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

Abstract

The invention provides an antibody specifically binding to IL-1R3. The antibody comprises an antibody heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2. Also provided is a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent, carrier or excipient. It also relates to said antibodies 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

[0001] Sequence Listing

[0002] This application contains a Sequence Listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. Technical Field

[0003] An antibody that specifically binds to IL-1R3 and comprises the heavy chain amino acid sequence of the antibody of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2. Also provided is a method for treating a disease or disorder in a subject in need thereof, the method comprising administering the antibody to the subject. Background Art

[0004] Interleukin-1 (IL-1) is a central mediator of innate immunity and inflammation. The type 1 IL-1 receptor (IL-1R1) and the 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, of the IL-1-induced effects. In addition to IL-1R1, IL-1R3 also serves as a receptor subunit of the heterodimeric IL-33 and IL-36 receptor complexes. Thus, IL-1R3 functions 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 that serve 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 numerous inflammatory syndromes, autoimmune disorders, and cancers. Tight regulation of the IL-1 family cytokine signaling pathways by receptor antagonists, decoy receptors, and signaling inhibitors ensures a balance between amplification of innate immunity and uncontrolled inflammation. There are human gene validations that link the IL-1 family signaling pathway to autoimmune diseases. Since inhibition of IL-1R3 can inhibit all three signaling pathways, blocking IL-1R3 is a multi-targeted strategy that will neutralize three cytokine pathways (IL-1, IL-33, and IL-36) and confer efficacy in indications where single cytokine targeting may not be sufficient. Accordingly, there is a need to develop therapeutic anti-IL-1R3 antibodies. For several years, attempts have been made to generate functional monoclonal antibodies (mAbs) against human IL-1R3. However, there is a need for improved anti-IL-1R3 antibodies. In particular, there is a need for anti-IL-1R3 antibodies that are advantageous in potently inhibiting the three signaling pathways without affecting other cellular pathways. Summary of the Invention

[0005] In a first aspect, an antibody that specifically binds to IL-1R3 is provided, which antibody comprises the heavy chain amino acid sequence of the antibody of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0006] In a second aspect, a pharmaceutical composition is provided, which pharmaceutical composition comprises the antibody of the first aspect and a pharmaceutically acceptable diluent, carrier or excipient.

[0007] In a third aspect, an isolated nucleic acid molecule is provided, which isolated nucleic acid molecule encodes the antibody of the first aspect.

[0008] In a fourth aspect, an expression vector is provided, which expression vector comprises the nucleic acid molecule of the third aspect.

[0009] In a fifth aspect, a host cell is provided, which host cell comprises the expression vector of the fourth aspect.

[0010] A sixth aspect relates to a method of making the antibody of the first aspect, the method comprising the steps of: (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 permit expression of the antibody; (iii) recovering the antibody; and (iv) optionally further purifying and / or modifying and / or formulating the antibody.

[0011] A seventh aspect relates to an antibody produced by the method of making according to the sixth aspect.

[0012] 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.

[0013] 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.

[0014] In a tenth aspect, a method for treating a disease or disorder in a subject in need thereof is provided, the method comprising administering to the subject the antibody of the first aspect or the pharmaceutical composition of the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments in conjunction with the accompanying drawings.

[0016] Figure 1Depicts the anti-IL-1R3 antibody titers in the 50 nM methotrexate (MTX) selection pool after transfection. These pools express anti-IL-1R3 antibodies containing the Fc region of IgG1 (IgG1-LALA) with L234A and L235A mutations or IgG4 (IgG4-P-FALA) with S228P, F234A, and L2345A mutations.

[0017] Figure 2 shows surface plasmon resonance (SPR) sensorgrams that demonstrate that the anti-IL-1R3 antibody in the IgG1-LALA form has residual binding activity to FcγR III ( Figure 2A and 2B [V176F mutant]) and FcγRI ( Figure 2C ) compared to the control (IgG1 antibody), while the same anti-IL-1R3 antibody in the IgG4-P-FALA form does not bind to Fcγ receptors I or III. In all Figure 2A 、 2B and 2C, the x-axis depicts time in seconds (s), and the y-axis depicts the response in response units (RU), where 0 represents the capture baseline.

[0018] Figure 3 shows a cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay that determines the binding of effector cells to the Fc region of the antibody. Although the anti-IL-1R3 antibody in the IgG1-LALA form ( Figure 3A and B[EFF-20-074-1]) shows significantly reduced ADCC activity compared to the IgG1 form (i.e., WT)[FF-20-2050-1], the IgG4-P-FALA form [EFF-20-075-1] results in complete silencing, with effector cells not binding to the Fc receptor. Detailed Description

[0019] Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector function is desired.

[0020] Certain mutations in the Fc region of IgG4 Abs can further reduce effector function. The IgG4 residues 234 and 235 according to the EU index (Proc Natl Acad Sci US A. [Proceedings of the National Academy of Sciences of the United States of America] 1969, 63(1), 78 - 85; Kabat et al., Sequences of proteins of immunological interest [Sequences of Proteins of Immunological Interest], 1991 fifth edition) can be mutated such that the phenylalanine at position 234 is changed to alanine (F234A) and the leucine at position 235 is changed to alanine (L235A) (Parekh et al. 2012 - see the cited references section). Such antibody mutations are referred to as FALA mutations. However, IgG4 Abs are dynamic molecules capable of undergoing Fab arm exchange (FAE), resulting in functionally monovalent bispecific antibodies (bsAbs), the therapeutic efficacy of which may be reduced. The amino acid residues serine (S228) at position 228 and arginine at position 409 of IgG4 drive FAE (Labrijn et al., 2011 - see the cited references section). Substituting 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 cited references section). The above FALA mutations and S228P mutations can be introduced into the constant region of the antibody simultaneously. The IgG4 heavy chain with the FALA mutation is referred to as the "IgG4 FALA" type heavy chain, the IgG4 heavy chain with the S228P mutation is referred to as the "IgG4-P" type heavy chain, and the IgG4 heavy chain with both the FALA mutation and the S228P mutation is referred to as the "IgG4-P-FALA" Ab.

[0021] WO 2017191325 A1 (Fischer et al., MAB Discovery GmbH) relates to 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" form) or at S228P and L235E in the human IgG4 Fc region ("IgG4-PE" form).

[0022] As outlined in the Background section herein, it has been extremely difficult to identify mAbs that have high affinity, high specificity, and potent neutralizing activity against IL-1R3. Surprisingly, the inventors have developed such antibodies, and these antibodies have further advantages. This disclosure encompasses humanized IL-1R3 antibodies that have high affinity and specificity for IL-1R3, have potent IL-1R3 neutralizing activity, reduced effector function, and improved stability. The antibodies disclosed herein show reduced or no Fcγ receptor signaling and do not induce antibody-dependent cell-mediated cytotoxicity (ADCC). Surprisingly, the antibodies disclosed herein have significantly higher yields in the IgG4-P-FALA format compared to the more common IgG1-LALA format.

[0023] For the antibodies disclosed herein to be useful industrially, they need to be able to be incorporated into compositions with certain favorable properties. For example, the composition should remain stable over time, as preservatives can be used without affecting the antibody. The viscosity and opalescence of the composition should be maintained within specific parameters and not change over time (e.g., during storage). The 3D structure of the antibody is crucial for its function and binding to its target. Therefore, it should not aggregate (self-associate) or should minimally aggregate in the composition, 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 value, both achieved with the antibody in a standard dilution composition, and the opalescence value in a concentrated composition. To enable the antibody to be formulated on a commercial scale, benchmark tolerance windows for various parameters need to be met.

