity across the BBB. For example, the therapeutic entity may comprise any therapeutic entity which needs to be taken across the BBB. For example, the therapeutic entity may be a functional protein which needs to be taken across the BBB. For example, the therapeutic entity may be any kind of protein or its functional fragment, variants thereof. For example, the therapeutic entity may be an antibody or the antigen-binding fragment thereof.

In the present application, the therapeutic entity may be linked to the antibody or the antigen-binding fragment thereof directly or indirectly. For example, the therapeutic entity may be linked to the antibody or the antigen-binding fragment thereof via a linker. For example, the linker may be a peptide linker. For example, the linker may be a flexible linker. For example, the linker may comprise an amino acid sequence as set forth in SEQ ID NO: 76 or SEQ ID NO: 84.

In some embodiments, the fusion protein may comprise a CD98HC antibody heavy chain, a CD98HC antibody light chain, a therapeutic entity linked to a Fc region. For example, the CD98HC heavy chain may comprise a Fc-knob. For example, the Fc-knob may comprise an amino acid sequence as set forth in SEQ ID NO: 60. For example, the Fc region which is linked to the therapeutic entity may comprise a Fc-hole. For example, the Fc-hole may comprise an amino acid sequence as set forth in SEQ ID NO: 61.

In some embodiments, the fusion protein may comprise two arms, one arm comprises CD98HC antigen-binding fragments, and the other arm comprises a therapeutic entity.

In some embodiments, the fusion protein may comprise three polypeptide chain, wherein the first polypeptide chain comprises a light chain of the antibody or the antigen-binding fragment of the present disclosure, the second polypeptide chain comprises a heavy chain of the antibody or the antigen-binding fragment of the present disclosure, the third polypeptide chain comprises the therapeutic entity and an immunoglobulin Fc region.

In some embodiments, the therapeutic entity may comprise a GLP-1 receptor agonist. In some cases, the therapeutic entity may comprise a GLP-1 protein or a functional fragment thereof. For example, the GLP-1 protein or a functional fragment thereof may be original GLP-1 (7-37) . For example, the GLP-1 protein or a functional fragment thereof may be GLP-1 mutant, which is the precursor of Ligraglutide and Semeglutide. For example, the GLP-1 protein or a functional fragment thereof may be Exenatide or Lixisenatide. For example, the GLP-1 protein or a functional fragment thereof may comprise the amino acid sequence as set forth in any one of SEQ ID NO: 75, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105.

In some embodiments, the fusion protein may comprise heavy chain 1, heavy chain 2 and light chain. For example, the heavy chain 1 may be 20A3E2-Knob, the heavy chain 2 may be GLP-1-Hole, and the light chain may be Chi20A3E2 light chain. For example, the heavy chain 1 may be 43F8C6-Knob, the heavy chain2 may be GLP-1-Hole, and the light chain may be Chi43F8C6 light chain. For example, the heavy chain 1 may be 44D5D6-Knob, the heavy chain 2 may be GLP-1-Hole, and the light chain may be Chi44D5D6 light chain. For example, the heavy chain 1 may be 15F8F8-Knob, the heavy chain 2 may be GLP-1-Hole, and the light chain may be Chi15F8F8 light chain.

In some embodiments, the fusion protein may comprise three polypeptide chain, and the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 63, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 70.

In some embodiments, the fusion protein may comprise three polypeptide chain, and the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 64, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 71.

In some embodiments, the fusion protein may comprise three polypeptide chain, and the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 65, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 72.

In some embodiments, the fusion protein may comprise three polypeptide chain, and the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 73.

In another aspect, the present application provides a protein conjugate, comprising the antibody or the antigen-binding fragment of the present disclosure.

In another aspect, the present application provides a pharmaceutical molecule, comprising the antibody or the antigen-binding fragment of the present disclosure. For example, the pharmaceutical molecule may further comprise a protein, a polypeptide, a nucleotide molecule, or a chemical drug, etc.

Nucleic acid, Vector, Cell, Preparation method, and Composition

In another aspect, the present disclosure provides isolated nucleic acid or molecules, encoding for the antibody or the antigen-binding fragment thereof, or the fusion protein.

The isolated nucleic acids may comprise one or more nucleic acid molecules, with each encoding for at least a part of the antibody of the present disclosure or an antigen-binding fragment thereof. For example, the isolated nucleic acids may comprise at least two nucleic acid molecules, with one encoding for the antibody heavy chain or a fragment thereof, and one encoding for the antibody light chain or a fragment thereof. In some cases, the isolated nucleic acids may encode for a fusion protein.

The isolated nucleic acid or isolated nucleic acids may be synthesized using recombinant techniques well known in the art. For example, the isolated nucleic acid or isolated nucleic acids may be synthesized with an automated DNA synthesizer. Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987) . Briefly, the subject nucleic acids may be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which may be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and RT-PCR.

Direct chemical synthesis of nucleic acids typically involves sequential addition of 3’ -blocked and 5’ -blocked nucleotide monomers to the terminal 5’ -hydroxyl group of a growing nucleotide polymer chain, wherein each addition is affected by nucleophilic attack of the terminal 5’ -hydroxyl group of the growing chain on the 3’ -position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. See for example, Matteuci et al., Tet. Lett. 521: 719 (1980) ; U.S. Pat. No. 4,500,707 to Caruthers et al. ; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al.

In another aspect, the present disclosure provides a vector or vectors, comprising the isolated nucleic acid molecule or molecules.

The vector may be any linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector may include a retrovirus, an adenovirus and an adeno-associated virus. In some cases, the vector is an expression vector, e.g., a phage display vector.

An expression vector may be suitable for use in particular types of host cells and not others. For example, the expression vector can be introduced into the host organism, which is then monitored for viability and expression of any genes/polynucleotides contained in the vector.

The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or otherwise identifying host cells that carry the expression vector. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

The subject vectors can be introduced into a host cell stably or transiently by a variety of established techniques. For example, one method involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, for example calcium phosphate, may also be used following a similar procedure. In addition, electroporation (that is, the application of current to increase the permeability of cells to nucleic acids) may be used. Other examples of transformation methods include microinjection, DEAE dextran mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers may also be employed to transfect the host cells.

In another aspect, the present disclosure provides a cell (e.g., an isolated cell, such as a host cell) , comprising the isolated nucleic acid molecule or molecules of the present disclosure or the vector or vectors of the present disclosure.

The cell may express the antibody, or the antigen-binding fragment thereof of the present disclosure, or the fusion protein of the present disclosure. The cell may be a eukaryotic cell or a prokaryotic cell. An appropriate cell may be transformed or transfected with the nucleic acid (s) or vector (s) of the present disclosure and utilized for the expression and/or secretion of the antibody, the antigen-binding fragment thereof, or the fusion protein. For example, the cell may be E. coli cells, other bacterial host cells, yeast cells, or various higher eukaryotic cells.

