Humanized anti-IL-1R3 antibodies and methods of use

Abstract

An antibody that specifically binds to IL-1R3, the antibody comprising the antibody heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2. A pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent, carrier, or excipient is also provided. Also provided is the antibody for use in treating a disease or disorder in a subject in need thereof. The disease or disorder may be an autoimmune or autoinflammatory disease or disorder.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety.

[0002] An antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2. Also, a method for treating a disease or disorder in a subject in need thereof, the method comprising administering the antibody to the subject. [Background technology]

[0003] Interleukin-1 (IL-1) is a central mediator of innate immunity and inflammation. The type 1 IL-1 receptor (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1-induced effects. In addition to IL-1R1, IL-1R3 also functions as a receptor subunit of the heterodimeric IL-33 and IL-36 receptor complex. IL-1R3 therefore plays a role in three signaling pathways involving six cytokines of the IL-1 family (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ). IL-1 family cytokines are potent mediators of inflammation and act to coordinate local and systemic immune responses to a wide range of stimuli. However, aberrant signaling by IL-1 family cytokine members is associated with many inflammatory syndromes, autoimmune conditions, and cancer. Tight regulation of IL-1 family cytokine signaling pathways through receptor antagonists, decoy receptors, and signaling inhibitors ensures a balance between amplifying innate immunity and uncontrolled inflammation. Human genetic evidence has linked IL-1 family signaling pathways to autoimmune diseases. Because IL-1R3 inhibition can inhibit all three signaling pathways, blocking IL-1R3 is a multitargeting strategy that neutralizes three cytokine pathways (IL-1, IL-33, and IL-36) and confers efficacy in indications where single cytokine targeting is insufficient. Therefore, there is a need for the development of therapeutic anti-IL-1R3 antibodies. Attempts to generate functional monoclonal antibodies (mAbs) against human IL-1R3 have been ongoing for several years. However, improved anti-IL-1R3 antibodies are needed. In particular, there is a need for anti-IL-1R3 antibodies that offer the advantage of superior inhibition of three signaling pathways without affecting other cellular pathways. Summary of the Invention [Means for solving the problem]

[0004] In a first aspect, there is provided an antibody that specifically binds to IL-1R3, the antibody comprising the antibody heavy chain amino acid sequence of SEQ ID NO:1 and the antibody light chain amino acid sequence of SEQ ID NO:2.

[0005] In a second aspect, there is provided a pharmaceutical composition comprising the antibody of the first aspect and a pharmaceutically acceptable diluent, carrier or excipient.

[0006] In a third aspect, provided is an isolated nucleic acid molecule encoding the antibody of the first aspect.

[0007] In a fourth aspect, there is provided an expression vector comprising the nucleic acid molecule of the third aspect.

[0008] In a fifth aspect, there is provided a host cell comprising an expression vector of the fourth aspect.

[0009] A sixth aspect relates to a method of producing an antibody of the first aspect, the method comprising: (i) optionally transfecting a host cell with the isolated nucleic acid molecule of the third aspect or the expression vector of the fourth aspect; (ii) culturing the host cell under conditions that allow expression of the antibody; (iii) recovering the antibody; and (iv) optionally further purifying, and / or modifying, and / or formulating the antibody.

[0010] The seventh aspect relates to an antibody produced by the production method according to the sixth aspect.

[0011] An eighth aspect relates to the antibody of the first aspect for use in treating a disease or disorder in a subject in need thereof, preferably wherein the disease or disorder is an autoimmune or autoinflammatory disease or disorder.

[0012] A ninth aspect relates to the pharmaceutical composition of the second aspect for use in treating a disease or disorder in a subject in need thereof, preferably wherein the disease or disorder is an autoimmune or autoinflammatory disease or disorder.

[0013] In a tenth aspect, provided is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an antibody of the first aspect or a pharmaceutical composition of the second aspect.

[0014] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] This shows the anti-IL-1R3 antibody titers of 50 nM methotrexate (MTX)-selected pools after transfection, which express anti-IL-1R3 antibodies containing either the Fc region of IgG1 with L234A and L235A mutations (IgG1-LALA) or the Fc region of IgG4 with S228P, F234A, and L2345A mutations (IgG4-P-FALA). [Figure 2A] 2A and 2B show surface plasmon resonance (SPR) sensorgrams demonstrating that, compared to the control (IgG1 antibody), an anti-IL-1R3 antibody in the IgG1-LALA format has residual binding activity to FcγRIII (FIGS. 2A and 2B [V176F mutant]) and FcγRI (FIG. 2C), whereas the same anti-IL-1R3 antibody in the IgG4-P-FALA format does not bind to Fcγ receptors I or IIII. In all of FIGS. 2A, 2B, and 2C, the x-axis represents time in seconds, the y-axis represents response in response units (RU), and 0 represents the baseline capture. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A]Figure 3 shows a cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay to determine effector cell binding to the Fc region of an antibody. The anti-IL-1R3 antibody in the IgG1-LALA format (Figures 3A and B [EFF-20-074-1]) showed significantly reduced ADCC activity compared to the IgG1 format (i.e., WT) [FF-20-2050-1], whereas the IgG4-P-FALA format [EFF-20-075-1] resulted in complete silencing of effector cell binding to the Fc receptor. [Figure 3B] Figure 3 shows a cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay to determine effector cell binding to the Fc region of an antibody. The anti-IL-1R3 antibody in the IgG1-LALA format (Figures 3A and B [EFF-20-074-1]) showed significantly reduced ADCC activity compared to the IgG1 format (i.e., WT) [FF-20-2050-1], whereas the IgG4-P-FALA format [EFF-20-075-1] resulted in complete silencing of effector cell binding to the Fc receptor. DETAILED DESCRIPTION OF THE INVENTION

[0016] Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector function is desirable.

[0017] Certain mutations in the Fc region of IgG4 Abs can further reduce effector function. IgG4 residues 234 and 235, according to the EU index (Proc Natl Acad Sci USA. 1969, 63(1), 78-85; Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition), can be mutated to change the phenylalanine at position 234 to alanine (F234A) and the leucine at position 235 to alanine (L235A) (Parekh et al. 2012—see the references section). Such antibody mutations are called FALA mutations. However, IgG4 Abs are dynamic molecules that can undergo Fab arm exchange (FAE), resulting in functionally monovalent bispecific antibodies (bsAbs) that may have reduced therapeutic efficacy. The amino acid residue serine at position 228 (S228) and the amino acid residue arginine at position 409 of IgG4 drive FAE (Labrijn et al., 2011—see the references section). Substitution of S228 with proline (S228P) has been shown to prevent IgG4 FAE and thus stabilize IgG4 Abs (Angal et al., 1993; Silva et al., 2015—see the references section). The above-mentioned FALA mutation and S228P mutation can be simultaneously introduced into the antibody constant region. An IgG4 heavy chain with a FALA mutation is referred to as an "IgG4 FALA"-type heavy chain, an IgG4 heavy chain with an S228P mutation is referred to as an "IgG4-P"-type heavy chain, and an IgG4 heavy chain with both the FALA mutation and the S228P mutation is referred to as an "IgG4-P-FALA" Ab.

[0018] WO 2017191325A1 (Fischer et al., MAB Discovery GmbH) describes humanized antibodies that specifically bind to IL-1R3 or a fragment or derivative thereof, and specifically describes substitutions at L234A and L235A in the human IgG1 Fc region ("IgG1-LALA" format) or at S228P and L235E in the human IgG4 Fc region ("IgG4-PE" format).

[0019] As outlined in the background section of this specification, it is extremely difficult to identify a mAb with high affinity, high specificity, and potent neutralizing activity for IL-1R3. Surprisingly, the present inventors have developed such an antibody, which has additional advantages. The present disclosure encompasses humanized IL-1R3 antibodies with high affinity and specificity for IL-1R3, which have potent IL-1R3 neutralizing activity, reduced effector function, and improved stability. The antibodies disclosed herein exhibit reduced or no Fcγ receptor signaling and do not induce antibody-dependent cell-mediated cytotoxicity (ADCC). Surprisingly, the antibodies disclosed herein have significantly higher production yields in the IgG4-P-FALA format compared to the more common IgG1-LALA format.