[0024] Generally, a higher isoelectric point is associated with higher antibody stability, a lower self-association kD value is associated with higher affinity and thus a higher likelihood of self-association, and a higher turbidity value (NTU) is less desirable as a clear solution is important. As understood from the median values for a series of different antibodies in the IgG1 or IgG4 Fc format (see Table 1 below), the latter format tends to result in less desirable values for the formulation parameters. Surprisingly, the anti-IL-1R3-IgG4-P-FALA Ab disclosed herein meets the benchmark window as the values are close enough to the median to be feasible on a commercial scale.

[0025] Table 1: Median values for various parameters of antibodies in the IgG1 Fc or IgG4 Fc format.

[0026]

[0027] Notably, several anti-IL-1R3 antibodies in the art and other antibodies targeting the IL-1 / IL-33 / IL-36 receptor superfamily do not employ the IgG4-P-FALA Fc of the present disclosure. Instead, many of these antibodies employ IgG1-LALA Fc. For example, antibody CAN04 (described in U.S. Patent 9,796,783, 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. [Immunology and Cell Biology] 2020.98(4):287-304) all employ IgG1-LALA Fc. This also holds true for the anti-IL-1R3 antibodies disclosed in WO 2022 / 053715 A1 (Macoin et al.), WO 2022 / 170008 A2 (Bigwarfe et al.), and WO 2022 / 243536A1 (Urso et al.), which all employ IgG1-LALA Fc. The inventors surprisingly found that the antibodies of the present invention have significantly higher yields compared to the IgG1-LALA form commonly used for anti-IL-1R3 antibodies.

[0028] The present disclosure describes an anti-IL-1R3 antibody that comprises at least the amino acid substitutions S228P, F234A, and L235A in the Fc region of human IgG4. In particular, the present disclosure relates to an antibody that specifically binds IL-1R3 and exhibits reduced or no FCγ receptor signaling and comprises the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0029] Aspects of the present disclosure are described in more detail below.

[0030] I. Definitions and Antibodies of the First Aspect

[0031] Unless otherwise indicated, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Any methods and materials similar or equivalent to those described herein can be used in the methods of the techniques of the present disclosure. For the purpose of describing and disclosing the methods, reagents, and tools reported in publications that can be used in conjunction with the present disclosure, all publications cited herein are incorporated by reference in their entirety.

[0032] Unless otherwise indicated, the methods and techniques of the present application are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Gennaro, A.R. (ed.) (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J.G., Limbird, L.E., and Gilman, A.G. (eds.) (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al. (eds.), Methods In Enzymology, Academic Press, Inc.; Weir, D.M. and Blackwell, C.C. (eds.) (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al. (eds.) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (eds.) (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons; Ream et al. (eds.) (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C.R. and Graham, A. (eds.) (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer-Verlag.

[0033] Unless otherwise specified, the term "antibody" is used to refer to whole antibodies as well as antigen-binding fragments of such antibodies. For example, the term encompasses four-chain IgG molecules as well as antibody fragments.

[0034] As used herein, the term "antibody fragment" refers to portions of a whole full-length antibody, e.g., as further described below.

[0035] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (˜24 kDa) and two identical heavy chains (˜55 or 70 kDa) form a tetramer. The amino-terminal portion of each chain is referred to as the variable (V) region and is distinguishable from the more conserved constant (C) region of the remainder of each chain.

[0036] Most of the amino acid sequence variability in immunoglobulins is confined to each of three separate positions within each of the V regions, which are referred to as hypervariable regions or complementarity-determining regions (CDRs), that are directly involved in antigen binding. Starting from the amino terminus, these regions are named CDR1, CDR2, and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Starting from the amino terminus, these regions are named FR1, FR2, FR3, and FR4, respectively. Starting from the amino terminus, these combined regions contained within the V region are named FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The positions and numbering systems of the CDR and FR regions have been defined by Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, U.S. Government Printing Office (1991), and its updates available online), and the CDR region boundaries have been further defined by the IMGT nomenclature.

[0037] As used herein, a "humanized mAb" is an antibody composed of a human antibody framework in which the complementarity determining regions (CDRs) from a non-human antibody have been grafted. The human receptor framework may also be altered. Procedures for designing and producing humanized antibodies are well known in the art and have been described, for example, in US 4816397 (Boss et al.), US 4816567 (Cabilly et al.), US 5225539 (Winter, MRC), EP 0120694 A2 (Boss et al.), EP 0125023 A1 (Cabilly et al.), EP 0194276 B1 (Neuberger and Rabbitts), EP 0239400 A2 (Winter, MRC), EP0519596A1 (Padlan et al.) and WO 1986001533 (Neuberger and 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 patents and patent application publications listed above are hereby incorporated by reference.

[0038] The constant region may be derived from any human antibody constant region. The variable region genes may be cloned in-frame with the constant region genes into an expression vector for the expression of immunoglobulin heavy and light chains. Such expression vectors may be transfected into host cells that produce antibodies for antibody synthesis.

[0039] Human antibody variable and constant regions may 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). Glycosylated antibodies may have a wide range of modified functionalities; see Boyd et al. (1996) Mol. Immunol. 32:1311-1318. As used herein, a "deltaab" or Δab modification is an Fc modification as described in Armour et al. (1999) Eur. J. Immunol. 29:2613-2624.

[0040] As used herein in the context of a binding polypeptide, the terms "stable", "stability", and "stabilized" refer to the resistance of a binding polypeptide to thermal and chemical degradation or fragmentation under given manufacturing, preparation, transportation, and storage conditions. Under given manufacturing, preparation, transportation, and storage conditions, a "stable" composition retains greater than or equal to 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of its biological activity. For example, the stability of a binding polypeptide can be evaluated using methods and measurements known to those of skill in the art, compared to a control or to starting material, based on the degree of degradation or fragmentation, or the level of specific fragments, or the type or size of aggregates. Such methods and measurements include, but are not limited to, a reduced area under the curve (AUC) compared to a reference, 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).

[0041] As used herein, the term "nucleic acid" includes a DNA molecule encoding an antibody described herein. A preferred DNA molecule encoding an antibody described herein is an expression vector suitable for expressing the antibody gene in a host cell. Expression vectors and host cells for antibody gene expression are known in the art; see, for example, Morrow, K. J. Genetic Engineering & Biotechnology News (June 15, 2008) 28(12) and Backliwal, G. et al. (2008) Nucleic Acids Res. 36(15): e96 - e96.

[0042] As used herein, the terms "treat" and "treatment" refer to the care of a patient or subject having a disease, disorder, or condition. Treatment can involve, but is not limited to, any one or any combination of the following: cure of the disease, disorder, or condition; amelioration of at least one symptom of the disease, disorder, or condition; and / or prophylactic or preventive action aimed at preventing or reducing the occurrence of the disease, disorder, or condition. In certain embodiments, treatment can involve, but is not limited to, cure of the disease, disorder, or condition; or amelioration of at least one symptom of the disease, disorder, or condition.

[0043] As used herein, the term "subject" refers to any mammal, including mice, rats, gerbils, hamsters, guinea pigs, rabbits, cats, dogs, sheep, goats, pigs, cows, horses, and primates. 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.

[0044] The first aspect relates to an antibody that specifically binds to IL-1R3, which antibody comprises the heavy chain amino acid sequence of the antibody of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0045] In at least one embodiment, the antibody is a humanized mAb. In at least one embodiment, the antibody is an anti-IL-1R3 antibody.

