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WO2024067810A1 - Anticorps anti-gpr183 et leurs utilisations - Google Patents

Anticorps anti-gpr183 et leurs utilisations Download PDF

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Publication number
WO2024067810A1
WO2024067810A1 PCT/CN2023/122615 CN2023122615W WO2024067810A1 WO 2024067810 A1 WO2024067810 A1 WO 2024067810A1 CN 2023122615 W CN2023122615 W CN 2023122615W WO 2024067810 A1 WO2024067810 A1 WO 2024067810A1
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seq
antibody
antigen
amino acid
binding fragment
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Guohuang FAN
Jianfei Wang
Na Wang
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Nanjing Immunophage Biotech Co Ltd
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Nanjing Immunophage Biotech Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • novel anti-GPR183 antibodies or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a vector and a host cell including the nucleic acid, a method for producing the antibody or the antigen-binding fragment thereof, a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof as an active ingredient, and the use of the antibody in treating the diseases mediated by GPR183.
  • GPR183 also known as EBI2
  • EBI2 is a member of the rhodopsin-like subfamily of 7TM receptors, which forms homodimers and heterodimers when it associates with CXCR5.
  • GPR183 is expressed in B cells, a subset of T cells, monocytes and macrophages, natural killer cells, dendritic cells (DCs) , neutrophils, eosinophils, platelets, and osteoclasts, and is highly upregulated by Epstein–Barr virus infection.
  • EBI2 expression has been characterized in astrocytes and the early developmental stages of immune cells, including hematopoietic stem and progenitor cells and thymocytes.
  • GPR183 ligands were identified as oxysterols which arise from the oxidation of cholesterol. 7 ⁇ ,25-dihydroxycholesterol (7 ⁇ , 25-diHC) has the strongest affinity to GPR183, 7 ⁇ , 27-diHC shows the second highest affinity whereas other oxysterols (monohydroxylated oxysterols 25-HC and 7 ⁇ -HC) show substantially lower activity. Synthesis of the GPR183 ligand 7 ⁇ , 25-diHC requires two hydroxylation steps at position 25 by the enzyme cholesterol 25-hydroxylase (CH25H) and at position 7 ⁇ , by cytochrome P450 family 7 subfamily member B1 (CYP7B1) . Degradation of 7 ⁇ , 25-diHC is catalyzed by the enzyme hydroxy- ⁇ -5-steroid dehydrogenase, 3 ⁇ -and steroid ⁇ isomerase.
  • CH25H cholesterol 25-hydroxylase
  • CYP7B1 cytochrome P450 family 7 subfamily member B1
  • GPR183 The binding of oxysterols to GPR183 leads to the release of intracellular calcium, suppression of cAMP and internalization of GPR183.
  • the most important consequence of GPR183 activation is a migration of GPR183-expressing cells towards higher 7 ⁇ , 25-diHC concentrations.
  • GPR183 contributes at various levels to the coordination of cell-cell encounters by modulating the migration and positioning of DCs, T cells and B cells. Besides supporting the migration of lymphocytes and DCs, GPR183 coordinates the migration of innate lymphoid cells (ILCs) .
  • ILCs innate lymphoid cells
  • GPR183 also contributes to the organization of intestinal lymphoid tissue.
  • GWAS Genome wide association studies
  • IBD inflammatory bowel diseases
  • GPR183-oxysterol also promotes osteoclast precursor migration to bone surfaces and regulates bone mass homeostasis.
  • the study shows that GPR183 enhances the development of large osteoclasts by promoting osteoclast precursor motility, facilitating cell-cell interactions and fusion in vitro and in vivo.
  • GPR183 is also necessary and sufficient for guiding osteoclast precursors (OCPs) toward bone surfaces.
  • OCPs osteoclast precursors
  • GPR183-oxysterol axis in the spinal cord also contributes to neuropathic pain.
  • GPR183-oxysterol axis is also implicated in playing a role in Non-alcoholic fatty liver disease.
  • GPR183 is expressed in human hepatoma cell lines and its expression is induced in vivo in mouse livers after high-fat diet feeding. Activation of GPR183 inhibits fat accumulation in primary mouse hepatocytes and HepG2 cells through Gi/o proteins, p38 MAPKs, PI3K, and AMPK.