In another aspect, the present disclosure provides a method for producing the antibody or the antigen-binding fragment thereof, or the fusion protein of the present disclosure, comprising culturing the cell of the present disclosure under conditions enabling expression of the antibody, the antigen-binding fragment thereof, or the fusion protein.

The method optionally may further comprise harvesting the antibody or the antigen-binding fragment thereof, or the fusion protein of the present disclosure.

In another aspect, the present disclosure provides a composition, comprising the antibody or the antigen-binding fragment thereof, the fusion protein, the protein conjugate, the isolated nucleic acid molecule or molecules, the vector or vectors, and/or the cell of the present disclosure, and optionally a pharmaceutically acceptable excipient.

In some cases, the pharmaceutically acceptable excipient may comprise a buffer. In some cases, the pharmaceutically acceptable excipient may comprise an amino acid.

In some embodiments, the pH of the pharmaceutical composition may be 1-13, for example, the pH may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

In some cases, the pharmaceutical composition may further comprise an effective amount of an additional therapeutically active component, for example, an additional therapeutically active component for treating a disease or a disorder. Each of the active components may be present in the pharmaceutical composition in a pharmaceutically active amount. In the composition, the antibody, the fragment thereof of the present application may or may not be associated with the additional active component.

Described below are non-limiting exemplary pharmaceutical compositions and methods for preparing the same. The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. In some cases, the pharmaceutical composition may be a liquid pharmaceutical composition.

Pharmaceutical compositions of the disclosure can be presented as discrete dosage forms, with each dosage containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid. Such dosage forms can be prepared by any of the methods known to a skilled person, for example, it may include the step of bringing the active ingredient into association with the carrier, which constitutes one or more other ingredients. In general, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The antibody, the antigen-binding fragment thereof, or the fusion protein, the protein conjugate, the pharmaceutical molecule of the present disclosure can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration.

The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and/or mixtures thereof.

The pharmaceutical compositions of the present disclosure may comprise a therapeutically effective amount of the active agent (e.g., the antibody, the antigen-binding fragment thereof, the fusion protein, or the pharmaceutical molecule of the present disclosure) . A therapeutically effective amount is an amount of the subject pharmaceutical composition capable of preventing and/or curing (at least partially) a condition or disorder (e.g., a chronic pain) and/or any complications thereof in a subject suffering from or having a risk of developing said condition or disorder. The specific amount/concentration of the active agent comprised may vary according to the method of administration and the need of a patient, and can be determined based on e.g., volume, viscosity, and/or body weight of a patient etc. It shall be understood that these specific doses may be conveniently adjusted by a skilled person in the art (e.g., a doctor or a pharmacist) based on conditions of a specific patient, formulation, and/or disease.

Medical Use and Methods of Treatment

In another aspect, the present application provides a method of taking a therapeutic entity across BBB, comprising using the antibody or the antigen-binding fragment of the present disclosure.

In another aspect, the present application provides a use of the antibody or the antigen-binding fragment of the present disclosure.

In another aspect, the present application provides a use of the antibody or the antigen-binding fragment thereof, the fusion protein, the protein conjugate, the pharmaceutical molecule, the isolated nucleic acid molecule or molecules, the vector or vectors, the cell, and/or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for preventing and/or treating a disease or disorder.

In another aspect, the present application provides a method for preventing and/or treating a disease or disorder, comprising administering the antibody or the antigen-binding fragment thereof, the fusion protein, the protein conjugate, the pharmaceutical molecule, the isolated nucleic acid molecule or molecules, the vector or vectors, the cell, and/or the pharmaceutical composition to a subject in need thereof.

In another aspect, the present application provides the antibody or the antigen-binding fragment thereof, the fusion protein, the protein conjugate, the pharmaceutical molecule, the isolated nucleic acid molecule or molecules, the vector or vectors, the cell, and/or the pharmaceutical composition for use in preventing and/or treating a disease or disorder.

In some embodiments, the disease or disorder is determined by the kind of the therapeutic entity which the antibody or the antigen-binding fragment linked to.

For example, the fusion protein comprising the antibody or the antigen-binding fragment thereof may be used for preventing and/or treating a neurodegenerative disease, cerebrovascular disease, mental disorder, CNS regulating metabolic or endocrine disease. For example, the neurodegenerative disease may comprise Alzheimer's disease, Parkinson's disease, and/or Amyotrophic Lateral Sclerosis. For example, the Cerebrovascular disease may be stroke. For example, the Mental disorder may be Depression, Schizophrenia. For example, the CNS regulating metabolic or endocrine disease may be Obesity, Pituitary dwarfism.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Example 1 CD98HC-ECD recombinant protein preparation

To construct the expression plasmid, the gene of human CD98HC extracellular domain (ECD) consisting of the amino acid (AA) 206 -630, cynomolgus monkey CD98HC ECD consisting of the amino acid (AA) 206 -630 and mouse CD98HC extracellular domain consisting of the amino acid (AA) 100 -526 were synthesized and subcloned into pcDNA3.4 vector with N-terminus fused to an 8xhis tag. Subsequently, the plasmid was transiently transfected into 293F or CHO-K1 cells and cultured for 5-7 days in a CO₂ incubator. Following incubation, the supernatant was harvested and purified through one-step immobilized metal affinity chromatography. The purified protein was used for immunization and antibody screening.

The sequence of CD98HC ECDs is listed below.

Example 2 Hybridoma antibody generation

6 mice in each group (3 BALB/c mice and 3 C57bl/6 mice) were subjected to immunization according to the schedule outlined in Table 2. In brief. Human and mouse CD98HC ECD proteins were mixed with either Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA) in a 1: 1 ratio, creating a stable oil-in-water emulsion. Subsequently, the emulsion was injected into the mice via subcutaneous (s.c. ) or intraperitoneal (i.p) routes. Following immunization, serum samples were collected to assess the antibody response (titer) through indirect ELISA and fluorescence-activated cell sorting (FACS) assay. Four days after the final immunization, the splenocytes were isolated and fused with SP2/0 myeloma cells using electro-cell fusion techniques. This process induced cell fusion and formed hybridomas.

Table 3 Immunization Schedule

The binding activity of hybridoma supernatants or purified antibodies to human, mouse and monkey CD98HC ECD was assessed using ELISA. Antibody clones that demonstrated positive results in the ELISA were subsequently validated through a fluorescence-activated cell sorting (FACS) assay. For the FACS assay, human CD98HC overexpressed CHO-K1 cells were cultured and harvested. Then 50 uL of the cell suspension was distributed into 96-well plate at a density of 100,000 cells per well and incubated with 50 μL hybridoma supernatant cells for 40 min at 4 ℃. Following washing the cells, 100 μL of diluted Alexa 647 AffiniPure^TM Goat Anti-Mouse IgG, Fcγ fragment specific (Jackson, 115-605-071) was added to each well and incubated for 30 min at 4 ℃. Subsequently, the cells were washed three times and resuspended into 50 μL assay buffer. The fluorescence signals from the cells were analyzed using the flow cytometer.