[0020] For the antibodies disclosed herein to be industrially useful, they must be capable of being incorporated into compositions with certain desirable properties. For example, the composition should be stable over time, allowing the use of preservatives without affecting the antibody. The viscosity and opalescence of the composition should remain within certain parameters and not change over time, for example, during storage. The 3D structure of the antibody is important for its function and binding to its target. Therefore, the antibody should not aggregate (self-associate) in the composition, or aggregation should be minimized, and other excipients in the composition should not induce changes in the 3D structure of the antibody or increase self-association. Of particular importance are the isoelectric point and self-association values ​​(both performed on the antibody in a standard dilution composition) and the opalescence value in a concentrated composition. To enable an antibody to be formulated for use on a commercial scale, benchmark tolerance windows must be met for various parameters.

[0021] Generally, a higher isoelectric point indicates higher antibody stability, a lower kD value for self-association indicates higher affinity and therefore a higher likelihood of self-association, and a higher turbidity value (NTU) is less desirable since a clear solution is important. As can be seen for the median values ​​of a series of different antibodies in either IgG1 Fc format or IgG4 Fc format (see Table 1 below), the latter format tends to have less desirable formulatability parameters. Surprisingly, the anti-IL-1R3-IgG4-P-FALA Ab disclosed herein met the benchmark window, in that this value was close enough to the median value to be viable on a commercial scale.

[0022] [Table 1]

[0023] It is worth noting that several anti-IL-1R3 antibodies and other IL-1 / IL-33 / IL-36 receptor superfamily-targeting antibodies in the art do not use the IgG4-P-FALA Fc of the present disclosure. Rather, many of these antibodies use IgG1-LALA Fc. For example, antibody CAN04 (described in U.S. Pat. No. 9,796,783, Ågerstam et al.), antibody CAN10 (described in WO 2022 / 136569A1, Liberg et al.), and spesolimab (anti-IL-36R, described in Chenoweth et al. Immunol Cell Biol. 2020.98(4):287-304) all use IgG1-LALA Fc. The same is true for the anti-IL-1R3 antibodies disclosed in WO 2022 / 053715A1 (Macoin et al.), WO 2022 / 170008A2 (Bigwarfe et al.), and WO 2022 / 243536A1 (Urso et al.), all of which use an IgG1-LALA Fc. The inventors have surprisingly discovered that the antibodies of the invention have substantially higher production yields compared to the IgG1-LALA format commonly used for anti-IL-1R3 antibodies.

[0024] The present disclosure describes anti-IL-1R3 antibodies comprising at least the amino acid substitutions S228P, F234A, and L235A in a human IgG4 Fc region. Specifically, the present disclosure relates to antibodies that specifically bind to IL-1R3, exhibit reduced or no Fcγ receptor signaling, and comprise the antibody heavy chain amino acid sequence of SEQ ID NO: 1 and the light chain amino acid sequence of SEQ ID NO: 2.

[0025] Aspects of the disclosure are described in more detail below.

[0026] I. Definitions and Antibodies of the First Aspect Unless otherwise specified, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the methods of the technology of this disclosure. All publications cited herein are incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that can be used in connection with this disclosure.

[0027] The methods and techniques of the present application are generally performed according to conventional methods well known in the art, unless otherwise indicated, and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, JG, Limbird, LE, and Gilman, AG, eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods. In Enzymology,Academic Press,Inc.;Weir,DMand Blackwell,CC,eds.(1986)Handbook of Experimental Immunology,Vols.I-IV,Blackwell Scientific Publications;Maniatis,T.et al.,eds.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Vols.I-III,Cold Spring Harbor Laboratory Press;Ausubel,FMet al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CRand Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer-Verlag.

[0028] The term "antibody" is used to refer to whole antibodies and antigen-binding fragments of such antibodies, unless otherwise indicated. For example, the term encompasses four-chain IgG molecules and antibody fragments.

[0029] As used herein, the term "antibody fragment" refers to a portion of an intact full-length antibody, for example, as further described below.

[0030] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kDa) and two identical heavy chains (about 55 or 70 kDa) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region, which can be distinguished from the more conserved constant (C) regions of the remainder of each chain.

[0031] The majority of amino acid sequence variation in immunoglobulins is confined to three distinct positions in each of the V regions known as hypervariable or complementarity-determining regions (CDRs), which are directly involved in antigen binding. Starting from the amino terminus, these regions are designated CDR1, CDR2, and CDR3, respectively. These CDRs are held in place by more conserved framework regions (FRs). Starting from the amino terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. Starting from the amino terminus, these combined regions within the V region are designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The locations and numbering system for the CDR and FR regions are defined by Kabat et al. (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, US Government Printing Office (1991) and updates thereof which can be found online). In addition, the CDR region boundaries are further defined by the IMGT nomenclature.

[0032] A "humanized mAb," as used herein, is an antibody composed of a human antibody framework onto which complementarity-determining regions (CDRs) from a non-human antibody have been grafted. Modifications of the human acceptor framework may also be made. Procedures for designing and producing humanized antibodies are well known in the art and are described, for example, in U.S. Pat. Nos. 4,816,397 (Boss et al.); 4,816,567 (Cabilly et al.); 5,225,539 (Winter, MRC); EP 0120694 A2 (Boss et al.); EP 0125023 A1 (Cabilly et al.); EP 0194276 B1 (Neuberger & Rabbitts); EP 0239400 A2 (Winter, MRC); EP 0519596 A1 (Padlan et al.); and WO 1986001533 (Neuberger & Rabbitts). Further details regarding antibodies, humanized antibodies, human engineered antibodies and methods for their preparation can be found in Kontermann, R. and Dijbel, S. eds. (2001, 2010) Antibody Engineering, 2nd ed., Springer-Verlag, New York, NY The entire contents of each of the above-listed patents and patent application publications are incorporated herein by reference.

[0033] The constant region can be derived from any human antibody constant region. The variable region genes can be cloned in frame with the constant region genes into expression vectors to express heavy and light immunoglobulin chains. Such expression vectors can be transfected into antibody-producing host cells for antibody synthesis.

[0034] Human antibody variable and constant regions can be derived from sequence databases. For example, immunoglobulin sequences are available in the IMGT / LIGM database (Giudicelli et al.,

[2006] Nucleic Acids Res. 34[suppl. 1]:D781-D784) or VBase 30 (vbase.mrc-cpe.cam.ac.uk). Deglycosylated antibodies can have significantly altered functionality; see Boyd et al. (1996) Mol. Immunol. 32:1311-1318. "Delta ab" or Δab modification, as used herein, refers to an Fc modification as described in Armour et al. (1999) Eur. J. Immunol. 29:2613-2624.

[0035] The terms "stable," "stability," and "stabilized," as used herein in reference to a binding polypeptide, refer to the resistance of the binding polypeptide to thermal and chemical degradation or fragmentation under given conditions of manufacture, preparation, shipping, and storage. A "stable" composition retains 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% or greater biological activity under given conditions of manufacture, preparation, shipping, and storage. The stability of a binding polypeptide can be assessed, for example, in terms of the degree of degradation or fragmentation or the level of specific fragments, types, or aggregate sizes compared to a control or compared to the starting material using methods and measurements known to those of skill in the art. Such methods and measurements include, but are not limited to, reduction in area under the curve (AUC), size exclusion chromatography (SEC), high-performance (or high-pressure) size exclusion chromatography (HPSEC), liquid chromatography-mass spectrometry (LC-MS), capillary gel electrophoresis (CGE), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) compared to a standard.

[0036] The term "nucleic acid," as used herein, includes DNA molecules encoding the antibodies described herein. Preferred DNA molecules encoding the antibodies described herein are expression vectors suitable for expressing antibody genes in host cells. Expression vectors and host cells for antibody gene expression are known in the art; see, for example, Morrow, KJ Genetic Engineering & Biotechnology News (June 15, 2008) 28(12) and Backliwal, G. et al. (2008) Nucleic Acids Res. 36(15):e96-e96.

[0037] The terms "treat" and "treatment," as used herein, refer to the care of a patient or subject having a disease, disorder, or condition. Treatment may be directed to any one or any combination of the following, but is not limited to: curing the disease, disorder, or condition; ameliorating at least one symptom of the disease, disorder, or condition; and / or prophylactic or preventative action aimed at preventing or reducing the occurrence of the disease, disorder, or condition. In certain embodiments, treatment may be directed to, but is not limited to, curing the disease, disorder, or condition or ameliorating at least one symptom of the disease, disorder, or condition.

[0038] The term "subject," as used herein, refers to any mammal, such as a mouse, rat, gerbil, hamster, guinea pig, rabbit, cat, dog, sheep, goat, pig, cow, horse, and primate. In certain embodiments, the subject is a mammal other than a human. In certain embodiments, the subject is a non-human primate. In certain embodiments, the subject is a human.

