阅读说明：本技术 人单克隆抗il-32抗体、多核苷酸、载体、宿主细胞、制备方法、组合物及应用 (Human monoclonal anti-IL-32 antibody, polynucleotide, vector, host cell, preparation method, composition and application ) 是由 艾德里安·海迪 凯·金萨德 凯·克朗 安娜莉莎·玛卡吉诺 石莫比·奥诺 帕特·彼得森 麦 于 2014-07-03 设计创作，主要内容包括：本发明提供了一种人单克隆抗IL-32抗体、多核苷酸、载体、宿主细胞、制备方法、组合物及应用。所述人单克隆抗IL32抗体或其IL-32结合片段相对于IL-32α,优先结合IL-32γ和/或不与IL-32α结合,在其可变区中,所述人单克隆抗IL32抗体或其IL-32结合片段包括以下氨基酸序列的可变重链和可变轻链的六个互补决定区：SEQ ID NO:18的31-35位,SEQ ID NO:18的50-66位,SEQ ID NO:18的99-105位,SEQ ID NO:20的23-33位,SEQ ID NO:20的49-55位,和SEQ ID NO:20的88-97位。(The invention provides a human monoclonal anti-IL-32 antibody, a polynucleotide, a vector, a host cell, a preparation method, a composition and an application. The human monoclonal anti-IL 32 antibody or IL-32 binding fragment thereof preferentially binds IL-32 γ and/or does not bind IL-32 α relative to IL-32 α, and in its variable region the human monoclonal anti-IL 32 antibody or IL-32 binding fragment thereof comprises the six complementarity determining regions of the variable heavy chain and variable light chain of the amino acid sequences: positions 31-35 of SEQ ID NO. 18, positions 50-66 of SEQ ID NO. 18, positions 99-105 of SEQ ID NO. 18, positions 23-33 of SEQ ID NO. 20, positions 49-55 of SEQ ID NO. 20, and positions 88-97 of SEQ ID NO. 20.)

1. A human monoclonal anti-IL-32 antibody or IL-32-binding fragment thereof, wherein the human monoclonal anti-IL 32 antibody or IL-32-binding fragment thereof preferentially binds IL-32 γ and/or does not bind IL-32 α relative to IL-32 α, and in its variable region, the human monoclonal anti-IL 32 antibody or IL-32-binding fragment thereof comprises a variable heavy chain (V) of amino acid sequence_H) And variable light chain (V)_L) Six Complementarity Determining Regions (CDRs) of (a):

VH-CDR1 SEQ ID NO 18 at positions 31-35,

VH-CDR2 SEQ ID NO 18 at positions 50-66,

VH-CDR3 SEQ ID NO 18 at positions 99-105,

VL-CDR1 SEQ ID NO:20 at positions 23-33,

VL-CDR2 SEQ ID NO:20 at positions 49-55, and

VL-CDR3 positions 88-97 of SEQ ID NO 20.

2. The human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof of claim 1, which

(i) Can be combined with recombinant human IL-32 gamma; and/or

(ii) Can neutralize IL-32 gamma.

3. Human monoclonal anti-IL-32 antibody or IL-32-binding fragment thereof according to claim 1 or 2, comprising in its variable region a V selected from SEQ ID NO 18 and 20_HAnd/or V_LThe amino acid sequence of the region.

4. The human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof of claim 1 or 2, comprising: c_HAnd/or C_LConstant region of said C_HAnd/or C_LConstant area packetComprises a nucleotide sequence selected from SEQ ID NO: 22 and 24.

5. The human monoclonal anti-IL-32 antibody or IL-32-binding fragment thereof according to claim 1 or 2, selected from the group consisting of single-chain Fv fragments (scFv), F (ab ') fragments, F (ab')₂Fragments and single domain antibody fragments.

6. The human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof of claim 1 or 2, which:

(i) detectably labeled, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a peptide, and a heavy metal; or

(ii) Is attached to the drug.

7. The human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof of claim 1 or 2, wherein the human monoclonal anti-IL-32 antibody is IgG1 or IgG 3.

8. One or more polynucleotides encoding the human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof according to any one of claims 1 to 5 and 7.

9. One or more vectors comprising the polynucleotide of claim 8.

10. A host cell comprising the polynucleotide of claim 8 or the vector of claim 9, wherein the polynucleotide is a cDNA encoding at least part of the variable and constant regions.

11. A method for preparing an anti-IL-32 antibody or one or more immunoglobulin chains thereof, the method comprising:

(a) culturing the host cell of claim 10; and

(b) isolating the antibody or one or more immunoglobulin chains thereof from the culture of the host cell.

12. An anti-IL-32 antibody or one or more immunoglobulin chains thereof encoded by a polynucleotide according to claim 8 or obtained by a method according to claim 11.

13. The anti-IL-32 antibody of claim 12, which:

(i) detectably labeled, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a peptide, and a heavy metal; or

(ii) Is attached to the drug.

14. A composition comprising the human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof of any one of claims 1-7, the anti-IL-32 antibody or one or more immunoglobulin chains thereof of claim 12, the anti-IL-32 antibody of claim 13, the polynucleotide of claim 8, the vector of claim 9, or the cell of claim 10.

15. The composition of claim 14, wherein the composition is:

(i) a pharmaceutical composition, and further comprising a pharmaceutically acceptable carrier; or

(ii) A diagnostic composition or kit, and further comprising reagents conventionally used in immune-based or nucleic acid-based diagnostic methods.

16. The composition of claim 15, wherein the pharmaceutical composition further comprises an additional agent for treating an inflammatory disease.

17. Use of a human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof according to any one of claims 1 to 7, an anti-IL-32 antibody or one or more immunoglobulin chains thereof according to claim 12, an anti-IL-32 antibody according to claim 13, or a composition according to any one of claims 14-16 in the manufacture of a medicament for:

(a) treating or preventing the progression of an immune-mediated or autoimmune disease or disorder;

(b) ameliorating a symptom associated with an immune-mediated or autoimmune disease or disorder; and/or

(c) Diagnosing or screening for the presence of an immune-mediated or autoimmune disease or disorder in a subject or determining the risk of a subject developing an immune-mediated or autoimmune disease or disorder;

wherein the disease is associated with expression of IL-32 in the patient, wherein the disease is selected from the group consisting of inflammatory bowel disease, psoriasis, rheumatoid arthritis, ankylosing spondylitis and other forms of spondyloarthritis, psoriatic arthritis, myasthenia gravis, chronic obstructive pulmonary disease, asthma, tuberculosis, cancer including leukemia, vascular inflammation, and atherosclerosis, wherein inflammatory bowel disease includes crohn's disease, ulcerative colitis, and celiac disease.

18. Use of a human monoclonal anti-IL-32 antibody or IL-32 binding fragment thereof according to any one of claims 1 to 7, an anti-IL-32 antibody or one or more immunoglobulin chains thereof according to claim 12, an anti-IL-32 antibody according to claim 13 in the manufacture of a medicament for diagnosing an immune-mediated or autoimmune disease or disorder associated with expression of IL-32 in a subject.

19. The use according to claim 18, wherein IL-32 is IL-32 γ.

20. A method for manufacturing a pharmaceutical composition for treating a disease associated with a level of expression and/or activity of IL-32, the method comprising:

(a) culturing the host cell of claim 10; preparing an anti-IL-32 antibody or IL-32-binding fragment thereof from a culture of said host cell;

(b) purifying the antibody or IL-32 binding fragment thereof from the culture of the host cell to pharmaceutical grade; and

(c) mixing the antibody or IL-32 binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Technical Field

The present invention relates generally to novel molecules of mammalian, preferably human, origin, that bind interleukin-32 (IL-32), particularly human monoclonal antibodies, as well as fragments, derivatives, and variants thereof. In particular, the invention relates to recombinant human patient-derived anti-IL-32 antibodies and IL-32 binding fragments thereof. In addition, compositions comprising these binding molecules, antibodies and mimetics thereof that are useful in the treatment and diagnosis of diseases (disorders ) are described. Furthermore, the present invention relates to anti-IL-32 antibodies and the mentioned equivalents thereof for use in immunotherapy and as targets in therapeutic interventions for autoimmune and autoinflammatory diseases and malignancies, such as various forms of arthritis, Inflammatory Bowel Disease (IBD), Myasthenia Gravis (MG), Chronic Obstructive Pulmonary Disease (COPD), asthma, vascular inflammation and atherosclerosis, atopic dermatitis and cancer.