  • GPR183 inhibitors e.g., J. Med. Chem. 2014, 57, 3358-3368
  • no antibodies that bind to GPR183 and inhibit GPR183 activity have been identified so far.
  • GPR183 plays in the pathogenesis of various diseases, it is desirable to prepare antibodies that inhibit GPR183 activity so as to be used in the treatment of a disease mediated by GPR183, such as cancer, autoimmune diseases, liver diseases, osteoporosis and neuropathic pain.
  • a disease mediated by GPR183 such as cancer, autoimmune diseases, liver diseases, osteoporosis and neuropathic pain.
  • novel anti-GPR183 antibody or an antigen-binding fragment thereof specifically binding GPR183 is disclosed herein.
  • a pharmaceutical composition comprising the anti-GPR183 antibody or the antigen-binding fragment thereof disclosed herein as an active ingredient.
  • nucleic acid encoding the anti-GPR183 antibody or the antigen-binding fragment thereof, a vector and a host cell containing the nucleic acid.
  • Figure 1A shows that Mab16 blocks GPR183-mediated chemotaxis
  • Figure 1B shows that Mab18 blocks GPR183-mediated chemotaxis.
  • subject as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) , and most preferably a human.
  • patient refers to a human patient.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • Treatment of a cell encompasses contact of a reagent to the cell, as well as the contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • administration and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
  • treating any disease or disorder refer to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof) ; or alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient; or modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom) , physiologically, (e.g., stabilization of a physical parameter) , or both; or preventing or delaying the onset or development or progression of the disease or disorder.
  • affinity refers to the strength of interaction between antibody and antigen. Within the antigen, the variable regions of the antibody interact through non-covalent forces with the antigen at numerous sites. In general, the more interactions, the stronger the affinity.
  • antibody refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen non-covalently, reversibly, and in a specific manner.
  • a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2, and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL are composed of three CDRs and four framework regions (FRs) arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the variable regions of each light/heavy chain (VL/VH) pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the two binding sites are, in general, the same in the primary sequence.
  • the CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.
  • the antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) .
  • the anti-GPR183 antibodies comprise at least one antigen-binding site, at least a variable region. In some embodiments, the anti-GPR183 antibodies comprise an antigen-binding fragment from a GPR183 antibody described herein.
  • the term “monoclonal antibody” or “mAb” or “Mab” as used herein means a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts.
  • conventional (polyclonal) antibody preparations typically include a multitude of different antibodies comprising different amino acid sequences in their variable domains, particularly their complementarity determining regions (CDRs) , which are often specific for different epitopes.
  • CDRs complementarity determining regions
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the production of the antibody by any particular method.
  • Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256: 495-497; U.S. Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993.
  • a hybridoma producing a monoclonal antibody can be cultivated in vitro or in vivo.
  • High titers of monoclonal antibodies can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired antibodies.
  • Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair comprising one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa) .
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of the heavy chain can define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the antibody's isotypes as IgA, IgD, IgE, IgG, and IgM, respectively.
  • the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
  • the positions of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, AbM and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29: 205-206 (2001) ; Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987) ; Chothia et al., Nature, 342: 877-883 (1989) ; Chothia et al., J. Mol. Biol., 227: 799-817 (1992) ; Al-Lazikani et al., J. Mol.
  • ImMunoGenTics (IMGT) numbering (Lefranc, M. -P., The Immunologist, 7, 132-136 (1999) ; Lefranc, M. -P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ( “IMGT” numbering scheme) ) .
  • Definitions of antigen combining sites are also described in the following: Ruiz et al., Nucleic Acids Res., 28: 219-221 (2000) ; and Lefranc, M.P., Nucleic Acids Res., 29: 207-209 (2001) ; MacCallum et al., J. Mol.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) , 50-65 (HCDR2) , and 95-102 (HCDR3) ; and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1) , 50-56 (LCDR2) , and 89-97 (LCDR3) .