[0039] The first aspect relates to an antibody that specifically binds to IL-1R3, the antibody comprising the antibody heavy chain amino acid sequence of SEQ ID NO:1 and the antibody light chain amino acid sequence of SEQ ID NO:2.

[0040] In at least one embodiment, the antibody is a humanized mAb. In at least one embodiment, the antibody is an anti-IL-1R3 antibody.

[0041] A humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) may comprise an antibody constant region (e.g., a human IgG4 constant region) that mediates one or more effector functions. For example, binding of the C1 component of complement to an antibody constant region can activate the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, IgG4 antibodies bind to receptors on various cells via their Fc region, and the Fc receptor binding site on the antibody Fc region binds to Fc receptors (FcRs) on cells. Binding of an antibody to an Fc receptor on the cell surface triggers many important and diverse biological responses, such as phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production.

[0042] In certain embodiments, a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) may comprise a constant region that is incapable of directing one or more effector functions (e.g., ADCC activity) and / or is incapable of binding to Fcγ (Fc gamma) receptors.

[0043] Certain embodiments described herein provide humanized mAbs or humanized mAb fragments (e.g., anti-IL-1R3 antibodies or fragments thereof) in which at least one amino acid in one or more of the constant region domains has been deleted or otherwise altered to confer desired biochemical properties, such as reduced effector function.

[0044] In certain other embodiments, the humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) comprises an Fc region or portion thereof from a human IgG4 molecule and a Ser228Pro (S228P) mutation (EU numbering) in the core hinge region of the molecule.

[0045] In certain exemplary embodiments, the Fc portion of a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) may be mutated to increase or decrease effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (via point mutation or other means) may reduce Fc receptor binding of the circulating modified antibody. In other cases, constant region modifications consistent with the present disclosure may attenuate complement binding and thus reduce the serum half-life and nonspecific association of the conjugated cytotoxin. Still other modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications (e.g., tumor localization, biodistribution, and serum half-life) may be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0046] In certain embodiments, the Fc domain used in a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) is an Fc variant. As used herein, the term "Fc variant" refers to an Fc domain that has at least one amino acid substitution relative to the wild-type Fc domain from which it is derived.

[0047] The amino acid substitutions in the Fc variants can be located at any position within the Fc domain (i.e., at any EU amino acid position). In one embodiment, the Fc variant comprises a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH2 domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH3 domain or a portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0048] In addition to the P-FALA Fc mutations, the antibodies described herein may employ any other art-recognized Fc variant known to provide improved (e.g., reduced) effector function and / or FcR binding, such as those described in International PCT Publication Nos. WO 88 / 07089 A1, WO 96 / 14339 A1, WO 98 / 05787 A1, WO 98 / 23289 A1, WO 99 / 51642 A1, WO 99 / 58572 A1, WO 00 / 09560 A2, WO 00 / 32767 A1, WO 00 / 42072 A2, WO 02 / 44 Brochure No. 215A2, Brochure No. 02 / 060919A2, Brochure No. 03 / 074569A2, Brochure No. 04 / 016750A2, Brochure No. 04 / 029207A2, Brochure No. 04 / 035752A2, Brochure No. 04 / 063351A2, Brochure No. 04 / 074455A2, Brochure No. 04 / 099249A2, Brochure No. 05 / 040217A2, Brochure No. 05 / 070963A1 brochure, 05 / 077981A2 brochure, 05 / 092925A2 brochure, 05 / 123780A2 brochure, 06 / 019447A1 brochure, 06 / 047350A2 brochure, and 06 / 085967A2 brochure, or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; and 6,096,871. and 7,083,784 (each of which is incorporated herein by reference).

[0049] In certain embodiments, a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) may comprise an Fc variant containing an amino acid substitution that alters the antigen-independent effector function of the antibody (particularly the circulating half-life of the antibody). Such antibodies, when compared to antibodies lacking this substitution, exhibit either increased or decreased binding to the neonatal Fc receptor (FcRn), and therefore increased or decreased serum half-life, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals in which a longer half-life of the administered antibody is desired, e.g., treating chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are useful, e.g., for administration to mammals in which a shorter circulation time may be advantageous, for example, for in vivo diagnostic imaging, or in situations in which the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful for treating diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desirable include applications in which localization to the brain, kidney, and / or liver is desired.

[0050] In a first aspect, an antibody is provided that specifically binds to IL-1R3 and comprises the antibody heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0051] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 3, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 4, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0052] In an alternative embodiment, an antibody that specifically binds to IL-1R3 and comprises an antibody heavy chain variable (VH) domain, an antibody light chain variable (VL) domain, and an IgG4 Fc domain, wherein the VH domain is a CDR-H1 sequence comprising the amino acid sequence of SYDMS (SEQ ID NO: 5) or GFSLSSYD (SEQ ID NO: 6); A CDR-H2 sequence comprising the amino acid sequence of TIYIGGTTAYASWPKG (SEQ ID NO: 7) or IYIGGTT (SEQ ID NO: 8); a CDR-H3 sequence comprising the amino acid sequence of LQGANYYNSLAL (SEQ ID NO: 9) or ARLQGANYYNSLAL (SEQ ID NO: 10); and the VL domain comprises A CDR-L1 sequence comprising the amino acid sequence of QASQSIYSFLS (SEQ ID NO: 11) or QSIYSF (SEQ ID NO: 12); A CDR-L2 sequence comprising the amino acid sequence of ASDLES (SEQ ID NO: 13) or AAS (SEQ ID NO: 14); A CDR-L3 sequence comprising the amino acid sequence of QSNYIIDYGA (SEQ ID NO: 15 or SEQ ID NO: 16); wherein the IgG4 Fc domain comprises an F234A substitution and an L235A substitution according to EU numbering.

[0053] In an alternative embodiment, an antibody that specifically binds to IL-1R3 and comprises an antibody heavy chain variable (VH) domain, an antibody light chain variable (VL) domain, and an IgG4 Fc domain, wherein the VH domain is a CDR-H1 sequence comprising the amino acid sequence of SYDMS (SEQ ID NO: 5) or GFSLSSYD (SEQ ID NO: 6); A CDR-H2 sequence comprising the amino acid sequence of TIYIGGTTAYASWPKG (SEQ ID NO: 7) or IYIGGTT (SEQ ID NO: 8); a CDR-H3 sequence comprising the amino acid sequence of LQGANYYNSLAL (SEQ ID NO: 9) or ARLQGANYYNSLAL (SEQ ID NO: 10); and the VL domain comprises A CDR-L1 sequence comprising the amino acid sequence of QASQSIYSFLS (SEQ ID NO: 11) or QSIYSF (SEQ ID NO: 12); A CDR-L2 sequence comprising the amino acid sequence of ASDLES (SEQ ID NO: 13) or AAS (SEQ ID NO: 14); A CDR-L3 sequence comprising the amino acid sequence of QSNYIIDYGA (SEQ ID NO: 15 or SEQ ID NO: 16); wherein the IgG4 Fc domain comprises F234A, L235A and S228P substitutions according to EU numbering.

[0054] [Table 2]

[0055] [Table 3]

[0056] [Table 4]

[0057] [Table 5]

[0058] [Table 6]

[0059] [Table 7]

[0060] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 24, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 29, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises an F234A substitution, an L235A substitution, and an S228P substitution according to EU numbering.

[0061] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 25, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0062] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 25, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0063] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 25, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0064] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 26, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0065] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 26, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0066] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 26, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0067] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 27, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0068] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 27, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0069] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 27, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0070] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 28, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0071] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 28, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0072] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 28, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises an F234A substitution, an L235A substitution, and an S228P substitution according to EU numbering.

[0073] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 33, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 34, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0074] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 35, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 36, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises an F234A substitution, an L235A substitution, and an S228P substitution according to EU numbering.

[0075] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 37, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 44, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0076] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 38, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 45, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises an F234A substitution, an L235A substitution, and an S228P substitution according to EU numbering.

[0077] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 39, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 46, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises an F234A substitution, an L235A substitution, and an S228P substitution according to EU numbering.

[0078] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 40, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 47, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0079] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 41, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 48, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0080] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 42, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 49, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0081] In an alternative embodiment, there is provided an antibody that specifically binds to IL-1R3 and comprises the antibody heavy chain variable (VH) domain amino acid sequence of SEQ ID NO: 43, the antibody light chain variable (VL) domain amino acid sequence of SEQ ID NO: 50, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A, L235A, and S228P substitutions according to EU numbering.