Background

Inappropriate responses by the immune system can cause stress symptoms (stressful symptoms) in the body involved. An exaggerated immune response to foreign substances or physical conditions that do not normally have a significant impact on the health of an animal or human may cause allergies with symptoms ranging from mild reactions (such as skin irritation) to fatal situations (such as anaphylactic shock or various vasculitis). The immune response to endogenous antigens can cause autoimmune diseases such as systemic lupus erythematosus, idiopathic autoimmune hemolytic anemia, pernicious anemia, type I diabetes, blistering skin disease (blistering skin disease), and different kinds of arthritis.

Immune responses occur in a coordinated manner, involving several cell types and requiring communication between the involved cell types by signaling molecules such as cytokines. This communication can be affected or inhibited by, for example, signal interception or blockade of individual receptors.

Cytokines are secreted soluble proteins, peptides and glycoproteins at nanomolar to picomolar concentrations as humoral regulators, which behave like classical hormones as they act at the systemic level, and they modulate the functional activity of individual cells and tissues under normal or pathological conditions. Cytokines differ from hormones in that: they are not produced by specialized cells of tissues in specialized glands, i.e. there is no single organ or cell source for these mediators (mediators), as they are expressed by virtually all cells involved in innate and adaptive immunity (such as epithelial cells, macrophages, Dendritic Cells (DC), Natural Killer (NK) cells), and in particular by T cells, among which helper T (th) lymphocytes are the predominant.

Cytokines can be classified into three functional categories according to their respective functions: modulation of innate immune response, modulation of adaptive immune response, and stimulation of hematopoiesis. Due to the pleiotropic activities of cytokines within these three classes, e.g., involving cell activation, proliferation, differentiation, recruitment or other physiological responses (e.g., secretion of proteins characteristic of inflammation by target cells), it has been discovered that interference of cell signaling mediated by abnormally regulated cytokine production is responsible for many diseases associated with defective immune responses (e.g., inflammation and cancer).

Interleukin-32 (IL-32, also known as natural killer cell protein 4) is a recently discovered cytokine with important functions in host defense and innate immunity. The human IL-32 gene is located on chromosome 16p 13.3. Apart from human and simian homologues, bovine, porcine and equine homologues have been found so far, whereas to date no mouse homologues are known. 6 isoforms of IL-32 that are known to arise from alternative splicing (Chen et al, Vitam Horm 74(2006), 207-. The longest isoform, IL-32gamma (IL-32. gamma. or IL-32g), comprises 234 aa (amino acids) (UniProtKB/Swiss-Prot ID: P24001-1). The second isoform, also known as IL-32beta (IL-32 β or IL-32 b; UniProtKB/Swiss-Prot ID: P24001-2) has 188 aa. The third isoform with 178 aa is also known as IL-32delta (IL-32. delta. or IL-32 d; UniProtKB/Swiss-Prot ID: P24001-3). IL-32alpha (IL-32 α or IL-32a) with 131 aa is the fourth isoform (UniProtKB/Swiss-Prot ID: P24001-4). Isoform 5(UniProtKB/Swiss-Prot ID: P24001-5) and isoform 6(UniProtKB/Swiss-Prot ID: P24001-6) have 168 aa and 179 aa, respectively. However, other isoforms may also exist, for example, Imaeda et al have reported a potentially new isoform with 112 aa (Mol Med Rep.4(2011), 483-487).

To date, the receptor for IL-32 is unknown. However, there are some data indicating that IL-32 can be bound and cleaved at the cell membrane by protease 3, suggesting this molecule as a possible receptor, where the generated fragment can have biological activity and activate macrophage inflammatory proteins-2 and IL-8(Dinarello and Kim, Ann Rheum Dis.65suppl.3 (2006); iii 61-64). It is suggested that IL-32 is the primary controller of the inflammatory pathway, with a significant synergistic effect with TNF α in the form of self-sustaining loop(s), where IL-32 promotes TNF α expression and vice versa, leading to the amplification of pro-inflammatory mediators. It has been reported that various cytokines such as TNFA/TNF-alpha, IL-1 beta, IL-6, IL-8 and macrophage inflammatory protein-2 (MIP-2) are induced to activate the typical cytokine signaling pathway of NF-. kappa.B and p38 MAPK, and this is an IL-18 inducible gene (Kim et al, Immunity 22(2005), 131-. Recently, IL-32 has also been shown to increase IFN- γ production by Peripheral Blood Mononuclear Cells (PBMCs) (Nold et al, JImmunal.181 (2008), 557-.

IL-32 has been reported to be produced primarily by NK cells, T lymphocytes, epithelial cells and blood monocytes stimulated by IL-2 or IFN- γ (Dahl et al, J Immunol.148(1992), 597-603; Kim et al, (2005)). Furthermore, IL-32 was observed to be overexpressed in Rheumatoid Arthritis (RA) synovial tissue biopsies, where the level of IL-32 expression was positively correlated with the severity of inflammation (Alsaleh et al, Arthritis Res Ther.12(2010), R135; Cagnard et al, EurCytokine Net.16 (2005), 289-292). In addition to various forms of arthritis belonging to the spondyloarthropathies family, such as Rheumatoid Arthritis (RA) or ankylosing spondylitis (Ciccia et al, Rheumatology 51(2012), 1966-. The high rate of atherosclerosis in RA also indicates a possible role of IL-32 in vascular inflammation and the inflammatory pathway of atherosclerosis, and this suggestion has also been confirmed, for example, by detection of IL-32 expression, while the expression of IL-32 β and IL-32 γ mRNA is significantly elevated in the arterial vessel wall of human atherosclerosis (Kobayashi et al, PLoS one.5 (2010); e 9458; Heinhuis et al, Cytokine. (2013), S1043-4666). IL-32 may also play a role in the immune response to tuberculosis (Kundu and Basu, PLoS Med.,3(2006), e 274; Neetea et al, 2006). Furthermore, increased transcription of IL-32 following infection with bacteria and viruses such as Mycobacterium tuberculosis (Neete et al, 2006) or influenza A (Li et al, PLoS one.3(2008), e1985) has been observed, suggesting a possible role for IL-32 in host defense.

Thus, IL-32 represents a novel therapeutic target that is not fully understood but important and requires IL-32 specific binding molecules that neutralize the function of all IL-32 isoforms, selected subregions thereof (sub-ranges) or individual IL-32 isoforms such as IL-32 γ.

First attempts to provide such molecules have been made. For example, U.S. Pat. No. US7,641,904B2 to Kim et al provides murine IL-32 monoclonal antibodies, wherein one of the antibodies selectively recognizes IL-32 α, and wherein the other antibody binds IL-32 α, IL-32 β, and IL-32 γ. International application WO 2005/047478 describes the production of murine antibody fragments specific for IL-32 α and IL-32 β. However, it is clear that no antibody specific for IL-32. gamma. has been provided.

Furthermore, humanized versions of the antibodies are mostly used in current therapeutic approaches (Chan and Carter, Nature Reviews Immunology 10(2010), 301-316; Nelson et al, Nature Reviews drug Discovery 9(2010),767-774) because of the immune response in humans to foreign antibodies such as mouse antibodies (human anti-mouse antibody-response (HAMA-response); Schroff et al, Cancer Res.45(1985), 879-885; Shawler et al, J.Immunol.135(1985), 1530-1535). One method of obtaining such antibodies is to graft Complementarity Determining Regions (CDRs) into a fully human framework, a method known as antibody humanization (Jones et al, Nature 321(1986), 522-525). This method is often complicated by the fact that: the mouse CDRs are not readily transferred to the human variable domain framework, resulting in a humanized antibody with lower affinity relative to its parent mouse antibody. Thus, additional elaborate mutagenesis experiments are often required to increase the affinity of the antibodies so engineered. Another way to achieve humanization of antibodies is to immunize mice that have had their innate antibody genes replaced with human antibody genes, and to isolate the antibodies produced by these animals. However, this method still requires immunization with antigens, but it is impossible to immunize with all antigens because of toxicity of some antigens. Furthermore, this method is limited to the production of transgenic mice with specific strains.

