Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • 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
  • C07K2317/72—Increased effector function due to an Fc-modification
• • 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

• the invention relates generally to porcine antibody mutants and uses thereof. Specifically, the invention relates to one or more mutations in the Fc constant region of porcine antibody for improving various characteristics.
• Porcine IgG monoclonal antibodies can be effective therapeutics in veterinary medicine.
• mAbs porcine IgG subclasses were identified. However, only a limited work has been done to improve the chraceteristics of porcine IgGs.
• the neonatal Fc receptor prolongs the half-life of an IgG in a pH-dependent interaction with its fragment crystallizable (Fc) region.
• Fc fragment crystallizable
• the Fc region spanning the interface of CH2 and CH3 domains interacts with the FcRn on the surface of cells to regulate IgG homeostasis. This interaction is favored by an acidic interaction after IgG pinocytosis and thus IgG is protected from degradation.
• the endocytosed IgG is then recycled back to the cell surface and released into the blood stream at a slightly alkaline pH thereby maintaining sufficient serum IgG for proper function. Accordingly, the pharmacokinetic profile of IgGs depend on the structural and functional properties of their Fc regions.
• the invention relates to mutant porcine IgGs that exhibit desired characteristics, relative to wild-type porcine IgGs.
• the inventors of the instant application have found that substituting an amino acid residue at position 252, 254, 256, 286, 293, 307, 311, 312, 378, 426, 428, 434, or 436 (numbered according to the Eu index as in Kabat) with another amino acid surprisingly and unexpectedly exhibited a desired effect.
• the unexpected desired effects include, but not limited to, enhanced affinity to FcRn.
• the invention provides a modified IgG comprising: a porcine IgG constant domain comprising at least one amino acid substitution relative to a wild-type porcine IgG constant domain, wherein said substitution is at amino acid residue 252, 254, 256, 286, 293, 307, 311, 312, 378, 426, 428, 434, or 436.
• the porcine IgG constant domain is an IgGl constant domain that comprises one or more of mutations of M252A, M252C, M252D, M252E, M252F, M252G, M252H, M252I, M252K, M252L, M252N, M252P, M252Q, M252R, M252S, M252T, M252V, M252W, M252Y, S254T, T256E, T256F, T256N, T286A, T286C, T286D, T286E, T286F, T286G, T286H, T286I, T286K, T286L, T286M, T286N, T286P, T286Q, T286R, T286S, T286V, T286W, T286Y, E293delete, P307Q, E
• the invention provides a polypeptide comprising: a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.
• the invention provides an antibody or a molecule comprising: a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.
• the invention provides a method for producing or manufacturing an antibody or a molecule, the method comprising: providing a vector or a host cell having a nucleic acid sequence that encodes an antibody, wherein said antibody comprises a porcine IgG constant domain comprising one or more amino acid substitutions of the invention described herein.
• FIG. 1 illustrates domain structure of IgG.
• FIG. 2 shows the alignment of the amino acid sequences of human IgGl and porcine IgGla, IgG2, IgG3, IgG4a, IgG5a, and IgG6a.
• CHI, hinge, CH2, and CH3 domains are as follows: CHI : residues 118-215; hinge: 216-230; CH2: 231-340; CH3: 341-447.
• the amino acid residues are numbered according to the Eu index as in Kabat.
• FIG. 3 Protein modeling of Porcine FcRn and IgG6 Fc regions with a zoomed-in subpanel showing amino acid residue positions are shown in ball-and-stick form.
• FIG. 4 Protein modeling of Porcine FcRn, IgG4a-Fc and IgG4b-Fc regions with a zoomed-in sub-panel showing amino acid residue positions are shown in ball-and-stick form.
• SEQ ID NO.: 3 refers to the amino acid sequence of porcine IgG2a wildtype constant region.
• SEQ ID NO.: 4 refers to the amino acid sequence of porcine IgG2b wildtype constant region.
• SEQ ID NO.: 5 refers to the amino acid sequence of porcine IgG3 wildtype constant region.
• SEQ ID NO.: 9 refers to the amino acid sequence of porcine IgG5b wildtype constant region.
• SEQ ID NO.: 12 refers to the amino acid sequence of human IgGl wildtype constant region.
• the numbering of the amino acid residues in an immunoglobulin heavy chain is that of the Eu index as in Kabat, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
• the "Eu index as in Kabat” refers to the residue numbering of the IgG antibody and is reflected herein in FIG. 2.
• isolated when used in relation to a nucleic acid is a nucleic acid that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is in a form or setting different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
• An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide encoded therein where, for example, the nucleic acid molecule is in a plasmid or a chromosomal location different from that of natural cells.
• the isolated nucleic acid may be present in single-stranded or double-stranded form.