[0046] A humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) may comprise an antibody constant region (e.g., a human IgG4 constant region) that mediates one or more effector functions. For example, the binding of the C1 component of complement to the antibody constant region can activate the complement system. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates an inflammatory response and may also be involved in autoimmune hypersensitivity reactions. Further, IgG4 antibodies bind to receptors on various cells via the Fc region, wherein the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell. The binding of the antibody to the Fc receptor on the cell surface triggers many important and diverse biological responses, including the phagocytosis and destruction of antibody-coated particles, the clearance of immune complexes, the lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity or ADCC), the release of inflammatory mediators, placental transfer, and the control of immunoglobulin production.

[0047] In certain embodiments, a humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) may comprise a constant region that does not direct one or more effector functions (e.g., ADCC activity) and / or does not bind to the Fcγ (Fc gamma) receptor.

[0048] Certain embodiments described herein provide a humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) in which at least one amino acid in one or more constant region domains has been deleted or otherwise altered to provide desired biochemical properties such as reduced effector function.

[0049] In certain other embodiments, a humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) comprises the Fc region of a human IgG4 molecule or a portion thereof and the Ser228Pro (S228P) mutation (EU numbering) in the core hinge region of the molecule.

[0050] In certain exemplary embodiments, techniques known in the art can be used to mutate the Fc portion of a humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) to increase or decrease effector function. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce Fc receptor binding of the modified antibody in circulation. In other cases, constant region modifications consistent with the present disclosure may temper complement binding and thus reduce the serum half-life and non-specific association of the conjugated cytotoxin. Other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties, thereby allowing enhanced localization due to increased antigen specificity or flexibility. The resulting physiological characteristics, bioavailability, and other biochemical effects of the modification (such as tumor localization, biodistribution, and serum half-life) can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0051] In certain embodiments, the Fc domain employed in a humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a 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 the Fc domain is derived.

[0052] The amino acid substitution of the Fc variant can be located at any position within the Fc domain (i.e., any EU conventional 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.

[0053] In addition to the P-FALA Fc mutations, the antibodies described herein may also employ any other well-known in the art Fc variants that are known to improve (e.g., reduce) effector function and / or FcR binding. Such Fc variants may include, for example, any of the amino acid substitutions disclosed in the following patents: International PCT Publications WO 88 / 07089A1, WO 96 / 14339A1, WO 98 / 05787A1, WO 98 / 23289A1, WO 99 / 51642A1, WO 99 / 58572A1, WO 00 / 09560A2, WO 00 / 32767A1, WO 00 / 42072A2, WO 02 / 44215A2, WO 02 / 060919A2, WO 03 / 074569A2, WO 04 / 016750A2, WO 04 / 029207A2, WO 04 / 035752A2, WO 04 / 063351A2, WO 04 / 074455A2, WO 04 / 099249A2, WO 05 / 040217A2, WO 05 / 070963A1, WO 05 / 077981A2, WO 05 / 092925A2, WO 05 / 123780A2, WO 06 / 019447A1, WO 06 / 047350A2, and WO 06 / 085967A2 or U.S. Patent Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784, each of which is incorporated herein by reference.

[0054] In certain embodiments, a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or fragment thereof) can comprise an Fc variant that contains amino acid substitutions that alter the antigen-independent effector functions of the antibody, particularly the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to the neonatal Fc receptor (FcRn) compared to antibodies lacking these substitutions and thus have an extended or shortened serum half-life, respectively. Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals in which a long half-life of the administered antibody is desired, e.g., to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules can also be used, e.g., for administration to a mammal in which a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging or in cases where the starting antibody has toxic side effects when present in the circulation for an extended period. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and can thus also be used to treat diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desired include those that require targeting to the brain, kidney, and / or liver.

[0055] In a first aspect, there is provided an antibody that specifically binds 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.

[0056] In an alternative embodiment, there is provided an antibody that specifically binds 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 the F234A substitution, L235A substitution, and S228P substitution according to EU numbering.

[0057] In an alternative embodiment, there is provided an antibody that specifically binds IL-1R3 and comprises an antibody heavy chain variable (VH) domain, an antibody light chain variable (VL) domain, and an IgG4 Fc domain, wherein:

[0058] the VH domain comprises

[0059] a CDR-H1 sequence containing SYDMS (SEQ ID NO:5) or GFSLSSYD (SEQ ID NO:6);

[0060] A CDR-H2 sequence comprising the amino acid sequence TIYIGGTTAYASWPKG (SEQ ID NO:7) or IYIGGTT (SEQ ID NO:8); and

[0061] A CDR-H3 sequence comprising the amino acid sequence LQGANYYNSLAL (SEQ ID NO:9) or ARLQGANYYNSLAL (SEQ ID NO:10);

[0062] The VL domain comprises

[0063] A CDR-L1 sequence comprising the amino acid sequence QASQSIYSFLS (SEQ ID NO:11) or QSIYSF (SEQ ID NO:12);

[0064] A CDR-L2 sequence comprising the amino acid sequence ASDLES (SEQ ID NO:13) or AAS (SEQ ID NO:14); and

[0065] A CDR-L3 sequence comprising the amino acid sequence QSNYIIDYGA (SEQ ID NO:15 or SEQ ID NO:16); and the IgG4 Fc domain comprises the F234A substitution and the L235A substitution according to EU numbering.

[0066] In an alternative embodiment, an antibody is provided 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:

[0067] The VH domain comprises

[0068] A CDR-H1 sequence comprising the amino acid sequence SYDMS (SEQ ID NO:5) or GFSLSSYD (SEQ ID NO:6);

[0069] A CDR-H2 sequence comprising the amino acid sequence TIYIGGTTAYASWPKG (SEQ ID NO:7) or IYIGGTT (SEQ ID NO:8); and

[0070] A CDR-H3 sequence comprising the amino acid sequence LQGANYYNSLAL (SEQ ID NO:9) or ARLQGANYYNSLAL (SEQ ID NO:10);

[0071] The VL domain comprises

[0072] A CDR-L1 sequence comprising the amino acid sequence of QASQSIYSFLS (SEQ ID NO:11) or QSIYSF (SEQ ID NO:12);

[0073] A CDR-L2 sequence comprising the amino acid sequence of ASDLES (SEQ ID NO:13) or AAS (SEQ ID NO:14); and

[0074] A CDR-L3 sequence comprising the amino acid sequence of QSNYIIDYGA (SEQ ID NO:15 or SEQ ID NO:16); and the IgG4 Fc domain comprises F234A substitution, L235A substitution and S228P substitution according to EU numbering.

[0075] Table 2. Amino acid sequences of the VH, VL and CDR regions of exemplary anti-IL-1R3 IgG4-P-FALA antibodies.

[0076]

[0077]

[0078]

[0079]

[0080] Table 3. Amino acid sequences of the VH and VL regions of additional exemplary anti-IL-1R3 antibodies that can be used in IgG4-P-FALA Fc.

[0081]

[0082]

[0083]

[0084]

[0085] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO:24, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO:29, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A substitution, L235A substitution and S228P substitution according to EU numbering.

[0086] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 25, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 30, 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.

[0087] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 25, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 31, 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.

[0088] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 25, the amino acid sequence of the antibody light chain variable (VL) domain 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.

[0089] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 26, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 30, 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.

[0090] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 26, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 31, 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.