Another method for generating antibodies is to use a human antibody library, such as phage display technology as described in International application WO 2005/007699, for example for the generation of IL-13 specific antibodies. Here, the bacteriophage is engineered to display human scFv/Fab fragments on its surface by inserting human antibody genes into the phage population. Unfortunately, this approach also suffers from a number of disadvantages, including size limitations of the protein sequences used for multivalent display, the need for secretion of proteins from bacteria, i.e. antibody scFv/Fab fragments, size limitations of the library, limited number of possible antibodies produced and tested, a reduced proportion of antibodies with somatic hypermutations resulting from natural immunity; and all phage-encoded proteins are fusion proteins, which may limit activity or accessibility (accessibility) for some protein binding. Another serious drawback of this technique is that the antibodies so produced risk undesired cross-reactivity to self-antigens and lack the properties of evolutionarily optimized natural human antibodies produced by the human immune system. Furthermore, such antibodies may not be sufficiently specific due to their cross-reactivity with other proteins and/or with target proteins under normal physiological circumstances and functions. Similarly, european patent application EP0616640a1 describes the generation of autoantibodies from a pool of antibody fragments displayed on phage. In this regard, phage libraries were generated from non-immunized humans (see, e.g., example 1 at page 16, lines 43-51, example 2 at page 17, paragraph [0158], lines 57-58). However, the method described in this patent application also suffers from the above-mentioned general disadvantages of antibodies generated from phage libraries, as compared to antibodies generated and matured in mammals, i.e. humans.

In view of the above, there remains a need for further novel compounds for use in the treatment and diagnosis of diseases or disorders associated with unfavorable IL-32 activity, such as binding molecules with high specificity for IL-32 or for a selected range or single IL-32 isotype specificity, in particular for antibodies specific for IL-32 γ, which are tolerable in humans both for monotherapy and for combinatorial approaches.

The solution to this problem is further provided below by means of embodiments of the invention as characterized in the claims and disclosed in the description and illustrated in the examples and figures.

Disclosure of Invention

The present invention relates to IL-32 specific human monoclonal antibodies and IL-32 binding fragments and derivatives thereof. In particular, as shown in the accompanying examples and figures, human monoclonal anti-IL-32 antibodies are provided which have selective binding properties (profile) for the IL-32 isotype and which exhibit binding and neutralizing activity in vitro and in vivo. Due to their neutralizing properties, the antibodies of the invention have therapeutic, prognostic and diagnostic efficacy, which makes them particularly valuable in applications associated with: a wide variety of autoimmune and inflammatory diseases and disorders associated with/associated with IL-32 activity in the initiation and/or maintenance of an undesirable immune response, such as various forms of arthritis (e.g., Rheumatoid Arthritis (RA) or spondyloarthropathy), Myasthenia Gravis (MG), Inflammatory Bowel Disease (IBD), pulmonary diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma, crohn's disease, psoriasis, vascular inflammation and atherosclerosis, atopic dermatitis and cancer; for these and other possible therapeutic and diagnostic indications against IL-32, reference is additionally made to the background section of the invention of the text above.

The antibodies of the invention have been isolated from mammals, particularly humans, which are affected by impaired central and/or peripheral tolerance or loss of self-tolerance, which may be caused by or associated with a disrupted or abnormal self-tolerance gene, preferably caused by a monogenic autoimmune disease. Examples of mammals which provide particularly suitable sources for autoantibodies according to the invention are mammals such as humans suffering from diseases associated with mutations in the AIRE (autoimmune regulator) gene such as autoimmune multiple endocrine adenosis syndrome type 1 (APS1) (Peterson et al, nat. rev. immunoadenosis syndrome.8 (2008),948-957), autoimmune multiple endocrine adenosis syndrome type ii (APS2) (Baker et al, j. clin. endocrine. metab.95(2010), E263-E270) and immune dysfunction multiple endocrine adenosis enteropathy and X chromosome linkage syndrome (IPEX) (Powell et al, j. pediatr.100(1982), 731-737; Ochs et al, immunol. rev.203 (156-164). Preferably, the patient from whom the antibody is isolated exhibits seroreactivity (seroactivity) against at least one of the human IL-32 isoforms, most preferably against IL-32 γ.

In particular, the experiments performed according to the present invention were successful in the isolation of IL-32, in particular IL-32 γ -specific antibodies from APS1 patients. Thus, the present invention relates generally to high affinity IL-32 neutralizing monoclonal antibodies. Thus, the present invention provides monoclonal human antibodies (MABs or MABs) against several or a single IL-32 isotype, as will be described in detail below, which are considered safe and effective therapies for disorders in which those cytokines are involved.

Naturally, the invention extends to nucleic acids, in particular cdnas encoding at least one variable, constant and/or complementarity determining region of an antibody of the invention, and to vectors, antibody-producing cell lines and recombinant cells comprising such nucleic acids. The invention also relates to pharmaceutical compositions, diagnostic assays and kits comprising binding molecules or peptides recognized by antibodies isolated according to the invention, and to therapeutic methods based thereon.

Other embodiments of the invention will be apparent from the ensuing description and examples. When doing so, the terms "monoclonal antibody", "mAb" and "mAb" are used interchangeably herein, if not otherwise indicated.

Furthermore, while the invention has been illustrated and described by reference to the human antibodies initially obtained in the experiments performed according to the invention and described in the examples, it is to be understood that the antibodies or antibody fragments of the invention include synthetic and biotechnological derivatives of antibodies, which represent any engineered antibody or antibody-like IL-32 binding molecule synthesized by chemical or recombinant techniques that retains one or more functional properties of the subject antibody, in particular its neutralizing activity against IL-32. Thus, although the invention may be described for the sake of brevity by reference to antibodies, unless otherwise indicated, synthetic and biotechnological derivatives of antibodies and equivalent IL-32 binding molecules are also meant and included within the meaning of the term antibodies.

Drawings

FIG. 1: the variable regions of IL-32-specific human antibodies of the invention (i.e., heavy chain and kappa/lambda light chain (V)_H、V_L) ) of a polypeptide. A: antibody 2C2(IgG3, λ); b: antibody 14B3(IgG1, λ); c: antibody 19a1(IgG1, λ); d: antibodies26A6(IgG1, lambda). Framework (FR) and Complementarity Determining Regions (CDR) are underlinedCDRAnd (4) showing.

FIG. 2: comparison of ELISA serum reactivity of IL-32 α (filled diamonds) and IL-32 γ (filled squares) in sera isolated from APS1 patients. The X-axis represents individual patients and the Y-axis represents OD450 measurements of MAB binding.

FIG. 3: exemplary anti-IL-322C 2, 14B3, 19a1, and 26a6 antibodies react with a: IL-32 γ (R & D company) or B: IL-32 α (Immunotools Inc.) bound to EC50 ELISA. All antibodies tested bound to high affinity IL-32. Antibody 2C2 also binds to low affinity IL-32 α. The remaining antibodies 14B3, 19a1 and 26a6 did not show any substantial binding to IL-32 α.

FIG. 4: binding and neutralizing properties of exemplary antibodies of the invention. A: exemplary anti-IL-322C 2 antibodies were tested in an EC50ELISA for binding to IL-32 γ (R & D) and IL-32 α (Immunotools), BSA being used as a control for non-specific binding. Exemplary antibody 2C2 binds to high affinity IL-32 γ and binds to very low affinity IL-32 α. B: exemplary anti-IL-32 antibodies 2C2 and 19A1 neutralize IL-32 γ activity.

FIG. 5: for each cohort, the CytoEar thickness measurements were calculated as fold changes from day 0 measurements and then normalized to the relevant PBS control (normalized to the relevant PBS control). Mean. + -. standard deviation (Mean. + -. SEM), indicated by N on the figure. The P value is obtained by a bidirectional A nO VA test, and ns (not significant) is P > 0.05; p ≦ 0.05; p is less than or equal to 0.01; p < 0.001, P < 0.0001. IP-intraperitoneal and ID-intradermal injection.

FIG. 6: for each group, the CytoEar thickness measurements are shown as absolute values (mm). Mean ± standard deviation, indicated by N on the graph. P values were obtained by ANOVA test. IP-intraperitoneal and ID-intradermal injection. The P value indicates the same as in fig. 5.

FIG. 7: CytoEar assay-weight monitoring. No significant weight change was observed in any of the animals tested in the experiment.

FIG. 8: related IL-32 anddetailed analysis of sensorgrams (sensorgrams) of binding of the anti-IL-322C 2 antibody of the present invention. Non-1: 1 behavior was observed, which allowed a best fit with heterologous ligands. (A) An overlay of the experimental data for langmuir fitting (Langmuirfit) and binding reactions shows a good fit to the 1.1 langmuir model. The ratio of A1: 100nM, A2: 33.33nM, A3: 11.11nM, A4: IL-32 was injected at a concentration of 3.70nM, A5:1.23 nM. The residual plot (residual scatter) (B) shows random scatter with a noise level, indicating a good residual fit. (C) The table below the figure shows the values for association (ka), dissociation (kd), R_{Maximum value}And a power parameter obtained from a curve fitted to the calculated dissociation constant KD. The KD of the exemplary anti-IL-322C 2 antibodies of the present invention appears to be in the nM range.