Hybridoma fusion clones that exhibited positive results in both the ELISA and FACS assay were chosen for further cloning of the antibody variable region gene.

Example 3 Cloning of antibody variable region gene

Hybridoma cells were cultured in a 10 cm dish and harvested during the logarithmic growth phase. Total cellular RNA was extracted using Trizol (Invitrogen, 15596-018) according to the manufacturer’s instructions. The RNA was resuspended in nuclease-free water. The concentration of RNA was determined by measuring the absorbance at 260 nm. For the generation of cDNA templates, 4 ug of each RNA sample was reverse transcribed using HiFiScript cDNA Synthesis Kit (CWBIO, CW2569) . Subsequently, the variable heavy (VH) or variable light (VL) fragments were amplified through degenerate primers based on previously reported sequences (Wang, Z et al, J Immunol Methods. 2000; 233 (1-2) : 167-177) and subjected to sequencing. The obtained sequencing results were analyzed using IMGT/V-QUEST web program (Hybridoma cells were cultured in a 10 cm dish and harvested during the logarithmic growth phase. Total cellular RNA was extracted using Trizol (Invitrogen, 15596-018) according to the manufacturer’s instructions. The RNA was resuspended in nuclease-free water. The concentration of RNA was determined by measuring the absorbance at 260 nm.For the generation of cDNA templates, 4 ug of each RNA sample was reverse transcribed using HiFiScript cDNA Synthesis Kit (CWBIO, CW2569) . Subsequently, the variable heavy (VH) or variable light (VL) fragments were amplified through degenerate primers based on previously reported sequences (Wang, Z et al, J Immunol Methods. 2000; 233 (1-2) : 167-177) and subjected to sequencing. The obtained sequencing results were analyzed using IMGT/V-QUEST web program (http: //www. imgt. org/IMGT_vquest/analysis) . Antibody complement-determinant regions (CDRs) were identified by utilizing the ANARCI tools (Dunbar J et al, Bioinformatics. 2016; 32 (2) : 298-300) , based on the Kabat, Enhanced Chothia, and IMGT systems, Hybridoma cells were cultured in a 10 cm dish and harvested during the logarithmic growth phase. Total cellular RNA was extracted using Trizol (Invitrogen, 15596-018) according to the manufacturer’s instructions. The RNA was resuspended in nuclease-free water. The concentration of RNA was determined by measuring the absorbance at 260 nm.For the generation of cDNA templates, 4 ug of each RNA sample was reverse transcribed using HiFiScript cDNA Synthesis Kit (CWBIO, CW2569) . Subsequently, the variable heavy (VH) or variable light (VL) fragments were amplified through degenerate primers based on previously reported sequences (Wang, Z et al, J Immunol Methods. 2000; 233 (1-2) : 167-177) and subjected to sequencing. The obtained sequencing results were analyzed using IMGT/V-QUEST web program (http: //www. imgt. org/IMGT_vquest/analysis) . Antibody complement-determinant regions (CDRs) were identified by utilizing the ANARCI tools (Dunbar J et al, Bioinformatics. 2016; 32 (2) : 298-300) , based on the Kabat, Enhanced Chothia, and IMGT systems, http: //www. imgt. org/IMGT_vquest/analysis) . Antibody complement-determinant regions (CDRs) were identified by utilizing the ANARCI tools (Dunbar J et al, Bioinformatics. 2016; 32 (2) : 298-300) , based on the Kabat, Enhanced Chothia, and IMGT systems.

The heavy and light chain variable region sequences of murine mAb are as follows:

Chi43F8C6 VH SEQ ID NO: 17

Chi43F8C6 VL SEQ ID NO: 18

Chi20A3E2 VH SEQ ID NO: 32

Chi20A3E2 VL SEQ ID NO: 33

Chi44D5D6 VH SEQ ID NO: 43

Chi44D5D6 VL SEQ ID NO: 44

Chi15F8F8 VH SEQ ID NO: 58

Chi15F8F8 VL SEQ ID NO: 59

Example 4 Expression and purification of monoclonal antibodies

To generate antibodies from hybridoma cells, the positive hybridomas were initially adapted to Hybridoma-SFM medium. Subsequently, the cells were cultured in this medium at 5%CO₂ and 37 ℃ in an incubator for 4 days to produce antibodies.

For transient transfection of HEK293 cells to express recombinant antibodies, the VH and VL genes were subcloned into the pCDNA3.4 vector along with human IgG4 and Kappa constant region respectively. The subcloned constructs were verified by DNA sequencing. A total of 5 μg of plasmids (3 μg for the light chain and 2 μg for the heavy chain) were diluted in 1 ml of I Reduced Serum Medium (GIBCO, 31985-070) and transfected into human 293F cells using 20 μg of PEI. The cells were cultured in serum-free medium (Shanghai opmbiosciences, OPM-293CD03) at 5%CO₂, 37 ℃, and 125 rpm/min. The culture medium was replenished on day 1 and cultured for another 4 days.

The antibodies were purified using Protein A MagBeads (GenScript, L00273) according to the manufacturer's instructions. Briefly, the clarified supernatant was incubated with the MagBeads at room temperature with gentle rocking for 2 hours. Subsequently, the MagBeads were washed three times with PBS buffer. The purified antibodies were eluted from the beads using Elution Buffer (0.1 M glycine, pH 3.0) and neutralized using Neutralization Buffer (1 M Tris, pH 8.5) .

Example 5 Measuring the dissociation constant of CD98HC antibodies with human CD98HC ECD.

To measure the dissociation constant of CD98HC antibodies with human CD98HC ECD, we employed the bio-layer interferometry (BLI) method using the Octet Red384 instrument. Briefly, Fc-coated biosensor AHC tips (ForteBio, 18-5060) were first pre-wet in an assay buffer consisting of PBS with 0.1%w/v bovine serum albumin and 0.05%Tween-20 (assay buffer) for at least 10 minutes in the pre-wetting plate. The purified antibodies, at a concentration of 100 nM in assay buffer, were then immobilized onto AHC biosensors to achieve capture levels of approximately 1 nm.

A series of 2-fold dilutions (ranging from 3.125 nM to 200 nM) of the human CD98HC ECD was used to determine the association and dissociation rates. All measurements were referenced against a control sensor exposed solely to the assay buffer to correct for baseline drift. By applying a 1: 1 interaction model (fitting local, full) through the ForteBio data analysis software, association rate constants (K_on) , dissociation rate constants (K_off) , and K_D values for each antibody were calculated. The dissociation constant of chimeric antibodies was listed in Table 4.

