Anti-IL-2 Antibody, and Antigen-Binding Fragment Thereof and Medical Use Thereof
20230089620 · 2023-03-23
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C07K2319/01
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are an anti-IL-2 antibody, and an antigen-binding fragment thereof and the medical use thereof. Further provided are a complex (including a fusion protein) of the anti-IL-2 antibody, the antigen-binding fragment thereof and IL-2, and the use of the complex as a drug for treating autoimmune diseases and inflammatory diseases.
Claims
1. An anti-IL-2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein: the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 9, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 10; the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2; the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 5, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 6; the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 3, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 4; the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 7, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 8; or the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 11, and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 12; the above CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
2. The anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1, comprising: a VH set forth in one of SEQ ID NOs: 9, 1, 5, 3, 7 and 11, or a VH having at least 90% or at least 95% identity thereto; and a VL set forth in one of SEQ ID NOs: 10, 2, 6, 4, 8 and 12, or a VL having at least 95% identity thereto.
3. The anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1, comprising: an HC set forth in one of SEQ ID NOs: 165, 157, 161, 159, 163 and 167, or an HC having at least 80%, 90% or 95% identity thereto; and an LC set forth in one of SEQ ID NOs: 166, 158, 162, 160, 164 and 168, or an LC having at least 80%, 90% or 95% identity thereto.
4. The anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof is a fully human antibody or an antigen-binding fragment thereof, and/or wherein the antigen-binding fragment is an scFv, Fv, Fab or Fab′ fragment.
5. (canceled)
6. The anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof is an IgG antibody or an antigen-binding fragment thereof.
7. A complex, comprising: the anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1 and IL-2.
8. The complex according to claim 7, wherein the complex selectively enhances the activity of or promotes the proliferation of cells highly expressing IL-2Ra, and/or the complex selectivity enhances the effect of the IL-12 on the activity of Tregs.
9. The complex according to claim 7, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof and the IL-2 are bound by covalent or non-covalent force.
10. The complex according to claim 7, wherein the complex is a fusion protein formed by the anti-IL-2 antibody or the antigen-binding fragment thereof and the IL-2.
11. The complex according to claim 10, wherein in the fusion protein, the IL-2 is linked to a light chain (LC) or a heavy chain (HC) of the anti-IL-2 antibody or the antigen-binding fragment thereof.
12. The complex according to claim 10, wherein in the fusion protein, the IL-2 is linked to the anti-IL-2 antibody or the antigen-binding fragment thereof via a linker.
13. The complex according to claim 7, wherein the IL-2 has an amino acid substitution at position 3 or an N-terminal deletion.
14. The complex according to claim 10, wherein the fusion protein comprises an HC and an LC selected from the group consisting of any one of the following groups: an HC set forth in SEQ ID NO: 169 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 158 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 171 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 160 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 159 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 172 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 173 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 162 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 175 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 164 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 163 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 176 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 177 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 166 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 179 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 168 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 167 or an HC having at least 90% sequence identity thereto, and an LC set forth in SEQ ID NO: 180 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 161 or an HC having at least 90% sequence identity thereto, and an LC set forth in any one of SEQ ID NOs: 174 and 181-186 or an LC having at least 90% sequence identity thereto; an HC set forth in SEQ ID NO: 157 or an HC having at least 90% sequence identity thereto, and an LC set forth in any one of SEQ ID NOs: 170 and 187 or an LC having at least 90% sequence identity thereto; and an HC set forth in SEQ ID NO: 165 or an HC having at least 90% sequence identity thereto, and an LC set forth in any one of SEQ ID NOs: 178, 188 and 189 or an LC having at least 90% sequence identity thereto.
15.-20. (canceled)
21. A pharmaceutical composition, comprising: the anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1, and one or more pharmaceutically acceptable excipients, diluents or carriers.
22. An isolated polynucleotide, encoding the anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1.
23. A host cell, transformed with or comprising the polynucleotide according to claim 22.
24. A method for preparing an anti-IL-2 antibody or an antigen-binding fragment thereof, or a complex thereof, comprising: expressing the anti-IL-2 antibody or the antigen-binding fragment thereof, or the complex thereof, in the host cell according to claim 23, and isolating the anti-IL-2 antibody or the antigen-binding fragment thereof, or the complex thereof, from the host cell.
25. A method for treating or delaying progression of an autoimmune disease, comprising: administering to a subject the anti-IL-2 antibody or the antigen-binding fragment thereof according to claim 1 in an effective amount for treating or delaying the disease.
26. A method for increasing the ratio of Tregs to non-Tregs in a population of T cells, or increasing the number of Tregs, or increasing the activity of Tregs, comprising: contacting the population of T cells with an effective amount of the complex according to claim 7.
27. A method for increasing the ratio of Tregs to non-Tregs in peripheral blood of a subject, or increasing the number of Tregs, or increasing the activity of Tregs, comprising: administering to the subject an effective amount of the complex according to claim 7.
28. A pharmaceutical composition, comprising: the complex according to claim 7, and one or more pharmaceutically acceptable excipients, diluents or carriers.
29. An isolated polynucleotide, encoding the complex according to claim 7.
30. A method for treating or delaying progression of an autoimmune disease, comprising: administering to a subject the complex according to claim 7 in an effective amount for treating or delaying the disease.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] The foregoing and other objects, aspects, features, and advantages of exemplary embodiments will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings.
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DETAILED DESCRIPTION
Terms
[0104] In order to facilitate the understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined elsewhere herein, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present disclosure belongs.
[0105] The three-letter and single-letter codes for amino acids used in the present disclosure are described as in J. Biol. Chem, 243, p 3558 (1968).
[0106] “Interleukin-2” or “IL-2” is intended to be interpreted broadly and includes any IL-2 related product, including but not limited to, human and non-human IL-2 homologs, fragments or truncations, fusion proteins (e.g., fused to a signal peptide or other active components (e.g., an antibody or antigen-binding fragment thereof) and inactive components), modified forms (e.g., PEGylated, glycosylated, albumin conjugated/fused, Fc conjugated and/or fused, and hydroxyethylated forms), conservatively modified proteins, etc. The term encompasses any natural IL-2 of any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term also encompasses an unprocessed IL-2 as well as any form of processed IL-2 derived from cells; naturally occurring IL-2 variants, such as splice variants or allelic variants; and conservatively modified variants of IL-2.
[0107] “IL-2Rα”, “CD25” or “α subunit of IL-2 receptor” refers to any natural CD25 of any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses an unprocessed “full-length” CD25 as well as any form of processed CD25 derived from cells; and naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25, with an illustrative sequence set forth in SEQ ID NO: 37.
[0108] A “high affinity IL-2 receptor” refers to the heterotrimer form of the IL-2 receptor that consists of a receptor γ subunit (also known as a common cytokine-receptor γ subunit, γc, or CD132), a receptor β subunit (also known as CD122, p70, or IL-2Rβ), and a receptor α subunit (also known as CD25, p55, or IL-2R). In contrast, a “moderate affinity IL-2 receptor” refers to an IL-2 receptor that comprises only γ and β subunits but no α subunit (see, e.g., Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008)).
[0109] “Conservative modifications” is applicable to amino acid and nucleotide sequences. For particular nucleotide sequences, conservative modifications refer to those nucleic acids encoding identical or substantially identical amino acid sequences, or, in the case of nucleotides not encoding amino acid sequences, to substantially identical nucleotide sequences. For amino acid sequences, “conservative modifications” refer to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in the art recognize that, generally speaking, a single amino acid replacement in a non-essential region of a polypeptide does not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p224, (4th edition)).
[0110] “Amino acid mutations” include amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to obtain a final construct that possesses desired properties, such as enhanced stability and increased activity. Amino acid sequence deletions and insertions include amino-terminal and/or carboxyl-terminal deletions and amino acid insertions. Preferred amino acid mutations are amino acid substitutions. To alter the binding properties of, for example, an anti-IL-2 antibody or an antigen-binding fragment thereof, non-conservative amino acid substitutions may be made, i.e., one amino acid is replaced with another amino acid having different structural and/or chemical properties. Preferred amino acid substitutions include the replacement of hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include the replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, and 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.
[0111] An “antibody” is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); and full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions) so long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, which is of a tetrapeptide chain structure formed by two heavy chains and two light chains linked by interchain disulfide bonds. The heavy chain constant regions of an immunoglobulin differ in their amino acid composition and arrangement, and thus in their antigenic specificity. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being μ chain, δ chain, γ chain, α chain and E chain, respectively. Ig of the same class can be divided into different subclasses according to differences in the composition of amino acids of the hinge regions and the number and positions of disulfide bonds of the heavy chains; for example, IgG can be divided into IgG.sub.1, IgG.sub.2, IgG.sub.3, and IGg.sub.4. Light chains are classified into κ or λ chains according to differences in the constant regions. Each of the five classes of Ig may have a κ chain or λ chain. In the heavy and light chains of antibody, the sequences of about 110 amino acids near the N-terminus vary considerably and thus are referred to as variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and thus are referred to as constant regions (C regions). The variable regions comprise 3 hypervariable regions (CDRs) and 4 framework regions (FRs) with relatively conservative sequences. The 3 hypervariable regions determine the specificity of the antibody and thus are also known as complementarity determining regions (CDRs). Each of the light chain variable regions (VLs) and the heavy chain variable region (VHs) consists of 3 CDR regions and 4 FR regions arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; and the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
[0112] For determination or definition of CDRs, the deterministic depiction of CDRs can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex, and identifying residues involved in the antigen binding site. This can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. A variety of analysis methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, and conformational definition.
[0113] The Kabat numbering system is a standard for numbering residues in antibodies and is generally used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8).
[0114] The Chothia numbering system is similar to the Kabat numbering system, except that it takes into account the location of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83).
[0115] The AbM numbering system adopts a computer program integration suite for modeling antibody structures manufactured by Oxford Molecular Group (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86: 9268-9272; “AbMTM, A Computer Program for Modeling Variable Regions of Antibodies”, Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system adopts a combination of a knowledge database and the de-novo method to model the tertiary structure of antibodies from basic sequences (see those described in Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach”, PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198).
[0116] The contact definition is based on the analysis of the available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45).
[0117] In the conformational definition, the position of the CDRs can be identified as residues that contribute enthalpy to the antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).
[0118] Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or a particular group of residues do not significantly affect the antigen binding.
[0119] As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. In the methods used herein, CDRs defined according to any of those methods may be used. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of Kabat, Chothia, extended, AbM, IMGT, contact, and/or conformational definitions.
[0120] A “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is to say, the antibodies comprised in the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, and target a single antigen site. Furthermore, unlike polyclonal antibodies, each monoclonal antibody targets a single determinant on the antigen. The modifier “monoclonal” indicates the characteristic of the antibody as obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used according to the present disclosure can be prepared by the hybridoma methods firstly described in Kohler and Milstein, 1975, Nature 256: 495, or can be prepared by the recombinant DNA method described in U.S. Pat. No. 4,816,567. For example, monoclonal antibodies can also be isolated from the generated phage library using the technique described in McCafferty et al., 1990, Nature 348: 552-554.
