Bispecific molecule and preparation and use thereof
20220233710 · 2022-07-28
Inventors
Cpc classification
A61K47/65
HUMAN NECESSITIES
A61K47/6845
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/2866
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
International classification
A61K47/68
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
Abstract
Provided are a bispecific molecule and preparation and use thereof. The bispecific molecule includes a molecule that specifically binds an interleukin-1 receptor (IL-1R) and an antibody that targets a free inflammatory factor. The molecule that specifically binds the cell surface interleukin-1 receptor (IL-1R) aggregates the antibody that targets the free inflammatory factor linked thereto on or near the cell surface, thereby the local concentration of the bispecific molecule on or near the cell surface is increased, adverse reactions are avoided, treatment effectiveness is increased and the infection risk of patients is also reduced.
Claims
1. A bispecific molecule, comprising: a) a molecule that specifically binds IL-1R; and b) an antibody that targets a free inflammatory factor; wherein, the molecule that specifically binds IL-1R and the antibody that targets the inflammatory factor are connected by a linker peptide.
2. The bispecific molecule according to claim 1, wherein the molecule that specifically binds IL-1R is a non-immunoglobulin polypeptide; preferably, the non-immunoglobulin polypeptide that specifically binds IL-1R is IL-1RA; preferably, the non-immunoglobulin polypeptide that specifically binds IL-1R comprises an amino acid sequence having 85%-100% of identity with SEQ ID NO: 50.
3. (canceled)
4. (canceled)
5. The bispecific molecule according to claim 1, wherein the molecule that specifically binds IL-1R is an anti-IL-1R antibody.
6. The bispecific molecule according to claim 5, wherein the anti-IL-1R antibody is a single domain antibody, a chimeric antibody, a humanized antibody, a human antibody, or a recombinantly modified part of the above antibodies.
7. The bispecific molecule according to claim 5, wherein the anti-IL-1R antibody comprises CDR groups: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein, a) HCDR1, HCDR2 and HCDR3 have the HCDR1, HCDR2 and HCDR3 sequences as contained in SEQ ID NO: 52 respectively; LCDR1, LCDR2 and LCDR3 have the LCDR1, LCDR2 and LCDR3 sequences as contained in SEQ ID NO: 54, respectively; b) HCDR1, HCDR2 and HCDR3 have the HCDR1, HCDR2 and HCDR3 sequence as contained in SEQ ID NO: 56 respectively; LCDR1, LCDR2 and LCDR3 have the LCDR1, LCDR2 and LCDR3 sequences as contained in SEQ ID NO: 54 respectively; or c) HCDR1, HCDR2 and HCDR3 have the HCDR1, HCDR2 and HCDR3 sequences contained in SEQ ID NO: 58 respectively; LCDR1, LCDR2 and LCDR3 have the LCDR1, LCDR2 and LCDR3 sequences as contained in SEQ ID NO: 60.
8. The bispecific molecule according to claim 5, wherein the anti-IL-1R antibody has a heavy chain and a light chain of the following: a) a heavy chain shown in SEQ ID NO: 52 and a light chain shown in SEQ ID NO: 54; b) a heavy chain shown in SEQ ID NO: 56 and a light chain shown in SEQ ID NO: 54; or c) a heavy chain shown in SEQ ID NO: 58 and a light chain shown in SEQ ID NO: 60.
9. The bispecific molecule according to claim 1, wherein the free inflammatory factor is selected from one of the group consisting of: IL-1 superfamily (IL-la, IL-1β, IL-18, IL-33, IL-36a, IL-36β, IL-36γ), IL-4, IL-13, IL-17A, IL-17E, IL-6, IL-12, IL-23, TNF superfamily (TNFa, TNFβ, TNFγ, OX40L (TNFSF4), CD40L (CD154), FasL (CD178, CD95L), CD27L (CD70), CD30L(CD153), 4-1BBL, CD253 (APO-2L), CD254, APO-3L(DR3L), CD256(TALL-2), CD257(B1yS), LIGHT(CD258), TL1 (TNFSF18, AITRL), ED1-A1), BAFF, IFN or GM-CSF.