Table 4 The binding affinities of the selected murine antibodies to human CD98HC

Example 6 The binding activity of CD98HC antibodies with human CD98HC overexpressed CHO-K1

The binding activity of CD98HC antibodies with cell surface-expressed CD98HC was assessed using fluorescence-activated cell sorting (FACS) assay on human CD98HC overexpressed CHO-K1 cells. Briefly, human CD98HC overexpressed CHO-K1 cells were cultured and harvested. Then the cell suspension was distributed into wells of the 96-well assay plate at a density of 200,000 cells per well. 100 μL antibody solution at various concentrations was added to each well and incubated for 45 min at 4 ℃. Following washing the cells, 100 μL diluted Goat anti-human IgG PE (SouthernBiotech, 2040-09) was added to each well and incubated for 30 min at 4 ℃. Subsequently, the cells were washed three times and resuspended into 150 μL assay buffer. The fluorescence signals were analyzed using the flow cytometer. The raw data was analyzed using a Four-Parameter Fit model in GraphPad Prism software. As shown in Fig. 1, 4 candidate antibodies all specifically bind with cell surface-expressed CD98HC.

Example 7 Evaluation of antibody transcytosis capability on iPSC-induced brain endothelial cells

To construct the in vitro blood-brain barrier (BBB) model, astrocytes (Procell, CP-R137) were digested and replated at a density of 100,000 cells/ml in 1 ml of medium in a 24-well plate. Prior to plating, the 24-well plate was coated with 50 μg/ml PDL and left for 1-2 hours. After 24 hours, the complete medium was exchanged, and the cells were further cultured for 24 hours to establish the in vitro BBB model.

For the differentiation of brain endothelial cells from iPSCs, Matrigel-coated culture dishes were prepared on day 0. iPSCs (Help) were digested and replated at a density of 200,000 cells/ml in 2 ml of mTeSR1 medium (Stem Cell, 85850) supplemented with a final concentration of 10 μM Y- 27632 (MCE, HY10071) . On day 1 and day 2, the old medium was replaced with mTeSR1 medium supplemented with 20 ng/ml bFGF. On day 3, the old medium was replaced with UM medium (DMEM/Ham’s F-12 containing 20%knockout Serum Replacement, 1%minimum essential medium Non-Essential Amino Acid (NEAA) , 1%L-glutamine, and 0.836 μmol/L beta-mercaptoethanol) . After 48 hours, the UM medium was replaced once, and the cells were maintained for 6 days. On day 9, the old medium was replaced with EM medium (Human Endothelial-Serum-free Medium (SFM) (Gibco, 11111044) , supplemented with 1%Human Platelet Lysate (PTS) (Biological Industries, PLTGOLD50R) and 20 ng/ml basic fibroblast growth factor (bFGF) with 20 μmol/L RA and 1.3 μmol/L hydrocortisone) . After another 48 hours, the EM medium was replaced once, and the cells were maintained for 13 days. On day 21, the endothelial cells were digested with 0.25%trypsin for 8-10 minutes, resuspended in EM medium with 10 μM Y-27632 after centrifugation, and then passed through a 100 μm cell strainer to obtain a single-cell suspension. The filtered cells were seeded at a density of 500,000 cells per well into Transwell inserts with a pore size of 0.47 μm. Prior to plating, the Transwell inserts were coated overnight with a mixture of collagen IV (400 μg/ml final concentration) and fibronectin (100 μg/ml final concentration) . After overnight culture, the wells without leakage underwent a 90%replacement of EM medium and were transferred to a 24-well plate coated with astrocytes for co-culture. The values of transendothelial electrical resistance (TEER) were measured at 24 hours and 48 hours post-co-culture. Wells with a TEER value higher than 600 Ω·cm² at 48 hours were selected for the next step of the antibody screening experiment.

For the antibody transport assay, the inserts were washed three times in preheated HBSS at 37℃ for 5-10 minutes each time. Next, 500 μl of the candidate antibody (10 nM) and negative antibody (10 μg/ml) solutions, diluted with preheated transport buffer (DMEM + 4500 mg/L D-glucose, 4 mmol/L L-glutamine, and 25 mmol/L HEPES, without sodium pyruvate and phenol red, pH 7.4) , were added to the inserts. The inserts were placed into a 24-well plate containing 1 ml of preheated transport buffer and incubated at 37℃ for 5-10 minutes. The Transwell was then placed on a 37℃ shaker at 20 rpm for 60 minutes. At 30 minutes, 100 μl of solution was collected from the upper and lower chambers and immediately replaced with 100 μl of preheated transport buffer. At 60 minutes, all the solution was collected from the upper and lower chambers. The concentration of the candidate antibody and the negative antibody was quantitated using ELISA. The exact permeability (P_exact, nm/s) of the Transwell was calculated using the following formula:

VD: Volume in the lower chamber; A: Surface area of the insert; VR: Volume in the upper chamber; t: Transport time (s) ; CR (t) : Concentration in the lower chamber at 30/60 min; CD (t) : Concentration in the upper chamber at 30/60 min.

If the permeability of the negative control is less than 50 nM/s, it indicates that the monolayer of brain endothelial cells was not damaged during the antibody transport process. In such cases, the acquired data can be used to evaluate the transcytosis capability of the antibody.

As shown in Fig. 2, compared with negative control antibody, all anti-CD98HC antibodies exhibited robust transcytosis capability.

Example 8 The impact of CD98HC Monoclonal Antibody on Amino Acid Transport of endothelial cells

To assess the effect of CD98HC monoclonal antibodies on amino acid transport in brain endothelial cells, iPSC-induced human endothelial cells were resuspended in ECM medium (ECM +5%FBS + 1%PS + 1x ECGS, Scien Cell, Catalog No. 1001) and plated at a density of 200,000 cells/ml in a 96-well black/clear bottom cell culture plate pre-coated with Matrigel matrix (37 ℃, 30 minutes) , with 100 μL per well. The plate was then incubated overnight at 37 ℃/5%CO₂.

Next, the cells were washed three times with pre-warmed HBSS. 200nM CD98HC antibodies and 1mM BCH diluted with pre-warmed HBSS were added to the wells and incubated at 37℃/5%CO₂ for 30 minutes. After the incubation, the supernatant was replaced with 150 μL of pre-warmed BPA uptake solution (Tongren Chemical, Catalog No. UP04) containing 1mM BCH, 200nM CD98HC antibodies or HBSS (Blank cell) and the cells were incubated at 37 ℃/5%CO₂ for 5 minutes, followed by three washes with pre-warmed HBSS. Subsequently, 150 μL of pre-warmed working solution (37 ℃) was added to each well, and the cells were incubated at 37℃/5%CO₂ for 5 minutes. After the incubation, the fluorescence intensity was directly measured using a fluorescence plate reader (Ex/Em=360/460 nm) .

As shown in Fig. 3, compared with BCH, which significantly interferes with the amino acid uptake, all anti-CD98HC antibodies do not interfere with the normal amino acid transportation function of endothelial cells.