[0121] A “human antibody” includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, a “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as mice) have been grafted into a human framework sequence (i.e., “humanized antibodies”).
[0122] A “human antibody” or “recombinant human antibody” includes human antibodies prepared, expressed, created, or isolated by recombinant methods, involving techniques and methods well known in the art, such as:
(1) antibodies isolated from transgenic human immunoglobulin genes, transchromosomal animals (e.g., mice), or hybridomas prepared therefrom;
(2) antibodies isolated from host cells that have been transformed to express the antibodies, such as transfectomas;
(3) antibodies isolated from a recombinant combinatorial human antibody library; and
(4) antibodies prepared, expressed, created or isolated by a method such as splicing human immunoglobulin gene sequences to other DNA sequences.
[0123] Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, and also include subsequent rearrangements and mutations which occur, for example, during antibody maturation.
[0124] An “antigen (Ag)” refers to a molecule used to immunize an immunologically active vertebrate to produce an antibody (Ab) that recognizes Ag; or a molecule or mimic used for screening in an expression library, e.g., especially a phage, yeast, or ribosome display library. Ag is defined more broadly herein, and is generally intended to include target molecules recognized by Abs. Therefore, Ag includes molecules, or portions or mimics thereof, used in immunization processes for producing Abs or in library screening for selecting Abs. Therefore, for the antibody of the present disclosure that binds to IL-2, full-length IL-2 from mammalian species (e.g., human, monkey, mouse, and rat IL-2), including monomers and polymers thereof (e.g., dimers, trimers, etc.), as well as truncated variants and other variants of IL-2, are all referred to as antigens.
[0125] An “epitope” refers to a site on an antigen to which an immunoglobulin (or antibody) specifically binds. An epitope may be formed from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. An epitope formed from contiguous amino acids are generally retained after exposure to a denaturing solvent, while an epitope formed by tertiary folding are generally lost after a denaturing solvent treatment. An epitope generally comprise, for example, 3 to 15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art and include an immunoblotting assay, an immunoprecipitation assay, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
[0126] An “antigen-binding fragment” include a single-chain antibody (i.e., full-length heavy and light chains); Fab, a modified Fab, Fab′, a modified Fab′, F(ab′).sub.2, Fv, Fab-Fv, Fab-dsFv, a single domain antibody (e.g., VH or VL or VHH), scFv, a bivalent or trivalent or tetravalent antibody, Bis-scFv, a diabody, a tribody, a triabody, a tetrabody and an epitope-binding fragment of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23 (9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2 (3), 209-217). Methods for producing and preparing such antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Fab-Fv was first disclosed in WO2009/040562, and its disulfide-stabilized form Fab-dsFv was first disclosed in WO2010/035012. The antigen-binding fragment of the present disclosure also include Fab and Fab′ fragments described in WO2005/003169, WO2005/003170 and WO2005/003171. Multivalent antibodies may comprise multiple specificities (e.g., bispecificites) or may be monospecific (see, e.g., WO92/22583 and WO05/113605), and an example of the latter is Tri-Fab (or TFM) described in WO92/22583.
[0127] “Binding to IL-2” refers to the ability to interact with IL-2 or an epitope thereof, wherein the IL-2 or the epitope thereof may be derived from human.
[0128] An “antigen-binding site” refers to a continuous or discontinuous three-dimensional spatial site on an antigen that is recognized by an antibody or an antigen-binding fragment of the present disclosure.
[0129] A “regulatory T cell” or “Treg” refers to a specialized CD4.sup.+ T cell type that can inhibit the response of other T cells. Tregs are characterized by expression of the α subunit of IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and play a key role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg requires IL-2 for its function and development as well as for the induction of its inhibitory characteristics.
[0130] “Effector function” refers to those biological activities attributable to the Fc region of an antibody (either the natural sequence Fc region or the amino acid sequence variant Fc region) and which vary with the antibody isotype. Examples of effector functions of an antibody include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. “ADCC” refers to the direct killing of antibody-coated target cells by Fc receptor-expressing cells through recognition of the Fc segment of the antibody. The ADCC effector function of the antibody may be reduced or eliminated by modification of the Fc segment of the IgG. The modification refers to mutations in the heavy chain constant region of the antibody, such as mutations selected from the group consisting of N297A, L234A, L235A and P329G of IgG.sub.1, IgG4.sub.2/4 chimera, and F234A/L235A of IgG.sub.4. “CDC” refers to the activation of the cytotoxic form of the complement cascade by binding the complement component C1q to the antibody Fc. Methods for detecting ADCC and CDC activity of an antibody are known in the art. For example, CDC can be evaluated by determining the binding activity between an antibody to be tested and an Fc receptor (e.g., C1q).
[0131] An “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a human FcR. In addition, a preferred FcR is an FcR (γ receptor) binding to an IgG antibody and includes receptors of FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. An FcγRII receptor includes FcγRIIA (“activated receptor”) and FcγRIIB (“inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activated receptor FcγRIIA comprises an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review M. Daeron, Annu. Rev. Immunol., 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9: 457-92 (1991); Capel et al., Immunomethods, 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126: 330-41 (1995). The term “FcR” herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol., 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)).
[0132] A “polynucleotide” or “nucleic acid” refers to a nucleotide strand of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a strand by a DNA or RNA polymerase.
[0133] “Specific binding” or “selective binding” refers to binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody binds to a predetermined antigen or epitope thereof with an equilibrium dissociation constant (K.sub.D) of about less than 10.sup.−7 M or even less and with an affinity that is at least twice as high as its affinity for binding to a non-specific antigen other than the predetermined antigen (or epitope thereof) or a closely related antigen (e.g., BSA, etc.), when determined by surface plasmon resonance (SPR) techniques in an instrument using recombinant human IL-2 or an epitope thereof as the analyte and an antibody as the ligand. The term “antigen-recognizing antibody” is used interchangeably herein with the term “specifically bound antibody”.
[0134] “Affinity” is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or an antigen-binding fragment thereof and an antigen). The term “affinity” is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules can be quantified by determining the dissociation constant (K.sub.D). K.sub.D can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K.sub.D is related to ka and kd by the equation K.sub.D=kd/ka. The value of the dissociation constant can be determined directly by well-known methods and can be calculated by methods such as those described by Caceci et al (1984, Byte 9: 340-362) even for complex mixtures. For example, K.sub.D can be determined by using a dual filtration nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for evaluating the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, western blot, RIA and flow cytometry, as well as other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore™ system or KinExA. The binding avidities associated with different molecular interactions, e.g., the binding avidities of different antibodies to a given antigen, can be compared by comparing the K.sub.D values of antibodies/antigen complexes. Similarly, the specificity of an interaction can be evaluated by determining and comparing the K.sub.D value for the interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the K.sub.D value for the non-interaction of interest (e.g., a control antibody known not to bind to IL-2). In some embodiments, an anti-IL-2 antibody or an antigen-binding fragment of the present disclosure is capable of binding to its target with an affinity at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold greater than its affinity for binding to another non-IL-2 molecule, without limitation herein.
[0135] A “complex” of an anti-IL-2 antibody or an antigen-binding fragment thereof and IL-2 refers to a complex comprising at least one anti-IL-2 antibody or antigen-binding fragment thereof of the present disclosure, which specifically binds to IL-2 or cells expressing IL-2. The complex comprises an anti-IL-2 antibody or an antigen-binding fragment thereof and an IL-2 molecule that are bound by covalent force, non-covalent force, or any other force. In some embodiments, the anti-IL-2 antibody or the antigen-binding fragment thereof forms a fusion protein with IL-2. In other embodiments, the anti-IL-2 antibody or the antigen-binding fragment thereof binds to IL-2 by non-covalent force. In some embodiments, the anti-IL-2 antibody or the antigen-binding fragment thereof and the IL-2 may still be maintained as a complex even after administration of the complex. It should be understood that, among other variables, the anti-IL-2 antibody or the antigen-binding fragment thereof and the IL-2 will form complexes that differ in binding ability based on the different K.sub.D values of their binding interactions.
[0136] “Inhibition” and “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition and blocking are also intended to include any measurable reduction in binding affinity and cell (e.g., T cell) proliferation-promoting activity of IL-2 in contact with an anti-IL-2 antibody, as compared to IL-2 not in contact with an anti-IL-2 antibody.
[0137] An “antibody that competes for binding with” a reference antibody refers to an antibody that blocks binding of the reference antibody to an antigen by 50% or more, or an antibody whose binding to an antigen is blocked by 50% or more by the reference antibody, in a competition assay. The antigen-binding activity of the anti-IL-2 antibody of the present disclosure can be tested by known methods such as ELISA and western blot. For example, antibodies that compete for binding to IL-2 can be identified by a competition assay. In certain embodiments, the competitive antibody binds to the same epitope (e.g., a linear or conformational epitope) as the antigen-binding molecule or the anti-IL-2 antibody. For example, a high-throughout method for obtaining antibodies that bind to the same epitopes based on cross-competition of antibodies is described in International Patent Publication No. WO03/48731. Therefore, an antibody and an antigen-binding fragment thereof that competes with the antibody molecule of the present disclosure for binding to the same epitope on IL-2 can be obtained using conventional techniques known to those skilled in the art.
[0138] “Giving”, “administering” and “treating”, when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluid, refer to contact of an exogenous drug, a therapeutic agent, a diagnostic agent or a composition with the animals, humans, subjects, cells, tissues, organs or biological fluid. “Giving”, “administering,” and “treating” can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with fluid, wherein the fluid are in contact with the cells. “Giving”, “administering” and “treating” refers to treating, e.g., a cell, by a reagent, diagnosis, a binding composition, or by another cell in vitro and ex vivo. “Treating”, when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or prophylactic measures, and research and diagnostic applications.
[0139] “Treating” or “treatment” refers to administering a therapeutic agent, such as a composition comprising any of the IL-2 antibodies or the antigen-binding fragments thereof of the present disclosure or complexes thereof with IL-2, either internally or externally to a subject who has had, is suspected of having, or is predisposed to having one or more immune-related diseases or symptoms thereof on which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also referred to as the “therapeutically effective amount”) may vary depending on factors such as the disease state, age and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a symptom of the disease has been alleviated can be evaluated by any clinical testing methods commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may be ineffective in alleviating symptoms of a disease of interest in a certain subject, they shall alleviate the symptoms of the disease of interest in a statistically significant number of subjects as determined by any statistical test method known in the art, such as the Student's t-test, Chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and Wilcoxon test.
[0140] An “effective amount” comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the factors such as the condition to be treated, the general health of the subject, the method and route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
[0141] “Homology” or “identity” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The homology percentage between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared×100%. For example, if 6 out of 10 positions are matched or homologous when two sequences are optimally aligned, the two sequences are 60% homologous. Generally, the comparison is made when the two sequences are aligned to give the greatest homology percentage.