10. The bispecific molecule according to claim 9, wherein the free inflammatory factor is IL-1β.
11. The bispecific molecule according to claim 10, wherein the antibody that targets the free inflammatory factor IL-1β has the HCDR1, HCDR2 and HCDR3 sequences contained in a heavy chain amino acid sequence shown in SEQ ID NO: 2, and the LCDR1, LCDR2 and LCDR3 sequences contained in a light chain amino acid sequence shown in SEQ ID NO: 4; or the HCDR1, HCDR2 and HCDR3 sequences contained in a heavy chain amino acid sequence shown in SEQ ID NO: 6, and the LCDR1, LCDR2 and LCDR3 sequences contained in a light chain amino acid sequence shown in SEQ ID NO: 8.
12. The bispecific molecule according to claim 9, wherein the free inflammatory factor is IL-17.
13. The bispecific molecule according to claim 12, wherein the antibody that targets the free inflammatory factor IL-17 has the HCDR1, HCDR2 and HCDR3 sequences contained in a heavy chain amino acid sequence shown in SEQ ID NO: 26, and the LCDR1, LCDR2 and LCDR3 sequences contained in a light chain amino acid sequence shown in SEQ ID NO: 28.
14. The bispecific molecule according to claim 9, wherein the free inflammatory factor is IL-6.
15. The bispecific molecule according to claim 14, wherein the antibody that targets the free inflammatory factor IL-6 has the HCDR1, HCDR2 and HCDR3 sequences contained in a heavy chain amino acid sequence shown in SEQ ID NO: 38, and the LCDR1, LCDR2 and LCDR3 sequences contained in a light chain amino acid sequence shown in SEQ ID NO:40.
16. The bispecific molecule according to claim 1, wherein the antibody that targets the free inflammatory factor is a chimeric antibody, a humanized antibody, a human antibody, or a recombinantly modified part of the above antibodies.
17. The bispecific molecule according to claim 2, wherein the bispecific molecule comprises a heavy chain and a light chain having the amino acid sequences selected from any one of the group consisting of: SEQ ID NO: 10 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 16; SEQ ID NO: 18 and SEQ ID NO: 4; SEQ ID NO: 2 and SEQ ID NO: 20; SEQ ID NO: 6 and SEQ ID NO: 22; SEQ ID NO: 6 and SEQ ID NO: 24; SEQ ID NO: 62 and SEQ ID NO: 4; SEQ ID NO: 64 and SEQ ID NO: 4; SEQ ID NO: 66 and SEQ ID NO: 8; SEQ ID NO: 6 and SEQ ID NO: 68; SEQ ID NO: 70 and SEQ ID NO: 4; SEQ ID NO: 72 and SEQ ID NO: 4; SEQ ID NO: 74 and SEQ. NO: 4; SEQ ID NO: 76 and SEQ ID NO: 4; SEQ ID NO: 34 and SEQ ID NO: 28; SEQ ID NO: 26 and SEQ ID NO: 36; SEQ ID NO: 30 and SEQ ID NO: 28; SEQ ID NO: 26 and SEQ ID NO: 32; SEQ ID NO: 46 and SEQ ID NO: 40; SEQ ID NO: 38 and SEQ ID NO: 48; SEQ ID NO: 38 and SEQ ID NO: 44; SEQ ID NO: 42 and SEQ ID NO: 40.
18. (canceled)
19. (canceled)
20. A nucleic acid, encoding the bispecific molecule according to claim 1.
21. An expression vector, comprising the nucleic acid according to claim 20.
22. A host cell, comprising the expression vector according to claim 21.
23. A pharmaceutical composition, comprising the bispecific molecule according to claim 1 and a pharmaceutically acceptable carrier or preparation.
24. A method for treating an inflammatory disease and/or an autoimmune disease in a subject in need thereof, the method comprises administering a therapeutically effective amount of a composition to the subject, and the composition comprises the bispecific molecule in a pharmaceutically acceptable form according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Drawings constituting the present application are used to provide further understanding of the present invention, exemplary embodiments of the present invention and descriptions thereof are used to explain the present invention and do not constitute improper limitation to the present invention. In the drawings:
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0046] The present invention is described in detail here by references using the following definitions and embodiments. The contents of all patents and disclosed documents mentioned in this article, including all sequences disclosed in these patents and disclosures, are expressly incorporated herein by reference.