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1) , 52-56 (HCDR2) , and 95-102 (HCDR3) ; and the amino acid residues in VL are numbered 26-32 (LCDR1) , 50-52 (LCDR2) , and 91-96 (LCDR3) .
  • the CDRs are numbered 26-35 (HCDR1) , 50-65 (HCDR2) , and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1) , 50-56 (LCDR2) , and 89-97 (LCDR3) in human VL.
  • the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1) , 51-57 (HCDR2) and 93-102 (HCDR3)
  • the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1) , 50-52 (LCDR2) , and 89-97 (LCDR3) (numbering according to Kabat) .
  • the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
  • hypervariable region means the amino acid residues of an antibody that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “CDR” (e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain) .
  • CDR e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain
  • CDR e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain
  • CDR e.g., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain
  • an “antigen-binding fragment” means antigen-binding fragments of antibodies, i.e., antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions.
  • antigen-binding fragments include, but not limited to, Fab, Fab', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single chain Fv (ScFv) ; nanobodies and multispecific antibodies formed from antibody fragments.
  • an antibody “specifically binds” to a target protein, meaning the antibody exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity.
  • An antibody “specifically binds” or “selectively binds, ” is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, or antigen binding antibody fragment, which refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, blood, serum, plasma or tissue sample.
  • the antibodies or antigen-binding fragments thereof specifically bind to a particular antigen at least two times greater when compared to the background level and do not specifically bind in a significant amount to other antigens present in the sample.
  • the antibody or antigen-binding fragment thereof specifically bind to a particular antigen at least ten (10) times greater when compared to the background level of binding and does not specifically bind in a significant amount to other antigens present in the sample.
  • human antibody herein means an antibody that comprises human immunoglobulin protein sequences only.
  • a human antibody can contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell.
  • mouse antibody or “rat antibody” means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.
  • humanized or “humanized antibody” means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the prefix “hum, ” “hu, ” “Hu, ” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies.
  • the humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions can be included to increase affinity, increase the stability of the humanized antibody, remove a post-translational modification or for other reasons.
  • corresponding human germline sequence refers to the nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences.
  • the corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences.
  • the corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementary determining regions, a variable segment (as defined above) , or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art.
  • the corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the reference variable region nucleic acid or amino acid sequence.
  • the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296: 57-86, 2000.
  • HSPs high scoring sequence pairs
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0) . For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W word length
  • E expectation
  • B B- 50
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787, 1993) .
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ) , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P (N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 (E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988) ) .
  • the percent identity between two amino acid sequences can be determined using the algorithm which has been incorporated into the GAP program in the GCG software package using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol. 48: 444-453, (1970) ) .
  • Equilibrium dissociation constant refers to the dissociation rate constant (kd, time -1 ) divided by the association rate constant (ka, time -1 , M -l ) .
  • Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies disclosed herein generally will have an equilibrium dissociation constant of less than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some aspects, less than about 10 -11 M, 10 -12 M or 10 -13 M.
  • cancer or “tumor” as used herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context disclosed herein, the cancer is not limited to a certain type or location.
  • the term “conservative substitution” refers to a modification of a polypeptide including substituting one or more amino acids by one or more amino acids having similar biological or biochemical properties that do not cause loss of the biological or biochemical functions of the polypeptide.
  • the term “conservative amino acid substitution” refers to a substitution to replace an amino acid residue by an amino acid residue having a similar side chain. Classes of the amino acid residue having a similar side chain are defined and well-known in the art.
  • Such classes include amino acids with basic side chains (e.g., lysine, arginine, histidine) , amino acids with acidic side chains (e.g., aspartic acid, glutamic acid) , amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . It is considered that the antibody according to the present invention has a conservative amino acid substitution and still retains activity.
  • GPR183 antibody refers to an antibody that binds to GPR183, and to inhibit the biological activity of GPR183.
  • the term is used herein interchangeably with “anti-GPR183 antibody” .
  • anti-GPR183 antibody includes both a polyclonal antibody and a monoclonal antibody, is preferably a monoclonal antibody and may have a whole antibody.