FIG. 9: the effect of 19A1 blocking antibody compared to 2C2 after hIL-32 γ induced inflammation in the CytoEar assay. To induce inflammation, hIL-32. gamma. was injected at a concentration of 6.25. mu.g/ml, i.e., 125 ng/ear. A: exemplary 10 day experimental timeline. B: overview of experimental treatments of experimental animal groups a to F. C-F: a group of mice (C57/BL6, 8 weeks) were IP injected with a defined amount of 2C2 or 19a1 antibody (or IgG control) on the day of initiation of the experiment, while 20 μ l PBS containing 125nghrIL-32 γ cytokine was injected intradermally into the mouse ear every 48-72 hours (or PBS control). Ear thickness measurements were performed using a Mitutoyo digital micrometer. For each cohort, CytoEar thickness measurements were calculated as fold changes relative to the starting day measurements and then normalized to the relevant PBS control. Mean ± standard deviation, indicated by N on the graph. P values were obtained by ANOVA test. IP, i.e. intraperitoneal antibody injection, ID, i.e. intra-auricular intradermal injection, NT, i.e. untreated control.

FIG. 10: the effect of varying doses of 19A1 antibody after hIL-32 γ induced inflammation in the CytoEar assay. To induce inflammation, hIL-32. gamma. was injected at a concentration of 6.25. mu.g/ml, i.e., 125 ng/ear. A: exemplary 10 day experimental timeline. B: overview of experimental treatments of experimental animal groups a to K. C-F: a group of mice (C57/BL6, 8 weeks) were IP injected with a defined amount of 2C2 or 19a1 antibody (or IgG control) on the day of initiation of the experiment, while 20 μ l PBS containing 125nghrIL-32 γ cytokine was injected intradermally into the mouse ear every 48-72 hours (or PBS control). Ear thickness measurements were performed using a Mitutoyo digital micrometer. For each cohort, CytoEar thickness measurements were calculated as fold changes from the measurement on the starting day and then normalized to the relevant PBS control. Mean ± standard deviation, indicated by N on the graph. P values were obtained by ANOVA test. IP, i.e. intraperitoneal antibody injection, ID, i.e. intra-auricular intradermal injection, NT, i.e. untreated control.

FIG. 11: dose dependence of IL-32 in inducing inflammation in the CytoAnkle assay. To induce inflammation, mice were injected intraluminally into the ankle joint with 10 μ l of hIL-32 γ in the right ankle joint, while the left ankle joint was injected with PBS. A: exemplary time axis for 13 day experiments. B: overview of experimental treatments of experimental animal cages a1 to D2. C-D: mice in the mouse cohort (C57/BL6, 8 weeks) were injected with prescribed amounts of 10 μ l PBS containing hrIL-32 γ cytokine (or PBS control) every 48-72 hours at ankle IA. Axial ankle thickness measurements were made using a Mitutoyo digital micrometer. For each cohort, CytoAnkle thickness measurements were calculated as fold changes from the measurements on the starting day and then normalized to the relevant PBS control. Mean ± standard deviation, indicated by N on the graph. P values were obtained by ANOVA test. IA is an intracavitary injection of the ankle joint.

FIG. 12: the effect of 2C2 antibody after hIL-32 γ induced inflammation in the CytoAnkle assay. CytoAnkle test: +/-IL-32 γ +/-2C 2. A: exemplary 10 day experimental timeline. B: overview of experimental treatments of experimental animal cages a1 to D2. C-D: a group of mice (C57/BL6, 8 weeks) was IP-injected with 200 μ g of 2C2 antibody (or IgG control) on the day of initiation of the experiment, while 10 μ l PBS containing 500ng of hrIL-32 γ cytokine (or PBS control) was injected into the mouse ankle IA every 48-72 hours. Axial ankle thickness measurements were made using a Mitutoyo digital micrometer. For each cohort, CytoAnkle thickness measurements were calculated as fold changes from the measurements on the starting day and then normalized to the relevant PBS control. Mean ± standard deviation, indicated by N on the graph. P values were obtained by ANOVA test. IP ═ intraperitoneal antibody injection, IA ═ intraankle joint injection.

Detailed Description

The present invention relates generally to a novel molecule of mammalian (preferably human) origin that binds to IL-32, in particular to human monoclonal antibodies and fragments, derivatives and variants thereof, which recognize different isotypes of IL-32.

As described in the background section, efforts have been made to provide IL-32 specific antibodies, however, as indicated above, the antibodies currently provided and used are not of human origin, which greatly impairs their therapeutic use in humans due to the immunogenicity of non-human antibodies. Furthermore, although murine antibodies against IL-32 α or murine antibodies with broader specificity against several IL-32 isoforms may be used, due to their broad binding specificity, they may have undesirable side effect profiles and thus may cause adverse reactions and diseases. Furthermore, it is clear that no human IL-32. gamma. specific antibody is available.

As described in the examples, the subject antibodies of the invention are isolated by the method disclosed in the applicant's co-pending international application WO2013/098419a1, which is based on screening autoantibodies against IL-32 protein in the serum of patients with impaired central and/or peripheral tolerance or loss of self-tolerance, such as APECED/APS1 patients. In these screens, the presence of autoantibodies recognizing a specific IL-32 isotype was unexpectedly observed in these sera. This cytokine has only recently been identified as a pro-inflammatory cytokine, and its role in disease is still less well known (at least compared to many other pro-inflammatory cytokines). In this regard, the absence of the presence of IL-32 autoantibodies in APS1 patients developing many common autoimmune and inflammatory disorders and diseases such as RA further supports the important role that this cytokine plays in the pathology of these diseases and confirms the method of targeted antibody-based inhibition of therapeutic intervention of the present invention and its use in diagnostic applications.

In view of the above, experiments conducted according to the present invention are aimed at providing IL-32 binding molecules, in particular antibodies that show a binding specificity for all IL-32 isoforms or only a sub-range of IL-32 isoforms or even only for a single isoform, preferably specifically binding to IL-32. gamma. this immunoreactivity has been shown in APECED/APS1 patients to prevent, for example, the onset of RA and/or IBD. Preferably, the IL-32 binding molecules are capable of neutralizing the biological activity of IL-32. The problem underlying the present invention has been solved as indicated by the exemplary anti-IL-322C 2 antibody of the present invention in the accompanying examples and figures (in particular fig. 3 and 4A showing binding affinity, fig. 4B and 5, 6 and 9 to 12 showing the neutralizing activity and therapeutic efficacy of the subject antibody in an animal model).

Thus, in its broadest aspect, the present invention relates to recombinant human monoclonal anti-interleukin-32 (IL-32) antibodies and IL-32 binding fragments thereof that bind to one or more of the IL-32 isoforms and biotechnological derivatives thereof; for a description of IL-32 isoforms including, for example, IL-32 γ, IL-32 α, IL-32 β, and IL-32 δ, see also the background section above. In one embodiment, the human monoclonal anti-IL-32 antibody, or IL-32 binding fragment thereof, is capable of neutralizing the biological activity of IL-32 γ and/or IL-32 α. With respect to the binding and neutralizing properties of the antibodies of the invention, they are substantially equivalent in targeting different IL-32 isoforms, or may have preferential binding and/or neutralizing activity for each IL-32 isoform.

In a preferred embodiment of the invention, the human monoclonal anti-IL-32 antibody, or IL-32 binding fragment thereof

(i) Can bind with recombinant human IL-32gamma (IL-32 gamma);

(ii) preferentially binds to human IL-32 γ and/or does not substantially bind to IL-32 α relative to IL-32alpha (IL-32 α); and/or

(iii) Can neutralize the bioactivity of IL-32 gamma.