[0047] Bispecific Molecule
[0048] The “bispecific molecule” of the present invention is a binding molecule with two binding specificities. It may be a non-immunoglobulin polypeptide with antigen binding specificity or an antibody with antigen binding specificity.
[0049] Antibody
[0050] The “antibody” of the present invention refers to an immunoglobulin molecule, a fragment of the immunoglobulin molecule, or a derivative of any one of them, which has the ability to specifically bind an antigen under typical physiological conditions to induce, promote, enhance and/or regulate the physiological response associated with the binding of the antibody to the antigen. Unless otherwise specified or clearly contradictory in the context, the “antibody” of the present invention includes fragments of antibodies, which are antigen-binding fragments, namely, retains the ability to specifically bind the antigen.
[0051] Molecule that Binds IL-1R
[0052] The “molecule that specifically binds IL-1R” in the present invention refers to a non-immunoglobulin polypeptide or antibody that may compete with IL-1α and/or IL-1β to bind IL-1R and inhibit the biological activity of IL-1R. Other single domain antibodies or heavy chain antibodies that may compete with IL-1α and/or IL-1β to bind IL-1R and inhibit the biological activity of IL-1R are also included in the present invention.
[0053] Inflammatory Factors
[0054] As used in the present invention, the “inflammatory factor” is a general term for cytokines involved in the occurrence and development of inflammation. The “free inflammatory factor” refers to an inflammatory factor that exists in a body in the form of solution and is not expressed on the cell surface. The common free inflammatory factor includes but is not limited to the following examples: IL-1 superfamily (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ), IL-4, IL-13, IL-17A, IL-17E, IL-6, IL-12, IL-23, TNF superfamily (TNFα, TNFβ, TNFγ, OX40L (TNFSF4), CD40L (CD154), FasL (CD178, CD95L), CD27L (CD70), CD30L (CD153), 4-1BBL, CD253 (APO-2L)), CD254, APO-3L (DR3L), CD256 (TALL-2), CD257 (BlyS), LIGHT (CD258), TL1 (TNFSF18, AITRL), ED1-A1), BAFF, IFN or GM-CSF and the like. The inflammatory factor receptor refers to a molecule that may bind to the inflammatory factor and is expressed on the cell surface.
[0055] CDR
[0056] The term “CDR” refers to a complementarity determining region within an immunoglobulin variable region sequence. For each heavy and light chain variable region, there are three CDRs, which are named as CDR1, CDR2, and CDR3. A term “CDR set” refers to a set of three CDRs that appear in a single variable region capable of binding antigen. Exact boundaries of these CDRs are defined differently according to different systems. A system described by Kabat (Kabat et al. (1987) and (1991)) not only provides a clear residue numbering system that may be applied to any variable region of an antibody or binding protein, but also provides a precise definition of residue boundaries of the three CDRs in each heavy chain or light chain sequence. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883) found that certain sub-parts in Kabat CDR adopt almost the same peptide framework although with great diversity at the amino acid sequence level. These sub-parts are named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have borders that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan (1995) FASEB J. 9: 133-139 and MacCallum (1996) J. Mol. Biol. 262(5): 732-45). There are other CDR boundary definitions that may not strictly follow one of the systems in this article, but may still overlap with Kabat CDRs, although they may be shortened or lengthen by considering the fact that a specific residue or a residue group or even the entire CDRs do not significantly affect the prediction of antigen binding or experimental discovery. The methods used herein may utilize CDRs defined according to any of these systems, although some embodiments use Kabat or Chothia defined CDRs (CN105324396 A).
[0057] A term “homology” or “identity” refers to sequence identity of primary unit between two polymer molecules (for example, between two nucleic acid molecules (for example, between two DNA molecules or two RNA molecules) or between two polypeptide molecules). While the position of the primary unit in these two molecules is occupied by the same monomeric subunit, for example, if a certain position in each molecule of two DNA molecules is occupied by adenine, they are homologous or the same at that position. The identity between two sequences varies directly with the number of matching positions or homologous positions. For example, if half of the positions in two sequences (for example, five positions in a polymer of ten subunits in length) are homologous, the two sequences are 50% homologous (also called 50% homology or 50% identity); if 90% of the positions (for example, 9 positions in 10 positions) are matched or homologous, these two sequences are 90% homologous (also called 90% homology or 90% identity).