Example 9 Construction of GLP-1-CD98HC antibody fusion proteins

To generate GLP-1-CD98HC antibody fusion proteins the variable region of anti-CD98HC antibodies heavy chain was combined with human IgG4 Fc with mutation S228P, F234A, L235A, F296Y, R409K, and K439E (SEQ ID NO: 60, Eu numbering) . This resulted in the production of Chi20A3E2-Knob, Chi43F8C6-Knob, Chi44D5D6-Knob, and Chi15F8F8-Knob (the heavy chain 1) . Additionally, the antibody B578, as described in patent WO2021205361A1, was also combined with human IgG4 Fc with mutation S228P, F234A, L235A, F296Y, R409K, and K439E (SEQ ID NO: 60, Eu numbering) to obtain the heavy chain 1 of B578-Knob. Furthermore, the GLP-1 (SEQ ID NO: 75) peptide with the linker (SEQ ID NO: 76) at the C terminal was fused with human IgG4 Hinge and CH2-CH3 region with mutation S228P, F234A, L235A, F296Y, E356K, R409K and H435R (SEQ ID NO: 61, Eu numbering) , resulting in the production of GLP-1-Hole (the heavy chain 2) . The two heavy chains, along with the associated anti-CD98 light chain were co-expressed in the HEK293 and CHO-K1 cells and purified through Protein A chromatography followed by Size Exclusion Chromatography or Ion Exchange Chromatography, depending on the protein purity (Table 5) .This process allowed us to obtain GLP-1-CD98HC antibody fusion proteins.

Table 5 The composition of GLP-1-CD98HC antibody fusion proteins

The CD98HC binding affinity of GLP-1-CD98HC antibody fusion proteins was confirmed by FACS assay as shown in Fig. 4

The in vitro transcytosis efficacy of GLP-1-CD98HC antibody fusion proteins on human brain endothelial cells was confirmed by in vitro transcytosis assay as described in Example 7. As shown in Fig. 5, all GLP-1-CD98HC antibody fusion proteins exhibited robust transcytosis efficacy in vitro. We also calculated the P_exact, and the result (Fig. 6) showed that the P_exact of Chi43F8C6 and Chi43F8C6-GLP-1 are significantly higher than the negative control antibody.

Example 10: Measurement of GLP-1-CD98HC antibody fusion protein activation activity

To evaluate the activation activity of GLP-1-CD98HC antibody fusion proteins, GLP1R/CRE-LUC HEK293 cells (Cobioer, Cat No. CBP71117) expressing the human GLP-1 receptor and a cyclic AMP (cAMP) -responsive CRE4-luciferase system were used.

GLP1R/CRE-LUC HEK293 cells were seeded into a PDL-coated 96-well cell culture plate, with 100 μL of cells per well. The plate was then incubated overnight. Next, Dulaglutide and GLP-1-CD98HC antibody fusion proteins were diluted with DMEM with 10%FBS and transferred to the respective wells, followed by 5 hours incubation @37℃/5%CO₂. After incubation, the luciferase assay reagent substrate solution (RhinoBio, RA-GL07) was added to each well and incubated at room temperature for 5 minutes. The luminescent signal was then detected using a luminescence detection instrument.

The EC₅₀ and the dose-response curve were generated using Prism GraphPad software with a four-parameter logic regression model.

As shown in Fig. 7, GLP-1-CD98HC antibody fusion proteins exhibited similar GLP-1 receptor activation activity compared with Dulaglutide.

Example 11 The GLP-1-CD98HC antibody fusion proteins rain penetration study

Human CD98HC knock-in (KI) mice (C57BL/6N strain) were employed to evaluate the ability of GLP-1-CD98HC antibody fusion proteins to penetrate the brain. In these mice, the endogenous extracellular region of mouse Slc3a2 (amino acids 100 to 526) was replaced with the corresponding extracellular domain of human Slc3a2 (amino acids 206 to 630) . The animals were housed in a controlled environment within a barrier system, adhering to the animal facility's Standard Operating Procedures (SOP) .

GLP-1-CD98HC antibody fusion proteins and Dulaglutide were diluted to the desired experimental concentrations using 1X PBS and administered intravenously. At the experimental endpoint, the animals were euthanized according to the experimental requirements and subsequently perfused with physiological saline. Blood and tissue samples were collected based on the experimental design. The collected tissues were either weighed and stored at -80℃ or fixed with 10%neutral buffered formalin at a volume at least ten times greater than the tissue volume, intended for subsequent research purposes.

To isolate the brain parenchyma and microvessels, the brains of the mice were homogenized using a Tissue Homogenizer. Briefly, 1 part (based on brain tissue weight) was added to 2/3 parts (based on buffer volume) of Homogenization Buffer A (containing 10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl₂, 1 mM MgSO₄·7H₂O, 1 mM NaH₂PO₄·7H₂O, and 10 mM D-glucose) . The mixture was homogenized for 8-10 rotations. Next, 3/4 parts of Homogenization Buffer B (Homogenization Buffer A supplemented with 26%Dextran) were added, followed by homogenization for 3 rotations. The supernatant and pellet were separated after centrifugation at 5200 x g for 15 minutes. The supernatant, representing brain parenchyma, was further processed by adding Triton X-100 to a final concentration of 1%and PMSR to a final concentration of 1x. This mixture was then centrifuged at 11,000 rpm for 15 minutes. The pellet, which consisted of brain microvessels, was resuspended by adding 5 ml of Homogenization Buffer A, 5 ml of Homogenization Buffer B, and 100 μL of Triton X-100, followed by centrifugation at 11,000 rpm for 15 minutes.

The concentration of GLP-1-CD98HC antibody fusion proteins in the brain parenchyma and microvessels was quantified using the Human IgG Total ELISA Kit (Thermo BMS2091TEM) . To measure the concentration of Dulaglutide in the serum, brain parenchyma, and microvessels, Dulaglutide was captured using Anti-Human IgG (Fc Specific) (Sigma, I2136-1ML) and detected using biotin-labeled GLP-1 Monoclonal Antibody (Thermo Fisher, ABS 033-10B-005) .

For Immunohistochemical (IHC) staining, the frozen mouse brain was embedded in the optimal cutting temperature (OCT) compound and cut into 7 μm tissue sections. The brain sections were subsequently fixed in acetone for 10 minutes, followed by a 30-minute incubation at room temperature with Goat Anti-Human IgG Fc (HRP) (Abcam, ab97225) . DAB staining was performed for 5 minutes, followed by a 20-second hematoxylin staining procedure.

As shown in Fig. 8 and Fig. 9, a notable enhancement in the concentration of all GLP-1-CD98HC antibody fusion proteins within the brain parenchyma and microvessels was observed when compared to Dulaglutide. Specifically, the Chi43F8C6-GLP-1 exhibited a concentration 20-fold higher than Dulaglutide. These findings were further supported by IHC staining, which revealed enhanced penetration of GLP-1-CD98HC antibody fusion proteins into the brain parenchyma, while Dulaglutide demonstrated limited penetration (Fig. 10) .