[0091] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 26, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0092] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 27, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0093] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 27, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0094] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 27, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0095] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 28, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 30, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0096] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 28, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 31, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0097] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 28, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 32, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0098] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 33, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 34, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0099] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 35, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 36, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0100] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 37, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 44, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0101] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 38, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 45, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0102] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 39, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 46, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0103] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 40, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 47, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0104] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 41, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 48, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0105] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the antibody heavy chain variable (VH) domain of SEQ ID NO: 42, the amino acid sequence of the antibody light chain variable (VL) domain of SEQ ID NO: 49, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises the F234A substitution, the L235A substitution, and the S228P substitution according to EU numbering.

[0106] In an alternative embodiment, an antibody is provided that specifically binds to IL-1R3 and comprises the amino acid sequence of the variable heavy (VH) domain of the antibody of SEQ ID NO: 43, the amino acid sequence of the variable light (VL) domain of the antibody of SEQ ID NO: 50, and an IgG4 Fc domain, wherein the IgG4 Fc domain comprises F234A substitution, L235A substitution, and S228P substitution according to EU numbering.

[0107] II. Antibody production and additional related aspects

[0108] Antibody production can be carried out by any technique known in the art. Antibodies can be produced by chemical synthesis or by expressing 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).

[0109] A third aspect relates to an isolated nucleic acid molecule that encodes the antibody of the first aspect. For further details regarding the antibody of the first aspect, see the disclosure in Part I above.

[0110] A fourth aspect relates to an expression vector that comprises the nucleic acid molecule of the third aspect.

[0111] A fifth aspect relates to a host cell that comprises the expression vector of the fourth aspect. In one embodiment, the host cell comprises a nucleic acid molecule encoding the antibody of the first aspect. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell, preferably wherein the host cell is a CHO DXB11 cell.

[0112] A sixth aspect relates to a method of making the antibody of the first aspect, the method comprising the steps of: (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 permit expression of the antibody; (iii) recovering the antibody; and (iv) optionally further purifying and / or modifying and / or formulating the antibody.

[0113] In one embodiment, the manufacturing method comprises:

[0114] (i) Transfecting a host cell that is a Chinese hamster ovary (CHO) cell, preferably wherein the host cell is a CHO DXB11 cell, with the isolated nucleic acid molecule of the third aspect or the expression vector of the fourth aspect.

[0115] In one embodiment of the manufacturing method, the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising a human IgG1 Fc region, preferably wherein the human IgG1 Fc region comprises the amino acid substitutions L234A and L235A according to EU numbering. In one embodiment of the manufacturing method, the expression level of the 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. In one embodiment of the manufacturing method, the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising:

[0116] - the amino acid sequence of the human IgG1 Fc region of SEQ ID NO:18, and

[0117] - the amino acid sequence of the light chain of SEQ ID NO:4.

[0118] In one embodiment of the manufacturing method, the expression level of the antibody is greater than the expression level of an anti-IL-1R3 antibody comprising:

[0119] - the amino acid sequence of the human IgG1 Fc region of SEQ ID NO:18, and

[0120] - the amino acid sequence of the light chain of SEQ ID NO:2.

[0121] In one embodiment, the manufacturing method comprises:

[0122] (iv) further purifying and / or modifying and / or formulating the antibody. In at least one embodiment, formulating comprises combining the antibody with a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, step (iv) produces a pharmaceutical composition according to the second aspect.

[0123] In one embodiment, the manufacturing method produces an antibody according to the first aspect. In one embodiment of the manufacturing method, the expression level of the antibody is greater than the expression level of the same anti-IL-1R3 antibody with the following difference: the heavy chain is a human IgG1 Fc region having the amino acid substitutions L234A and L235A according to EU numbering.

[0124] The seventh aspect relates to an antibody produced by the manufacturing method according to the sixth aspect. In one embodiment, the antibody produced is according to the first aspect.

[0125] Isolate polynucleotides encoding a humanized mAb or a humanized mAb fragment (e.g., the anti-IL-1R3 antibody or a fragment thereof disclosed herein), and insert them 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 a humanized mAb fragment according to the embodiments (e.g., an anti-IL-1R3 antibody or a fragment thereof) are available in the art. A variety of vectors are available, including vectors that are maintained in a host cell as a single copy or multiple copies, or vectors that are 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 the expression of the humanized immunoglobulin.

[0126] Polynucleotides encoding a humanized mAb or a humanized mAb fragment (e.g., the anti-IL-1R3 antibody or a fragment thereof disclosed herein) are readily isolated and sequenced using conventional procedures (e.g., oligonucleotide probes). Vectors that can be used include plasmids, viruses, bacteriophages, transposons, minichromosomes, with plasmids being a typical example. Generally, such vectors further include a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence, which are operably linked to the light chain and / or heavy chain polynucleotides to facilitate expression. The polynucleotides encoding the light chain and the heavy chain can be inserted into separate vectors and introduced (e.g., by transformation, transfection, electroporation, or transduction) into the same host cell simultaneously or sequentially, or, if desired, both the heavy chain and the light chain can be inserted into the same vector prior to such introduction.

[0127] A promoter can be provided for expression in a suitable host cell. The promoter can be constitutive or inducible. For example, the promoter can be operably linked to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain such that it directs the expression of the encoded polypeptide. A variety of promoters suitable for prokaryotic and eukaryotic hosts are available. Prokaryotic promoters include the lac, tac, T3, T7 promoters of Escherichia 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, iso-cytochrome C, acid phosphatase, metallothionein, and enzymes responsible for nitrogen metabolism or maltose / galactose utilization; RNA polymerase II promoters, including viral promoters such as polyomavirus, fowlpox virus, and adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus (especially the immediate early gene promoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter, and early or late simian virus 40, as well as non-viral promoters such as EF-1α (Mizushima and Nagata (1990) Nucleic Acids Res. [Nucleic Acid Research] 18(17):5322). Those skilled in the art will be able to select an appropriate promoter for expressing a humanized antibody or a portion thereof.

[0128] In appropriate cases, for example, for expression in cells of higher eukaryotes, additional enhancer elements can be included to replace or supplement the enhancer elements located in the above promoters. Suitable mammalian enhancer sequences include enhancer elements from globin, elastase, albumin, fetoprotein, metallothionein, and insulin. Alternatively, enhancer elements from eukaryotic cell viruses can be used, such as the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculovirus enhancer, or murine IgG2a locus (see, for example, Kallmeier & Gay WO2004009823). Although such enhancers are typically located at sites upstream of the promoter on the vector, they can also be located elsewhere, for example, within the untranslated region or downstream of the polyadenylation signal. The selection and location of the enhancer can be based on compatibility with the host cell used for expression.

[0129] In addition, a vector (e.g., an expression vector) may contain a selectable marker for selecting host cells carrying the vector and, in the case of a replicable vector, an origin of replication. Genes encoding products that confer antibiotic or drug resistance are common selectable markers and can be used in prokaryotic cells (e.g., the β-lactamase gene (ampicillin resistance), the tet gene (tetracycline resistance)) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin B resistance genes). The dihydrofolate reductase marker gene allows selection with methotrexate in a variety of hosts. Genes encoding the gene products of host auxotrophic markers (e.g., LEU2, URA3, HIS3) are commonly used as selectable markers in yeast. The use of viral (e.g., baculovirus) or phage vectors, as well as vectors capable of integrating into the host cell genome, such as retroviral vectors, is also contemplated.