[0058] Inflammatory Disease
[0059] The “inflammatory disease” as used in the present application refers to a disease related to an inflammatory response. Examples of the inflammatory disease include arthritis such as RA, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and other inflammatory diseases of the joints, inflammatory bowel diseases such as ulcerative colitis, Crohn's disease, Barrett's syndrome, ileitis, enteritis, gluten-sensitive enteropathy, inflammatory diseases of the respiratory system, such as asthma, adult respiratory distress syndrome, allergic rhinitis, silicosis, chronic respiratory obstructive diseases, hypersensitivity lung diseases, bronchiectasis; inflammatory diseases of the inflammatory skin, including psoriasis, scleroderma, and inflammatory skin diseases such as eczema, atopic dermatitis, urticaria and pruritus; diseases involving central and peripheral nervous system inflammation include multiple sclerosis, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy and neurodegenerative diseases such as Alzheimer's disease; systemic lupus erythematosus, immune-mediated kidney disease, such as glomerulonephritis and spondyloarthropathy; diseases with undesirable chronic inflammatory components, such as systemic sclerosis, characteristic inflammatory myopathy, Sjogren's syndrome, vasculitis, sarcomatoid disease, thyroiditis, gout, otitis, conjunctivitis, sinusitis, sarcomatoid disease, Behcet's syndrome, hepatobiliary diseases such as hepatitis, primary biliary cirrhosis, granulomatous hepatitis and sclerosing cholangitis; inflammation and ischemic damage of the cardiovascular system such as ischemic heart disease, stroke and atherosclerosis; and graft rejection, including allograft rejection and graft-versus-host disease. Various other inflammatory diseases include tuberculosis and chronic cholecystitis. For example, other chronic inflammatory diseases are also described in Harrison's Principles of Internal Medicine (12th Edition, Wilson, et al., eds., McGrawill, Inc.).
Pharmaceutical Composition
[0060] The pharmaceutical composition as described herein is prepared by mixing the bispecific antibody of the present invention with the desired purity and one or more optional pharmaceutically acceptable carriers, which is in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is generally non-toxic to the recipient at dose and concentration used.
[0061] The present invention may be administered as an active ingredient alone, or in combination with, for example, an adjuvant or with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, for example, for the treatment of an inflammatory disease and/or an immune disease.
EXAMPLES
[0062] It should be noted that, in the case of no conflict, the embodiments in the present application are only illustrative, and are not intended to limit the present invention in any manners.
Example 1: Construction, Expression, Purification and Functional Detection of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
Example 1.1: Cloning of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
[0063] Gene fragments encoding the heavy chain (HC) and the light chain (LC) of the aIL-1R-1, aIL-1R-2, aIL-1R-3, aIL-1β-1, aIL-1β-2 antibodies and IL-1RA were synthesized, respectively. IL-1RA was fused to the N-terminal or C-terminal of heavy chain or light chain of the aIL-1β antibody (aIL-1β-1 and aIL-1β-2) by a linker peptide. VH and VL of the aIL-1R antibody were fused to the N-terminal of the heavy and light chains of the aIL-1β antibody by the linker peptide, respectively, or VH and VL of the aIL-1β antibody were fused to the N-terminal of the heavy chain and N-terminal of the light chain of the aIL-1R by the linker peptide, respectively. All the sequences were verified by sequencing.