Example 12: The PK&PD study of neurotensin-CD98HC antibody fusion protein in human CD98HC knock-in mice

To further demonstrate the capability of Chi43F8C6 to deliver a peptide into the mouse brain. Neurotensin (8-13) , a short peptide that could not cross BBB but can induce the hypothermia effect when entered into the brain, was fused with Chi43F8C6 to produce neurotensin-CD98HC antibody fusion protein. The PK and neurotensin-CD98HC antibody fusion protein-induced hypothermia effect was evaluated in human CD98HC KI mice.

Fusion protein preparation: As described in Example 9, the gene of the heavy chain of Chi43F8C6 with mutation S228P, F234A, L235A, F296Y, R409K, and K439E (SEQ ID NO: 60, Eu numbering) was synthesized and cloned into pCDNA3.4 to generate Chi43F8C6-Knob (the heavy chain 1, SEQ ID NO: 64) . Furthermore, the neurotensin (8-13) (SEQ ID NO: 83) peptide with the linker (SEQ ID NO: 84) was fused with the C terminal of human IgG4 with mutation S228P, F234A, L235A, F296Y, E356K, R409K and H435R (SEQ ID NO: 85, Eu numbering) , resulting in the production of NT-hole (the heavy chain 2, SEQ ID NO: 82) . The two heavy chains along with the associated Chi43F8C6 light chain (SEQ ID NO: 71) were co-expressed in the HEK293 or CHO-K1 cells and purified through Protein A chromatography followed by Size Exclusion Chromatography or Ion Exchange Chromatography, depending on the protein purity. This process allowed us to obtain neurotensin-CD98HC antibody fusion protein.

The PK study: The animals were housed in a controlled environment within a barrier system, adhering to the animal facility's Standard Operating Procedures (SOP) . The antibodies were diluted to the desired experimental concentrations using 1X PBS and administered intravenously at a dosage of 100 nmol/kg, 3 or 4 animals included in each group. At 24 and 72 hours after administration, the animals were euthanized according to the experimental design and subsequently perfused with physiological saline. Blood and tissue samples were collected based on the experimental design.

To prepare the brain homogenate, the collected brain tissues were rinsed with ice-cold saline first and then mixed with lysis buffer (NP-40 (Beyotime, P0013F) containing 1x Protease Inhibitor Cocktail (Beyotime, P1006-1) ) at a ratio of 1 mg brain tissue to 5 μL lysis buffer. Next, the mixture was homogenized using a tissue homogenizer, followed by centrifugation at 14,000 rpm, 4℃ for 20 minutes to obtain supernatant. To measure the concentration of antibodies in the brain homogenate, antibodies were captured using Anti-Human IgG (Fab specific) (Sigma, I5260) and detected with Goat Anti-Human IgG Fc (Biotin) preadsorbed (Abcam, ab98618) and Streptavidin HRP (BD, 554066) .

The protein concentration in microvessel and parenchyma was determined as described in Example 11.

The brain concentration of Chi43F8C6-NT is 6.7 times higher than that of isotype control-NT at 24 hours after administration and 9.4 times higher at 72 hours after administration (Figure 11) . In microvessel (Figure 12) and parenchyma (Figure 13) , a similar trend was observed.

Hypothermia effect evaluation: The fusion proteins were diluted to the desired experimental concentrations using 1X PBS and administered intravenously at the dose of 10-100 nmol/kg. 1 x PBS, neurotensin served as the control. The core temperature of animals was measured using an anus thermometer (Lab Animal Technology Develop Co., LAT-212) . Briefly, the animal was fixed, and the metal part of the anal thermometer probe was lubricated by petroleum jelly and submerged into the anus. The reading body temperature was recorded when the reading was stable and lasted more than 5 seconds. The basal core temperature was measured at 15-30 min before administration. After administration, the core temperature was monitored every 30 minutes until 6 hours and 24 hours. The data was analyzed in GraphPad prism and the statistical significance was determined by the two-way ANOVA followed by Dunnett’s multiple comparison, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 compared with the PBS group.

As shown in Figure 14, PBS, neurotensin and isotype control-NT cannot induce the body temperature decrease in mice. In contrast, Chi43F8C6-NT significantly decreased the body temperature in mice in a concentration-dependent manner.

Example 13: Humanization of CD98HC antibodies

Chi43F8C6 and Chi44D5D6 were selected for humanization due to their high brain penetration efficacy. Humanization of murine antibodies was carried out using CDR grafting approach. Briefly, the parental (murine antibody) variable region (VH and VL) frameworks were replaced with those of the selected human germline V and J genes of VH and VL. The germline genes were selected based on the homology between the parental antibody and the germline V and J genes. Human HC germline genes IGHV3-33*03 and IGHJ4*01 were selected as FR donors for the humanization of Chi443F8C6 VH; Human LC germline genes IGKV1-39*01 and IGKJ2*01 were selected as FR donors for the humanization of Chi43F8C6 VL. Three humanized antibodies hu43F8C6-V6、 hu43F8C6-V7 and Hu43F8C6-V8 were generated. Human HC germline genes IGHV3-71*01 and IGHJ4*01 were selected as FR donors for the humanization of Chi44D5D6 VH; Human LC germline genes IGKV4-1*01 and IGKJ2*01 were selected as FR donors for the humanization of Chi44D5D6 VL. Two humanized antibodies Hu44D5D6-V1 and hu44D5D6-V2 were generated.

The corresponding encoding nucleic acid sequences of humanized antibodies were synthesized and subcloned into a pCDNA3.4 vector. All recombinant antibodies were expressed and purified as described in Example 4. The affinity of the humanized antibodies was determined using a BIAcoreT200^TM SPR system. The humanized antibodies were immobilized on a protein A chip and human CD98HC ECD dilutions in 1 x HBS-EP+ buffer were injected over the antibody-bound surface at a flow rate of 30 μl/min. By applying a 1: 1 interaction model (fitting local, full) through the Biacore data analysis software, association rate constants (K_on) , dissociation rate constants (K_off) , and K_D values for each antibody were calculated. The results are summarized below:

Table 6 The binding affinities of the humanized antibodies

Example 14: The PK study of VHH-CD98HC antibody fusion protein in human CD98HC knock-in mice

To demonstrate the capability of CD98HC antibodies to deliver recombinant protein into the mouse brain. An anti-hel egg white lysozyme (HEL) single domain antibody (VHH) was fused with Chi43F8C6 and Hu43F8C6-V8 to produce VHH-CD98HC antibody fusion protein. The PK of the VHH-CD98HC antibody fusion proteins was evaluated in human CD98HC KI mice.