[0082] II. Antibody Production and Related Additional Aspects Production of antibodies can be carried out by any technique known in the art: they can be produced by chemical synthesis or by expression of a gene encoding the antibody in a host cell (e.g., a cell line such as a Chinese hamster ovary (CHO) cell line or a human embryonic kidney (HEK) cell line).

[0083] A third aspect relates to an isolated nucleic acid molecule encoding the antibody of the first aspect. For further details of the antibody of the first aspect, see the disclosure in Section I above.

[0084] A fourth aspect relates to an expression vector comprising the nucleic acid molecule of the third aspect.

[0085] A fifth aspect relates to a host cell comprising the expression vector of the fourth aspect. In one embodiment, the host cell comprises a nucleic acid molecule encoding an antibody of the first aspect. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell; preferably, the host cell is a CHO DXB11 cell.

[0086] A sixth aspect relates to a method of producing an antibody of the first aspect, the method comprising: (i) optionally transfecting a host cell with the isolated nucleic acid molecule of the third aspect or the expression vector of the fourth aspect; (ii) culturing the host cell under conditions that allow expression of the antibody; (iii) recovering the antibody; and (iv) optionally further purifying, and / or modifying, and / or formulating the antibody.

[0087] In one embodiment, the method of manufacture comprises: (i) transfecting a host cell which is a Chinese hamster ovary (CHO) cell with an isolated nucleic acid molecule of the third aspect or an expression vector of the fourth aspect, preferably the host cell is a CHO DXB11 cell. Includes:

[0088] In one embodiment of the production method, the expression level of this antibody is greater than the expression level of an anti-IL-1R3 antibody comprising a human IgG1 Fc region, preferably the human IgG1 Fc region comprises the amino acid substitutions L234A and L235A according to EU numbering. In one embodiment of the production method, the expression level of this antibody is greater than the expression level of an anti-IL-1R3 antibody comprising the amino acid sequence of the human IgG1 Fc region of SEQ ID NO: 18 ... - the amino acid sequence of a human IgG1 Fc region of SEQ ID NO: 18; - the light chain amino acid sequence of SEQ ID NO: 4; In one embodiment of this method of production, the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising: - the amino acid sequence of a human IgG1 Fc region of SEQ ID NO: 18; - the light chain amino acid sequence of SEQ ID NO: 2; The expression level of the anti-IL-1R3 antibody is greater than that of the anti-IL-1R3 antibody containing

[0089] In one embodiment, the method of manufacture comprises: (iv) further purifying, and / or modifying, and / or formulating the antibody; In at least one embodiment, the formulating step comprises combining the antibody with a pharmaceutically acceptable diluent, carrier, or excipient. In one embodiment, step (iv) results in a pharmaceutical composition according to the second aspect.

[0090] In one embodiment, the production method produces an antibody according to the first aspect, in which the expression level of the antibody is greater than the expression level of the same anti-IL-1R3 antibody, which differs only in that the heavy chain is a human IgG1 Fc region with amino acid substitutions L234A and L235A according to EU numbering.

[0091] A seventh aspect relates to an antibody produced by the production method according to the sixth aspect. In one embodiment, the produced antibody is according to the first aspect.

[0092] A polynucleotide encoding a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof disclosed herein) is isolated and inserted into a replicable construct or vector, such as a plasmid, for further propagation or expression in a host cell. Constructs or vectors (e.g., expression vectors) suitable for expressing a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) according to described embodiments are available in the art. A variety of vectors are available, including vectors that are maintained in a single copy or multiple copies in the host cell or that become integrated into the host cell chromosome. The construct or vector can be introduced into a suitable host cell, and cells expressing the humanized immunoglobulin can be generated and maintained in culture. A single vector or multiple vectors can be used for expression of the humanized immunoglobulin.

[0093] Polynucleotides encoding humanized mAbs or humanized mAb fragments (e.g., anti-IL-1R3 antibodies or fragments thereof disclosed herein) are readily isolated and sequenced using conventional procedures (e.g., oligonucleotide probes). Vectors that can be used include plasmids, viruses, phages, transposons, and minichromosomes, with plasmids being a typical embodiment. Generally, such vectors further comprise a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence operably linked to the light chain polynucleotide and / or the heavy chain polynucleotide to facilitate expression. Polynucleotides encoding the light and heavy chains can be inserted into separate vectors and introduced simultaneously or sequentially into the same host cell (e.g., by transformation, transfection, electroporation, or transduction), or, if desired, both the heavy and light chains can be inserted into the same vector prior to such introduction.

[0094] A promoter can be provided for expression in a suitable host cell. The promoter can be constitutive or inducible. For example, a promoter can be operably linked to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain to drive expression of the encoded polypeptide. A variety of promoters suitable for prokaryotic and eukaryotic hosts are available. Prokaryotic promoters include: lac, tac, T3, T7 promoters for E. coli; 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, and glucokinase. Eukaryotic promoters include inducible yeast promoters such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein, and enzymes involved in nitrogen metabolism or maltose / galactose utilization; RNA polymerase II promoters, such as viral promoters such as polyoma, fowlpox, and adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus (particularly immediate early gene promoters), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter, and early or late simian virus 40; and non-viral promoters such as EF-1α (Mizushima and Nagata (1990) Nucleic Acids Res. 18(17):5322). Those skilled in the art will be able to select an appropriate promoter for expressing a humanized antibody or portion thereof.

[0095] Where appropriate, for example, for expression in cells of higher eukaryotes, additional enhancer elements may be included instead of or in addition to those located in the promoters described above. Suitable mammalian enhancer sequences include enhancer elements derived from globin, elastase, albumin, fetoprotein, metallothionine, and insulin. Alternatively, enhancer elements derived from eukaryotic cell viruses, such as the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculovirus enhancer, or mouse IgG2a locus, may be used (see, e.g., Kallmeier & Gay, International Publication No. 2004009823). Such enhancers are often located in the vector at a site upstream of the promoter, but can also be located elsewhere, for example, in the untranslated region or downstream of the polyadenylation signal. The selection and positioning of the enhancer may be based on compatibility with the host cell used for expression.

[0096] In addition, vectors (e.g., expression vectors) may contain a selectable marker for selecting host cells carrying the vector and, in the case of replicable vectors, an origin of replication. Genes encoding products that confer antibiotic or drug resistance are common selectable markers and can be used in prokaryotes (e.g., the F3-lactamase gene (ampicillin resistance), the tet gene (tetracycline resistance)) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin 5 resistance genes). A dihydrofolate reductase marker gene allows for methotrexate selection in various hosts. Genes encoding the gene product of a host auxotrophic marker (e.g., LEU2, URA3, HIS3) are often used as selectable markers in yeast. The use of viral (e.g., baculovirus) or phage vectors, as well as vectors that can integrate into the genome of a host cell, such as retroviral vectors, is also contemplated.

[0097] In eukaryotic systems, polyadenylation and termination signals are operably linked to the polynucleotide encoding the antibody described herein. Such signals are typically located 3' to the open reading frame. In mammalian systems, non-limiting examples of polyadenylation / termination signals include those derived from growth hormone, elongation factor-1 alpha, and viral (e.g., SV40) genes or retroviral long terminal repeats. In yeast systems, non-limiting examples of polyadenylation / termination signals include those derived from the phosphoglycerate kinase (PGK) gene and the alcohol dehydrogenase 1 (ADH) gene. In prokaryotic systems, polyadenylation signals are typically not required; instead, shorter, more defined terminator sequences are commonly used. The selection of polyadenylation / termination sequences can be based on compatibility with the host cell used for expression. In addition to the above, other features that can be used to enhance yield include chromatin remodeling elements, introns, and host cell-specific codon modifications. The codon usage of the antibodies described herein can be altered to accommodate the codon bias of the host cell to enhance transcription and / or product yield (e.g., Hoekema, A. et al. (1987) Mol. Cell Biol. 7(8):2914-24). Codon selection can be based on compatibility with the host cell used for expression.

[0098] Thus, the present disclosure relates to isolated nucleic acid molecules encoding humanized immunoglobulins or heavy or light chains thereof. The present disclosure also relates to isolated nucleic acid molecules encoding antigen-binding portions of immunoglobulins and their chains.