[0130] In eukaryotic systems, polyadenylation and termination signals are operably linked to the polynucleotides encoding the antibodies described herein. Such signals are typically located 3' of the open reading frame. In mammalian systems, non-limiting examples of polyadenylation / termination signals include those derived from the growth hormone, elongation factor-1α, 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) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are generally not required, and instead, shorter and more defined terminator sequences are typically employed. The choice of polyadenylation / termination sequence 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 modified to accommodate the codon bias of the host cell, thereby increasing transcript and / or product yield (e.g., Hoekema, A. et al. (1987) Mol. Cell Biol. 7(8):2914-24). The choice of codons can be based on compatibility with the host cell used for expression.

[0131] Accordingly, the present disclosure relates to isolated nucleic acid molecules encoding a humanized immunoglobulin or a heavy or light chain thereof. The present disclosure also relates to isolated nucleic acid molecules encoding the antigen-binding portions of immunoglobulins and their chains.

[0132] A humanized mAb or a humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) can be produced, for example, by expressing 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 produced using any suitable method. For example, an expression construct described herein (e.g., one or more vectors, e.g., a mammalian cell expression vector) can be introduced into a suitable host cell, and the resulting cell can be maintained (e.g., in culture) under conditions suitable for the expression construct or vector. The host cell can be prokaryotic, including bacterial cells such as Escherichia coli (e.g., strain DH5α TM )(Invitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), Bacillus subtilis, and / or other suitable bacteria; eukaryotic cells such as cells of higher eukaryotes such as cells from mammals (e.g., COS cells such as COS-1 (ATCC accession number CRL-1650) and COS-7 (ATCC accession number CRL-1651), CHO (e.g., ATCC accession number CRL-9096), CHO DG44 (Urlaub, G. and Chasin, L.A. (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, W.S. et al. (1993) Proc. Natl. Acad. Sci. U.S.A., 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, F.M. 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, e.g., it is an isolated host cell or part of a cell culture.

[0133] Host cells can be cultured in spinner flasks, shake flasks, roller bottles, wave reactors (e.g., system 1000 from wavebiotech.com) or hollow fiber systems, but for large-scale production, stirred tank reactors or bag reactors (e.g., Wave Biotech, Somerset, New Jersey, USA) are preferably used, especially for suspension culture. Stirred tank reactors can be suitable for aeration using, for example, spargers, baffles or low-shear impellers. For bubble column and air-lift reactors, air or oxygen bubbles can be used directly for aeration. In the case where host cells are cultured in serum-free medium, the medium can be supplemented with cell protectants such as pluronic F-68 to help prevent cell damage due to the aeration process. Depending on the host cell characteristics, microcarriers can be used as a growth substrate for anchorage-dependent cell lines, or the cells can be adapted to suspension culture. The culture of host cells (especially 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 processing or perfusion culture. Although recombinant transformed mammalian host cells can be cultured in serum-containing media such as media containing fetal calf serum (FCS), preferably, they are cultured in serum-free media such as the media disclosed in Keen et al. (1995) Cytotechnology 17:153-163 or commercially available media such as ProCHO TM or UltraCHO TM (Cambrex NJ, USA), and these media are supplemented with an energy source (such as glucose) and synthetic growth factors (such as recombinant insulin) when necessary. Serum-free culture of host cells may require adapting those cells to grow under serum-free conditions. One method of adaptation is to culture such host cells in serum-containing medium and repeatedly exchange 80% of the medium with serum-free medium to enable the host cells to learn to adapt to serum-free conditions (see, for example, Scharfenberg, K. et al. (1995) Animal Cell Technology: Developments Towards the 21st Century (edited by Beuvery, E.C. et al.), pp. 619-623, Kluwer Academic publishers).

[0134] A humanized mAb or humanized mAb fragment (e.g., an antibody or a fragment thereof) according to the embodiments described herein can be secreted into the culture medium and recovered and purified therefrom using a variety of techniques to provide a degree of purification suitable for the intended use. For example, when compared to a culture medium containing a therapeutic antibody, the use of a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) for treating a human subject generally requires at least 95% purity as determined by reducing SDS-PAGE, and more typically 98% or 99% purity. In the first case, cell debris can be removed from the culture medium by centrifugation, and then a clarification step can be performed on the supernatant using, for example, microfiltration, ultrafiltration, and / or depth filtration. Alternatively, a humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof) can be harvested by microfiltration, ultrafiltration, or depth filtration without prior centrifugation. A variety of other techniques are available, such as dialysis and gel electrophoresis, as well as chromatographic techniques, such as hydroxyapatite (HA), affinity chromatography (optionally involving an affinity tag system, such as polyhistidine), and / or hydrophobic interaction chromatography (HIC) (see US 5,429,746). In one embodiment, after various clarification steps, protein A or protein G affinity chromatography is used, followed by further chromatographic steps (such as ion exchange and / or HA chromatography, anion or cation exchange, size exclusion chromatography, and ammonium sulfate precipitation) to capture the humanized mAb or humanized mAb fragment (e.g., an anti-IL-1R3 antibody or a fragment thereof). Various virus removal steps can also be employed (e.g., nanofiltration using, for example, a DV-20 filter). After these different steps, a purified preparation containing at least 10 mg / mL or more (e.g., 100 mg / mL or more) of the antibody described herein is provided, and thus another embodiment described herein is formed. Concentrations up to 100 mg / mL or higher can be generated by ultracentrifugation. Such preparations are substantially free of aggregated forms of the antibody.

[0135] Bacterial systems are particularly suitable for the expression of antibody fragments. Such fragments are localized intracellularly or periplasmically. 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. [Journal of Biotechnology] 72:13-20; Cupit, P.M. et al. (1999) Lett. Appl. Microbiol. [Letters in Applied Microbiology] 29:273-277.

[0136] The fifth aspect relates to a host cell comprising the expression vector of the fourth aspect. This disclosure also relates to a cell (host cell) comprising a nucleic acid (e.g., a vector) (e.g., an expression vector) described herein. For example, a nucleic acid (i.e., one or more nucleic acids) encoding the heavy and light chains of a humanized immunoglobulin according to the embodiments or a construct (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 suitable for the selected host cell (e.g., transformation, transfection, electroporation, infection), wherein the nucleic acid is operably linked to one or more expression control elements (e.g., in a vector, in a construct generated by a process in the cell, 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, in a suitable 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 from, for example, the host cell, the medium, or milk. This process encompasses the expression of transgenic animals or plants (e.g., tobacco) in host cells (e.g., mammary gland cells) (see, e.g., Lonberg & Kay, WO 1992003918).

[0137] Batch consistency and comparability are closely related to the successful drug development of recombinant mAbs and related products. Minor structural modifications can result in variants (or proteoforms) that differ in size, charge, or hydrophobicity. These modifications may or may not affect the stability, pharmacokinetics, and efficacy of the recombinant mAb. The presence of the same types of modifications found in endogenous immunoglobulin G (IgG) can significantly reduce the safety risk of the mAb.

[0138] The following post-translational and physicochemical modifications can occur in recombinant mAbs and are related to the expression system used: N-terminal modifications (N-terminal pyroglutamate, incomplete removal of the signal peptide, truncation); asparagine deamidation; aspartic acid isomerization; the presence of succinimide; degradation / oxidation of amino acid residues (especially methionine and tryptophan); cysteine-related modifications (free cysteine residues, alternative disulfide linkages (perturbations), trithio bonds, formation of thioethers, cysteine racemization); glycosylation; glycation; C-terminal modifications (cleavage of C-terminal lysine, amidation, sequence variations due to inherent errors in transcription or translation); and rare chemical modifications (such as oxidative carbonylation, histidine-histidine crosslinking, tyrosine sulfation, modification of the heavy chain N-terminus by maleic acid, modification of the N-terminal primary amine or lysine side chain by citric acid or its degradation products, and O-fucosylation of serine residues).