  • the whole antibody is a structure having two full-length light chains and two full-length heavy chains, and including a constant region, wherein each light chain is linked to the corresponding heavy chain by a disulfide bond.
  • the whole antibody of the anti-GPR183 antibody disclosed herein includes IgA, IgD, IgE, IgM and IgG forms, and IgG includes subtypes IgG1, IgG2, IgG3 and IgG4.
  • the anti-GPR183 antibody disclosed herein is preferably a fully human antibody screened from human antibody libraries, but the present invention is not limited thereto.
  • the term “antigen binding fragment” of the anti-GPR183 antibody refers to a fragment having a function capable of binding to an antigen of the anti-GPR183 antibody, that is, GPR183, and encompasses Fab, Fab', F (ab') 2 , scFv, (scFv) 2 , scFv-Fc, Fv, Fab, Fab', F (ab') 2 , minibody, and diabody and the like, which is used interchangeably with “antibody fragment” .
  • Fab includes a variable region of each of the heavy chain and the light chain, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, each having an antigen-binding site.
  • Fab' is different from Fab in that it further has a hinge region including at least one cysteine residue at a C-terminus of the CH1 domain of the heavy chain.
  • F (ab') 2 is formed by a disulfide bond between cysteine residues in the hinge region of Fab'.
  • An Fv (variable fragment) including a variable region of each of the heavy chain and the light chain is the minimal antibody fragment having original specificity of parent immunoglobulin.
  • Double chain Fv (dsFv, disulfide-stabilized Fv) is formed by binding the variable region of the light chain to the variable region of the heavy chain via a disulfide bond.
  • Single chain Fv (scFv) is an Fv wherein the respective variable regions of the heavy chain and the light chain are covalently linked via a peptide linker.
  • These antibody fragments can be obtained by treating the whole antibody with a protease (for example, Fab can be obtained by restriction-cleaving the whole antibody with papain, and the F (ab’) 2 fragment can be obtained by restriction-cleaving the whole antibody with pepsin) and are preferably constructed by genetic recombination technology (for example, by amplifying a DNA encoding the heavy chain of the antibody or a variable region thereof or a DNA encoding the light chain or a variable region thereof as a template by PCR (polymerase chain reaction) using a pair of primers, and amplifying using a combination of a pair of primers to link DNA encoding a peptide linker and each of both ends thereof to the heavy chain or a variable region thereof and the light chain or a variable region thereof) .
  • a protease for example, Fab can be obtained by restriction-cleaving the whole antibody with papain, and the F (ab’) 2 fragment can be obtained by restriction-cleaving the whole
  • nucleic acid as used herein is interchangeable with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • nucleic acid may be present in a cell or a cell lysate, or in a partially purified form or in a substantially pure form.
  • the nucleic acid may be “isolated” or “substantially pure” , when purified from other cellular components or other contaminants, for example, nucleic acids or proteins of other cells, by standard techniques including, for example, alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well-known in the art.
  • the nucleic acid of the present invention may, for example, be DNA or RNA, and may or may not include an intron sequence.
  • DNA encoding partial-or full-length light and heavy chains are obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the target antibody) , and the DNA may be “operably bound” to transcription and translation control sequences to be inserted into the expression vector.
  • operably bound may indicate that the gene encoding the antibody is ligated into the vector so that the transcription and translation control sequences can serve the intended function of regulating the transcription and translation of the antibody genes.
  • the expression vector and expression control sequences which are compatible with the host cell used for expression are selected.
  • the light chain genes of the antibody and the heavy chain genes of the antibody are inserted into separate vectors, or both the genes are inserted into the same expression vector.
  • Antibodies are inserted into expression vectors by standard methods (e.g., ligation of an antibody gene fragment and complementary restriction enzyme sites on vectors, or blunt end ligation when there is no restriction enzyme site) .
  • the recombinant expression vectors may encode signal peptides that facilitate the secretion of the antibody chains from host cells.
  • the antibody chain genes may be cloned into vectors such that signal peptides are attached to the amino terminus of the antibody chain genes in accordance with the frame.
  • the signal peptides may be immunoglobulin signal peptides or heterologous signal peptides (i.e., signal peptides derived from proteins excluding immunoglobulin) .