Fusion protein preparation: As described in Example 9, the gene of the heavy chain of anti-Chi43F8C6, Hu43F8C6-V8 with mutation S228P, F234A, L235A, F296Y, R409K, and K439E (SEQ ID NO: 60, Eu numbering) was synthesized and cloned into pCDNA3.4 to generate Chi43F8C6-Hole and HHu43F8C7-V8-Hole (the heavy chain 2, SEQ ID NO: 93 and SEQ ID NO: 94) . Furthermore, the anti-HEL VHH (SEQ ID NO: 95) was fused to the N terminal of human IgG4 with mutation S228P, F234A, L235A, F296Y, E356K, R409K and H435R (SEQ ID NO: 61, Eu numbering) , resulting in the production of the heavy chain 1. The two heavy chains along with the associated light chain were co-expressed in the HEK293 or CHO-K1 cells and purified through Protein A chromatography followed by Size Exclusion Chromatography or Ion Exchange Chromatography, depending on the protein purity. This process allowed us to obtain VHH-CD98HC antibody fusion protein.

The PK study: The PK study was performed as described in Example 12. The concentration of fusion protein in brain homogenates was determined by a sandwich ELISA. In brief, the fusion proteins in brain homogenates were captured by Lysozyme from chicken egg white (MCE, HY-B2237) coated on the plate and detected using mouse anti-human IgG4 pFc’ [HP6023] (Abcam, ab99817) .

As shown in Fig. 15, The brain concentration of Chi43F8C6-VHH is 14 times and Hu43F8C6-V8-VHH is 5.7 times higher than that of isotype control-VHH at 72 hours after administration.

Example 15: The PK study of VHH-CD98HC antibody fusion protein in non-human primate

The brain penetration capability of Chi43F8C6 and humanized antibodies was further evaluated in cynomolgus monkeys. Briefly, 3 test articles, Chi43F8C6-VHH, Hu43F8C6-V8-VHH and isotype control-VHH were administered via intravenous infusion at a dosage of 30 mg/kg. Each group includes 2 animals. The serum was collected at pre-dose, 0.083h, 2h, 6h, 24h, 48h, 72 D6 (120h) , D8(168h) , D15 (336h) , D22 (504h) , D29 (672h) post-administration. Additionally, cerebrospinal Fluid (CSF) was collected at 24h, 72h, D6 (120 h) , D8 (168h) , D15 (336h) , D22 (504h) , D29 (672h) post-administration.

The concentration of VHH-CD98HC antibody fusion protein in serum and CSF was determined by a sandwich ELISA. In brief, the fusion proteins in serum and CSF were captured by Lysozyme from chicken egg white (MCE, HY-B2237) coated on the plate and detected using mouse anti-human IgG4 pFc’ [HP6023] (Abcam, ab99817) .

The serum PK of VHH-CD98HC antibody fusion proteins in cynomolgus monkeys is shown in Figure 16. In CSF, the concentration of Chi43F8C6-VHH and Hu43F8C6-V8-VHH are significantly higher than isotype control-VHH, the Cmax of Chi43F8C6-VHH and Hu43F8C6-V8-VHH are 6 times of isotype control-VHH (Fig. 17) .

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody or an antigen-binding fragment thereof, which is capable of binding to CD98HC, said antigen-binding fragment has one or more characteristics selected from the group consisting of:

1) is capable of binding to CD98HC with a KD of less than about 5×10^-6M;

2) exhibits transcytosis capability;

3) does not interfere with the normal amino acid transportation function of CD98HC;

said antibody or the antigen-binding fragment is capable of mediating therapeutical molecules across the BBB (blood-brain barrier) .
The antibody or the antigen-binding fragment of claim 1, wherein said CD98HC is a human CD98HC.
The antibody or the antigen-binding fragment of any one of claims 1-2, wherein said CD98HC comprises the extracellular domain (ECD) of CD98HC.
The antibody or the antigen-binding fragment of any one of claims 1-3, wherein said antibody is selected from the group consisting of: a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody and a multi-specific antibody.
The antibody or the antigen-binding fragment of any one of claims 1-4, wherein said antigen-binding fragment is selected from the group consisting of: a Fab fragment, a Fab’ fragment, a F (ab) ₂ fragment, a Fv fragment, a VHH and an scFv.
The antibody or the antigen-binding fragment of any one of claims 1-5, which is capable of competing with a reference antibody for binding to said CD98HC, wherein said reference antibody comprises light chain CDR1-3 (LCDR1-3) and heavy chain CDR1-3 (HCDR1-3) , said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 80, SEQ ID NO: 15, or SEQ ID NO: 56, said LCDR2 comprises an amino acid sequence as ser forth in SEQ ID NO: 16, SEQ ID NO: 31, or SEQ ID NO: 57, said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50, said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 55.
The antibody or the antigen-binding fragment of any one of claims 1-6, which comprises at least one CDR of a heavy chain variable region (VH) , wherein said VH comprises an amino acid sequence as set forth in any one of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.
The antibody or the antigen-binding fragment of any one of claims 1-7, which comprises at least one CDR of a light chain variable region (VL) , wherein said VL comprises an amino acid sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92.
The antibody or the antigen-binding fragment of any one of claims 1-8, which comprises an HCDR1, wherein said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 77 or SEQ ID NO: 27.
The antibody or the antigen-binding fragment of any one of claims 1-9, which comprises an HCDR1, wherein said HCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 27, and SEQ ID NO: 53.
The antibody or the antigen-binding fragment of any one of claims 1-10, which comprises an HCDR2, wherein said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 78 or SEQ ID NO: 79.
The antibody or the antigen-binding fragment of any one of claims 1-11, which comprises an HCDR2, wherein said HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 13, SEQ ID NO: 28, SEQ ID NO: 40, and SEQ ID NO: 54.
The antibody or the antigen-binding fragment of any one of claims 1-12, which comprises an HCDR3, wherein said HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 55.
The antibody or the antigen-binding fragment of any one of claims 1-13, which comprises an LCDR1, wherein said LCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 80, SEQ ID NO: 15, and SEQ ID NO: 56.
The antibody or the antigen-binding fragment of any one of claims 1-14, which comprises an LCDR1, wherein said LCDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 42, and SEQ ID NO: 56.
The antibody or the antigen-binding fragment of any one of claims 1-15, which comprises an LCDR2, wherein said LCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 31, and SEQ ID NO: 57.
The antibody or the antigen-binding fragment of any one of claims 1-16, which comprises an LCDR3, wherein said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 81 or SEQ ID NO: 50.
The antibody or the antigen-binding fragment of any one of claims 1-17, which comprises an LCDR3, wherein said LCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9, SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 50.
The antibody or the antigen-binding fragment of any one of claims 1-18, which comprises HCDRs 1-3 and LCDRs 1-3, wherein said HCDR1-3 and LCDR1-3 comprises the amino acid sequences selected from the group consisting of:

1) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 12, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 13, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 14, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 15, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 9;

2) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 29, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 30, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 24;

3) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 40, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 41, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 42, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 38; and