[0139] III. Pharmaceutical Compositions and Administration Methods of Anti-IL-1R3 Antibodies

[0140] The second aspect relates to a pharmaceutical composition comprising the antibody of the first aspect and a pharmaceutically acceptable diluent, carrier or excipient. In certain embodiments, a pharmaceutical composition is provided that comprises a humanized mAb or a humanized mAb fragment as described herein (e.g., an anti-IL-1R3 antibody or a fragment thereof), or one or more ligands that can be identified by an assay method as defined in the previous aspects of the present disclosure. The ligand can be an immunoglobulin, a peptide, a nucleic acid or a small molecule, as discussed herein. In the following discussion, they are referred to as "compounds".

[0141] In one embodiment, the pharmaceutical composition described herein is a composition of matter comprising one or more compounds capable of modulating T cell activity as an active ingredient. The compound can be in the form of any pharmaceutically acceptable salt, or, for example, in appropriate cases, an analogue, free base form, tautomer, enantiomeric racemate or a combination thereof. It is contemplated that when administered in an amount depending on the particular circumstances, the active ingredient of the pharmaceutical composition comprising the active ingredient described herein will exhibit therapeutic activity, for example, in the treatment of graft-versus-host disease.

[0142] In certain embodiments, the pharmaceutical composition comprises a humanized mAb or a humanized mAb fragment as described herein (e.g., an anti-IL-1R3 antibody or a fragment thereof), and a pharmaceutically acceptable diluent, carrier or excipient. In at least one embodiment, the pharmaceutical composition is an aqueous composition.

[0143] In certain embodiments, one or more of the compounds described in the present disclosure can be used in combination with any compound recognized in the art as being suitable for treating a particular indication of any of the above-mentioned conditions. Thus, one or more of the compounds described herein can be combined with one or more compounds recognized in the art as being suitable for treating the aforementioned indications such that a convenient single composition can be administered to a subject. The dosage regimen can be adjusted to provide an optimal therapeutic response.

[0144] For example, several separate doses can be administered daily, or the dose can be proportionally reduced, as indicated by the exigencies of the treatment situation.

[0145] The active ingredient can be administered in a convenient manner, such as by oral, intravenous (in the case of water solubility), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implantation (e.g., using a slow-release molecule). 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 transplantation. This can be done to prevent, reduce, for example, the likelihood of graft-versus-host disease or to alleviate its symptoms.

[0146] Depending on the route of administration, the active ingredient may need to be encapsulated in a material to protect the ingredient from the action of enzymes, acids, and other natural conditions that may inactivate the ingredient.

[0147] For parenteral administration of the active ingredient, it will be 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 liposomes. The adjuvant is used in its broadest sense and includes any immunostimulatory compound such as interferon. Adjuvants contemplated herein include resorcinol, nonionic surfactants such as polyoxyethylene oleyl ether and cetyl polyethylene ether. Enzyme inhibitors include trypsin.

[0148] Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.

[0149] The active ingredient can also be administered parenterally or intraperitoneally.

[0150] Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and their mixtures, as well as in oils. Under ordinary storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0151] Dosage forms suitable for injectable use include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must have a degree of fluidity that permits easy injection. It must be stable under the conditions of manufacture and storage and must prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0152] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In certain cases, it may be preferred to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0153] A sterile injectable solution is prepared by incorporating the required amount of the active ingredient, together with several of the other ingredients enumerated above (as required), in a suitable solvent, followed by filtered sterilization. Generally, a dispersion is prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0154] Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. Of course, any material used in the preparation of any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained-release preparations and formulations.

[0155] As used herein, "pharmaceutically acceptable diluents, carriers or excipients" include 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 diluent is an aqueous fluid. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0156] Particularly advantageous is the formulation of parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to physically discrete units suitable as unit doses for the mammalian subject to be treated; each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the novel dosage unit forms described herein are dictated by and directly dependent on: (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active materials for the treatment of diseases in living subjects suffering from ill health as herein described. The principal active ingredient is compounded in effective amount with a suitable pharmaceutically acceptable carrier in dosage unit form for convenient and effective administration. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dosages and manner of administration of the said ingredients.

[0157] To facilitate the delivery of peptide compounds, including antibodies, to cells, the peptides can be modified to enhance their ability to cross cell membranes. For example, Chang et al. disclose in US 5149782 (Tanox Biosystems, Inc.) the use of fusion peptides, ion channel-forming peptides, membrane peptides, long-chain fatty acids, and other membrane blending agents for increasing the transmembrane transport of proteins. These and other methods are also described in WO 1997037016 by Wallach et al. and US 5108921 by Low et al., both of which are incorporated herein by reference.

[0158] 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 a compound recognized in the art as suitable for treating a specific indication. Accordingly, there is provided the use of the active ingredient as described herein in the manufacture of a medicament for treating a disease associated with an abnormal immune response.

[0159] Furthermore, there is provided a method for treating a condition associated with an abnormal immune response, the method comprising administering to a subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof.

[0160] IV: Methods for treating diseases or disorders

[0161] The 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.

[0162] The 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.

[0163] The tenth aspect relates to a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the antibody of the first aspect or the pharmaceutical composition of the second aspect.

[0164] 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.

[0165] 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.

[0166] In view of the evidence presented, those skilled in the art will understand that, since the antibodies of the present disclosure are capable of inhibiting the signaling of IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ, thereby targeting several IL-dependent pathways simultaneously, the antibodies of the present disclosure can be used for the prevention, treatment, alleviation, detection, and / or diagnosis of inflammatory and / or fibrotic and / or neoplastic disease conditions.

[0167] One embodiment relates to an antibody of the first aspect or a 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.

[0168] 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, wherein the inflammatory and / or fibrotic disease or disorder is selected from the group consisting of: rheumatoid arthritis, all types of arthritis, psoriatic arthritis, all types of juvenile arthritis, including 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), pyogenic arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult-onset Still's disease, hyper IgD syndrome, type 2 diabetes, macrophage activation syndrome, TNF receptor-associated periodic syndrome, Blau disease, ankylosing spondylitis, Sweet's disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, allergy, atherosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes, chronic obstructive pulmonary disease (COPD), Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis), hepatitis, hepatitis C, ischemia-reperfusion injury, multiple sclerosis, Neisseria or pneumococcal meningitis, tuberculosis, Behcet'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, varicose veins, polycystic ovary syndrome, gastroesophageal reflux disease (GERD), fatty liver disease, colorectal cancer, breast cancer, uterine cancer, chronic renal failure, stroke and hyperuricemia), disc disease, irritable bowel syndrome, stroke and hyperuricemia), disc disease, irritable bowel syndrome, Schnitzler syndrome, allergy / atopic dermatitis, acne (hidradenitis suppurativa), cardiac fibrosis, cardiovascular disease, cryopyrin-associated periodic syndrome, cystic fibrosis, Goodpasture's syndrome, Guillain-Barre syndrome, renal fibrosis, liver fibrosis, lung fibrosis (lung fibrosis / pulmonary fibrosis), skin fibrosis (skin fibrosis / dermal fibrosis), myocarditis, autoimmune myocarditis, organ dysfunction associated with organ transplantation, pancreatitis, peritonitis, uveitis, vasculitis, pneumonia, pulmonary hypertension, sclerodermatous chronic graft-versus-host disease, sepsis, Sjogren's syndrome ( syndrome), Takayasu's arteritis, and gout.