[0099] A humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) can be produced, for example, by expression of one or more recombinant nucleic acids encoding the antibody in a suitable host cell (e.g., a CHO cell line). The host cell can be made using any suitable method. For example, an expression construct (e.g., one or more vectors, e.g., mammalian cell expression vectors) described herein can be introduced into a suitable host cell, and the resulting cell can be maintained under conditions (e.g., in culture) suitable for expression of the construct or vector. Host cells can be prokaryotic, for example, bacterial cells, such as E. coli (e.g., DH5α™ strain) (Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), B. subtilis and / or other suitable bacteria; eukaryotic cells, for example, cells of higher eukaryotes, such as those of mammalian origin (e.g., COS cells, e.g., COS-1 (ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCC Accession No. CRL-9096), CHO DG44 (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA, 77(7):4216-4220), 293 (ATCC accession number CRL-1573), HEK, HeLa (ATCC accession number CCL-2), CVI (ATCC accession number CCL-70), WOP (Dailey, L., et al. (1985) J. Virol., 54:739-749), 3T3, 293T (Pear, WS, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:8392-8396), NS0 cells, SP2 / 0 cells, HuT 78 cells, etc., or plants (e.g., tobacco, Lemna (duckweed), and algae). See, e.g., Ausubel, FM et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc. (1993).In some embodiments, the host cell is not part of a multicellular organism, for example, an isolated host cell or part of a cell culture.

[0100] Host cells can be cultured in spinner flasks, shake flasks, roller bottles, wave reactors (e.g., System 1000 at wavebiotech.com), or hollow fiber systems; however, stirred tank or bag reactors (e.g., Wave Biotech, Somerset, New Jersey, USA), particularly for suspension culture, are preferred for large-scale production. Stirred tank reactors can be adapted for aeration, for example, using spargers, baffles, or low-shear impellers. For bubble column and airlift reactors, direct aeration with air or oxygen bubbles can be used. When host cells are cultured in serum-free culture medium, the medium can be supplemented with a cytoprotectant, such as pluronic F-68, to help prevent cell damage as a result of the aeration process. Depending on the characteristics of the host cells, microcarriers can be used as growth substrates for anchorage-dependent cell lines, or the cells can be adapted for suspension culture. Culturing host cells, particularly vertebrate host cells, can utilize various modes of operation, such as batch, fed-batch, repeated-batch processing (see Drapeau et al. (1994) Cytotechnology 15:103-109), extended-batch processes, or perfusion culture. While recombinantly transformed mammalian host cells can be cultured in serum-containing media, such as media containing fetal calf serum (FCS), it is preferable to culture such host cells in serum-free media, e.g., as disclosed in Keen et al. (1995) Cytotechnology 17:153-163, or commercially available media such as ProCHO™ or UltraCHO™ (Cambrex NJ, USA), supplemented with an energy source, such as glucose, and synthetic growth factors, such as recombinant insulin, as needed. Serum-free culturing of host cells can require that the cells be adapted to grow under serum-free conditions.One adaptation approach is to culture such host cells in serum-containing medium and repeatedly replace 80% of the culture medium with serum-free medium so that the host cells learn to adapt to serum-free conditions (see, e.g., Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21st Century (Beuvery, EC et al., eds), pp. 619-623, Kluwer Academic publishers).

[0101] The humanized mAb or humanized mAb fragment (e.g., an antibody or fragment thereof) according to the described embodiments can be secreted into the culture medium and recovered from the medium and purified using various techniques to obtain a degree of purity appropriate for its intended use. For example, use of a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) to treat a human subject typically requires at least 95% purity, and more typically 98% or 99% purity, as determined by reducing SDS-PAGE, compared to the culture medium containing the therapeutic antibody. In a first example, cellular debris from the culture medium can be removed using centrifugation, followed by a supernatant clarification step using, for example, microfiltration, ultrafiltration, and / or depth filtration. Alternatively, the humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) can be recovered by microfiltration, ultrafiltration, or depth filtration without prior centrifugation. Various other techniques, such as dialysis and gel electrophoresis, as well as chromatographic techniques such as hydroxyapatite (HA), affinity chromatography (optionally including an affinity tagging system such as polyhistidine), and / or hydrophobic interaction chromatography (HIC) (see U.S. Pat. No. 5,429,746), are available. In one embodiment, a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) is captured using Protein A or Protein G affinity chromatography after various clarification steps, followed by additional chromatographic steps such as ion exchange and / or HA chromatography, anion or cation exchange, size exclusion chromatography, and ammonium sulfate precipitation. Various virus removal steps (e.g., nanofiltration, e.g., using a DV-20 filter) may also be used. Following these various steps, a purified preparation comprising at least 10 mg / mL or more (e.g., 100 mg / mL or more) of the antibodies described herein is provided, thus forming another embodiment described herein. Concentrations of 100 mg / mL or more can be obtained by ultracentrifugation. Such preparations are substantially free of aggregated forms of the antibody.

[0102] Bacterial systems are particularly suitable for the expression of antibody fragments. Such fragments are localized intracellularly or within the periplasm. Insoluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art (see Sanchez et al. (1999) J. Biotechnol. 72:13-20; Cupit, PM et al. (1999) Lett. Appl. Microbiol. 29:273-277).

[0103] A fifth aspect relates to a host cell comprising the expression vector of the fourth aspect. The present disclosure also relates to cells (host cells) comprising a nucleic acid, e.g., a vector (e.g., an expression vector), described herein. For example, nucleic acids (i.e., one or more nucleic acids) encoding the heavy and light chains of a humanized immunoglobulin according to the described embodiments, or constructs (e.g., one or more constructs, e.g., one or more vectors) comprising such nucleic acids, can be introduced into a suitable host cell by a method appropriate for the selected host cell (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is or becomes operably linked to one or more expression control elements (e.g., expression control elements in a vector, expression control elements in a construct created by intracellular processes, expression control elements integrated into the host cell genome). The host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, which is a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. If desired, the encoded humanized antibody can be isolated, for example, from host cells, culture medium, or milk, a process that involves expression in host cells (e.g., mammary cells) of transgenic animals or plants (e.g., tobacco) (see, e.g., Lonberg & Kay, WO 1992003918).

[0104] Batch consistency and comparability are highly relevant to the successful pharmaceutical development of recombinant mAbs and related products. Small structural changes can result in variants (or proteoforms) that differ in size, charge, or hydrophobicity. These changes may or may not affect the stability, pharmacokinetics, and efficacy of the recombinant mAb. The presence of the same types of changes found in endogenous immunoglobulin G (IgG) can substantially reduce the safety risks of the mAb.

[0105] The following post-translational and physicochemical modifications can occur in recombinant mAbs and are a function of the expression system used: N-terminal modifications (N-terminal pyroglutamic acid, incomplete removal of signal peptide, truncation); asparagine deamidation; aspartate isomerization; presence of succinimide; degradation / oxidation of amino acid residues (especially methionine and tryptophan); cysteine-related modifications (free cysteine ​​residues, alternative disulfide bond ligation (recombinant), trisulfide bonds, formation of thioethers, cysteine ​​racemization); glycosylation; glycation; C-terminal modifications (C-terminal lysine clipping, amidation, sequence variation due to inherent errors in transcription or translation); and rare chemical modifications (e.g., oxidative carbonylation, histidine-histidine cross-links, tyrosine sulfation, heavy chain N-terminal modification with maleuric acid, modification of N-terminal primary amines or lysine side chains with citric acid or its degradation products, and O-fucosylation of serine residues).

[0106] III. Pharmaceutical Compositions and Administration Methods of Anti-IL-1R3 Antibodies A second aspect relates to a pharmaceutical composition comprising an antibody of the first aspect and a pharmaceutically acceptable diluent, carrier, or excipient. In certain embodiments, a pharmaceutical composition is provided comprising a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) described herein, or a ligand identifiable by the assay method defined in the previous aspect of the disclosure. The ligand can be an immunoglobulin, peptide, nucleic acid, or small molecule, as discussed herein. In the discussion below, these are referred to as "compounds."

[0107] In one embodiment, the pharmaceutical compositions described herein are compositions of matter comprising a compound capable of modulating T cell activity as an active ingredient. This compound may be in the form of any pharmaceutically acceptable salt, or, for example, an analog, free base form, tautomer, enantiomeric racemate, or combinations thereof, as appropriate. The active ingredient of the pharmaceutical compositions comprising the active ingredient described herein is intended to exhibit therapeutic activity when administered in an amount appropriate to the specific case, for example, in the treatment of graft-versus-host disease.