TABLE-US-00001 TABLE 1 Bispecific molecule nucleic acid and sequence Nucleotide Amino acid sequence sequence Construct Composition Seq ID No: Seq ID No: aIL1β-1 chain-1 1 2 chain-2 3 4 aIL1β-2 chain-1 5 6 chain-2 7 8 BSI-1 chain-1 9 10 chain-2 3 4 BSI-2 chain-1 1 2 chain-2 11 12 BSI-3 chain-1 13 14 chain-2 3 4 BSI-4 chain-1 1 2 chain-2 15 16 BSI-5 chain-1 17 18 chain-2 3 4 BSI-6 chain-1 1 2 chain-2 19 20 BSI-7 chain-1 5 6 chain-2 21 22 BSI-8 chain-1 5 6 chain-2 23 24 aIL-17A chain-1 25 26 chain-2 27 28 BSI-9 chain-1 33 34 chain-2 27 28 BSI-10 chain-1 25 26 chain-2 35 36 BSI-11 chain-1 29 30 chain-2 27 28 BSI-12 chain-1 25 26 chain-2 31 32 BSI-13 chain-1 45 46 chain-2 39 40 BSI-14 chain-1 37 38 chain-2 47 48 BSI-15 chain-1 37 38 chain-2 43 44 BSI-16 chain-1 41 42 chain-2 39 40 aIL-6 chain-1 37 38 chain-2 39 40 IL-IRA / 49 50 aIL-IR-1 chain-1 51 52 chain-2 53 54 aIL-IR-2 chain-1 55 56 chain-2 53 54 aIL-IR-3 chain-1 57 58 chain-2 59 60 BSI-17 chain-1 61 62 chain-2 3 4 BSI-18 chain-1 63 64 chain-2 3 4 BSI-19 chain-1 65 66 chain-2 7 8 BSI-20 chain-1 5 6 chain-2 67 68 BSI-21 chain-1 69 70 chain-2 3 4 BSI-22 chain-1 71 72 chain-2 3 4 BSI-23 chain-1 73 74 chain-2 3 4 BSI-24 chain-1 75 76 chain-2 3 4
Example 1.2: Expression and Purification of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
[0064] The heavy chain and light chain of the expression vector constructed in Example 1.1 were transiently transfected into Free Style™ HEK293 cells (Thermo Fisher) (the amount of a heavy chain plasmid and a light chain plasmid during transfection was at a molar ratio of 1:1):28 ml of Free Style™ HEK293 (a total of 3×10.sup.7 cells) were seeded in a 125 ml cell culture flask, plasmids were diluted with 1 ml of Opti-MEM (Invitrogen), and then added to 1 ml of Opti-MEM containing 60 μl 293Fectin (Invitrogen). After standing at room temperature for 30 min, the plasmid-293Fectin mixture was added to the cell culture medium, then incubated at 125 rpm, 37° C., 5% CO.sub.2. Cell culture supernatant was collected at 48 h and 96 h after transfection, respectively, and purified by Protein A Resin (Thermo Fisher Scientific). The purified proteins were analyzed by SDS-PAGE. Results are shown in
Example 1.3: Mass Spectrometry of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
[0065] The purified proteins in Example 1.2 were incubated with PNGase F (NEB) at 37° C. for 8 hours, and treated with 10 mM dithiothreitol (DTT), then the sample were injected into 300SB-C8, 2.1×50 mm column of HPLC-Q-TOF-MS (Agilent, USA) for mass spectrometry analysis. As shown in Table 2, the molecular weight of antibody fusion protein in different fusion forms detected by the mass spectrometry is consistent with the theoretical value.
TABLE-US-00002 TABLE 2 Theoretical molecular weight of bispecific molecule and MS detection molecular weight Chain-1 Chain-2 molecular weight (D) molecular weight (D) Bispecific Theoretical Detection Theoretical Detection molecule value value value value BSI-1 67919.69 67916.04 23357.97 23354.64 BSI-2 48912.7 48889.73 42365.39 42361.82 BSI-3 69656.64 69651.17 23357.97 23355.01 BSI-4 48912.7 48888.88 44102.44 44098.27 BSI-5 66910.52 66887.14 23357.97 23355.01 BSI-6 48912.7 48889.19 41228.15 41223.56 BSI-7 48799.94 48776.2 42131.15 42126.9 BSI-8 48799.94 48776.1 43868.1 43863.5
Example 1.4: Analysis of In Vitro Activity of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
[0066] Recombinant human IL-1β (Thermo Fisher Scientific), recombinant mouse IL-1β (Sino Bio), recombinant mouse IL-1R (Sino Bio), or recombinant human IL-1R (100 ng/well) (DPBS buffer, pH7.4) was coated in a 96-well plate, and incubated overnight at 4° C. After blocked by PBST (0.5% Tween-20 in PBS) containing 2% skimmed milk powder for 1 hour at room temperature, and being washed with PBST for 3 times, the gradient diluted bispecific molecules (0.006 nM-100 nM) were added and incubated at 37° C. for 1 h. After being washed with PBST for 3 times, anti-human Fc-HRP (KPL; 1:2500) secondary antibody diluted by 2% milk/PBST was added and incubated at room temperature for 1 h, and then washed by PBST for 5 times. QuantaBlu fluorescent peroxide enzyme substrate (Life technologies, Cat. 15169) was used for color development, and reading was performed at 325 nm and 420 nm, or TMB color reagent (Bio Legend) was used for color development, and the reading was performed at 650 nm. Data was analyzed by nonlinear regression using specific binding model in Prizm Graphpad software.