[0142] A “host cell” includes various cells or cell cultures and may be or has been the receptor of a vector for incorporation of the polynucleotide insert. The host cell includes progenies of a single host cell, and the progenies may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutations. The host cell includes cells transfected and/or transformed in vivo with polynucleotides of the present disclosure. “Cell”, “cell line” and “cell culture” are used interchangeably, and all such designations include their progenies. It should also be understood that all progenies may not be exactly identical in DNA content due to intentional or unintentional mutations. Mutant progenies with the same function or biological activity as those screened from the original transformed cells are included.
[0143] A “vector” refers to a construct capable of delivering and, in some embodiments, expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.
[0144] The term “optional” or “optionally” means that the event or circumstance subsequently described may, but not necessarily, occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, “optionally comprising 1-3 antibody heavy chain variable regions” means that an antibody heavy chain variable region of a particular sequence may, but not necessarily, be present.
[0145] The term “pharmaceutical composition” refers to a mixture containing one or more of the antibodies or the antigen-binding fragments described herein or a physiologically/pharmaceutically acceptable salt or pro-drug thereof, and other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to an organism, which facilitates the absorption of the active ingredient, thereby exerting biological activities.
[0146] A “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as a phosphate buffered saline solution, water, an emulsion such as an oil/water emulsion, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, eds., Mack Publishing Co., Easton, Pa., 1990; and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).
[0147] Herein, when “about” is applied to a value or parameter, it includes (and describes) embodiments involving the value or parameter itself. For example, a description referring to “about X” includes a description of “X”. A numerical range includes the numbers defining the range. Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical values inherently contain certain errors necessarily resulting from the standard deviation found in their respective tests or measurements. Moreover, all ranges disclosed herein should be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; i.e., all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.
EXAMPLES
[0148] The present disclosure is further described below with reference to examples, which, however, are not intended to limit the scope of the present disclosure.
[0149] Experimental procedures without specific conditions indicated in the examples or test examples are generally conducted according to conventional conditions, or according to conditions recommended by the manufacturer of the starting materials or commercial products, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents without specific origins indicated are commercially available conventional reagents.
Example 1. Screening of Anti-IL-2 Antibodies
[0150] A) Screening of Human Natural Phage Fab Library
[0151] The preserved natural library phage suspension was diluted and blocked with BSA, and incubated with magnetic bead Dynabeads (M-280, Invitrogen). The phages were collected after negative screening and incubation. The beads were bound and washed according to the Kingfisher magnetic bead screening system method (Thermo Scientific), and incubated with 5% BSA. Dynabeads were coated and blocked by biotin-labeled IL-2 with a human Fc-tag (Sanyou Biopharmaceutical, GenBank Accession No: P60568-1), incubated with a phage suspension collected after negative screening, and bound and washed according to the Kingfisher magnetic bead screening system method. The phages were eluted with pancreatin. After the elution, the phage solution was mixed well with E. coli SS320 cells (Sanyou Biopharmaceutical) in the logarithmic growth phase, and incubated at 37° C. for 30 min. E. coli SS320 cells were applied to a 2YT-Car.sup.+-Tet.sup.+ plate and cultured overnight in an incubator at 37° C. The phage input, output and the like were calculated, and the input phages were prepared by scraping plates and screened for 3 rounds.
[0152] B) Primary Screening for Monoclonal Phages Expressing Anti-IL-2 Fab Capable of Binding to IL-2 by ELISA
[0153] 2000 clones from the 3rd round of screening were picked and cultured overnight in a 96-well deep-well plate. The supernatant was collected by centrifugation. Anti-human IgG (STAR161, Bio-rad) was diluted to 2 μg/mL with PBS and added at 30 μL/well to coat an ELISA plate, and the plate was incubated overnight at 4° C. The plate was washed with PBST (PBS, pH 7.4+0.1% Tween 20) 3 times. The plate was blocked with 1% BSA for 1 h at room temperature and washed with PBST 3 times. 30 μL of the supernatant obtained by centrifugation was added, and the plate was incubated at room temperature for 2 h and washed with PBST 3 times. Biotin-labeled IL-2 with a human Fc-tag was diluted to 2 μg/mL with PBS and added to the plate at 30 μL/well. The plate was incubated at room temperature for 1 h and washed with PBST 3 times. 30 μL of secondary antibody NeutrAvidin-HRP diluted in a 1:8000 ratio was added. The plate was incubated at room temperature for 1 h and washed with PBST 9 times. 30 μL of TMB was added for color developing for 5-10 min at room temperature, then 30 μL of 2M HCl or H.sub.2SO.sub.4 was added to terminate the reaction. The plate was read for data by a microplate reader at OD 450. Finally, 364 clones with specific sequences were obtained.
[0154] C) Primary Screening for Anti-IL-2 Fab Capable of Blocking the Binding of IL-2 to IL-2Rβ by ELISA
[0155] The above 364 clones were inoculated into 50 mL of 2YT-Car.sup.+-Tet.sup.+ medium in a 1:1000 ratio, and cultured overnight at 37° C. The bacterial solution was transferred to a 50 mL centrifuge tube, and centrifuged at room temperature. The supernatant was discarded, and 0.5 mL of protein lysis buffer (10 mM Tris-HCl, pH 9.0, 1 mM EDTA, 5 mM MgCl.sub.2, 25 U/mL Bezonase nuclease (Merck)) pre-cooled to 4° C. was added thereto. The mixture was left to stand on ice and incubated for 1 h, and centrifuged at 4° C. The supernatant was collected for later use.
[0156] IL-2Rβ-Fc (CJ82, Novoprotein) (GenBank Accession No: NP_000869.1) was diluted to 8 μg/mL with PBS and added at 30 μL/well to coat an ELISA plate, and the plate was incubated overnight at 4° C. The plate was washed with PBST 3 times. The plate was blocked with 1% BSA for 1 h at room temperature and washed with PBST 3 times. The obtained Fab lysate was incubated with biotin-labeled IL-2 with a human Fc-tag (2.5 μg/mL) at room temperature for 1 h, and added to the plate at 30 μL/well. The plate was incubated at room temperature for 1 h and washed with PBST 3 times. 30 μL of secondary antibody NeutrAvidin-HRP diluted in a 1:8000 ratio was added. The plate was incubated at room temperature for 1 h and washed with PBST 9 times. 30 μL of TMB was added for color developing for 5-10 min at room temperature, then 30 μL of 2M HCl or H.sub.2SO.sub.4 was added to terminate the reaction. The plate was read for data by a microplate reader at OD 450.
[0157] 6 Fabs with strong capability of blocking the binding of IL-2 to IL-2Rβ were obtained. For 6 fully human antibodies, the sequences of the heavy chain and light chain variable regions are shown in Table 1, and the sequences of the complementarity determining regions (CDRs) according to different numbering systems are shown in Table 2.
TABLE-US-00001 TABLE 1 Sequences of heavy chain (HCVR) and light chain (LCVR) variable regions of fully human anti-IL-2 antibodies Antibody Amino acid sequences of heavy chain variable No. region (VH) and light chain variable region (VL) Sequence No. A2-22 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQ SEQ ID No: 1 APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVY MELSSLRSEDTAVYYCAREDWMEYEYYYGMDVWGQGTLV TVSS VL EIVLTQSPGTLSLSPGERATLSCRASQSVSNNYLAWYQQKPG SEQ ID No: 2 QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YSCQQYGSSPLTFGQGTRLEIK A3-21 VH EVQLLESGGGLAQPGGSLRLSCAGSGFTFSSYAMSWVRQAP SEQ ID No: 3 GKGLEWVSGITGTGYRTYYADSVKGRFTVSRDNSRNTVYL QMNSLRAEDTAVYYCVRESGHYYGWFDSWGQGTLVTVSS VL QSALTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHWYQQV SEQ ID No: 4 PGTVPKLLIDGKGNRPSGVPDRFSGSKSGTSASLAITGLQAE DEADYYCQSYDYMLSGSVVFGRGTKLTVL B2-15 VH EVQLLESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQA SEQ ID No: 5 PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYM ELSSLRSEDTAVYYCAREGNSSGYYNAFDIWGQGTMVTVSS VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG SEQ ID No: 6 QAPRLLIYGASNRATGIPDRFSGSGSGADFTLTISRLEPEDFA VYYCQQYGSSPLTFGGGTKVEIK C2-53 VH EVQLVESGGGLVQPGRSLILSCAASGFTFDDYAMHWVRQAP SEQ ID No: 7 GKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQ MNSLRAEDTALYYCAKDQLRWGTYYYYGMDVWGQGTTV TVSS VL DIQMTQSPSTLSASVGDRVTITCRASQSVSIWVAWYQQKPG SEQ ID No: 8 KAPKLLIYKASSLERGVPTRFSGSGSGTEFTLTISSLQPEDFAT YHCQQYDTGATFGQGTKVEMK D2-60 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQA SEQ ID No: 9 PGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTA YMELRSLRSDDTAVYYCAREYSSWYSGGMDVWGQGTTVT VSS VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGK SEQ ID No: 10 VPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVAT YYCQKYDSAPFTFGQGTRLEIK D3-68 VH QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYGISWVRRAP SEQ ID No: 11 GQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARDLQSPTYYYGSGVIDYWGQGTL VTVSS VL DIQLTQSPSTLSASVGDRVTISCRASRAVSPWLAWYQHRPG SEQ ID No: 12 QAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYRSYPFTFGPGTTVDIR
TABLE-US-00002 TABLE 2 Sequences of heavy chain and light chain complementarity determining regions (CDRs) of fully human anti-IL-2 antibodies Kabat Chothia IMGT AbM Antibody numbering numbering numbering numbering No. CDR system system system system A2-22 HCDR1 SYYMH GYTFTSY GYTFTSYY GYTFTSYYMH (SEQ ID No: 13) (SEQ ID No: 49) (SEQ ID No: 85) (SEQ ID No: 121) HCDR2 IINPSGGSTSYAQKFQG NPSGGS INPSGGST IINPSGGSTS (SEQ ID No: 14) (SEQ ID No: 50) (SEQ ID No: 86) (SEQ ID No: 122) HCDR3 EDWMEYEYYYGMDV EDWMEYEYYYGMDV AREDWMEYEYYYGMDV EDWMEYEYYYGMDV (SEQ ID No: 15) (SEQ ID No: 51) (SEQ ID No: 87) (SEQ ID No: 123) LCDR1 RASQSVSNNYLA RASQSVSNNYLA QSVSNNY RASQSVSNNYLA (SEQ ID No: 16) (SEQ ID No: 52) (SEQ ID No: 88) (SEQ ID No: 124) LCDR2 GASSRAT GASSRAT GA GASSRAT (SEQ ID No: 17) (SEQ ID No: 53) (SEQ ID No: 89) (SEQ ID No: 125) LCDR3 QQYGSSPLT QQYGSSPLT QQYGSSPLT QQYGSSPLT (SEQ ID No: 18) (SEQ ID No: 54) (SEQ ID No: 90) (SEQ ID No: 126) A3-21 HCDR1 SYAMS GFTFSSY GFTFSSYA GFTFSSYAMS (SEQ ID No: 19) (SEQ ID No: 55) (SEQ ID No: 91) (SEQ ID No: 127) HCDR2 GITGTGYRTYYADSVKG TGTGYR ITGTGYRT GITGTGYRTY (SEQ ID No: 20) (SEQ ID No: 56) (SEQ ID No: 92) (SEQ ID No: 128) HCDR3 ESGHYYGWFDS ESGHYYGWFDS VRESGHYYGWFDS ESGHYYGWFDS (SEQ ID No: 21) (SEQ ID No: 57) (SEQ ID No: 93) (SEQ ID No: 129) LCDR1 TGSSSNIGAGYNVH TGSSSNIGAGYNVH SSNIGAGYN TGSSSNIGAGYNVH (SEQ ID No: 22) (SEQ ID No: 58) (SEQ ID No: 94) (SEQ ID No: 130) LCDR2 GKGNRPS GKGNRPS GK GKGNRPS (SEQ ID No: 23) (SEQ ID No: 59) (SEQ ID No: 95) (SEQ ID No: 131) LCDR3 QSYDYMLSGSVV QSYDYMLSGSVV QSYDYMLSGSVV QSYDYMLSGSVV (SEQ ID No: 24) (SEQ ID No: 60) (SEQ ID No: 96) (SEQ ID No: 132) B2-15 HCDR1 SYYMH GYTFTSY GYTFTSYY GYTFTSYYMH (SEQ ID No: 25) (SEQ ID No: 61) (SEQ ID No: 97) (SEQ ID No: 133) HCDR2 IINPSGGSTSYAQKFQG NPSGGS INPSGGST IINPSGGSTS (SEQ ID No: 26) (SEQ ID No: 62) (SEQ ID No: 98) (SEQ ID No: 134) HCDR3 EGNSSGYYNAFDI EGNSSGYYNAFDI AREGNSSGYYNAFDI EGNSSGYYNAFDI (SEQ ID No: 27) (SEQ ID No: 63) (SEQ ID No: 99) (SEQ ID No: 135) LCDR1 RASQSVSSSYLA RASQSVSSSYLA QSVSSSY RASQSVSSSYLA (SEQ ID No: 28) (SEQ ID No: 64) (SEQ ID No: 100) (SEQ ID No: 136) LCDR2 GASNRAT GASNRAT GA GASNRAT (SEQ ID No: 29) (SEQ ID No: 65) (SEQ ID No: 101) (SEQ ID No: 137) LCDR3 QQYGSSPLT QQYGSSPLT QQYGSSPLT QQYGSSPLT (SEQ ID No: 30) (SEQ ID No: 66) (SEQ ID No: 102) (SEQ ID No: 138) C2-53 HCDR1 DYAMH GFTFDDY GFTFDDYA GFTFDDYAMH (SEQ ID No: 31) (SEQ ID No: 67) (SEQ ID No: 103) (SEQ ID No: 139) HCDR2 GISWNSGSIGYADSVKG SWNSGS ISWNSGSI GISWNSGSIG (SEQ ID No: 32) (SEQ ID No: 68) (SEQ ID No: 104) (SEQ ID No: 140) HCDR3 DQLRWGTYYYYGMDV DQLRWGTYYYYGMDV AKDQLRWGTYYYYGMDV DQLRWGTYYYYGMDV (SEQ ID No: 33) (SEQ ID No: 69) (SEQ ID No: 105) (SEQ ID No: 141) LCDR1 RASQSVSIWVA RASQSVSIWVA QSVSIW RASQSVSIWVA (SEQ ID No: 34) (SEQ ID No: 70) (SEQ ID No: 106) (SEQ ID No: 142) LCDR2 KASSLER KASSLER KA KASSLER (SEQ ID No: 35) (SEQ ID No: 71) (SEQ ID No: 107) (SEQ ID No: 143) LCDR3 QQYDTGAT QQYDTGAT QQYDTGAT QQYDTGAT (SEQ ID No: 36) (SEQ ID No: 72) (SEQ ID No: 108) (SEQ ID No: 144) D2-60 HCDR1 SYGIS GYTFTSY GYTFTSYG GYTFTSYGIS (SEQ ID No: 37) (SEQ ID No: 73) (SEQ ID No: 109) (SEQ ID No: 145) HCDR2 WISAYNGNTNYAQKLQG SAYNGN ISAYNGNT WISAYNGNTN (SEQ ID No: 38) (SEQ ID No: 74) (SEQ ID No: 110) (SEQ ID No: 146) HCDR3 EYSSWYSGGMDV EYSSWYSGGMDV AREYSSWYSGGMDV EYSSWYSGGMDV (SEQ ID No: 39) (SEQ ID No: 75) (SEQ ID No: 111) (SEQ ID No: 147) LCDR1 RASQGISNYLA RASQGISNYLA QGISNY RASQGISNYLA (SEQ ID No: 40) (SEQ ID No: 76) (SEQ ID No: 112) (SEQ ID No: 148) LCDR2 AASTLQS AASTLQS AA AASTLQS (SEQ ID No: 41) (SEQ ID No: 77) (SEQ ID No: 113) (SEQ ID No: 149) LCDR3 QKYDSAPFT QKYDSAPFT QKYDSAPFT QKYDSAPFT (SEQ ID No: 42) (SEQ ID No: 78) (SEQ ID No: 114) (SEQ ID No: 150) D3-68 HCDR1 SYGIS GYTFTSY GYTFTSYG GYTFTSYGIS (SEQ ID No: 43) (SEQ ID No: 79) (SEQ ID No: 115) (SEQ ID No: 151) HCDR2 WISAYNGNTNYAQKLQG SAYNGN ISAYNGNT WISAYNGNTN (SEQ ID No: 44) (SEQ ID No: 80) (SEQ ID No: 116) (SEQ ID No: 152) HCDR3 DLQSPTYYYGSGVIDY DLQSPTYYYGSGVIDY ARDLQSPTYYYGSGVIDY DLQSPTYYYGSGVIDY (SEQ ID No: 45) (SEQ ID No: 81) (SEQ ID No: 117) (SEQ ID No: 153) LCDR1 RASRAVSPWLA RASRAVSPWLA RAVSPW RASRAVSPWLA (SEQ ID No: 46) (SEQ ID No: 82) (SEQ ID No: 118) (SEQ ID No: 153) LCDR2 AASSLQS AASSLQS AA AASSLQS (SEQ ID No: 47) (SEQ ID No: 83) (SEQ ID No: 119) (SEQ ID No: 155) LCDR3 QQYRSYPFT QQYRSYPFT QQYRSYPFT QQYRSYPFT (SEQ ID No: 48) (SEQ ID No: 84) (SEQ ID No: 120) (SEQ ID No: 156)
Example 2. Preparation of Full-Length Fully Human Anti-IL-2 Antibodies
[0158] The heavy chain and light chain variable regions of the 6 antibodies of Example 1 were linked to the heavy chain constant region of human IgG.sub.1 and the kappa or lamda chain constant region, respectively, to construct full-length fully human anti-IL-2 antibodies. The heavy chain constant region carries L234A and L235A mutations (LALA mutation) to remove possible ADCC function of the antibody. The full-length sequences of the 6 antibodies are shown in Table 3.
TABLE-US-00003 TABLE 3 Full-length heavy and light chain sequences of fully human anti-IL-2 antibodies Antibody Full-length heavy chain (HC) and light No. chain (LC) amino acid sequences Sequence No. A2-22 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMH SEQ ID No: 157 WVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCAREDWMEYEY YYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK LC EIVLTQSPGTLSLSPGERATLSCRASQSVSNNYLAWY SEQ ID No: 158 QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYSCQQYGSSPLTFGQGTRLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC A3-21 HC EVQLLESGGGLAQPGGSLRLSCAGSGFTFSSYAMSW SEQ ID No: 159 VRQAPGKGLEWVSGITGTGYRTYYADSVKGRFTVSR DNSRNTVYLQMNSLRAEDTAVYYCVRESGHYYGWF DSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK LC QSALTQPPSVSGAPGQRVTISCTGSSSNIGAGYNVHW SEQ ID No: 160 YQQVPGTVPKLLIDGKGNRPSGVPDRFSGSKSGTSAS LAITGLQAEDEADYYCQSYDYMLSGSVVFGRGTKLT VLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPG AVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSY LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS B2-15 HC EVQLLESGAEVKKPGASVKVSCKASGYTFTSYYMH SEQ ID No: 161 WVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTM TRDTSTSTVYMELSSLRSEDTAVYYCAREGNSSGYY NAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ SEQ ID No: 162 QKPGQAPRLLIYGASNRATGIPDRFSGSGSGADFTLTI SRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC C2-53 HC EVQLVESGGGLVQPGRSLILSCAASGFTFDDYAMHW SEQ ID No: 163 VRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCAKDQLRWGTYY YYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK LC DIQMTQSPSTLSASVGDRVTITCRASQSVSIWVAWYQ SEQ ID No: 164 QKPGKAPKLLIYKASSLERGVPTRFSGSGSGTEFTLTI SSLQPEDFATYHCQQYDTGATFGQGTKVEMKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC D2-60 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISW SEQ ID No: 165 VRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTM TTDTSTSTAYMELRSLRSDDTAVYYCAREYSSWYSG GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK LC DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQ SEQ ID No: 166 QKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTI SSLQPEDVATYYCQKYDSAPFTFGQGTRLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC D3-68 HC QVQLVQSGPEVKKPGASVKVSCKASGYTFTSYGISW SEQ ID No: 167 VRRAPGQGLEWMGWISAYNGNTNYAQKLQGRVTM TTDTSTSTAYMELRSLRSDDTAVYYCARDLQSPTYY YGSGVIDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK LC DIQLTQSPSTLSASVGDRVTISCRASRAVSPWLAWYQ SEQ ID No: 168 HRPGQAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQYRSYPFTFGPGTTVDIRRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (Note: the underlined portion of HC is the heavy chain constant region of Human IgG.sub.1, and the underlined portion of LC is the kappa chain constant region)
[0159] The above sequences were synthesized, digested with BamHI and XhoI, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) through the BamHI/XhoI enzymatic digestion site. The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) in a 1:2 ratio, and the cells were placed in a C02 incubator and incubated for 4-5 days. The expressed antibodies were isolated by centrifugation, purified by a conventional method, and identified to obtain the full-length fully human antibodies of the present disclosure.
Example 3. ELISA Assay on Anti-IL-2 Antibodies Capable of Blocking or Reducing the Binding of IL-2 to IL-2Rα and Blocking the Binding of IL-2 to IL-2Rβ
[0160] A) ELISA Assay on the Blocking or Reducing of the Binding of IL-2 to IL-2Rα by Antibodies
[0161] IL-2Rα-Fc (CJ78, Novoprotein) (Accession #NP_000408) was diluted to 0.5 μg/mL with PBS and added at 30 μL/well to coat an ELISA plate, and the plate was incubated overnight at 4° C. The plate was washed with PBST 3 times. The plate was blocked with 1% BSA for 1 h at room temperature and washed with PBST 3 times. The anti-IL-2 antibody was diluted in a gradient to 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.63 μg/mL, 0.31 μg/mL, 0.16 μg/mL, and 0.08 μg/mL with PBS, pH 7.4, incubated with biotin-labeled IL-2 with a human Fc-tag (final concentration: 1 μg/mL) at room temperature for 15 min, added to the plate at 30 μL/well. The plate was incubated at room temperature for 1 h, and washed with PBST 3 times. 30 μL of secondary antibody NeutrAvidin-HRP diluted in a 1:6000 ratio was added, and the plate was incubated at room temperature for 1 h and washed with PBST 9 times. 30 μL of TMB was added for color developing for 5-10 min at room temperature, then 30 μL of 2 M HCl or H.sub.2SO.sub.4 was added to terminate the reaction. The plate was read for data by a microplate reader at OD 450.