• the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand, but may contain both the sense and anti-sense strands (i.e., may be double-stranded).
• a nucleic acid molecule is "operably linked” or “operably attached” when it is placed into a functional relationship with another nucleic acid molecule.
• a promoter or enhancer is operably linked to a coding sequence of nucleic acid if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence of nucleic acid if it is positioned so as to facilitate translation.
• the "CH3 domain" of a porcine IgG Fc region generally is the stretch of residues C- terminal to a CH2 domain in an Fc region, for example, residue 341 to the c-terminus in Figure 2.
• a "functional Fc region” possesses an "effector function" of a native sequence Fc region. Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
• Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable.
• Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.
• the purified FcRn/B2M was biotinylated as follows.
• the purified FcRn/B2M protein was dialyzed into 10 mM Tris-HCl, pH 8.0 and concentrated using AmiconUltra,10KMWCO (EMD Millipore, Billerica, MA).
• the Biotin Acceptor Peptide (BAP) AGLNDIFEAQKIEWHE which was expressed at the c-terminus of the receptor allowed for transfer of biotin to this stretch of amino acids using the biotin ligase BirA. Biotinylation reactions were carried out as described in the manufacturer protocol (Avidity, LLC, Aurora, CO).
• the FcRn/B2M receptor was then dialyzed into PBS to remove residual biotin.
• Plasmids containing sequence encoding for porcine constant regions for the IgG6a were utilized and VH/VL sequences for each mAh investigated herein were inserted upstream and in frame with the nucleotides encoding for the constant domains. Mutations were incorporated into either CH2 or CH3 domain positions of each plasmid by direct DNA synthesis of the constant region as gene fragment and were subsequently sub-cloned into respective variable region of interest.
• EXPICHO-S Choinese Hamster Ovary cells
• EXPICHO-S cells were maintained in EXPICHO expression medium (Gibco) between 0.14 and 8.0xl0e6 cells/ml.
• Cells were diluted following the ExpiCHO Protocol user manual on Day -1 and transfection day. Diluted cells were transfected as described in the protocol using reagents sourced from ExpiFectamine CHO Transfection Kit (Gibco) following Max Titer conditions. Following 12-14 days of incubation, the cultures were harvested and clarified.
• Non-reduced (nr) and reduced sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) was performed using 4-12% Bis-Tris NuPAGE gels in MES- SDS running buffer, and SeeBlue Plus 2 standards, all from Invitrogen.
• NEM alkylating agent N-ethylmaleimide
• DTT dithiothreitol
• Molecular Operating Environment developed by Chemical Computing Group (MOE2019.0102) provides a flexible and automated graphical user interface for protein modeling.
• the sequence-to-profile alignment algorithm uses a scoring algorithm to rank the sequence templates and scores higher than 85% ensure the selection of protein templates with physically realistic structures.
• the model was then optimized using the same pipeline and the structural stability of the models was verified using Ramachandran Plots, which checks the stereochemical quality of a protein structure.
• IgG4 IgG4a and IgG4b
• IgG2 and IgGl IgGla and IgGlb
• An RMSD plot was generated to calculate the root mean square deviation of the WT structures, Porcine IgGla, Procine-IgGlb, Porcine IgG2, Porcine-IgG4a, Porcine-IgG4b, Porcine-IgG6a and Porcine IgG6b relative to each other (Fig 6).
• An RMSD value of 2.0A or lower is considered the standard for considering two structures to be alike. Results indicated that the protein fold of porcine IgG4a construct was identical to the porcine IgG4b allotype with average RMSD value for the structure being 0.11 A (individual position RMSD in Table 3).
• Porcine IgG6a construct was identical to the porcine IgG6b allotype with an average RMSD value of 0.66 A (individual position RMSD in Table 4). Allotypes IgGla, IgGlb and IgG2 also showed RMSDs of 0.2 and 0.78 A (individual position RMSD in Table 5 and 6). RMSDs at the positions where mutational scanning was performed was also noted in Tables 7 through 10 with values ranging from 0.11-0.93 A.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Immunology (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=90098773

Family Applications (1)

Country Status (8)

Family Cites Families (3)

• 2023
  • 2023-09-01 JP JP2025512980A patent/JP2025529206A/ja active Pending
  • 2023-09-01 CA CA3265530A patent/CA3265530A1/en active Pending
  • 2023-09-01 CN CN202380066811.9A patent/CN119968390A/zh active Pending
  • 2023-09-01 WO PCT/US2023/073260 patent/WO2024050491A2/en not_active Ceased
  • 2023-09-01 AU AU2023334358A patent/AU2023334358A1/en active Pending
  • 2023-09-01 KR KR1020257010607A patent/KR20250054824A/ko active Pending
  • 2023-09-01 EP EP23861579.3A patent/EP4581053A2/de active Pending
• 2025
  • 2025-02-28 MX MX2025002522A patent/MX2025002522A/es unknown

Also Published As

Similar Documents

Legal Events