[0067] As shown in
Example 1.5: Analysis of Inhibition of IL-1β-Induced HEK-Blue-IL1R Cell Activation by Bispecific Molecules
[0068] 5×10.sup.4 HEK-Blue-IL1R cells (InvivoGene) were seeded on a 96-well plate, and 100 ul of IL-1β (R&D systems) (diluted with 5% FBS/DMEM medium) in gradient dilutions (0.024-50 ng/ml) was added into per well and cultured overnight. Cell supernatant was collected and mixed with QUANTI-Blue (InvivoGene) at a ratio of 1:9. After incubating at 37° C. for 4 hours, the absorbance at 655 nm was read at Spectramax fluorescence plate reader. Data was analyzed with log[agonist] vs response (3 parameters) model by nonlinear regression in Prizm Graphpad software. As shown in
Example 1.6: In Vivo Neutralization Efficacy of Bispecific Molecules that Specifically Binds IL-1R and IL-1β
[0069] DPBS or bispecific molecule (two doses: 5 mg/kg, 15 mg/kg) was injected intraperitoneally into female C57BL/6 mice (6-7 weeks). After 24 hours, each mouse was injected with 20 ng of recombinant human IL-1β (PHC0815, Thermo Fisher Scientific). After 2 hours, blood was taken from the posterior eye socket, and the level of mIL-6 in mouse plasma was detected by Mouse IL-6 ELISA MAX™ Deluxe (Cat. 431306, BioLegend) according to instructions provided by a manufacturer. Data was analyzed with Graphpad Prizm software. For statistical analysis, unpaired, and two-tailed t test was performed.
[0070] As shown in
Example 2: Construction, Expression, Purification and Functional Detection of Bispecific Molecules that Specifically Binds IL-1R and IL-17
Example 2.1: Cloning of Bispecific Molecules that Specifically Binds IL-1R and IL-17
[0071] Gene fragments encoding the heavy chain and light chain of aIL-17 antibody were synthesized, respectively, and IL-1RA was fused to N-terminal or C-terminal the heavy chain or light chain of the aIL-17 antibody through the linker peptide L1, L2 or L3 using standard molecular biology technology. VH and VL of the aIL-1R antibody was fused to the N-terminal of the heavy chain and light chain of the aIL-17 antibody through the linker peptides L4 and L5, respectively, or VH and VL of the aIL-17 antibody was fused to the N-terminal of the heavy chain and N-terminal of the light chain of the aIL-1R antibody, respectively. All the sequences were verified by sequencing.
Example 2.2: Expression, Purification and Analysis of Bispecific Molecules that Specifically Binds IL-1R and IL-17
[0072] The heavy chain and light chain of the expression vector constructed in Example 2.1 were transiently transfected into Free Style™ HEK293 cells (Thermo Fisher), and the amount of the heavy chain plasmid and the light chain plasmid during transfection was at a molar ratio of 1:1:28 ml of Free Style™ HEK 293 (a total of 3×10.sup.7 cells) were seeded in a 125 ml cell culture flask, the plasmid was diluted with 1 ml of Opti-MEM (Invitrogen) and then added to 1 ml of Opti-MEM containing 60 μl 293Fectin (Invitrogen). After standing at room temperature for 30 min, the plasmid-293Fectin mixture was added to the cell culture medium, and then incubated at 125 rpm, 37° C., 5% CO.sub.2. Cell culture supernatant was collected at 48 h and 96 h after transfection, respectively, and purified by Protein A Resin (Thermo Fisher Scientific). The purified proteins were analyzed by SDS-PAGE. As shown in
[0073] In order to further investigate the physical properties of the bispecific molecule in solution, GE's AKTA was used for size exclusion chromatography. The column used was HiLoad 16/600 Superdex 200, and the solution used was DPBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH7.4) with a flow rate at 0.4 ml/min.