[0162] The results are shown in
TABLE-US-00004 TABLE 4 IC.sub.50 values for anti-IL-2 antibodies blocking the binding of IL-2 to IL-2Rα Antibody No. IC.sub.50 (μg/mL) A2-22 N.A. A3-21 1.456 B2-15 N.A. C2-53 0.6591 D2-60 N.A. D3-68 1.196 (Note: N.A.: due to the weak blocking effect, no meaningful IC.sub.50 values could be obtained by fitting the curve.)
[0163] B) ELISA Assay on the Blocking of the Binding of IL-2 to IL-2Rβ by Antibodies
[0164] IL-2Rβ-Fc (CJ82, Novoprotein) was diluted to 8 μg/mL with PBS and added at 30 μL/well to coat an ELISA plate, and the plate was incubated overnight at 4° C. The plate was washed with PBST 3 times. The plate was blocked with 1% BSA for 1 h at room temperature and washed with PBST 3 times. The anti-IL-2 antibody was diluted in a gradient to 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.63 μg/mL, 0.31 μg/mL, 0.16 μg/mL, and 0.08 μg/mL with PBS, pH 7.4, incubated with biotin-labeled IL-2 with a human Fc-tag (final concentration: 5 μg/mL) at room temperature for 15 min, added to the plate at 30 μL/well. The plate was incubated at room temperature for 1 h, and washed with PBST 3 times. 30 μL of secondary antibody NeutrAvidin-HRP diluted in a 1:6000 ratio was added, and the plate was incubated at room temperature for 1 h and washed with PBST 9 times. 30 μL of TMB was added for color developing for 5-10 min at room temperature, then 30 μL of 2 M HCl or H.sub.2SO.sub.4 was added to terminate the reaction. The plate was read for data by a microplate reader at OD 450.
[0165] The results are shown in
TABLE-US-00005 TABLE 5 IC.sub.50 values for anti-IL-2 antibodies blocking the binding of IL-2 to IL-2Rβ IC.sub.50 Antibody No. (μg/mL) A2-22 2.728 A3-21 3.475 B2-15 3.045 C2-53 1.441 D2-60 8.272 D3-68 2.134
Example 4. Octet Assay on Anti-IL-2 Antibodies Blocking or Reducing the Binding of IL-2 to IL-2Rα and Blocking the Binding of IL-2 to IL-2Rβ
[0166] A) Octet Assay on the Blocking or Reducing of the Binding of IL-2 to IL-2Rα by Antibodies
[0167] A Streptavidin biosensor (Fortebio, #18-5020) was immersed in 200 μL of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 s for the wetting treatment. Then, biotinylated IL-2Rα-Fc (CJ78, Novoprotein) was diluted to 10 μg/mL with the KB buffer, and the sensor was immersed in 200 μL of the solution for 150 s. The sensor was immersed in the KB buffer for 60 s to elute excess IL-2Rα. IL-2-His (CX66, Novoprotein) was mixed with an anti-IL-2 antibody and diluted with KB buffer to final concentrations of 100 nM and 500 nM, and incubated at room temperature for 30 min. The sensor was immersed in the mixture for 300 s for association. The stronger the ability of the anti-IL-2 antibody to block or reduce the binding of IL-2 to IL-2Rα is, the lower the reading is. The values at 230 s after the beginning of the binding were read. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 The blocking or reducing of the binding of IL-2 to IL-2Rα by anti-IL-2 antibodies in Octet assay Values for blocking IL-2Rα Antibody No. at an Octet level C2-53 0.410 A3-21 0.428 D3-68 0.473 B2-15 0.700 D2-60 0.754 A2-22 0.848
[0168] B) Octet Assay on the Blocking of the Binding of IL-2 to IL-2Rβ by Antibodies
[0169] A Streptavidin biosensor (Fortebio, #18-5020) was immersed in 200 μL of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 s for the wetting treatment. Then, biotinylated IL-2-Fc (Sanyou Biopharmaceutical) was diluted to 10 μg/mL with the KB buffer, and the sensor was immersed in 200 μL of the solution for 300 s. The sensor was immersed in the KB buffer for 60 s to elute excess IL-2. The anti-IL-2 antibody was diluted to a final concentration of 500 nM with the KB buffer, and the sensor was immersed in the antibody solution for association for 300 s. The sensor was immersed in the KB buffer for 60 s to elute excess anti-IL-2 antibody. The IL-2Rβ-Fc (CJ82, Novoprotein) was diluted to a final concentration of 8000 nM with the KB buffer, and the sensor was immersed in the antibody solution for association for 300 s. The stronger the ability of the anti-IL-2 antibody to block the binding of IL-2 to IL-2Rβ is, the lower the reading is. The values at 230 s after the beginning of the binding of IL-2Rβ-Fc were read. The results are shown in Table 7.
TABLE-US-00007 TABLE 7 The blocking of the binding of IL-2 to IL- 2Rβ by anti-IL-2 antibodies in Octet assay Values for blocking IL-2Rβ Antibody No. at an Octet level A3-21 0.003 C2-53 0.017 D3-68 0.027 A2-22 0.042 B2-15 0.056 D2-60 0.086
Example 5. ELISA Assay on Binding of Anti-IL-2 Antibodies to IL-2
[0170] Anti-human IgG (STAR161, Bio-rad) was diluted to 2 μg/mL with PBS and added at 30 μL/well to coat an ELISA plate, and the plate was incubated overnight at 4° C. The plate was washed with PBST 3 times. The plate was blocked with 1% BSA for 1 h at room temperature and washed with PBST 3 times. The anti-IL-2 antibody was diluted in a gradient to 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL, 0.3125 μg/mL, and 0.15625 μg/mL with PBS. 30 μL of the antibody solution was added to an ELISA plate, and the plate was incubated at room temperature for 1 h and washed with PBST 3 times. Biotin-labeled IL-2 with a human Fc-tag (Sanyou Biopharmaceutical) was diluted to 2 μg/mL with PBS and added to the plate at 30 μL/well. The plate was incubated at room temperature for 1 h and washed with PBST 3 times. 30 μL of secondary antibody NeutrAvidin-HRP diluted in a 1:6000 ratio was added. The plate was incubated at room temperature for 1 h and washed with PBST 9 times. 30 μL of TMB was added for color developing for 5-10 min at room temperature, then 30 μL of 2M HCl or H.sub.2SO.sub.4 was added to terminate the reaction. The plate was read for data by a microplate reader at OD 450. The results are shown in
TABLE-US-00008 TABLE 8 EC50 values for anti-IL-2 antibodies binding to IL-2 EC.sub.50 Antibody No. (μg/mL) A2-22 1.289 A3-21 1.350 B2-15 2.117 C2-53 0.7301 D2-60 1.428 D3-68 1.205
Example 6. Biacore Assay on Affinity and Kinetics of Anti-IL-2 Antibodies for IL-2
[0171] Antibodies were captured with a Protein A sensor chip (GE, Cat #29127556) of a Biacore instrument (Biacore T200, GE), in which anti-IL-2 antibodies were diluted to 1 μg/mL with 1×HBS-EP, and flowed at a flow rate of 10 μL/min for 30 s. Then, IL-2 (C013, Novoprotein) solutions at a series of concentration gradients flowed over the chip surface at a flow rate of 30 μL/min for association for 120 s. The solutions flowed at a flow rate of 30 μL/min for dissociation for 300 s. The reaction signals were detected in real time to obtain association and dissociation curves. After the dissociation was completed for each assay cycle, the chip was washed and regenerated with 10 mM Gly-HCl, pH 2.0. The data obtained from the assay were fitted with a 1:1 binding model to obtain the affinity values of the anti-IL-2 antibodies for IL-2. The results are shown in Table 9. The results showed that all antibodies could bind to IL-2 with affinity values spanning from 0.267 nM for C2-53 to 39.8 nM for A2-22.
TABLE-US-00009 TABLE 9 Affinity of anti-IL-2 antibodies for IL-2 Kon Koff KD Antibody No. (1/Ms) (1/s) (M) C2-53 1.64E+07 4.37E−03 2.67E−10 D3-68 1.16E+07 5.46E−02 4.72E−09 D2-60 5.09E+05 6.51E−03 1.28E−08 B2-15 1.46E+06 4.48E−02 3.06E−08 A2-22 1.33E+06 5.27E−02 3.98E−08
Example 7. Determination of Activity of Non-Covalent Complexes of Anti-IL-2 Antibody and IL-2 for STAT5 Phosphorylation in Human Peripheral Blood PBMC
[0172] The ability of the antibodies to regulate IL-2 activity was detected based on the levels of STAT5 phosphorylation in various cell populations (including Tregs, CD4+ T cells, and CD8+ T cells) in human peripheral blood treated with different concentrations of non-covalent complexes of anti-IL-2 antibodies and IL-2.
[0173] Basic medium: RPMI 1640+10% fetal bovine serum.
[0174] Antibody mixture: CD3 APC-Cy7 (BD 557832), CD4 BB515 (BD 564419), CD8 BB700 (BD 566452), CD25 BV421 (BD 564033), and pSTAT5 AF647 (BD 562076).
[0175] Human PBMC STAT5 phosphorylation assay: freshly isolated human PBMC cells were adjusted to a density of 6.5×10.sup.6 cells/mL with a basic medium, and 80 μL of the mixture were added into a 96-well plate. The anti-IL-2 antibody at different concentrations was premixed with IL-2 at different concentrations for 30 min at room temperature, and 20 μL of the mixture was added to 90 μL of PBMC and stimulated at 37° C. for 20 min. Immediately thereafter, the cells were immobilized with a pre-warmed BD Cytofix buffer (BD, Cat No. 554655) at 37° C. for 15 min and then immobilized on ice for another 15 min. The mixture was centrifuged at 400 g for 7 min at 4° C. The supernatant was removed, and the plate was washed with 150 μL of PBS once. 150 μL of BD Phosflow Perm Buffer III (BD, Cat No. 558050) pre-cooled at −20° C. was added to perform membrane rupture overnight at −20° C. The mixture was centrifuged at 500 g for 7 min at 4° C. The supernatant was removed, and the plate was washed with 150 μL of PBS, pH 7.4 twice. 100 μL of FcR blocking reagent diluted in a 1:200 ratio was added, and the plate was incubated at 4° C. for 20 min. The mixture was centrifuged at 500 g for 7 min at 4° C. The supernatant was removed, 50 μL of the antibody mixture was added, and the cells were stained at 4° C. for 1 h. The mixture was centrifuged at 500 g for 7 min at 4° C. The supernatant was removed, and the plate was washed with 200 μL of PBS, pH 7.4 once. The cells were resuspended in 150 μL of PBS at pH 7.4 and detected by flow cytometry (BD FACSCelesta). CD8.sup.+ T cells were defined as CD3.sup.+CD4.sup.−CD8.sup.+ cells, and Tregs were defined as CD3.sup.+CD4.sup.+CD8.sup.+CD25.sup.+ cells.