  • the recombinant expression vectors have regulatory sequences that control the expression of the antibody chain genes in the host cells. “Regulatory sequences” may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of the antibody chain genes. It will be appreciated by those skilled in the art that the design of expression vectors can be varied by selecting different regulatory sequences depending on factors such as the choice of host cells to be transformed and the levels of protein expression.
  • host cell refers to any cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • the host cell disclosed herein is preferably selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli and insect cells, but the present invention is not limited thereto.
  • the host cell disclosed herein may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp.
  • the host cell may be selected from fungi such as Aspergillus sp., yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neurospora crassa, and other eukaryotic cells including lower eukaryotic cells, and higher eukaryotic cells derived from insects.
  • the host cell may also be derived from plants or mammals.
  • the host cell is selected from the group consisting of monkey kidney cells (COS7) , NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, but the present invention is not limited thereto.
  • COS7 monkey kidney cells
  • SP2/0 Chinese hamster ovary
  • CHO Chinese hamster ovary
  • W138 W138
  • baby hamster kidney (BHK) cells baby hamster kidney (BHK) cells
  • MDCK myeloma cell lines
  • HuT 78 cells HuT 78 cells
  • HEK293 cells HuT 78 cells
  • CHO cells are used.
  • Mammalian host cells are used to express and produce the anti-GPR183 polypeptides disclosed herein.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell
  • CHO cell lines capable of secreting intact immunoglobulins
  • various COS cell lines including the CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B-cells and hybridomas.
  • mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev.
  • expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter) , the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors” ) .
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the nucleic acid or the vector is transformed or transfected into a host cell.
  • Various techniques commonly used to introduce foreign nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transformation” or “transfection” include electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection or the like.
  • Various expression host/vector combinations may be used to express the anti-GPR183 antibody according to the invention.
  • the antibody When a recombinant expression vector capable of expressing the anti-GPR183 antibody or the antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody can be produced by incubation for a period of time sufficient to allow expression of the antibody in the host cell, more preferably, for a period of time sufficient to allow the antibody to be secreted into a culture medium.
  • the expressed antibody may be separated from the host cells and purified to homogeneity.
  • the separation or purification of the antibody can be carried out by separation and purification methods commonly used for proteins, for example, chromatography.
  • the chromatography may, for example, include affinity chromatography including a protein A column and a protein G column, ion exchange chromatography or hydrophobic chromatography.
  • the antibody can be separated and purified by a combination of filtration, ultrafiltration, salting out, dialysis or the like.
  • pharmaceutically acceptable carrier refers to a substance that can be added to the active ingredient to help formulate or stabilize the formulation and does not cause significantly harmful toxic effects on patients.
  • terapéuticaally effective amount refers to an amount of a combination of an anti-GPR183 antibody or an antigen-binding fragment thereof required to cause measurable benefits in vivo in a patient in need of treatment.
  • the exact amount will depend on a number of factors including, but not limited to, the ingredients and physical properties of the therapeutic composition, the population of intended patients and considerations of respective patients and can be readily determined by those skilled in the art. When fully taking these factors into consideration, it is important to administer a minimum amount sufficient to achieve maximum effects without causing adverse effects, and this dose can be easily determined by an expert in the field.
  • an antibody or an antigen-binding fragment thereof that specifically binds GPR183.
  • the antibody or the antigen-binding fragment thereof disclosed herein includes, but are not limited to, the antibody or the antigen-binding fragment thereof, generated as described, below.
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein is humanized or is a fully human antibody.
  • GPR183 is human GPR183.
  • the anti-GPR183 antibody is monoclonal.
  • the anti-GPR183 antibody disclosed herein is an antigen-binding fragment of the full-size monoclonal antibody.
  • the anti-GPR183 antibody is isolated or recombinant.
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a HCDR (heavy chain complementarity determining region) comprising an amino acid sequence of any one of the HCDRs listed in Table 1 (based on IMGT numbering scheme) or an amino acid sequence of any one of the HCDRs listed in Table 2 (based on Kabat numbering scheme) .