Preferably, the antibody of the invention or a binding fragment of IL-32 thereof or an equivalent binding molecule comprises in its variable region:

(a) (i) FIG. 1 (V)_H) (SEQ ID NO: 2. 10, 18 and 26) and (ii) FIG. 1 (V)_L) (SEQ ID NO: 4. 12, 20 and 28) are described_HAnd/or V_LAt least one Complementarity Determining Region (CDR) of the variable region amino acid sequence;

(b) v depicted in FIG. 1_HAnd/or V_LThe amino acid sequence of the region;

(c) at least one CDR consisting of an amino acid sequence resulting from a partial alteration of the amino acid sequence of any one of (a); and/or

(d) A heavy chain and/or light chain variable region comprising an amino acid sequence resulting from a partial alteration of the amino acid sequence of (b).

As described in more detail below, the antibodies or antigen-binding fragments thereof of the present invention can be or be derived from any type, species, or subclass of immunoglobulin molecule. However, in a preferred embodiment, the antibodies of the invention are provided in an IgG isotype, most preferably an IgG1 or IgG3 subtype.

In order to provide such humanized, chimeric and in particular fully human antibodies and natural Fab fragments thereof, in one embodiment the antibody or IL-32 binding fragment of the invention further comprises C_HAnd/or C_LA constant region comprising C selected from those described in Table 1_HAnd C_LAmino acid sequences of the amino acid sequences (SEQ ID NOS: 6, 8, 14, 16, 22, 24 and 30) or amino acid sequences which have at least 60% identity, preferably 70% identity, more preferably 80% identity, more preferably 90% identity, and particularly preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the reference sequence mentioned.

As described above, it has been found that IL-32 pro-inflammatory activity induces various cytokines, such as TNF α/TNF-alpha, IL-1 β, IL-6, IL-8, MIP-2 (see Kim et al, (2005); Neete et al, (2008); Joosten et al, (2006)). This activation mechanism has been used in the present invention for designing in vitro and in vivo assays to determine IL-32 activity, monitoring IL-6 expression by IL-32 stimulated RAW 264.7 macrophages as described in example 3 and fig. 4B, 5 and 6 and monitoring the neutralizing properties of the antibodies of the present invention as well as ear inflammation assays. As detailed therein, it has been found that the antibodies of the invention have potent neutralizing activity against IL-32 γ, one of which also exhibits residual binding activity to IL-32 α, as further detailed below. Thus, in one embodiment, the antibodies of the invention or IL-32 binding fragments thereof are capable of reducing the biological activity of human IL-32 (preferably IL-32. gamma.). In a preferred embodiment, the biological activity is human IL-32 γ -induced inflammation. Furthermore, in one embodiment of the invention, the biological activity is determined in an IL-6 induction assay and/or an otitis assay.

Furthermore, the binding affinity of the antibodies of the invention has been tested by ELISA as described herein, e.g., in example 2 and shown in figures 3 and 4A. Based on the results of these experiments, the present invention provides several exemplary anti-IL-32 antibodies and IL-32 binding fragments thereof that exhibit different binding affinities for different IL-32 isoforms, which exemplify the binding and neutralizing properties of the IL-32 binding molecules provided herein.

Since the exemplary anti-IL-32 antibodies described in the examples are derived from human patients, the present invention advantageously provides fully human antibodies that are particularly useful in therapeutic applications, which are substantially free of foreign antibodies (e.g., murine in human, HAMA-responses) or immune responses otherwise commonly observed in humanized and human-like antibodies.

Thus, referring additionally to the discussion below, in contrast to humanized and other human-like antibodies, the human antibodies of the invention are characterized by comprising CDRs that are already found in humans and thus have substantially no risk of developing immunity. Thus, an antibody of the invention may still be indicated as being of human origin if at least one, preferably two and most preferably all three CDRs of one or both of the variable light and heavy chains of the antibody are derived from a human antibody as specified herein.

Human antibodies may also be referred to as "human autoantibodies" to emphasize that those antibodies are actually initially expressed by the subject, rather than in vitro selected constructs generated, for example, by a human immunoglobulin expressing phage library, or xenogeneic antibodies generated in a transgenic animal expressing part of a human immunoglobulin library (human immunoglobulin repertoire), which to date represents the most common approach used in attempts to provide human-like antibodies. In another aspect, the human antibodies of the invention may be expressed as synthetic, recombinant, and/or biotechnological to distinguish them from human serum antibodies which may themselves be purified by protein a or affinity columns.

However, the present invention uses and envisions further studies of the antibodies of the invention in animal models (e.g., in transgenic mice expressing human IL-32). To avoid an immune effect in experimental animals that is similar to the HAMA-response in humans, in one aspect, an antibody or binding fragment of the invention is provided that is a chimeric antibody, preferably a chimeric rodent-human or rodent-derived (rodentized) antibody, most preferably a chimeric mouse-human or murine-derived (murinized) antibody.

The antibodies of the invention have been isolated from APECED/APS1 patients as described above. In this case, the experiments disclosed in the applicant's co-pending international application WO2013/098419a1 surprisingly show that: APECED/APS1 patients display autoimmunity (auto-immune), i.e. also include a broad spectrum of autoantibody profiles of binding molecules specific for different IL-32 isoforms. APS1 is a rare autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene. The AIRE protein governs expression in thymic medullary epithelial cells of many peripheral self-antigens (e.g., insulin) presented by MHC to tolerate development of thymocytes. In APS1, AIRE mutations cause abnormal negative selection, allowing autoreactive T cells to escape to the periphery. Thus, patients exhibit a widely varying clinical profile in APS1, but typically have several endocrine tissue autoimmune diseases. Typical APS1 triple markers (tripads) include chronic mucocutaneous candidiasis (chronic mucocutaneous candidiasis), hypoparathyroidism, and adrenal failure (Perheentupa, Endocrinol. Metab. Clin. North am.31(2002), 295-320). Other clinical conditions seen in patients with APECED include autoimmune diseases of the thyroid gland, diabetes, gonadal failure, vitiligo, alopecia areata, chronic hepatitis, chronic gastritis and pernicious anemia and other gastrointestinal symptoms of different forms. For further details on APECED/APS1 patients and their screening for autoimmunity, see the description of international application WO2013/098419a1 and the examples described therein, in particular the materials and methods section on page 112-; example 1 at page 117 and example 7 at page 128 and subsequent tables 1-14; and example 17 of page 168-171, the disclosure of which is incorporated herein by reference.

As mentioned above, in a preferred embodiment, the antibodies of the invention are obtained from or can be obtained from a sample of a human subject suffering from autoimmune polyendocrine adenopathy-candidiasis-ectodermal dystrophy (APECED/APS1) or from a patient suffering from a similar autoimmune disease, as described in international application WO2013/098419a1 and the examples therein, in particular the materials and methods section at page 112-; page 117-118 example 1; example 10 on page 156-161 (especially where the "patients and controls" section on page 156); and example 17, page 168-171, the disclosure of which is incorporated herein by reference. Furthermore, in a preferred embodiment, the APS1 subject is characterized by exhibiting serum reactivity to human IL-32 (preferably to IL-32 γ and/or IL-32 α).

In this context, it is to be noted that the subject anti-IL-32 antibodies of the present invention have been cloned by a novel proprietary method of isolating human antibodies disclosed in applicants' co-pending international application WO2013/098420 a1, the disclosure of which is incorporated herein by reference.

Briefly, a sample for isolating an antibody of interest comprises or consists of Peripheral Blood Mononuclear Cells (PBMCs) and serum for detecting possible antibody reactivity. The subject-derived sample may be used directly, e.g., to test for serum reactivity to one or more desired antigens, or may be further processed, e.g., enriched for B lymphocytes. In particular, preferably the sample comprises or is derived from B cells producing an antibody of interest, more preferably memory B cells. Memory B cells were cultured under the following conditions: it only allows a defined life cycle of B cells, usually not exceeding 1 to 2 weeks, until cells responding to the desired antigen are picked out of the B cell culture, followed by RT-PCR of the individual picked cells for obtaining the immunoglobulin gene bank; see examples 1 and 2 on pages 118-120 of WO2013/098419a1, and in particular examples 1 to 4 on pages 27-31 of WO2013/0984220 a1, the disclosures of which are incorporated herein by reference. Naturally, the invention extends to human B memory lymphocytes and B cells, respectively, that produce antibodies with significantly unique characteristics as defined above and below.

Thus, in addition to using a selected patient pool, APS1 subjects preferably exhibiting seroreactivity to at least one of the human IL-32 isoforms neutralized by exemplary antibodies of the present invention, anti-IL-32 antibodies have also been provided by using specific methods developed specifically for isolating human monoclonal antibodies from B cells of patients with autoimmune diseases, such as APECED/APS1 patients.