[0176] The fluorescence (MFI) values of pSTAT5 were counted for both cell populations, and the percentage of the value at each concentration to the maximum pSTAT5 MFI value in the fully activated state was calculated and fitted using a computer program or four-parameter regression calculation method. The results are shown in
[0177] The results showed that the IgG.sub.1 isotypes did not have any effect on the activation of pSTAT5 in Tregs and CD8.sup.+ T cells by IL-2 as antibody concentration increased. In contrast, A3-21 and C2-53 did not substantially affect the activity of IL-2 for Tregs, but significantly reduced the activity of IL-2 for CD8.sup.+ T cells over a wide concentration range. Only at high concentrations, such as 100 nM (A3-21) and 33.3 nM (C2-53), there was a certain decreased in the activity of IL-2 for Tregs, but under that condition, IL-2 completely had no activity for CD8.sup.+ T cells. In other words, A3-21 and C2-53 could inhibit or to a greater extent reduce, the activity of IL-2 for immune effector cells such as CD8.sup.+ T cells while not affecting or to a lesser extent reducing, the activity of IL-2 for Tregs. Similarly, D3-68 may also reduce the activity of IL-2 for CD8.sup.+ T cells to a greater extent than that for Tregs, although this ability may not be as significant as that of A3-21 and C2-53.
Example 8. Preparation of Fusion Proteins
[0178] Non-covalent complexes of anti-IL-2 antibody and IL-2 may be easily dissociated in vivo to produce free IL-2, resulting in toxicity. We obtained 12 fusion proteins of anti-IL-2 antibody and IL-2 by conjugating human IL-2 to the N-terminus of the heavy or light chain of anti-IL-2 antibody via a (G.sub.4S).sub.5 linker arm (i.e., GGGGSGGGGSGGGGSGGGGSGGGGS).
[0179] For antibody numbering, if IL-2 is at the N-terminus of the heavy chain of the antibody, the fusion protein is suffixed a; and if IL-2 is at the N-terminus of the light chain of the antibody, the fusion protein is suffixed b.
[0180] For fused IL-2, it carries a T3A mutation (i.e., mutation of the amino acid at position 3 from Thr to Ala) to remove possible glycosylation. The mature human IL-2 protein does not contain amino acid M at position 1, so the numbering starts at amino acid A at position 2. Amino acid sequences of the 12 fusion proteins are as follows:
TABLE-US-00010 TABLE 10 Full-length sequences of fusion proteins Antibody Full-length heavy chain (HC) and full- Sequence No. length light chain (HL) amino acid sequences No. A2-22-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 169 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTS YYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGR VTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDWME YEYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK LC SEQ ID No: 158 SEQ ID No: 158 A2-22-b HC SEQ ID No: 157 SEQ ID No: 157 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 170 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSNNY LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYSCQQYGSSPLTFGQGTRLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC A3-21-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 171 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGGGLAQPGGSLRLSCAGSGFTFSSY AMSWVRQAPGKGLEWVSGITGTGYRTYYADSVKGRF TVSRDNSRNTVYLQMNSLRAEDTAVYYCVRESGHYY GWFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK LC SEQ ID No: 160 SEQ ID No: 160 A3-21-b HC SEQ ID No: 159 SEQ ID No: 159 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 172 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSQSALTQPPSVSGAPGQRVTISCTGSSSNIGAGY NVHWYQQVPGTVPKLLIDGKGNRPSGVPDRFSGSKSG TSASLAITGLQAEDEADYYCQSYDYMLSGSVVFGRGT KLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFY PGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS B2-15-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 173 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGAEVKKPGASVKVSCKASGYTFTS YYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGR VTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGNSSG YYNAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK LC SEQ ID No: 162 SEQ ID No: 162 B2-15-b HC SEQ ID No: 161 SEQ ID No: 161 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 174 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSY LAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGAD FTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC C2-53-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 175 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGRSLILSCAASGFTFDDY AMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCAKDQLRWG TYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK LC SEQ ID No: 164 SEQ ID No: 164 C2-53-b HC SEQ ID No: 163 SEQ ID No: 163 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 176 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSVSIW VAWYQQKPGKAPKLLIYKASSLERGVPTRFSGSGSGTE FTLTISSLQPEDFATYHCQQYDTGATFGQGTKVEMKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC D2-60-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 177 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTS YGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREYSSW YSGGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK LC SEQ ID No: 166 SEQ ID No: 166 D2-60-b HC SEQ ID No: 165 SEQ ID No: 165 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 178 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYL AWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDF TLTISSLQPEDVATYYCQKYDSAPFTFGQGTRLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC D3-68-a HC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 179 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSQVQLVQSGPEVKKPGASVKVSCKASGYTFTS YGISWVRRAPGQGLEWMGWISAYNGNTNYAQKLQGR VTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLQSPT YYYGSGVIDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK LC SEQ ID No: 168 SEQ ID No: 168 D3-68-b HC SEQ ID No: 167 SEQ ID No: 167 LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTR SEQ ID No: MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS 180 KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGG SGGGGSDIQLTQSPSTLSASVGDRVTISCRASRAVSPWL AWYQHRPGQAPKLLIYAASSLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQYRSYPFTFGPGTTVDIRRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC
[0181] The above 12 fusion proteins were expressed and purified according to the method of Example 2. The expression level and purity in SEC-HPLC are shown in Table 11 below.
TABLE-US-00011 TABLE 11 Expression level and purity of fusion proteins Expression Purity Antibody No. level (mg/L) (%) A2-22-a 39 90.780 A2-22-b 104 96.308 A3-21-a 82 89.358 A3-21-b 2 82.095 B2-15-a 89 53.821 B2-15-b 70 99.839 C2-53-a N.A. 54.940 C2-53-b 6 26.598 D2-60-a 2.4 40.248 D2-60-b 129 97.540 D3-68-a 0.3 N.A. D3-68-b 106 100
[0182] The results showed that for different anti-IL-2 antibodies, IL-2 conjugated at the N-terminus of the heavy or light chain greatly affected the expression level and purity of the fusion protein. The low expression level or purity suggests that IL-2 and antibody may not form normal intramolecular binding but intermolecular binding in the fusion protein, thereby causing aggregation of the protein. The fusion proteins with higher expression level (at least 20 mg/L) and purity (at least 90%) were picked for subsequent identification.
Example 9. Octet Assay on Binding of Fusion Proteins to IL-2Rα and IL-2Rβ
[0183] A Protein A biosensor (Fortebio, #18-5010) was immersed in 200 μL of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 s for the wetting treatment. Then, the fusion protein was diluted to 10 μg/mL with the KB buffer, and the sensor was immersed in 200 μL of the solution until the reading was 1.2 nm. The sensor was immersed in the KB buffer for 100 s to elute excess antibody-IL-2 fusion protein. IL-2Rα (ILA-H52H9, Acrobiosystem) was diluted in a 2-fold gradient to 100-3.125 nM with the KB buffer. The sensor was immersed in the solution for 60 s for association. The sensor was immersed in the KB buffer for 60 s for dissociation. The data were fitted in a dynamic 1:1 binding mode. The affinity of fusion proteins for IL-2Rα is shown in Table 12. The results showed that IL-2 conjugated with anti-IL-2 antibodies had reduced binding to IL-2Rα as compared to IL-2, among which A3-21-a and D3-68-b did not bind to IL-2Rα at all.
TABLE-US-00012 TABLE 12 Affinity of fusion proteins for IL-2Rα Kon Koff KD Test sample (1/Ms) (1/s) (M) IL-2 1.40E+06 2.51E−02 1.80E−08 A2-22-a 9.22E+05 3.75E−02 4.06E−08 A2-22-b 1.63E+06 4.13E−02 2.53E−08 A3-21-a N.A. N.A. No binding B2-15-b 1.23E+06 3.30E−02 2.68E−08 D2-60-b 1.24E+06 5.61E−02 4.51E−08 D3-68-b N.A. N.A. No binding (Note: N.A.: no numerical values can be obtained by fitting due to no binding. In an assay on affinity of IL-2 for IL-2Rα, IL-2Rα was used as a stationary phase, and IL-2 was used as a mobile phase).
[0184] Similar to the above methods, the data were fitted in a steady-state mode by Octet. The affinity of antibody-IL-2 fusion proteins for IL-2Rβ (CD2-H5221, Acrobiosystem) determined is shown in Table 13. The antibody fusion protein was used as a stationary phase, and the IL-2Rβ was used as a mobile phase. The results showed that most of IL-2 conjugated with anti-IL-2 antibodies did not bind to IL-2Rβ at all as compared to IL-2, and individual antibody-IL2 fusion proteins, such as D3-68-b, were weakened in binding to IL-2Rβ, but still had a certain degree of binding. In an assay on affinity of IL-2 for IL-2Rβ, IL-2Rβ was used as a stationary phase, and IL-2 was used as a mobile phase.
TABLE-US-00013 TABLE 13 Affinity of fusion proteins of anti- IL-2 antibody and IL-2 for IL-2Rβ KD Test sample (nM) IL-2 4.6E−07 A2-22-a No binding A2-22-b No binding A3-21-a No binding B2-15-b No binding D2-60-b No binding D3-68-b 1.1E−06
Example 10. Determination of Activity of Fusion Proteins for STAT5 Phosphorylation in Human Peripheral Blood (PBMC)
[0185] The activity of fusion proteins for STAT5 phosphorylation in Tregs and CD8.sup.+ T cells in human peripheral blood (PBMC) was determined according to the method in Example 7. As shown in
[0186] However, for D3-68-b, its activity for Tregs was reduced more than that for CD8.sup.+ T cells (shown in
TABLE-US-00014 TABLE 14 EC50 values for fusion proteins activating pSTAT5 in Treg cells EC.sub.50 Test sample (nM) IL-2 0.0003714 A2-22-a 0.04439 A2-22-b 0.05677 B2-15-b 0.01072 D2-60-b 0.1940 D3-68-b 0.1731 A3-21-a 0.1923
TABLE-US-00015 TABLE 15 EC.sub.50 values for fusion proteins activating pSTAT5 in CD8.sup.+ T cells EC.sub.50 Test sample (nM) IL-2 0.4343 A2-22-a N.A. A2-22-b N.A. B2-15-b N.A. D2-60-b N.A. D3-68-b 31.06 A3-21-a N.A. (Note: N.A.: values at the highest test concentration were still low, and accurate EC.sub.50 values could not be obtained by fitting).