  • HCDR heavy chain complementarity determining region
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises (or alternatively, consists of) one, two, three, or more HCDRs comprising an amino acid sequence of any of the HCDRs listed in Table 1 (based on IMGT numbering scheme) or an amino acid sequence of any one of the HCDRs listed in Table 2 (based on Kabat numbering scheme) .
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a LCDR (light chain complementarity determining region) comprising an amino acid sequence of any one of the LCDRs listed in Table 1 (based on IMGT numbering scheme) or an amino acid sequence of any one of the LCDRs listed in Table 2 (based on Kabat numbering scheme) .
  • LCDR light chain complementarity determining region
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises (or alternatively, consists of) one, two, three, or more LCDRs comprising an amino acid sequence of any of the LCDRs listed in Table 1 (based on IMGT numbering scheme) or an amino acid sequence of any one of the LCDRs listed in Table 2 (based on Kabat numbering scheme) .
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in any row of Table 1 and/or a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in any row of Table 1.
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in any row of Table 1 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in the same row of Table 1.
  • the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 is presented in Table 1:
  • Table 1 Amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (based on IMGT numbering scheme)
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises
  • HCDR1 selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 52 and SEQ ID NO: 58,
  • HCDR2 selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 59 and SEQ ID NO: 64, and
  • HCDR3 selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 60;
  • LCDR1 selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 53 and SEQ ID NO: 61,
  • LCDR2 selected from the group consisting of SEQ ID NO: 41, and SEQ ID NO: 62, and
  • LCDR3 selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 63,
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in a row of Table 2 and/or a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in a row of Table 2.
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 in any row of Table 2 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 in the same row of Table 2.
  • the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (based on Kabat numbering scheme) is presented in Table 2:
  • Table 2 Amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (based on Kabat numbering scheme)
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises
  • HCDR1 selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 72, SEQ ID NO: 75, and SEQ ID NO: 84,
  • HCDR2 selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 73, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85 and SEQ ID NO: 89, and
  • HCDR3 selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 83 and SEQ ID NO: 86;
  • LCDR1 selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 87 and SEQ ID NO: 90,
  • LCDR2 selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 88, and
  • LCDR3 selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 63,
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 (Table 3 or 4 or 5) , or an amino acid sequence at least 95%, 96%, 97%, 98%or 99%identical to any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35; and/or (b) a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 (Table 3 or 4 or 5) , or an amino acid sequence at least 95%, 96%, 97%, 98%or 99%identical to any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.
  • the anti-GPR183 antibody or an antigen-binding fragment thereof disclosed herein comprises (a) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 (Table 3 or 4 or 5) , or an amino acid sequence with one, two, or three amino acid substitutions in the amino acid sequence of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35; and/or (b) a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 (Table 3 or 4 or 5) , or an amino acid sequence with one, two, or three amino acid substitutions in the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) in any row of Table 3 and/or a light chain variable region (VL) in any row of Table 3.
  • the antibody or an antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) in any row of Table 3 and a light chain variable region (VL) in the same row of Table 3.
  • the amino acid sequences of VH and VL is presented in Table 3:
  • the antibody of the present disclosure is of IgG1, IgG2, IgG3, or IgG4 isotype.
  • the antibody of the present disclosure comprises the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2.
  • amino acid sequences of the heavy chain (VH) and light chain (VL) and the variable region sequences of the heavy chain CDRs and light chain CDRs of the anti-GPR183 antibody or the antigen-binding fragment thereof disclosed herein are as shown in Tables 4 and 5.
  • Table 4 Amino acid sequences of the heavy chain (VH) and light chain (VL) and amino acid sequences of heavy chain CDRs and light chain CDRs of anti-GPR183 antibodies disclosed herein, wherein the CDRs are based on IMGT numbering scheme:
  • Table 5 Amino acid sequences of the heavy chain (VH) and light chain (VL) and amino acid sequences of heavy chain CDRs and light chain CDRs of anti-GPR183 antibodies disclosed herein, wherein the CDRs are based on Kabat numbering scheme:
  • composition comprising a compound disclosed herein or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, optionally together with a pharmaceutically acceptable excipient.