[0108] In certain embodiments, a pharmaceutical composition comprises a humanized mAb or humanized mAb fragment described herein (e.g., an anti-IL-1R3 antibody or fragment thereof) and a pharmaceutically acceptable diluent, carrier, or excipient. In at least one embodiment, the pharmaceutical composition is an aqueous composition.

[0109] In certain embodiments, one or more compounds described in the present disclosure may be used in combination with any art-recognized compound known to be suitable for treating a particular indication in the treatment of any of the above-mentioned conditions.Thus, one or more compounds described herein may be combined with one or more art-recognized compounds known to be suitable for treating the above-mentioned indication, so that a single convenient composition can be administered to the subject.Dosage regimens may be adjusted to obtain optimal therapeutic responses.

[0110] For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0111] The active ingredient can be administered in any convenient manner, such as orally, intravenously (if water-soluble), intramuscularly, subcutaneously, intranasally, intradermally, or via suppository route, or by implantation (e.g., implantation using sustained-release molecules). In the case of transplantation, the active ingredient can also be used to treat cells, tissues, or organs to be transplanted into a patient prior to the transplantation. This can be done, for example, to prevent graft-versus-host disease, reduce the likelihood of the disease, or alleviate the symptoms of the disease.

[0112] Depending on the route of administration, the active ingredient may need to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it.

[0113] To administer the active ingredient by a means other than parenteral administration, the active ingredient is coated with or administered with a material to prevent its inactivation. For example, the active ingredient can be administered in an adjuvant, co-administered with an enzyme inhibitor, or administered in a liposome. The term "adjuvant" is used in its broadest sense and includes any immunostimulating compound, such as interferon. Adjuvants contemplated herein include resorcinol, nonionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin.

[0114] Liposomes include water-in-oil-in-water emulsions and conventional liposomes.

[0115] The active ingredient may also be administered parenterally or intraperitoneally.

[0116] Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0117] Pharmaceutical forms suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0118] Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0119] Sterile injectable solution is prepared by incorporating the required amount of active ingredient into a suitable solvent with some of the other ingredients listed above, if necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains a basic dispersion medium and the other ingredients listed above that are required.For the preparation of sterile injectable solution, the preferred method of preparing sterile powder is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0120] Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active ingredient may be incorporated into sustained-release preparations and formulations.

[0121] As used herein, "pharmaceutically acceptable diluents, carriers, or excipients includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In certain embodiments, the pharmaceutically acceptable carrier or excipient is an aqueous liquid. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0122] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage for a mammalian subject to be treated, each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the novel dosage unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active substance and the specific therapeutic effect to be achieved, and (b) the limitations inherent in formulation technology, such as active substances for the treatment of diseases in living subjects with diseases or conditions that impair physical health. The main active ingredient is formulated in dosage unit form with a suitable pharmaceutically acceptable carrier in an effective amount for convenient and effective administration. In the case of compositions containing auxiliary active ingredients, the dosage is determined by referring to the usual dosage and administration method of the ingredient.

[0123] To facilitate the delivery of peptide compounds such as antibodies into cells, peptides can be modified to improve the peptide's ability to cross cell membranes. For example, U.S. Patent No. 5,149,782 to Chang et al. (Tanox Biosystems, Inc.) discloses the use of fusogenic peptides, ion channel-forming peptides, membrane peptides, long-chain fatty acids, and other membrane-mixing agents to increase protein transport across cell membranes. These and other methods are also described in International Publication No. WO 1997037016 to Wallach et al. and U.S. Patent No. 5,108,921 to Low et al. (which are incorporated herein by reference).

[0124] In a further aspect, there is provided an active ingredient as described herein for use in the treatment of a disease, either alone or in combination with an art-recognized compound known to be suitable for treating a particular indication. Thus, there is provided a use of an active ingredient as described herein for the manufacture of a medicament for the treatment of a disease associated with an aberrant immune response.

[0125] Further provided is a method for treating a condition associated with an aberrant immune response, comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof.

[0126] IV: Methods of Treating Diseases or Disorders An eighth aspect relates to the antibody of the first aspect for use in treating a disease or disorder in a subject in need thereof, preferably wherein the disease or disorder is an autoimmune or autoinflammatory disease or disorder.

[0127] A ninth aspect relates to the pharmaceutical composition of the second aspect for use in treating a disease or disorder in a subject in need thereof, preferably wherein the disease or disorder is an autoimmune or autoinflammatory disease or disorder.

[0128] A tenth aspect relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an antibody of the first aspect or a pharmaceutical composition of the second aspect.

[0129] Another aspect relates to the use of the antibody of the first aspect or the pharmaceutical composition of the second aspect in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

[0130] Another aspect relates to the use of the antibody of the first aspect or the pharmaceutical composition of the second aspect for treating a disease or disorder in a subject in need thereof.

[0131] In light of the evidence presented, one skilled in the art will understand that the antibodies of the present disclosure may be used in the prevention, treatment, mitigation, detection and / or diagnosis of inflammatory and / or fibrotic and / or neoplastic diseases and disorders due to their ability to inhibit IL-1α, IL-1β, IL-33, IL-36α, IL-36β and IL-36γ signaling, thereby simultaneously targeting several IL-dependent pathways.

[0132] One embodiment relates to the antibody of the first aspect or the pharmaceutical composition of the second aspect for use in treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is an inflammatory and / or fibrotic disease or disorder, preferably an inflammatory skin disease.

[0133] In some embodiments, the disease or disorder is an inflammatory and / or fibrotic disease or disorder. In some embodiments, the disease or disorder is an inflammatory and / or fibrotic disease or disorder, including rheumatoid arthritis, any type of arthritis, psoriatic arthritis, any type of juvenile arthritis, such as systemic-onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells disease, neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), septic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome, adult-onset Still's disease, hyper-IgD syndrome, type 2 diabetes, macrophage activation syndrome, TNF receptor-associated periodontitis, Blau's disease, ankylosing spondylitis, Sweet's disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, allergies, atherosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, IgD syndrome, rheumatoid arthritis ... Type 2 diabetes, chronic obstructive pulmonary disease (COPD), Helicobacter pylori-associated gastritis, inflammatory bowel disease (including ulcerative colitis), hepatitis, hepatitis C, ischemia-reperfusion injury, multiple sclerosis, Neisseria or pneumococcal meningitis, tuberculosis, Behçet's syndrome, septic shock, graft-versus-host disease, adult T-cell leukemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (e.g., metabolic syndrome, cardiac hypertrophy, congestive heart failure, myocardial infarction, The present invention relates to a method for treating varicose veins, polycystic ovary syndrome, gastroesophageal reflux disease (GERD), fatty liver, colorectal cancer, breast cancer, uterine cancer, chronic renal failure, stroke, and hyperuricemia, intervertebral disc disease, irritable bowel syndrome, Schnitzler's syndrome, allergic / atopic dermatitis, acne inversa (hidradenitis suppurativa), cardiac fibrosis, cardiovascular disease, cryopin-associated periodic syndrome, cystic fibrosis, Goodpasture's syndrome, Guillain-Barré syndrome, renal fibrosis, liver fibrosis, lung fibrosis (pulmonary fibrosis), skin fibrosis (dermal fibrosis), myocarditis, autoimmune myocarditis, organ failure associated with organ transplantation, pancreatitis, peritonitis, uveitis, vasculitis, pneumonia, pulmonary hypertension, scleroderma chronic graft-versus-host disease, sepsis, Sjogren's syndrome, Takayasu's arteritis, and gout.

[0134] In at least one embodiment, the disease or disorder is an inflammatory condition, such as a metabolic rheumatic disorder associated with hyperuricemia, which may be selected from the group consisting of gout, pseudogout, drug-induced gout, and chronic active (refractory) gout.