In one embodiment, the antibody or IL-32 binding molecule of the invention comprises V depicted in FIG. 1 or encoded by a corresponding nucleic acid shown in Table 1_HAnd/or V_LThe amino acid sequence of the region. In addition, in another embodiment, the invention relates to an anti-IL-32 antibody or IL-32 binding molecule that competes with an antibody of the invention as defined above for specific binding to human IL-32, preferably to IL-32 γ. In particular, anti-IL-32 antibodies are provided, demonstrating the immunological and/or biological properties outlined for the antibodies shown in the examples and figures. Currently, the term "immunological binding properties" or other binding properties of an antibody to an antigen (in all its grammatical forms) refers to the specificity, affinity, cross-reactivity and other binding properties of an antibody.

In one embodiment, the antibody of the invention is an antibody fragment. For example, the antibody or antibody fragment of the invention may be selected from the group consisting of single chain F_vFragments (scFv), F (ab ') fragments, F (ab')₂Fragments and single domain antibody fragments (sdabs).

Another advantage of the antibodies of the invention is that: due to the fact that a humoral immune response to the native antibody has been elicited in the physiological and cellular environment of the native antibody, autoantibodies are usually produced and can be isolated, which can recognize conformational epitopes of the antigen as they are present, for example, in an environment with other cellular components, on cell surface membranes and/or bind to receptors. In contrast, traditional methods of generating monoclonal antibodies (such as mouse monoclonal antibodies), humanized versions thereof, or antibodies obtained by phage display technology, typically employ antigenic fragments of the target protein for immunizing and detecting, respectively, non-human mammals, according to which antibodies are typically obtained that recognize linear or conformational epitopes restricted to the two-dimensional structure of the immunogen rather than recognizing the presence of the native protein in its physiological and cellular environment. Therefore, it is prudent to expect that the autoantibodies of the present invention are unique in their epitope specificity. Thus, the invention also relates to antibodies and binding-like molecules that exhibit substantially the same binding specificity as autoantibodies isolated according to the methods of the invention. Such antibodies can be readily tested by the following method: for example, competitive ELISA, or more suitably cell-based neutralization assays using autoantibodies of the invention and monoclonal derivatives thereof, respectively, as reference antibodies, and immunoassay methods described in the examples or known to those skilled in the art.

The present invention illustrates IL-32 binding molecules, i.e., antibodies and binding fragments thereof, that may be generally characterized as V's that include variable regions in their variable regions (i.e., binding domains)_HAnd/or V_L(ii) at least one Complementarity Determining Region (CDR) of (A), the variable region being comprised in (V) of FIG. 1_H) (SEQ ID NO: 2. 10, 18 and 26) and (V)_L) (SEQ ID NO: 4. 12, 20 and 28) -see the exemplary CDR sequences underlined in fig. 1 and indicated in table 1. However, as discussed below, the skilled person is clearly aware of the fact that CDRs of amino acids differing in their amino acid sequence by one, two, three or even more from those shown in figure 1 may additionally or alternatively be used, particularly in the case of CDR2 and CDR 3.

As further demonstrated for the antibodies of the invention, the antibodies are capable of neutralizing the biological activity of their target proteins; see, for example, the results of the IL-6 neutralization assay described in example 3, FIG. 4B and the IL-32 otitis assay (CytoEar assay) and ankle inflammation assay (CytoAnkle assay) described in example 4, FIGS. 5 and 6, and FIGS. 9 to 12. In this context, the term "neutralizing" means that the anti-IL-32 antibody or IL-32 binding fragment thereof of the invention is capable of interfering with the biological activity of its target protein in a biochemical cell-based or in vivo assay, as may be assessed by performing the respective assay in the presence of a subject antibody of the invention, wherein the biological activity of the target protein decreases with increasing levels of the antibody of the invention subjected to the assay, as compared to the biological activity of the protein in the absence of the antibody of the invention and in the presence of a compound, e.g., a control antibody, which is known to qualitatively not affect the biological activity of the target protein. Such biochemical in vitro and in vivo based assays may also be performed using a reference antibody known to be capable of neutralizing the biological activity of the target protein (such as shown for the anti-IL-32 antibody of the invention) and subjecting the candidate antibody to an assay sample, wherein either an additional neutralizing effect due to the combined activity of the reference and candidate antibodies may be observed or competition between the candidate and reference antibodies may be observed, as may be determined by labeling either antibody. Thus, in a preferred embodiment of the invention, the antibody obtained by the method of the invention is capable of neutralizing the biological activity of its antigen, e.g. at least one of the human IL-32 isoforms, preferably IL-32. gamma. The neutralizing effect can be determined, for example, by the amount of IL-32 activity that is reduced or the time at which such reduction is observed upon introduction of the IL-32 binding molecules of the invention, or of course, in terms of a combination of that amount and time.

As detailed below, the antibodies or antigen binding fragments of the invention, such as peptides, polypeptides or fusion proteins, may be provided by expression, for example, in a host cell or in an in vitro cell-free translation system. For expression of a peptide, polypeptide or fusion protein in a host cell, the nucleic acid molecule encoding the peptide, polypeptide or fusion protein may be inserted into an appropriate expression vector, i.e., a vector containing the essential elements for transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct expression vectors comprising sequences encoding the polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. This technique is described in Sambrook et al, Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al, Current Protocols in Molecular Biology (1989); for more details in this regard, see also the "polynucleotides" and "expression" sections, described further below, and the references cited in the examples section.

Suitable host cells for expressing the product may be any prokaryotic or eukaryotic cell; for example, bacterial cells (such as E.coli or Bacillus subtilis), insect cells (baculoviruses), yeast cells, plant cells or animal cells. However, mammalian cells are preferred for efficient processing. Typical mammalian cell lines that may be used for this purpose include CHO cells, HEK293 cells, COS cells and NSO cells.

Of course, the isolated antibodies of the invention cannot be applied to patients as such, but must generally be formulated pharmaceutically to ensure, for example, their stability, acceptability and bioavailability in patients. Thus, in one embodiment, there is provided a method of the invention further comprising the step of admixing the isolated monoclonal antibody or fragment thereof with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier will be described in further detail below.

As a means of obtaining a stable and permanent source of the binding molecules of the invention, the heterologous genes encoding these binding molecules can be isolated by direct cloning, PCR amplification or artificial synthesis, and introduced and expressed in a suitable host cell or organism. It is therefore also an object of the present invention to provide a method for preparing a recombinant cell useful for the production of a recombinant human anti-IL-32 antibody or IL-32 binding fragment thereof, comprising the steps of:

(a) preparing B cells by the method described above;

(b) sequencing nucleic acids and/or obtaining nucleic acids from B cells, the nucleic acids encoding:

(i) c listed in Table 1_HAnd C_LAt least one of the amino acid sequences (SEQ ID NOS: 6, 8, 14, 16, 22, 24, and 30) or an amino acid sequence having at least 60% identity;

(ii) in FIG. 1 (V)_H) (SEQ ID NO: 2. 10, 18 and 26) and fig. 1 (V)_L) (SEQ ID NO: 4. 12, 20 and 28) are described_HAnd/or V_LAt least one Complementarity Determining Region (CDR) in the variable region amino acid sequence;

(iii) v as depicted in FIG. 1_HAnd/or V_LThe amino acid sequence of the region;

(iv) at least one CDR consisting of an amino acid sequence resulting from a partial alteration of the amino acid sequence of any one of (a);

(v) (iii) a heavy and/or light chain variable region comprising an amino acid sequence resulting from a partial alteration of the amino acid sequence of (ii); and/or

(c) The nucleic acid is inserted into an expression host to allow expression of the antibody of interest in the host.

The host cells described herein may also be used in previous methods, and may be used as described in detail in the "host" section of the specification. In this aspect, in one embodiment, the above method is provided, wherein the expression host is a yeast cell, a plant cell or an animal cell.

With respect to the above-described methods for producing each antibody of interest, in one embodiment, the present invention provides a method wherein the nucleic acid is manipulated between steps (b) and (c) above to introduce restriction sites, alter codon usage, and/or increase or optimize transcriptional and/or translational regulatory sequences.