Example 11. Determination of Effect of Fusion Proteins on Immune Cells in Peripheral Blood of Balb/c Mice
[0187] Female BALB/c mice (purchased from Charles River Laboratories, Beijing) aged 6-8 weeks and weighing 18-20 g were subjected to adaptive feeding for 5 days before the experiment. All BALB/C mice were housed in an IVC constant temperature and pressure system in an SPF-grade animal room at a temperature of 20-26° C. with humidity at 40%-70%, in a 12112 hour light/dark cycle. Not more than 6 BALB/c mice were housed in each cage. Mice were grouped according to body weight, and administration was performed after the grouping. The type of drug administered, the dose of administration and the route of administration are shown in Table 16. The day of model grouping was day 0.
TABLE-US-00016 TABLE 16 Administration regimen for mice Dose of Route Mode Treat- administration of of Groups N ment (mg/kg) administration administration 1 3 A2-22-b 0.7 Subcutan- Single eously administration 2 3 B2-15-b 0.7 Subcutan- Single eously administration 3 3 D2-60-b 0.7 Subcutan- Single eously administration
[0188] Freshly collected anticoagulated blood at each time point was lysed with red blood cell lysis buffer and washed with PBS once. A mixed staining solution was prepared with PBS containing 1% FBS. The mixed staining solution comprises CD3 APC-Cy7 (Biolegend 100329), CD8 PE (Biolegend 100708), CD4 PE-Cy7 (eBioscience 25-0042-82) and CD25 PerCP-Cy5.5 (BD 561112). 100 μL of mixed staining solution was added to each sample, and the mixture was incubated at 4° C. for 30 min. The mixture was washed with PBS containing 1% FBS twice. Fixation and membrane rupture were performed using True-Nuclear™ Transcription Factor Buffer Set (Biolegend 424401) for 60 min, and 100 μL of anti-mouse Foxp3 antibody (Biolegend 126405) and anti-mouse Ki67 antibody (eBioscience, 25-5698-82) were incubated at room temperature for 60 min. The mixture was washed with PBS (pH 7.4) twice, finally resuspended in 500 μL of PBS washing buffer at pH 7.4, and analyzed using machine. CD8.sup.+ T cells were defined as CD3.sup.+CD4.sup.−CD8.sup.+ cells, and Tregs were defined as CD3.sup.+CD4.sup.+CD25.sup.+Foxp3.sup.+ cells.
[0189] The experimental results are shown in
Example 12. Determination of Effect of Fusion Proteins on Immune Cells in Spleen of Balb/c Mice Immunized with Chicken Ovalbumin OVA
[0190] Chicken ovalbumin (OVA, Sigma A5503) was dissolved in PBS to obtain a solution at 0.5 mg/mL. Freund's complete adjuvant (CFA, containing 1 mg/mL inactivated Mycobacterium tuberculosis, Sigma, F5881). Male C57B16/J mice (purchased from Shanghai Laboratory Animal Center) aged 6-8 weeks and weighing 18-20 g. Mice were grouped according to body weight. On day 1 of the experiment, 8 mL of OVA solution was taken, mixed well with 8 mL of CFA, and emulsified to form a water-in-oil solution. Each animal was immunized by intraperitoneal injection of 200 μL of the solution. The mice in all groups were each injected subcutaneously (sc) with 10 mL/kg test fusion protein or PBS control on Days 3 and 8. The type of drug administered, the dose of administration and the route of administration are shown in Table 17.
TABLE-US-00017 TABLE 17 Immunization regimen for mice Dose of Group Test Route of administration Time of No. Number Immunization sample administration (mpk) administration 1 4 OVA (50 μg) PBS Subcutaneously NA Days 3 and 8 2 4 OVA (50 μg) A2-22-b Subcutaneously 0.7 Days 3 and 8 3 4 OVA (50 μg) B2-15-b Subcutaneously 0.7 Days 3 and 8 4 4 OVA (50 μg) D2-60-b Subcutaneously 0.7 Days 3 and 8
[0191] On Day 10, mice were euthanized, and spleens were taken and processed for detection of relevant immune cells by flow cytometry. The procedures were as follows: after being immersed in 75% ethanol for 5 min, the spleen of the mouse was taken out under the aseptic condition, rinsed 1-2 times with PBS, and then cut into pieces. The filter mesh was placed in a 50 mL centrifuge tube, and spleen tissue pieces were transferred to the mesh and ground while fresh PBS was added continuously. The suspension after grinding was centrifuged at 1800 rpm for 3 min, and the supernatant was discarded. 10 mL of red blood cell lysis buffer was added, the mixture was left to stand on ice for 5 min before adding PBS to terminate the lysis. The mixture was centrifuged at 1800 rpm for 3 min twice. After staining according to the BioLegend flow cytometry antibody staining process, cells were resuspended in PBS twice, sieved, and detected by flow cytometry.
[0192] The detected cells and their markers were as follows:
splenic germinal center B cells (GCBs) (Fas.sup.+GL-7.sup.+B220.sup.+);
follicular T helper cells (Tfhs) (PD1-high, CXCR5-high, FOXP3.sup.−CD4.sup.+);
follicle regulatory T cells (Tfrs) (PD1-high, CXCR5-high, FOXP3.sup.+CD4.sup.+);
regulatory T cells (Tregs) (FOXP3.sup.+CD25.sup.+CD4.sup.+ CXCR5-low);
[0193] The experimental results are shown in
Example 13. Experiment on Efficacy of Fusion Proteins in Delayed Hypersensitivity Mouse Model
[0194] The experimental procedures were as follows: 2,4-dinitrofluorobenzene (DNFB, Sigma, 42085-50G) was dissolved with a mixture of acetone and olive oil (4:1) to obtain a 1% (1 g/100 mL) solution for later use, which was then diluted to a 0.5% (0.5 g/100 mL) solution for later use. Male ICR mice (purchased from Shanghai Laboratory Animal Center) aged 6 weeks and weighing 18-20 g. Mice were grouped according to body weight. On Day 0 of the experiment, the mice were immunized by applying 50 μL of 1% DNFB solution to the abdomen; on Day 5 of the experiment, the mice was challenged by applying 10 μL of 0.5% DNFB solution (20 μL in total) to the inside and outside of the right ear; and on Day 6 of the experiment, the left and right ear tissues were separately removed by using an 8 mm puncher and weighed, and the weight difference between left and right ear tissues was calculated. The test drug was subcutaneously administered once every 5 days from 2 days before the start of the experiment (Day −2).
[0195] The type of drug administered, the dose of administration and the route of administration are shown in Table 18.
TABLE-US-00018 TABLE 18 Immunization regimen for mice Dose of Route Time adminis- of of Test tration adminis- adminis- Group No. Number sample (mpk) tration tration 1 5 PBS NA Subcutan- Days −2 and 3 eously 2 5 A2-22-b 1 Subcutan- Days −2 and 3 eously 3 5 A2-22-b 0.3 Subcutan- Days −2 and 3 eously 4 5 A2-22-b 0.1 Subcutan- Days −2 and 3 eously 5 5 B2-15-b 1 Subcutan- Days −2 and 3 eously 6 5 B2-15-b 0.3 Subcutan- Days −2 and 3 eously 7 5 B2-15-b 0.1 Subcutan- Days −2 and 3 eously 8 5 D2-60-b 1 Subcutan- Days −2 and 3 eously 9 5 D2-60-b 0.3 Subcutan- Days −2 and 3 eously 10 5 D2-60-b 0.1 Subcutan- Days −2 and 3 eously
[0196] The experimental results are shown in
Example 14. Experiment on Efficacy of Fusion Proteins in Arthritis Mouse Model
[0197] Male DBA/1 inbred mice were purchased from Shanghai SLAC laboratory Animal Co., Ltd. The arthritis model was induced by immunization with bovine type II collagen (Biolead, 20022). On Day 0 of the experiment, 2.5 mL of bovine type II collagen/glacial acetic acid (2 mg/mL) was taken and mixed well with 2.5 mL of Freund's complete adjuvant to form a water-in-oil emulsion. 0.1 mL of the emulsion was injected intradermally into the tail root of each mouse under light ether anesthesia, followed by one booster injection 3 weeks later. The molded animals were randomly divided into following 5 groups: a normal control group, a model control group, an IL-2 (C013, novoprotein) group, an A2-22-b group, and a B2-15-b group, with 8 animals in each group. Meanwhile, normal control animals were included in the normal control group. The groups are shown in Table 19.
TABLE-US-00019 TABLE 19 Immunization regimen for mice Number of Dose of Volume for Group animals administration Frequency of Route of administration No. Groups (mice) (mg/kg) administration administration (mL/kg) 1 Normal control 8 — Q5D × 14 days i.p. — group 2 Model control 8 — Q5D × 14 days i.p. — group 3 IL-2 group 8 100000 IU Q2D × 14 days s.c. 5 4 A2-22-b group 8 0.7 Q5D × 14 days i.p. 5 5 B2-15-b group 8 0.7 Q5D × 14 days i.p. 5 (Note: Q5D, once every five days; Q2D, once every two days; i.p., intraperitoneal administration; s.c., subcutaneous administration.)
[0198] Evaluation of joint inflammation: mice were weighed and scored semi-quantitatively (clinical scores) for the severity of joint inflammation every 3 days starting from secondary immunization:
0 points: no redness and swelling;
1 point: inflammation or redness and swelling at one place on the foot;
2 points: mild inflammation on the foot or redness and swelling at two or more places on the whole foot;
3 points: moderate redness and swelling on the whole foot; and
4 points: severe redness and swelling, even joint stiffness, deformity and mobility disorder on the whole foot.
[0199] The maximum score is 4 points per foot and 16 points per mouse. The observation of the severity of joint inflammation continued until the end of the experiment.
[0200] Measurement of concentration of anti-bovine type II collagen antibody in serum: at the end of the experiment, blood was collected, and serum was separated and stored at −80° C. The level of the anti-bovine type II collagen antibody in serum was measured using an ELISA detection kit (Chondrex, 20322T). The specific steps were carried out according to the instructions.
[0201] The experimental results are shown in
Example 15: Effect of Different Lengths of Linker Arms on Expression Level and Purity of Fusion Protein
[0202] The (G.sub.4S).sub.5 linker arm between IL-2 and the light chain of anti-IL-2 antibody B2-15 in B2-15-b was shortened to 4 repeats of G.sub.4S while the heavy chain remained unchanged, so as to obtain a variant B2-15-b-(G.sub.4S).sub.4 of B2-15-b. The light chain sequence of the variant is shown in Table 20.
TABLE-US-00020 TABLE 20 Full-length light chain sequence of the antibody in the fusion protein Fusion protein Full-length light No. chain amino acid sequence Sequence No. B2-15-b- LC APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT SEQ ID No: 181 (G.sub.4S).sub.4 RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSG GGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL AWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGA DFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0203] After the variant of B12-15-b described above was expressed and purified according to the method of Example 2, the expression level could still reach 47 mg/L, and the purity was 99% by SEC-HPLC, indicating that different lengths of linker arms all can achieve the expression of the fusion protein.