[0169] In at least one embodiment, the disease or disorder is an inflammatory condition, such as a metabolic rheumatic disorder associated with hyperuricemia. The metabolic rheumatic disorder may be selected from the group consisting of: gout, pseudogout, drug-induced gout, and chronic active (refractory) gout.

[0170] In at least one embodiment, the disease or disorder is an IL-1-dependent inflammatory disease. For example, the disease may be a systemic or local inflammatory disease. In at least one embodiment, the disease or disorder is selected from the group consisting of: Schnitzler syndrome, Behçet's disease, secondary amyloidosis, Henoch-Schönlein purpura, idiopathic recurrent pericarditis, systemic-onset juvenile idiopathic arthritis, adult-onset Still's disease (AOSD), macrophage activation syndrome, Sweet syndrome / neutrophilic dermatosis (acute febrile neutrophilic dermatosis), neutrophilic panniculitis, Erdheim-Chester disease (histiocytosis), SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis), PFAPA (periodic fever, aphthous stomatitis, pharyngitis, adenitis), multicentric Castleman disease, Jessner-Kanof disease, primary Sjögren's syndrome (fatigue), Kawasaki disease, colitis in chronic granulomatous disease, hidradenitis suppurativa (acne inversa), autoimmune inner ear disease, and severe traumatic brain injury. In at least one embodiment, the disease or disorder is a hereditary systemic inflammatory disease, such as familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), tumor necrosis factor (TNF) receptor 1-associated periodic syndromes (TRAPSa), hyper-IgD syndrome (HIDS), PAPA (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, PASH (pyoderma gangrenosum, acne, and hidradenitis suppurativa) syndrome, PAPASH (pyogenic arthritis, acne, pyoderma gangrenosum, and hidradenitis suppurativa) syndrome, interleukin-1 receptor antagonist deficiency (DIRA), Blau syndrome / granulomatous arthritis, mevalonate kinase deficiency, Majeed syndrome, and NLRP12 (nucleotide-binding leucine-rich repeat-containing receptor 12) autoinflammatory syndrome.

[0171] In certain embodiments, the disease or disorder is selected from the group consisting of: atopic dermatitis, inverse acne (hidradenitis suppurativa), pyoderma gangrenosum 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 gangrenosum syndrome. In at least one embodiment, the disease or disorder is pyoderma gangrenosum.

[0172] 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 (inverse acne) and COPD, preferably hidradenitis suppurativa (inverse acne). 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.

[0173] One embodiment relates to an antibody of the first aspect or a 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.

[0174] In some embodiments, the disease or disorder is a neoplastic disease or disorder, wherein 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, wherein 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, wherein 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, urogenital organ cancer, biliary tract cancer (also known as cholangiocarcinoma), 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 breast cancer, colon cancer, lung cancer, pancreatic cancer, liver cancer, non-small cell lung cancer, colorectal cancer, gastric cancer (stomach cancer), gastric 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.

[0175] Another aspect relates to a method for treating a subject who is characterized by being resistant to or showing an inadequate response 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 one or more cytotoxic, cytostatic or targeted anti-cancer agents. In one embodiment, the antibody of the first aspect is administered sequentially with one or more cytotoxic, cytostatic or targeted anti-cancer agents. In the latter case, it is preferred that the antibody of the first aspect is administered after treatment with one or more cytotoxic, cytostatic or targeted anti-cancer agents. The cytotoxic or cytostatic anti-cancer agent can be a taxane, an anthracycline, an alkylating agent, a histone deacetylase inhibitor, a topoisomerase inhibitor, a kinase inhibitor, a nucleotide analogue, a peptide antibiotic and a platinum agent.

[0176] In at least one embodiment, the disease or disorder is cancer-related chronic inflammation.

[0177] Another 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.

[0178] The examples provided below are for illustrative purposes only and should not be considered as limiting the compositions and methods described herein.

[0179] Examples

[0180] Example 1: Generation of anti-IL-1R3 IgG4-P-FALA antibody

[0181] The anti-IL-1R3 mAb was humanized by CDR grafting using recombinant DNA technology. The human IgG backbone is an IgG4 containing the P-FALA (S228P, F234A, L235A) mutation in the Fc region or an IgG1 backbone containing the LALA (L234A, L235A) mutation in the Fc region. The resulting vectors encode the heavy and light chains of the humanized antibody anti-IL-1R3-IgG4-P-FALA or anti-IL-1R3-IgG1-LALA. Example 2: Selection of a CHO cell line clone expressing the anti-IL-1R3 IgG4-P-FALA antibody in a CHO cell line

[0182] The CHO DXB11 host cell line is directly derived from the CHO DXB11 cell line (Urlaub and Chasin, 1980, Proc Natl Acad Sci USA), by finally adapting the CHO DXB11 cell line to a serum-free and chemically defined commercial medium (CD DG44), followed by subcloning. A master cell bank of the host was established, and a working cell bank (WCB) of the host was generated using this cell bank. The WCB was used as the cell source for generating the anti-IL-1R3 expressing cell line.

[0183] The host cell line was propagated in a commercial medium (CD DG44, Thermo Fisher Scientific) supplemented with 4 mM L-glutamine and 0.18% (v / v) Pluronic F68 (Thermo Fisher Scientific). The cells were grown as a suspension culture at 37 °C, 5% CO2, and 80% relative humidity. The host cell line was cryopreserved in this CD DG44 growth medium (CD DG44, 4 mM L-glutamine, 0.18% (v / v) Pluronic F68) containing 7% (v / v) dimethyl sulfoxide (DMSO) as a cryoprotectant.

[0184] Table 4: Nucleotide sequences of each part of the expression cassette / vector for expressing the anti-IL-1R3 antibody

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] The DNA sequences of the expression cassettes / vectors encoding the heavy or light chains of the anti-ILR13 antibody are shown in Table 4. The vectors encoding the heavy and light chain polypeptides of anti-IL-1R3 IgG4-P-FALA Ab (aIL-1R3-IgG4) or anti-IL-1R3 IgG1LALA 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 the CD52 reporter gene. The electroporated cells were subjected to two rounds of selection in growth media containing 5 nM methotrexate (MTX) and 50 nM MTX, respectively. After the second round of selection, the antibody titers were measured in all five pools.

[0193] Figure 1 Titled "50 mM MTX Selection Pools - 7-Day Batch Titers", and shows the pool numbers and corresponding antibody titers for each of the five pools selected after the second round of MTX selection for the pools expressing aIL-1R3-IgG1 or aIL-1R3-IgG4 Ab. As Figure 1 shown, all five selected pools expressing the aIL-1R3-IgG1 antibody produced antibody titers below 0.1 g / L. Among the five selected pools expressing the aIL-1R3-IgG4 antibody, four pools (1, 2, 3, and 4) produced antibodies with higher titers and were thus viable for clone isolation.

[0194] Pools 2 and 4 expressing aIL-1R3-IgG4 were used for clone selection using a clone selection tool. The clonality of the clones was examined, and the verified clones were amplified for evaluation on AMBR (cell line screening software). The cell growth, titer, metabolites, and product quality of the top 21 clones were evaluated. The top six clones selected based on antibody titer were A136, A155, A101, A61, and B21. The "A" clones were from pool 2, while the "B" clones were from pool 4. Among all the selected clones, the top six clones also showed the highest antibody productivity.