As demonstrated in the appended examples 2 and 3 and summarized in table 3, binding molecules, i.e. antibodies, have been identified and cloned which exhibit a particularly high apparent binding affinity to human IL-32 (EC50/ED 50). To this end, in one embodiment of the invention, there is provided an antibody or binding fragment thereof as defined above, which has a high affinity for its corresponding target molecule (e.g. the human IL-32 isotype defined above, preferably for IL-32 γ), exhibiting EC50 at a concentration of less than 2000ng/ml or 1500ng/ml, preferably less than 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100ng/ml, more preferably less than 50, 20 or 10 ng/ml. Alternatively or additionally, in one embodiment, there is provided an antibody or antigen-binding fragment thereof as defined above, which has a high neutralizing capacity against the human IL-32 isotype, preferably against IL-32 γ, exhibiting an IC50 at a concentration of below 500 or 400ng/ml, preferably below 300, 200 or 100ng/ml, more preferably below 50, 20 or 10 ng/ml. For more details on the binding affinity of the antibodies of the invention, see for example the "binding properties" section below. In one embodiment, the antibodies or IL-32 binding fragments of the invention specifically bind to more than one isoform of IL-32, preferably wherein IL-32 γ is one of the isoforms identified. In one embodiment, the second isoform is IL-32 α. In a preferred embodiment, the anti-IL-32 antibody or IL-32 binding fragment thereof preferably binds IL-32 γ as compared to the second recognized isoform. Furthermore, in one embodiment, the anti-IL-32 antibodies or IL-32 binding fragments thereof of the invention bind to one IL-32 isotype and do not bind, or do not substantially bind, any other IL-32 isotype.

The invention also relates to polynucleotides encoding at least one variable region of an immunoglobulin chain of an antibody or antigen-binding fragment of the invention. Preferably, the variable regions comprise V of the variable regions listed in FIG. 1_HAnd/or V_LAt least one Complementarity Determining Region (CDR).

In the case of derivative sequences, the sequences show at least 60% identity, more preferably (in the following order) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and most preferably 95%, at least 96-99% or even 100% identity to sequences in the group consisting of those mentioned above and identified in the sequence listing. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Alignment of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms well known to those skilled in the art. The identities referred to herein are determined by using the BLAST program described further below.

As mentioned above, in a preferred embodiment, the invention relates to substantially fully human antibodies (Preferably IgG) comprising at least a constant region of a constant heavy chain I (C)_H1) And the corresponding light chain, i.e., γ -1, γ -2, γ -3 or γ -4 in combination with lambda (λ) or kappa (κ). In a particularly preferred embodiment, the nucleotide and amino acid sequences of those constant regions exemplified in the examples that were isolated for antibodies of subjects are used as described in table 1 below, and the nucleotide sequences are represented by SEQ ID NOs: 5. 7, 13, 15, 21, 23 and 29, and/or the amino acid sequences of SEQ ID NOs: 6. 8, 14, 16, 22, 24 and 30.

In view of the foregoing, in one embodiment, the invention also provides polynucleotides encoding at least the variable region of an immunoglobulin chain of an antibody or antigen-binding fragment of the invention. Typically, the variable region encoded by the polynucleotide comprises the V of the variable region of the antibody_HAnd/or V_LAt least one Complementarity Determining Region (CDR). The variable and constant regions of the antibody are described in more detail in the "IgG structure" section below. In a preferred embodiment of the invention, the polynucleotide comprises, consists essentially of, or consists of a nucleic acid having a polynucleotide sequence that is specific for V of an antibody of the invention as described in table 1 below_HOr V_LThe region is encoded. To this end, those skilled in the art will readily appreciate that polynucleotides encoding at least the variable domains of the light and/or heavy chains may encode multiple immunoglobulin chains or the variable domain of only one of them. In a preferred embodiment, the polynucleotide encodes an anti-IL-32 antibody or IL-32 binding fragment thereof as defined above.

Table 1: the variable and constant regions (V) of the IgG3, Lambda, IL-32 specific 2C2 antibodies and IgG1, Lambda, IL-32 specific 14B3, 19A1 and 26A6 antibodies of the invention_H、V_L、C_H、C_L) The nucleotide sequence of (a). Underlined and bolded nucleotides or amino acids indicate in the variable chain sequenceCDRA coding region. Underlined italic nucleotides or amino acids indicate that they have not been ordered but are notHas already been got from Obtaining in a databaseIn (1) orderAnd (4) columns. In the invariant chain, these regions are aligned with or tuned according to the relevant human germline variable region sequences in the database; see, e.g., Vbase (R) sponsored by MRC (Committee for medical research) protein engineering center (Cambridge university, UK) ((R))http://vbase.mrc-cpe.cam.ac.uk)。

Those skilled in the art will readily appreciate that the variable domains of antibodies having the above described variable domains can be used to construct other polypeptides or antibodies having the desired specificity and biological function. Thus, the invention also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and advantageously having substantially the same or similar binding properties as the antibodies described in the appended examples. The skilled person will readily understand that antibodies can be constructed by using the variable domains or CDRs described herein according to methods known in the art, for example as described in european patent applications EP0451216 a1 and EP0549581 a 1. Furthermore, it is known to those skilled in the art that binding affinity can be increased by amino acid substitutions within the CDRs or within hypervariable loops (Chothia and Lesk, j.mol.biol.196(1987), 901-. Thus, the invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more (preferably no more than two) amino acid substitutions. Preferably, the antibodies of the invention comprise a needle in one or both of their immunoglobulin chainsTo V_HThe region is as shown in SEQ ID NO: 2. 10, 18 and 26 and for V_LThe region is as shown in SEQ ID NO: 4. 12, 20 and 28 or as shown in figure 1.

The polynucleotides of the present invention encoding the above-described antibodies may be, for example, DNA, cDNA, RNA, or synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any of these polynucleotides, alone or in combination. In one embodiment, the polynucleotide is a cDNA encoding the variable region and at least a portion of the constant domain. In a preferred embodiment, there is provided a vector comprising the above-described polynucleotide, optionally in combination with said polynucleotide encoding the variable region of the other immunoglobulin chain of said antibody. These vectors may comprise further genes, such as marker genes, which allow the selection of the vector in a suitable host cell and under suitable conditions.

Preferably, the polynucleotide of the invention is operably linked to an expression control sequence allowing expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide includes transcription of the polynucleotide into a translatable mRNA. Regulatory elements which ensure expression in eukaryotic, preferably mammalian, cells are well known to the person skilled in the art. Regulatory elements typically include regulatory sequences that ensure initiation of transcription, and optionally, poly a signals that ensure termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers and/or naturally associated or heterologous promoter regions.

In this regard, one skilled in the art will readily appreciate that a polynucleotide encoding at least the variable domain of a light chain and/or a heavy chain may encode the variable domain of two immunoglobulin chains or only one immunoglobulin chain.

Likewise, the polynucleotides may be under the control of the same promoter, or may be separately controlled for expression. Possible regulatory elements which allow expression in prokaryotic host cells include, for example, P in E.coli_LLac, trp or tac promoter, and examples of regulatory elements which allow expression in eukaryotic host cells are yeastAOX1 or GAL1 promoter in (b), or CMV promoter, SV40 promoter, RSV promoter, CMV enhancer, SV40 enhancer, or globular intron in mammalian and other animal cells.

In addition to the elements responsible for the initiation of transcription, such regulatory elements include transcription termination signals downstream of the polynucleotide, such as the SV40 polya site or the tk polya site. In addition, a leader sequence capable of directing the polypeptide to a cellular compartment or capable of secreting the polypeptide into a medium may be added to the coding sequence of the polynucleotide of the present invention, depending on the expression system used, and is well known in the art. One or more leader sequences are assembled at appropriate stages with translation, initiation and termination sequences, and preferably, leader sequences that direct secretion of the translated protein, or a portion thereof, to the periplasmic space or extracellular medium. Alternatively, the heterologous sequence may encode a fusion protein comprising a C-terminal or N-terminal identification peptide that confers a desired characteristic of the expressed recombinant product, such as stability or simple purification. In this case, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORT1(GIBCO BRL).

Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence, and collection and purification of the immunoglobulin light chain, heavy chain, light/heavy chain dimer or whole antibody, binding fragment or other immunoglobulin form may follow, as desired; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y. (1979).

Furthermore, the present invention relates to vectors, in particular plasmids, cosmids, viruses and bacteriophages commonly used in genetic engineering, comprising polynucleotides encoding an antigen or preferably encoding a variable domain of the immunoglobulin chain of an antibody of the invention; optionally, it is combined with a polynucleotide of the invention encoding a variable domain of another immunoglobulin chain of an antibody of the invention. Preferably, the vector is an expression vector and/or a gene transfer or targeting vector.