Example 16. Determination of Activity of Fusion Proteins of Anti-IL-2 Antibody and Different IL-2 Variants for STAT5 Phosphorylation in Human Peripheral Blood (PBMC)
[0204] IL-2 was mutated to introduce T3E or T3Q, or to remove the first 3 amino acids at the N-terminus of IL-2 (1B2-15-b-Del3), or to remove the first 7 amino acids at the N-terminus of IL-2 (1B2-15-b-Del7). Alternatively, wild-type IL-2 was directly conjugated with anti-IL-2 antibodies. The following fusion proteins were produced (the heavy chain sequence of each antibody was kept constant), shown in Table 21.
TABLE-US-00021 TABLE 21 Full-length light chain sequences of antibodies in fusion proteins Fusion protein Full-length light No. chain amino acid sequence Sequence No. B2-15-b-T3E LC APESSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT SEQ ID No: 182 RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSG GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSV SSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGS GSGADFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC B2-15-b-WT LC APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT SEQ ID No: 183 (wild-type RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA IL-2) QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSG GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSV SSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGS GSGADFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC B2-15-b-T3Q LC APQSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT SEQ ID No: 184 RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSG GGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSV SSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGS GSGADFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC B2-15-b-Del3 LC SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML SEQ ID No: 185 TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGG GSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGS GADFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC B2-15-b-Del7 LC KKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK SEQ ID No: 186 FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFH LRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL NRWITFAQSIISTLTGGGGSGGGGSGGGGSGGGGSGG GGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLA WYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGADF TLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC A2-22-b-Del3 LC SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML SEQ ID No: 187 TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGG GSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSN NYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYSCQQYGSSPLTFGQGTRLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC D2-60-b-Del3 LC SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML SEQ ID No: 188 TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISN YLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGS GTDFTLTISSLQPEDVATYYCQKYDSAPFTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC D2-60-b-T3E LC APESSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT SEQ ID NO: 189 RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLTGGGGSGGGGSGGGGSG GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQG ISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQKYDSAPFTFGQGT RLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
[0205] The activity of the fusion proteins of anti-IL-2 antibody and IL-2 variants described above for STAT5 phosphorylation in Tregs and CD8.sup.+ T cells in human peripheral blood (PBMC) was determined according to the method in Example 7. As shown in
TABLE-US-00022 TABLE 22 EC.sub.50 values for fusion proteins of anti-IL-2 antibody and different IL-2 variants activating pSTAT5 in Treg cells EC.sub.50 Fusion protein No. (nM) A2-22-b 0.168 A2-22-b-Del3 0.144 B2-15-b 0.016 B2-15-b-T3E 0.031 B2-15-b-WT 0.041 B2-15-b-T3Q 0.029 B2-15-b-Del3 0.038 B2-15-b-Del7 0.055 D2-60-b 0.114 D2-60-b-Del3 0.229 D2-60-b-T3E 0.168
Example 17. Experiment on Efficacy of Fusion Proteins in MRL/lpr Spontaneous Lupus Erythematosus Mouse Model
[0206] Female BALB/c mice and female MRL/lpr mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Female BALB/c mice were included in a normal control group, and female MRL/lpr mice were randomly divided into the following 3 groups according to proteinuria level and body weight: an MRL/lpr control group (model control group), a 1 mg/kg administration group and a 3 mg/kg administration group. The mice in the two administration groups, when aged at 8 weeks, were each subcutaneously injected with D2-60-b-T3E at doses of 1 mg/kg and 3 mg/kg, respectively, once every 3 days for consecutive 8 weeks.
TABLE-US-00023 TABLE 23 Experimental design and grouping Dose of Frequency Route Number adminis- of of of tration adminis- adminis- animals Mouse Groups (mg/kg) tration tration (mice) Normal Normal control — Once every sc. 10 Balb/c group 3 days MRL/ Model control — Once every sc. 10 lpr group 3 days D2-60-b-T3E 1 Once every sc. 10 administration 3 days group 3 Once every sc. 10 3 days
[0207] During the treatment period, the urine protein content of mice in each group was determined every two weeks; at the end of 8 consecutive weeks of treatment, the following tests were performed: (1) determination of the content of anti-double-stranded DNA IgM and IgG antibodies in the serum of mice in each group; (2) determination of the content of serum creatinine (CRE) and blood urea nitrogen (BUN) in the serum of mice in each group; (3) determination of the content of IL-6, IL-10 and IFN-γ in the serum of mice in each group; and (4) pathological analysis and scoring of renal tissue of mice in each group.
[0208] 1) Determination of the Urine Protein Content of Mice
[0209] In the experiment, the urine of mice was collected by the tail-lift reflex method, and the urine protein concentration of mice was determined using a urine protein detection kit (CBB method, NanJing JianCheng Bioengineering Institute, #C035-2-1). During the pathogenesis of the lupus erythematosus in MRL/lpr mice, kidney damage occurred due to the deposition of dsDNA antibodies, which in turn led to decreased glomerular filtration function and reabsorption capacity and increased urine protein concentration. As lupus nephritis in MRL/lpr mice progressed, the urine protein level of mice would gradually increase. The determination of the urine protein level of the mice performed every two weeks showed that initially there was no significant difference in the urine protein level among the groups, but as the experiment progressed, the urine protein level of the mice in the MRL/lpr control group (model control group) was gradually increased and significantly higher than that in the normal group, indicating that the MRL/lpr mice suffer from severe spontaneous renal injury due to the deposition of autoantibodies. The average urine protein content of mice in the 1 mg/kg and 3 mg/kg D2-60-b-T3E administration groups was lower than that in the model control group and showed a dose-dependent relationship, but there was no statistical difference between the administration groups and the model control group due to the large individual differences in the data. The data are shown in
[0210] 2) Determination of the Content of Anti-Double-Stranded DNA IgM and IgG Antibodies in Serum
[0211] The content of anti-double-stranded DNA (anti-dsDNA) IgG and IgM antibodies in serum was determined by the ELISA method. Refer to the instructions of ELISA kit (Alpha Diagnostic, #5120 and #5130) for specific procedures. The average content of anti-dsDNA IgG and IgM antibodies in the serum of mice in the 3 mg/kg D2-60-b-T3E administration group was significantly lower than that in the model control group (P<0.01), and there was no statistically significant difference between the 1 mg/kg group and the model control group although the content of IgG and IgM in the 1 mg/kg group tended to be lower than that in the model control group. The experimental results are shown in
[0212] 3) Effect of D2-60-b-T3E on Renal Function of MRL/lpr Mice
[0213] The content of serum creatinine and blood urea nitrogen in the serum of mice at the end of the experiment was detected using a creatinine (CRE) determination kit (NanJing JianCheng Bioengineering Institute, #C011-2-1) and a blood urea nitrogen (BUN) determination kit (NanJing JianCheng Bioengineering Institute, #C013-2-1).
[0214] Blood urea nitrogen and creatinine are metabolic products of human proteins and muscles, respectively, and the kidney is the final excretory organ. After the renal function is damaged, blood urea nitrogen and creatinine in the serum cannot be effectively excreted, and the concentration of blood urea nitrogen (BUN) and serum creatinine (CRE) is gradually increased due to retention. Therefore, the levels of BUN and CRE in serum are the major indicators of renal function clinically. The content of serum creatinine (CRE) and blood urea nitrogen (BUN) in the serum of mice in the MRL/lpr control group (model control group) was significantly increased as compared to that of normal mice (P<0.0001), suggesting that the mice in the MRL/lpr model group have functional renal injury.
[0215] The content of creatinine in the serum of mice in the 1 mg/kg and 3 mg/kg administration groups was significantly lower than that in the model control group (P<0.01 and P<0.0001), indicating that the renal function of MRL/lpr mice can be improved to a certain extent. In addition, the content of creatinine in the serum of mice in the 3 mg/kg administration group was significantly lower than that in the 1 mg/kg administration group. The content of blood urea nitrogen in the serum of mice in the 3 mg/kg administration group was also significantly lower than that in the model control group (P<0.05), indicating that the D2-60-b-T3E at 3 mg/kg can reverse the spontaneous renal injury of MRL/lpr mice. The results are shown in
[0216] 4) Effect of D2-60-b-T3E on the Content of IL-6, IL10 and INF-γ in Serum of MRL/lpr Mice
[0217] The content of the cytokines IFN-γ, IL-6 and IL-10 in the serum was determined by the ELISA method.
[0218] The content of IL-6 and IFN-γ in the serum of mice in the MRL/lpr control group (model group) was significantly increased as compared to that of normal mice (P<0.0001). The content of IL-6 in the serum of mice in the 3 mg/kg administration group was significantly lower than that in the model control group and the 1 mg/kg administration group (P<0.0001), and showed a dose-dependent relationship. The content of IFN-γ in the serum of mice in the 3 mg/kg administration group was significantly lower than that in the model control group (P<0.01). The results are shown in
[0219] The content of IL-10 in the serum of mice in the MRL/lpr control group (model control group) was significantly decreased as compared to that of normal mice (P<0.0001). The content of IL-10 in the serum of mice in the 3 mg/kg D2-60-b-T3E administration group was significantly higher than that in a model control group (P<0.001). The results are shown in
[0220] 5) Effect of D2-60-b-T3E on Renal Lesions of MRL/lpr Mice
[0221] At the end of the experiment, the left kidney of the mouse was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and then stained by H&E. The section was observed under a microscope and subjected to pathological scoring to grade and score pathological injuries of glomerulus, renal interstitium, blood vessel and the like. The scoring criteria are shown in Table 24.
TABLE-US-00024 TABLE 24 Renal pathological scoring criteria Interstitial Inflammation of Scoring Glomerulonephritis nephritis blood vessels 1+ Focal, mild or early 1-3 foci (5-10 cells) Monocytes around the renal hyperplasia monocytes pelvis/blood vessels 2+ Multifocal hyperplasia Mild monocyte infiltration Monocyte infiltration around with increased stromal around the single tubule; large arteries; monocyte foci of and inflammatory isolated atrophic tubules 10-20 cells around interlobular cells arteries 3+ Diffuse More extensive infiltration with More extensive monocyte lesions hyperplasia large tubular atrophic lesions around small arterial branches 4+ Extensive Extensive monocyte infiltration Monocyte infiltration into the sclerosis/crescent between tubules; extensive surrounding parenchyma/ shape; tubular atrophy necrosis involvement of most proteinuria of blood vessels/vasculitis
[0222] The mice in the MRL/lpr control group (model control group) showed severe pathological injuries of glomerulus, renal interstitium and blood vessel in kidney, with glomerular sclerosis and partial inward depression, massive cell infiltration in the perivascular and interstitial regions, and severe glomerular swelling and hyperplasia. The renal pathological scores of the mice in the MRL/lpr control group (model control group) were significantly increased as compared to those of normal mice (P<0.001), suggesting that the mice in the model control group have significant renal lesions.
[0223] The pathological injuries of glomerulus, renal interstitium and blood vessel of the mice in the administration groups were relieved to a certain extent. The renal pathological scores of the mice in the 1 mg/kg and 3 mg/kg administration groups were significantly lower than those of the model control group (P<0.05 and P<0.0001), indicating that D2-60-b-T3E can significantly reduce spontaneous renal lesions in MRL/lpr mice. The results are shown in