[0074] Results from
Example 2.3: In Vitro Activity of Bispecific Molecule that Specifically Binds IL-1R and IL-17
[0075] Recombinant human IL-17 (SinoBiological) (100 ng/well) (PBS buffer, pH7.4) was coated in a 96-well plate, and incubated overnight at 4° C. After blocked with PBST containing 2% skimmed milk powder (0.5% Tween-20 in PBS) at room temperature for 1 hour and followed by being washed for 3 times with PBST, the gradient diluted bispecific molecules (0.04 nM-30 nM) were added and incubated at 37° C. for 1 h. After being washed for 3 times with PBST, anti-human Fc-HRP (KPL, 1:2500) secondary antibody diluted by 2% milk/PBST was added and incubated at room temperature for 1 h. After being washed for 5 times with PBST, TMB (BioLegend) was used for color development, and readings were performed in Thermal VARIOSKAN FLASH plate reader at 650 nm. Data was analyzed with a log[agonist] vs response (3 parameters) model by “non-linear regression” in Graphpad Prizm software.
[0076] The ELISA results in
Example 3: Construction, Expression, Purification and Functional Detection of Bispecific Molecules that Specifically Binds IL-1R and IL-6
Example 3.1: Cloning of Bispecific Molecules that Specifically Binds IL-1R and IL-6
[0077] Gene fragments encoding a heavy chain and a light chain of the aIL-6 antibody were synthesized, and IL-1RA was fused to N-terminal or C-terminal of the heavy chain or the light chain of the aIL-6 antibody through the linker peptide L1, L2 or L3 using standard molecular biology technology. VH and VL of the aIL-1R antibody was fused to the N-terminal of the heavy chain and light chain of the aIL-6 antibody through the linker peptides L4 and L5, respectively, or VH and VL of the aIL-6 antibody was fused to the N-terminal of the heavy chain and N-terminal of the light chain of the aIL-1R antibody, respectively. All the sequences were verified by sequencing.
Example 3.2: Expression, Purification and Analysis of Bispecific Molecules that Specifically Binds IL-1R and IL-6
[0078] The heavy chain and light chain of the expression vector constructed in Example 3.1 were transiently transfected into Free Style™ HEK293 cells (Thermo Fisher), and the amount of the heavy chain plasmid and the light chain plasmid during transfection was at a molar ratio of 1:1:28 ml of Free Style™ HEK 293 (a total of 3×10.sup.7 cells) were seeded in a 125 ml cell culture flask, the plasmid was diluted with 1 ml of Opti-MEM (Invitrogen) and then added to 1 ml of Opti-MEM containing 60 μl 293Fectin (Invitrogen). After standing at room temperature for 30 min, the plasmid-293Fectin mixture was added to the cell culture medium, and incubated at 125 rpm, 37° C., 5% C02. Cell culture supernatant was collected at 48 h and 96 h after transfection, respectively, and purified by Protein A Resin (Thermo Fisher Scientific). The purified proteins were analyzed by SDS-PAGE. As shown in
[0079] In order to further investigate the physical properties of the bispecific molecule in solution, GE′ AKTA was used for gel exclusion chromatography. The column used was HiLoad 16/600 Superdex 200, and the solution used was DPBS buffer (0.010M phosphate buffer, 0.0027M KCl, 0.14M NaCl, pH7.4) with a flow rate at 0.4 ml/min.
[0080] The size exclusion chromatography results of
Example 3.3: In Vitro Activities of Bispecific Molecule that Specifically Binds IL-1R and IL-6
[0081] Recombinant human IL-6 (SinoBiological) (100 ng/well) (PBS buffer, pH7.4) was coated in a 96-well plate, and incubated overnight at 4° C. After blocked with PBST containing 2% skimmed milk powder (0.5% Tween-20 in PBS) at room temperature for 1 hour, followed by being washed for 3 times with PBST, the bispecific molecules (0.04 nM-30 nM) in gradient dilutions were added and incubated at 37° C. for 1 h. After being washed for 3 times with PBST, anti-human Fc-HRP (KPL, 1:2500) secondary antibody diluted by 2% milk/PBST was added and incubated at room temperature for 1 h, followed by being washed for 5 times with PBST. TMB (BioLegend) was used for color development, and readings was performed in Thermal VARIOSKAN FLASH plate reader at 650 nm. Data was analyzed with a log[agonist] vs response (3 parameters) model in “non-linear regression” by Graphpad Prizm software.
[0082] The ELISA results in