[0135] In at least one embodiment, the disease or disorder is an IL-1 dependent inflammatory disease. For example, the disease can be a systemic or local inflammatory disease. In at least one embodiment, the disease or disorder is selected from the group consisting of Schnitzler's syndrome, Behcet's disease, secondary amyloidosis, Henoch-Schönlein purpura, idiopathic relapsing pericarditis, systemic juvenile idiopathic arthritis, adult-onset Still's disease (AOSD), macrophage activation syndrome, Sweet's syndrome / neutrophilic dermatosis (acute febrile neutrophilic dermatosis), neutrophilic panniculitis, Erdheim-Chester disease (histiocytosis), SAPHO syndrome (synovitis, acne, pustulosis, osteophytosis, osteitis), PFAPA (periodic aphthous stomatitis, pharyngitis, adenitis), multicentric Castleman's disease, Jessner-Kanof disease, primary Sjogren's syndrome (fatigue), Kawasaki disease, colitis in chronic granulomatous disease, hidradenitis suppurativa (acne suppurativa inversa), autoimmune inner ear disease, and severe traumatic brain injury. In at least one embodiment, the disease or disorder is a genetic systemic inflammatory disease, such as Familial Mediterranean Fever (FMF), Cryopyrin-Associated Periodic Syndrome (CAPS), Tumor Necrosis Factor (TNF) Receptor 1-Associated Periodic Syndrome (TRAPSa), Hyper IgD Syndrome (HIDS), PAPA (Septic Arthritis, Pyoderma Gangrenosum, and Acne) Syndrome, PASH (Pyoderma Gangrenosum, Acne, and Hidradenitis Suppurativa) Syndrome, PAPASH (Septic Arthritis, Acne, Pyoderma Gangrenosum, and Hidradenitis Suppurativa) Syndrome, Deficiency of Interleukin-1 Receptor Antagonist (DIRA), Blau Syndrome / Granulomatous Arthritis, Mevalonate Kinase Deficiency, Majeed Syndrome, and NLRP12 (Nucleotide-Binding Lectin-Rich Repeat-Containing Receptor 12) Autoinflammatory Syndrome.

[0136] In certain embodiments, the disease or disorder is selected from the group consisting of atopic dermatitis, acne inversa (hidradenitis suppurativa), pyoderma syndrome, pyoderma gangrenosum, pustular psoriasis, asthma, idiopathic pulmonary fibrosis, peritonitis, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD). In at least one embodiment, the disease or disorder is pyoderma syndrome. In at least one embodiment, the disease or disorder is pyoderma gangrenosum.

[0137] In at least one embodiment, the disease or disorder is asthma. In at least one embodiment, the disease or disorder is idiopathic pulmonary fibrosis. In certain embodiments, the disease or disorder is selected from the group consisting of hidradenitis suppurativa (acne inversa) and COPD, preferably selected from the group consisting of hidradenitis suppurativa (acne inversa). In at least one embodiment, the disease or disorder is atopic dermatitis. In certain embodiments, the disease or disorder is a respiratory disease. In certain embodiments, the disease or disorder is an inflammatory skin disease.

[0138] One embodiment relates to the antibody of the first aspect or the pharmaceutical composition of the second aspect for use in treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is a neoplastic disease or disorder.

[0139] In some embodiments, the disease or disorder is a neoplastic disease or disorder, and the neoplastic disease or disorder is a hematological disease or disorder or a solid tumor. In some embodiments, the neoplastic disease or disorder is a hematological disease or disorder, and the neoplastic hematological disease or disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). In some embodiments, the neoplastic disease or disorder is a solid tumor, and the solid tumor is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colon cancer, colorectal cancer, melanoma, bladder cancer, brain / CNS cancer, cancer of the urinary system, cancer of the ureter (also known as bile duct cancer), cervical cancer, esophageal cancer, gastric cancer, head and neck cancer (head and neck squamous cell carcinoma), kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, sarcoma, skin cancer, and uterine cancer. Preferably, the neoplastic disease or disorder is selected from the group consisting of breast cancer, colon cancer, lung cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, colorectal cancer, stomach cancer, gastric cancer, estrogen receptor positive breast cancer, head and neck squamous cell carcinoma, mesothelioma, gallbladder cancer, ovarian cancer, bladder cancer, prostate cancer, thyroid cancer, Hodgkin's disease, MALT lymphoma, salivary gland cancer or melanoma.

[0140] A further aspect relates to a method of treating a subject, wherein the subject is resistant to or responds poorly to treatment with one or more cytotoxic, cytostatic, or targeted anti-cancer agents. In one embodiment, the antibody of the first aspect is administered in combination with one or more cytotoxic, cytostatic, or targeted anti-cancer agents. In one embodiment, the antibody of the first aspect is administered simultaneously with the one or more cytotoxic, cytostatic, or targeted anti-cancer agents. In one embodiment, the antibody of the first aspect is administered sequentially with the one or more cytotoxic, cytostatic, or targeted anti-cancer agents. In the latter case, the antibody of the first aspect is preferably administered after treatment with the one or more cytotoxic, cytostatic, or targeted anti-cancer agents. The cytotoxic or cytostatic anti-cancer agents can be taxanes, anthracyclines, alkylating agents, histone deacetylase inhibitors, topoisomerase inhibitors, kinase inhibitors, nucleotide analogs, peptide antibiotics, and platinum-based drugs.

[0141] In at least one embodiment, the disease or disorder is cancer-associated chronic inflammation.

[0142] A further aspect relates to the non-therapeutic use of the antibody of the first aspect or the pharmaceutical composition of the second aspect for cosmetic purposes.

[0143] The examples presented below are for illustrative purposes only and should not be construed as limitations on the compositions and methods described herein. [Example]

[0144] Example 1: Generation of anti-IL-1R3 IgG4-P-FALA antibodies Anti-IL-1R3 mAbs were humanized by CDR grafting using recombinant DNA technology. The human IgG backbone was either an IgG4 backbone containing P-FALA (S228P, F234A, L235A) mutations in the Fc region or an IgG1 backbone containing LALA (L234A, L235A) mutations in the Fc region. The resulting vectors encoded the heavy and light chains of the humanized antibodies anti-IL-1R3-IgG4-P-FALA or anti-IL-1R3-IgG1-LALA.

[0145] Example 2: Selection of CHO cell line clones expressing anti-IL-1R3 IgG4-P-FALA antibodies in CHO cell lines The CHO DXB11 host strain was derived directly from the CHO DXB11 cell line (Urlaub and Chasin, 1980, Proc Natl Acad Sci USA) by final adaptation to a commercially available animal-component-free, chemically defined medium (CD DG44) followed by subcloning. A host master cell bank was created, and this cell bank was used to generate a host working cell bank (WCB). The WCB was used as the cell source for generating anti-IL-1R3-expressing cell lines.

[0146] The host cell line is grown in a commercially available medium (CD DG44, ThermoFisher Scientific) supplemented with 4 mM L-glutamine and 0.18% (v / v) poloxamer 188 (Pluronic F68, ThermoFisher Scientific). The cells are grown as suspension cultures at 37°C, 5% CO2, and 80% relative humidity. The host cell line is cryopreserved in this CD DG44 growth medium (CD DG44, 4 mM L-glutamine, 0.18% (v / v) poloxamer 188) with 7% (v / v) dimethyl sulfoxide (DMSO) as a cryoprotectant.

[0147] [Table 8]

[0148] [Table 9]

[0149] [Table 10]

[0150] [Table 11]

[0151] The DNA sequences of the expression cassettes / vectors encoding the heavy or light chains of the anti-ILR13 antibodies are shown in Table 4. Vectors encoding the heavy and light chain polypeptides of the anti-IL-1R3 IgG4-P-FALA Ab (aIL-1R3-IgG4) or anti-IL-1R3 IgG1 LALA Ab (aIL-1R3-IgG1) were electroporated into the CHO DXB11 cell line in five replicate pools using dihydrofolate reductase (DHFR) selection. The vector carrying the heavy chain also contained a CD52 reporter. The electroporated cells were subjected to two rounds of selection in growth medium containing 5 nM methotrexate (MTX) and 50 nM MTX, respectively. Antibody titers were measured in all five pools after the second round of selection.

[0152] Figure 1, titled "50 mM MTX Selection Pools - 7-Day Batch Titers," shows the pool number and corresponding antibody titers for each of the five pools selected after the second round of MTX selection for pools expressing either aIL-1R3-IgG1 or aIL-1R3-IgG4 Abs. As shown in Figure 1, all five selected pools expressing aIL-1R3-IgG1 antibodies produced antibody titers of less than 0.1 g / L. Of the five selected pools expressing aIL-1R3-IgG4 antibodies, four pools, 1, 2, 3, and 4, produced higher titers of antibody and were therefore feasible for clonal isolation.

[0153] Pools 2 and 4 expressing aIL-1R3-IgG4 were used for clone selection using a clone selection tool. Clones were reviewed for clonality, and validated clones were expanded for evaluation by AMBR (cell line screening software). The top 21 clones were evaluated for cell growth, titer, metabolites, and product quality. The top six clones selected based on antibody titer were A136, A155, A101, A61, and B21. "A" clones were from pool 2, and "B" clones were from pool 4. The top six clones also showed the highest antibody productivity among all selected clones.