  • the disease mediated by GPR183 is cancer, autoimmune diseases, liver diseases, osteoporosis, and neuropathic pain.
  • the cancer is blood, brain, breast, colorectal, gastrointestinal, liver, lung, ovarian, pancreatic, prostate, skin, or uterine cancer.
  • the cancer produces molecules involved in Epstein-Barr virus (EBV) -induced G-protein coupled receptor 2 (EBI2) mediated signaling.
  • EBV Epstein-Barr virus
  • EBI2 G-protein coupled receptor 2
  • Anti-GPR183 monoclonal antibodies were developed by immunizing SD rats with 293T cells overexpressing GPR183. Briefly, 293T Cells were transfected with lentiviral vectors encoding full-length human GPR183 (Gene ID: 1880) by polybrene (8 ⁇ g/mL) , selected in media containing puromycin (2 ⁇ g/mL) , and tested for the expression of GPR183 by FACS. Individual clones with the greatest MFI to GPR183 were selected for subsequent studies.
  • Spleen and lymph node cells from these rats were fused with myeloma cells (SP20) by standard methods to generate hybridomas producing unique antibodies.
  • Supernatants containing antibodies produced by pools of these cells were tested by cell-based ELISA for reactivity with GPR183 overexpressing cells.
  • Cell-based ELISA was generally carried out as follows. Approximately 3 ⁇ 10 4 CHOK1-GPR183 cells per well were seeded in a 96-well plate, and after overnight culture, the cells were washed with 1 ⁇ PBS-T, followed by the addition of 100 ⁇ L of 4%paraformaldehyde solution to fix and cross-link the cells to the microplate. The cells were washed with 1 ⁇ PBS-T twice.
  • the cells were incubated with neat supernatants taken from hybridoma cultures including blank and positive control at 37°C for 60 minutes. Subsequently, the cells were washed with 1 ⁇ PBS-T four times. The cells were then incubated with goat anti-rat IgG second antibody at 1: 10000 dilution in 100 ⁇ L PBS at 37°C for 60 minutes, followed by four times washing with 1 ⁇ PBS-T. One hundred ⁇ L TMB was added to the 96-well plate. After 10-12 minutes of incubation, the plate was scanned with detection at 450 nm wavelength.
  • FACS analyses were generally carried out as follows. Approximately 5 ⁇ 10 5 CHOK1-GPR183 cells per sample were prepared and blocked with Mouse BD Fc Block. The cells were distributed into 96-well round-bottom polystyrene plates and incubated with neat supernatants taken from hybridoma cultures on ice for 20-30 minutes. Next, the cells were washed with PBS/0.5%BSA and were centrifuged.
  • the pelleted cell samples were then incubated with the second antibody of anti-mouse IgG labelled with FITC at 1: 300 dilution in 100 ⁇ L PBS/0.5%BSA on ice for 30 minutes, and then washed with PBS/0.5% BSA and spun down.
  • the cell pellets were re-suspended in PBS/0.5%BSA, and the samples were analyzed on a CYTOFLEX (Beckman) .
  • the same supernatants were tested on non-transfected parental cells to confirm that the reactive antibody recognized GPR183 specifically.
  • the affinity of anti-GPR183 antibodies was evaluated by FACS. 293T-GPR183, which had high level of GPR183 expressed on the cell surface, was used in the binding test. Affinity analyses were generally carried out as follows. Approximately 5 ⁇ 10 5 293T-GPR183 cells per sample were prepared and blocked with Human BD Fc Block. The test antibodies were diluted with PBS/0.5%BSA (1: 3 series dilution from 225 ⁇ g/mL to 0.00381 ⁇ g/mL) . The transfected cells along with parent non-transfected cells were distributed into 96-well round-bottom polystyrene plates and incubated with the diluted antibodies on ice for 20-30 minutes.
  • the samples were washed with PBS/0.5%BSA and the cells were centrifuged.
  • the pelleted cell samples were incubated with the second antibody of anti-mouse IgG-FITC-at 1: 300 dilution in 100 ⁇ L PBS/0.5%BSA on ice for 30 minutes, and then washed with PBS/0.5%BSA, followed by pelleting the cells.