4) said HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 53, said HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 54, said HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 55, said LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 56, said LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 57, and said LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 50.
The antibody or the antigen-binding fragment of any one of claims 1-19, which comprises a heavy chain variable region (VH) , wherein said VH comprises an amino acid sequence as set forth in any one of SEQ ID NO: 17, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 91.
The antibody or the antigen-binding fragment of any one of claims 1-19, which comprises a VH, wherein said VH comprises an amino acid sequence as set forth in SEQ ID NO: 97 or SEQ ID NO: 98.
The antibody or the antigen-binding fragment of any one of claims 1-21, which comprises a light chain variable region (VL) , wherein said VL comprises an amino acid sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 59, SEQ ID NO: 86, SEQ ID NO: 90, and SEQ ID NO: 92.
The antibody or the antigen-binding fragment of any one of claims 1-21, which comprises a VL, wherein said VL comprises an amino acid sequence as set forth in SEQ ID NO: 98 or SEQ ID NO: 100.
The antibody or the antigen-binding fragment of any one of claims 1-23, which comprises a VH and a VL, wherein said VH and VL comprises amino acid sequences selected from the group consisting of:

1) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 17, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 18;

2) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 32, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 33;

3) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 43, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 44; and

4) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 58, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 59;

5) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 85, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

6) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 87, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

7) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 88, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 86;

8) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 89, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 90;

9) said VH comprises an amino acid sequence as set forth in SEQ ID NO: 91, and said VL comprises an amino acid sequence as set forth in SEQ ID NO: 92.
The antibody or the antigen-binding fragment of any one of claims 1-24, which comprises a heavy chain constant region.
The antibody or the antigen-binding fragment of claim 25, wherein said heavy chain constant region comprises a human IgG constant region.
The antibody or the antigen-binding fragment of any one of claims 1-26, which comprises a light chain constant region.
A fusion protein, comprising the antibody or the antigen-binding fragment of any one of claims 1-27.
The fusion protein of claim 28, which further comprises a therapeutic entity.
The fusion protein of any one of claims 28-29, wherein said therapeutic entity is a functional protein.
The fusion protein of any one of claims 28-30, wherein said therapeutic entity is an antibody or the antigen-binding fragment thereof.
The fusion protein of any one of claims 28-31, wherein said therapeutic entity is linked to said antibody or the antigen-binding fragment thereof directly or indirectly.
The fusion protein of claim 32, wherein the said therapeutic entity is linked to said antibody or the antigen-binding fragment thereof via a linker.
The fusion protein of claim 33, wherein said linker comprises an amino acid sequence as set forth in SEQ ID NO: 76 or SEQ ID NO: 84.
The fusion protein of any one of claims 28-34, comprising three polypeptide chain, wherein the first polypeptide chain comprises a light chain of the antibody or the antigen-binding fragment of any one of claims 1-27, the second polypeptide chain comprises a heavy chain of the antibody or the antigen-binding fragment of any one of claims 1-27, the third polypeptide chain comprises the therapeutic entity and an immunoglobulin Fc region.
The fusion protein of claim 35, wherein said therapeutic entity comprises a GLP-1 protein or a functional fragment thereof.
The fusion protein of claim 36, wherein said GLP-1 protein or a functional fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 75.
The fusion protein of any one of claims 36-37, wherein said polypeptide chains comprise amino acid sequences respectively selected from the group consisting of:

1) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 63, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 70;

2) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 64, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 71;

3) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 65, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 72; and

4) said first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 66, said second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 68, and said third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 73.
A protein conjugate, comprising the antibody or the antigen-binding fragment of any one of claims 1-27, or the fusion protein of any one of claims 28-38.
A pharmaceutical molecule, comprising the antibody or the antigen-binding fragment of any one of claims 1-27, or the fusion protein of any one of claims 28-38.
Isolated nucleic acid molecule or molecules, encoding for the antibody or the antigen-binding fragment of any one of claims 1-27, or the fusion protein of any one of claims 28-38.
Vector or vectors, comprising the isolated nucleic acid molecule or molecules of claim 41.
A cell, comprising the isolated nucleic acid molecule or molecules of claim 41, or the vector or vectors of claim 42.
A pharmaceutical composition, comprising the antibody or the antigen-binding fragment of any one of claims 1-27, the fusion protein of any one of claims 28-38, the protein conjugate of claim 39, the pharmaceutical molecule of claim 40, the isolated nucleic acid molecule or molecules of claim 41, the vector or vectors of claim 42, and/or the cell of claim 43, and optionally a pharmaceutically acceptable excipient.
Use of the antibody or the antigen-binding fragment of any one of claims 1-27, in taking a therapeutic entity across BBB.
Use of the fusion protein of any one of claims 28-38, the nucleic acid molecule or molecules of claim 41, the vector or vectors of claim 42, the cell of claim 43, the protein conjugate of claim 39, the pharmaceutical molecule of claim 40, or the pharmaceutical composition of claim 44 in manufacture of a medicament for preventing and/or treating a disease, wherein said medicament has neuroprotective effects.
The use of claim 46, wherein said disease comprises a neurodegenerative disease, cerebrovascular disease, mental disorder, CNS regulating metabolic or endocrine disease.
The use of any one of claims 46-47, wherein said neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, and/or Amyotrophic Lateral Sclerosis, said cerebrovascular disease comprises stroke, said mental disorder comprises Depression, and/or Schizophrenia, said CNS regulating metabolic or endocrine disease comprises Obesity, and/or Pituitary dwarfism.
A method for preventing and/or treating a disease, comprising administering the fusion protein any one of claims 28-38, the nucleic acid molecule or molecules of claim 41, the vector or vectors of claim 42, the cell of claim 43, the protein conjugate of claim 39, the pharmaceutical molecule of claim 40, or the pharmaceutical composition of claim 44 to a subject in need thereof.
The method of claim 49, wherein said disease comprises a neurodegenerative disease, cerebrovascular disease, mental disorder, CNS regulating metabolic or endocrine disease.
The method of any one of claims 49-50, wherein said neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, and/or Amyotrophic Lateral Sclerosis, said cerebrovascular disease comprises stroke, said mental disorder comprises Depression, and/or Schizophrenia, said CNS regulating metabolic or endocrine disease comprises Obesity, and/or Pituitary dwarfism.
The fusion protein any one of claims 28-38, the nucleic acid molecule or molecules of claim 41, the vector or vectors of claim 42, the cell of claim 43, the protein conjugate of claim 39, the pharmaceutical molecule of claim 40, or the pharmaceutical composition of claim 44, for use in preventing and/or treating a disease.
The fusion protein, the nucleic acid molecule or molecules, the vector or vectors, the cell, the protein conjugate, the pharmaceutical molecule or the pharmaceutical composition of claim 52, wherein said disease comprises a neurodegenerative disease, cerebrovascular disease, mental disorder, CNS regulating metabolic or endocrine disease.
The fusion protein, the nucleic acid molecule or molecules, the vector or vectors, the cell, the protein conjugate, or the pharmaceutical composition of any one of claims 52-53, wherein said neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, and/or Amyotrophic Lateral Sclerosis, said cerebrovascular disease comprises stroke, said mental disorder comprises Depression, and/or Schizophrenia, said CNS regulating metabolic or endocrine disease comprises Obesity, and/or Pituitary dwarfism.