[0195] Example 3: Inhibition of IL-1α / β, IL33, and IL-36α / β / γ induced the release of IL-8 from skin-derived carcinoma A-431 cells

[0196] The in vitro potency of the anti-IL-1R3 antibody to interfere with the activities of multiple cytokines for IL8 cytokine release was tested in the A-431 stable cell line.

[0197] In 20 μl of DMEM, 10% heat-inactivated FCS medium, A-431 cells were seeded at a cell density of 20,000 cells / well in a 384-well black flat-bottom tissue culture-treated microplate (Corning catalog number 3764). Antibodies serially diluted in DMEM, 1% heat-inactivated FCS were immediately added at a volume of 5 μl, and the plate was incubated at 37 °C / 5% CO2 for 60 minutes. Thereafter, 5 μl of the corresponding recombinant human IL-1α / 1β, IL33, or IL-36α / 36β / 36γ (R&D Systems) protein prepared in DMEM, 1% heat-inactivated FCS was added to the 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 plate was incubated at 37 °C / 5% CO2 for 24 hours. Each condition was technically tested in triplicate. The level of secreted human IL-8 in the supernatant was measured using the CisBio HTRF IL8 assay kit (catalog number 62HIL08PEG) according to the manufacturer's instructions. Curve fitting and EC50 calculations were performed using XLfit. The results are summarized in Table 5 below. Anti-IL-1R3-IgG4-P-FALA antibody was compared with anti-IL-1R3-IgG1-LALA antibody. Both antibodies showed robust activity against the three pathways.

[0198]

[0199] Example 4: Fcγ receptor binding activity of anti-IL-1R3 antibodies in IgG1-LALA and IgG4-P-FALA forms.

[0200] Human Fcγ receptors were obtained from R&D Systems. Binding studies were performed by surface plasmon resonance (SPR) using a Biacore T200 instrument. According to the manufacturer's instructions, Fcγ receptors were captured on a CM5 sensor chip using an anti-his capture antibody at a receptor concentration of 0.1 μg / ml. Antibodies were flowed over the so-captured receptors at 3.0 μM for 300 seconds (FcγRI) and 420 seconds (FcγRIII), respectively. Then buffer was flowed over the chip surface to monitor the dissociation of the antibody-receptor complex.

[0201] Figure 2A It was shown that anti-IL-1R3-IgG1-LALA showed residual binding to human FcγRIIIa, and Figure 2B the human FcγRIIIa V176F mutant was shown, while anti-IL-1R3-IgG4-P-FALA showed no binding.

[0202] Figure 2C Anti-IL-1R3-IgG1-LALA showed residual binding to human FcγRI (CD64), while anti-IL-1R3-IgG4-P-FALA showed no binding.

[0203] In all Figure 2A 、 2B and 2C, the x-axis depicts time in seconds (s), while the y-axis depicts the response in response units (RU), where 0 represents the capture baseline.

[0204] The same trend was observed for the cynomolgus monkey receptor (data not shown).

[0205] For mouse Fcγ receptors, no binding of anti-IL-1R3-IgG1-LALA or anti-IL-1R3-IgG4-P-FALA was detected (data not shown).

[0206] Example 5: Cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-IL-1R3 antibodies in IgG1-LALA and IgG4-P-FALA forms.

[0207] In antibody-dependent cell-mediated cytotoxicity (ADCC), effector cells actively lyse target cells bound by specific antibodies. The process begins with the binding of the antibody to the surface of the target cell. Effector cells recognize and bind to the Fc region of the antibody using Fc receptors found on their cell surface. After binding, the effector cells release cytotoxic factors, which ultimately kill the target cell.

[0208] The iLite ADCC Bioassay #BM5001, SVAR Life Sciences, ) was used to test any ADCC activity of anti-IL-1R3 antibodies in IgG1-LALA and IgG4-P-FALA forms. This assay uses effector cells carrying a reporter gene (Jurkat cells expressing FcγIIIa (V158)), which are used in combination with target cells that express a specific antigen at a constant high level (i.e., CHO IL1R3 target cells). The binding of the antibody to the target cell and the binding of the effector cell to the Fc receptor of the antibody result in the expression of the firefly luciferase reporter gene in the effector cell. The promoter of firefly luciferase in the effector cell includes binding sites for NfkB, AP1, NFAT, CRE, and STAT, and thus includes five major transcription factors of the FcγRIII signal transduction pathway. The effector cells further contain Renilla luciferase for normalization purposes.

[0209] Figure 3APresents the raw data comparing the anti-IL-1R3 antibodies of the present invention in IgG1-LALA and IgG4-P-FALA forms, and the IgG1 control antibody, and Figure 3B presents the normalized data. Although the IgG1 control antibody used as a positive control showed a strong reporter gene signal indicative of ADCC, the IgG1-LALA form significantly reduced the reporter gene signal. Surprisingly, the IgG4-P-FALA form completely abolished the reporter gene signal, indicating a complete absence of Fcγ receptor signaling.

[0210] Cited references

[0211] 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.

[0212] 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

[0213] 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.

[0214] Silva JP, Vetterlein O, Jose J, Peters S, Kirby H. The S228P mutationprevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using acombination of novel quantitative immunoassays and physiological matrixpreparation. 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 the heavy chain amino acid sequence of the antibody of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:

2.

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

3. An isolated nucleic acid molecule, the isolated nucleic acid molecule encoding the antibody as claimed in claim 1.

4. An expression vector, the expression vector comprising the nucleic acid molecule as claimed in claim 3.

5. A host cell, the host cell comprising the expression vector as claimed in claim 4.

6. The host cell as claimed in claim 5, the host cell being a Chinese hamster ovary (CHO) cell, preferably wherein the host cell is a CHO DXB11 cell.

7. A method of making the antibody as claimed in claim 1, the method comprising the steps of: (i) Optionally transfecting a host cell with the isolated nucleic acid molecule as claimed in claim 3 or the expression vector as claimed in claim 4; (ii) Culturing the host cell under conditions that permit expression of the antibody; (iii) Recovering the antibody; and (iv) Optionally further purifying and / or modifying and / or formulating the antibody.

8. The method of making as claimed in claim 7, wherein the method comprises: (i) Transfecting a host cell, which is a Chinese hamster ovary (CHO) cell, preferably wherein the host cell is a CHO DXB11 cell, with the isolated nucleic acid molecule as claimed in claim 3 or the expression vector as claimed in claim 4.

9. The method of making as claimed in any one of claims 7 and 8, wherein the method comprises: (iv) Further purifying and / or modifying and / or formulating the antibody, wherein the formulation comprises combining the antibody with a pharmaceutically acceptable diluent, carrier or excipient.

10. The method of making as claimed in any one of claims 7 to 9, 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, preferably wherein the human IgG1 Fc region comprises the amino acid substitutions L234A and L235A according to EU numbering.

11. An antibody, the antibody produced by the method of making as claimed in any one of claims 7 to 10.

12. The antibody as claimed in claim 1, the antibody 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.

13. The pharmaceutical composition as claimed in claim 2, the pharmaceutical composition 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.

14. The antibody as claimed in claim 12 or the pharmaceutical composition as claimed in claim 13, 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.

15. The antibody according to claim 12 or the pharmaceutical composition according to claim 13 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.

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