[0154] Example 3: Inhibition of IL-1α / β, IL33, and IL-36α / β / γ-induced IL-8 release by skin-derived carcinoma A-431 cells The in vitro efficacy of anti-IL-1R3 antibodies to block multiple cytokine activity was tested in the A-431 stable cell line for IL8 cytokine release.

[0155] A-431 cells were seeded into 384-well black, flat-bottom, tissue-culture-treated microplates (Corning #3764) at a cell density of 20,000 cells / well in 20 μl of DMEM, 10% heat-inactivated FCS medium. Serially diluted antibodies in DMEM, 1% heat-inactivated FCS, were immediately added in a volume of 5 μl, and the plates were incubated for 60 minutes at 37°C / 5% CO2. Five μl of each recombinant human IL-1α / 1β, IL-33, or IL-36α / 36β / 36γ (R&D Systems) protein, prepared in DMEM, 1% heat-inactivated FCS, was then added to 5 μl of medium to a final concentration of 3 ng / ml (IL-1α / β), 125 ng / ml (IL-33), or 30 ng / ml (IL-36α / β / γ). The plates were incubated for 24 hours at 37°C / 5% CO2. Each condition was tested in three technical replicates. Secreted human IL-8 levels in the supernatant were measured using the CisBio HTRF IL8 Detection Kit (Cat. No. 62HIL08PEG) according to the manufacturer's instructions. Fitting curves and EC50 calculations were performed using XLfit fitting. A summary of the results is shown in Table 5 below. The anti-IL-1R3-IgG4-P-FALA antibody was compared with the anti-IL-1R3-IgG1-LALA antibody. Both antibodies showed robust activity against the three pathways.

[0156] [Table 12]

[0157] Example 4: Fcγ receptor binding activity of anti-IL-1R3 antibodies in IgG1-LALA and IgG4-P-FALA formats. Human Fcγ receptors were obtained from R&D Systems. Binding studies were performed by surface plasmon resonance (SPR) using a Biacore T200 instrument. Fcγ receptors were captured on a CM5 sensor chip using an anti-his capture antibody according to the manufacturer's instructions, using a receptor concentration of 0.1 μg / ml. Antibodies were flowed over the thus-captured receptors at 3.0 μM for 300 s (Fcγ RI) and 420 s (Fcγ RIII), respectively. A buffer solution was then flowed over the chip surface to monitor dissociation of the antibody-receptor complex.

[0158] Figure 2A shows that anti-IL-1R3-IgG1-LALA showed residual binding to human FcγRIIIa, and Figure 2B shows that human FcγRIIIa V176F mutant, but anti-IL-1R3-IgG4-P-FALA, showed no binding.

[0159] FIG. 2C shows that anti-IL-1R3-IgG1-LALA, but not anti-IL-1R3-IgG4-P-FALA, showed residual binding to human Fcγ RI (CD64).

[0160] In all of Figures 2A, 2B and 2C, the x-axis represents time in seconds and the y-axis represents response in response units (RU), with 0 representing the capture baseline.

[0161] The same trend was observed for the cynomolgus monkey receptor (data not shown).

[0162] For mouse Fcγ receptors, no binding was detected for either anti-IL-1R3-IgG1-LALA or anti-IL-1R3-IgG4-P-FALA (data not shown).

[0163] Example 5: Cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-IL-1R3 antibodies in IgG1-LALA and IgG4-P-FALA formats. In antibody-dependent cell-mediated cytotoxicity (ADCC), effector cells actively lyse target cells bound by specific antibodies. The process begins with antibody binding to the surface of the target cell. Effector cells use Fc receptors found on the cell surface to recognize and bind to the Fc region of the antibody. Upon binding, the effector cells release cytotoxic factors that ultimately kill the target cell.

[0164] Anti-IL-1R3 antibodies in the IgG1-LALA and IgG4-P-FALA formats were tested for any ADCC activity using a reporter gene assay (iLite ADCC Bioassay #BM5001, SVAR Life Sciences, Malmo). This assay uses reporter gene-bearing effector cells (Jurkat cells expressing Fc gamma RIIIa (V158)) in combination with target cells expressing a specific antigen at a constant, high level (i.e., CHO IL1R3 target cells). Binding of the antibody to the target cells and binding of the effector cells to the antibody's Fc receptor results in expression of a firefly luciferase reporter gene in the effector cells. The firefly luciferase promoter in the effector cells contains binding sites for NfkB, AP1, NFAT, CRE, and STAT, thus encompassing five key transcription factors of the FcγRIII signaling pathway. The effector cells also contain Renilla luciferase for normalization purposes.

[0165] Figure 3A shows the raw data comparing an anti-IL-1R3 antibody of the invention in IgG1-LALA and IgG4-P-FALA formats with an IgG1 control antibody, and Figure 3B shows the normalized data. The IgG1 control antibody, used as a positive control, shows a strong reporter gene signal indicative of ADCC, while the IgG1-LALA format significantly reduces the reporter signal. Surprisingly, the IgG4-P-FALA format completely abolishes the reporter gene signal, indicating a complete absence of Fcγ receptor signaling.

[0166] Cited references Angal S, King DJ, Bodmer MW, Turner A, Lawson AD, Roberts G, Pedley B, Adair JR. A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody. Mol Immunol. 1993 Jan;30(1):105-8. doi:10.1016 / 0161-5890(93)90432-b. PMID:8417368. Labrijn A.F., Rispens T., Meesters J., Rose R.J., den Bleker T.H., Loverix S., van den Bremer E.T., Neijssen J., Vink T., Lasters I., Aalberse R.C., Heck A.J., van de Winkel J.G., Schuurman J., Parren P.W. (2011) Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength. J. Immunol. 187, 3238-3246 Parekh BS, Berger E, Sibley S, Cahya S, Xiao L, LaCerte MA, Vaillancourt P, Wooden S, Gately D. Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay. MAbs. 2012 May-Jun;4(3):310-8. doi:10.4161 / mabs.19873. Epub 2012 Apr 26. PMID:22531445; PMCID:PMC3355484. Silva JP,Vetterlein O,Jose J,Peters S,Kirby H.The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.J Biol Chem.2015 Feb 27;290(9):5462-9.doi:10.1074 / jbc.M114.600973.Epub 2015 Jan 7.PMID:25568323;PMCID:PMC4342462.

Claims

1. An antibody that specifically binds to IL-1R3, the antibody comprising an antibody heavy chain comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:

2.

2. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable diluent, carrier, or excipient.

3. An isolated nucleic acid molecule encoding an antibody that specifically binds to IL-1R3, said antibody comprising a heavy chain comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:

2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. The host cell of claim 5, which is a Chinese hamster ovary (CHO) cell.

7. 1. A method for producing an antibody that specifically binds to IL-1R3, comprising: (i) transfecting a host cell with an isolated nucleic acid molecule encoding the antibody or an expression vector comprising a nucleic acid molecule encoding the antibody; (ii) culturing the host cells under conditions that allow expression of the antibody; (iii) recovering the antibody; (iv) optionally further purifying and / or modifying and / or formulating the antibody; wherein the antibody comprises a heavy chain comprising SEQ ID NO: 1 and a light chain comprising SEQ ID NO:

2.

8. 8. The method of claim 7, wherein the host cell is a Chinese hamster ovary (CHO) cell.

9. (v) further formulating the antibody. wherein said formulating comprises dissolving said antibody in a pharmaceutically acceptable diluent, carrier or excipient.

8. The method of claim 7, comprising combining with an agent.

10. 8. The method of claim 7, wherein the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising a human IgG1 Fc region, and the human IgG1 Fc region comprises amino acid substitutions L234A and L235A according to EU numbering.

11. 9. The method of claim 8, wherein the host cell is a CHO DXB11 cell.

12. (v) further formulating the antibody.

9. The method of claim 8, wherein said formulating comprises combining said antibody with a pharmaceutically acceptable diluent, carrier, or excipient.

13. (v) further formulating the antibody.

12. The method of claim 11 , wherein said formulating comprises combining said antibody with a pharmaceutically acceptable diluent, carrier, or excipient.

14. 9. The method of claim 8, wherein the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising a human IgG1 Fc region, and the human IgG1 Fc region comprises amino acid substitutions L234A and L235A according to EU numbering.

15. 12. The method of claim 11, wherein the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising a human IgG1 Fc region, the human IgG1 Fc region comprising amino acid substitutions L234A and L235A according to EU numbering.

16. The host cell of claim 5 , wherein the host cell is a CHO DXB11 cell.

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