  • the cell pellets were re-suspended in PBS/0.5%BSA for reading, and samples were analyzed using CYTOFLEX (Beckman) .
  • U937 cells expressed with human GPR183 were maintained in suspension in RPMI 1640 containing 1%heat-inactivated FBS, 100U/ml penicillin and 100 ⁇ g/ml streptomycin for about 3 hours. Then, the cells were washed once in PBS and were re-suspended at 2.66 x10 6 cells/ml in assay buffer (RPMI 1640, 0.5%endotoxin-free BSA, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin) . The test antibodies were diluted with assay buffer (1: 5 series dilution from 60 ⁇ g/mL to 0.000768 ⁇ g/mL) .
  • the diluted antibodies were pre-incubated with 100 ⁇ l of cells for 30 minutes at 37°C and placed into the upper chamber of a transwell plate with 5 ⁇ m pores (Corning Life Sciences) . In the lower chambers, 150 nM 7 ⁇ , 25-diHC was added and the assay was incubated at 37°C for 4 hours (5%CO2) . Live cells migrating to the lower chamber were counted using CYTOFLEX (Beckman) .
  • Mab16 and Mab18 have been shown to block GPR183-mediated chemotaxis.
  • the IC50 of Mab16 and Mab18 is 0.6452 nM and 4.627 nM.

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Abstract

L'invention concerne de nouveaux anticorps anti-GPR183, ou un fragment de liaison à l'antigène de ceux-ci, un acide nucléique codant pour l'anticorps ou le fragment de liaison à l'antigène de celui-ci, un vecteur et une cellule hôte comprenant l'acide nucléique, un procédé de production de l'anticorps ou du fragment de liaison à l'antigène de celui-ci, une composition pharmaceutique contenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci en tant que principe actif, et l'utilisation de l'anticorps dans le traitement des maladies médiées par GPR183.
PCT/CN2023/122615 2022-09-29 2023-09-28 Anticorps anti-gpr183 et leurs utilisations Ceased WO2024067810A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103608030A (zh) * 2011-06-21 2014-02-26 昂科发克特公司 用于治疗和诊断癌症的组合物和方法
WO2018029586A1 (fr) * 2016-08-07 2018-02-15 Novartis Ag Procédés d'immunisation à médiation par arnm.
WO2018209324A2 (fr) * 2017-05-11 2018-11-15 The Broad Institute, Inc. Procédés et compositions d'utilisation de sous-types de lymphocytes infiltrant les tumeurs cd8 + et leurs signatures géniques
WO2021148884A1 (fr) * 2020-01-24 2021-07-29 Tychan Pte. Ltd. Anticorps anti-coronavirus du wuhan
US20210253722A1 (en) * 2018-03-12 2021-08-19 Zoetis Services Llc Anti-NGF Antibodies and Methods Thereof
WO2022159718A1 (fr) * 2021-01-22 2022-07-28 The Brigham And Women's Hospital, Inc. Modulation d'un phénotype pathogène dans des cellules th1

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103608030A (zh) * 2011-06-21 2014-02-26 昂科发克特公司 用于治疗和诊断癌症的组合物和方法
WO2018029586A1 (fr) * 2016-08-07 2018-02-15 Novartis Ag Procédés d'immunisation à médiation par arnm.
WO2018209324A2 (fr) * 2017-05-11 2018-11-15 The Broad Institute, Inc. Procédés et compositions d'utilisation de sous-types de lymphocytes infiltrant les tumeurs cd8 + et leurs signatures géniques
US20210253722A1 (en) * 2018-03-12 2021-08-19 Zoetis Services Llc Anti-NGF Antibodies and Methods Thereof
WO2021148884A1 (fr) * 2020-01-24 2021-07-29 Tychan Pte. Ltd. Anticorps anti-coronavirus du wuhan
WO2022159718A1 (fr) * 2021-01-22 2022-07-28 The Brigham And Women's Hospital, Inc. Modulation d'un phénotype pathogène dans des cellules th1

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