Expression vectors derived from viruses (such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses) can be used to deliver the polynucleotides or vectors of the invention to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, e.g., techniques described in Sambrook, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994)). Alternatively, the polynucleotides and vectors of the invention may be recombined into liposomes for delivery to target cells. Vectors comprising a polynucleotide of the invention (e.g., the heavy and/or light chain variable domains of an immunoglobulin chain, coding sequences, and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for transformation of other cellular hosts; see Sambrook section above.

In relation to the above, the present invention also relates to a host cell comprising said polynucleotide or vector. The host cell may be a prokaryotic or eukaryotic cell. The polynucleotide or vector of the invention present in the host cell may be integrated into the genome of the host cell or may be maintained extrachromosomally. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell; suitable host cells and methods for producing the antibodies of the invention are described in more detail in the "host cell" section below.

Using the above-described host cells, the antibodies of the invention can be produced and prepared, e.g., for pharmaceutical use or as targets for therapeutic intervention. Accordingly, in one embodiment, it is also an object of the present invention to provide a method for preparing an anti-IL-32 antibody or IL-32 binding fragment thereof, said method comprising:

(a) culturing a cell as defined above; and

(b) isolating the antibody or IL-32 binding fragment thereof from the culture.

Thus, the present invention relates to recombinant, preferably human, anti-IL-32 antibodies and IL-32-binding fragments thereof, one or more immunoglobulin chains thereof, encoded by the polynucleotides of the invention or obtainable by the above-described methods for the preparation of an anti-IL-32 antibody or one or more immunoglobulin chains thereof. Means and methods for recombinant production of antibodies and mimetics thereof, and methods of screening for competing binding molecules (which may or may not be antibodies) are known in the art. However, as described herein, particularly with respect to therapeutic applications in humans, the antibodies of the invention are human antibodies in the following sense: administration of the antibody is substantially free of immune responses to such antibodies, which are observed for chimeric and even human antibodies.

The binding molecules, antibodies or fragments thereof may be used directly as therapeutic agents. However, in one embodiment, the antibody or antigen-binding fragment provided herein is detectably labeled or attached to a drug, preferably wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a peptide, and a heavy metal. The labeled antibodies or antigen-binding fragments of the invention may be used to detect specific targets in vivo or in vitro, including in vitro "immunochemical/immunolabeling" type assays. In vivo, they can be used to detect tissues, cells or other materials expressing an antigen of interest in a manner similar to nuclear medicine imaging techniques. The labels, their use in diagnosis and their conjugation to the binding molecules of the invention are described in further detail below in the "labels and diagnostics" section.

The antibodies of the invention are isolated from an animal or human suffering from an autoimmune disease. On the other hand, IL-32 specific antibodies identified in the present invention may be involved in severely damaging the immune system of affected individuals, which is associated with symptoms observed, for example, in APECED patients. Thus, another aspect of the present invention is to eliminate or at least reduce the pathological response of a subject suffering from an autoimmune disease by providing means and measures to minimize the number of autoantibodies and/or their effect in the diseased human patient or animal. Thus, in one embodiment, the invention also relates to a peptide or peptide-based compound comprising an epitope specifically recognized by an autoantibody of the invention. As described in further detail below, a similar effect can be obtained by anti-idiotype antibodies as by the use of competing antigens, sequestering and thus preventing the binding of autoantibodies to their respective targets. Thus, in one embodiment, the invention also provides anti-idiotype antibodies to the autoantibodies of the invention.

As already indicated above, the present invention also relates to an anti-idiotype antibody or peptide-based compound of the invention for use in the treatment of a disease as defined above, i.e. a disease associated with the disruption or uncontrolled development of self-tolerance. The isolated antibodies or fragments thereof of the present invention can be used as an immunogene to generate a panel of monoclonal anti-idiotypic antibodies (panel). For suitable methods for generating anti-idiotypic antibodies see Raychadhuri et al, J.Immunol.137(1986),1743, and for T cells, Ertl et al, J.exp.Med.159(1985), 1776. Anti-idiotype antibodies will be characterized based on the expression of an intra-or non-intra-image idiotype (idiotype) using standard assays that are routinely performed in the art, as described in detail in Raychaudhuri et al, J.Immunol.137(1986), 1743. An anti-idiotype antibody is referred to as an "internal image" of an antigen if it structurally mimics the antigen to which it binds or to which it binds.

Described in the art are methods of providing molecules that mimic the idiotype of an autoantibody (autoantibody) associated with an autoimmune disease; see, for example, International application WO03/099868, the disclosure of which is incorporated herein by reference. For example, such a method may comprise the steps of: (a) providing an autoantibody according to the method of the invention; (b) binding the autoantibodies to a solid phase to form an affinity matrix; (c) contacting the pooled plasma or B-cells comprising immunoglobulins with an affinity matrix followed by removal of unbound plasma components; (d) eluting the bound immunoglobulin as an anti-idiotype antibody (anti-Id) of the autoantibody from the matrix; (e) providing a library of molecules comprising a plurality of molecular members; and (e) contacting the anti-Id with a library of molecules and isolating those binding molecules bound by the anti-Id that are molecules that mimic the idiotype of the autoantibody. Methods of isolating idiotypic autoantibodies are disclosed in international application WO2010/136196, the disclosure of which is incorporated herein by reference, describing immunoglobulin preparations comprising native polyclonal IgG-reactive antibodies (abs) isolated from Normal Human Serum (NHS) for use in the treatment of autoimmune diseases and immune system disorders. The IgG-reactive Ab strongly neutralizes disease-associated or pathogenic autoantibodies present in the serum of patients with autoimmune disease by binding to its antigenic determinant located within or near (e.g., overlapping) the antigen-combining site.

The present invention also relates to compositions comprising any of the above-described anti-IL-32 antibodies or IL-32 binding fragments thereof, polynucleotides, vectors, cells, peptides or peptide-based compounds of the invention and/or to a cocktail of anti-IL-32 antibodies or IL-32 binding fragments thereof (cocktail) that, in combination, exhibits the characteristics of the anti-IL-32 antibodies or IL-32 binding fragments thereof of the invention. Additionally or alternatively, in one embodiment, the composition or kit of the invention comprises an anti-idiotypic antibody of the invention. In one embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, routes of administration and dosage regimens can be obtained from the corresponding literature known to those skilled in the art and are also described in more detail in the "pharmaceutical carriers" and "dosage regimens" section below.

In addition, the present invention relates to a method for the manufacture of a composition comprising an anti-IL-32 monoclonal antibody or an IL-32 binding fragment or biotechnological derivative thereof, the manufacture comprising the steps of: the antibody, IL-32 binding fragment thereof or biotechnological derivative is prepared by expression in a recombinant host organism transformed with DNA encoding the antibody, IL-32 binding fragment thereof or biotechnological derivative. In one embodiment, the composition is a pharmaceutical composition, wherein, in the manufacture of the pharmaceutical composition, the antibody, IL-32 binding fragment thereof, or biotechnological derivative is mixed with a pharmaceutically acceptable carrier after the step of preparing the antibody, IL-32 binding fragment thereof, or biotechnological derivative, optionally after one or more steps in between. For example, the antibody or IL-32 binding fragment thereof may be purified from cell culture to pharmaceutical grade and/or derivatized (e.g., pegylated or conjugated to a diagnostic marker or drug) to obtain a pharmaceutical composition prior to formulation of the pharmaceutical composition.

In addition to biochemical and cell-based in vitro assays, the therapeutic efficacy of the antibodies of the invention can be demonstrated in appropriate animal models as described in detail in the examples section below.

In one embodiment, the pharmaceutical composition further comprises an additional agent useful for treating inflammation or autoimmune disease, preferably wherein said agent is selected from the group consisting of a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, an antihistamine, and combinations thereof. Additionally or alternatively, in another embodiment, the pharmaceutical composition further comprises an agent useful for treating an inflammation-related disorder selected from the group consisting of immunosuppressive and anti-inflammatory or "anti-rheumatic" agents.

In another embodiment, the composition is a diagnostic composition or kit and further comprises reagents commonly used in immune or nucleic acid based diagnostic methods.

Furthermore, the present invention provides the aforementioned anti-IL-32 antibodies and IL-32 binding fragments thereof or the composition as defined above for use in a method comprising:

(a) treating or preventing the development of an immune-mediated or autoimmune disease or disorder;

(b) ameliorating a symptom associated with an immune-mediated or autoimmune disease or disorder; and/or

(c) Diagnosing or screening a subject for the presence of an immune-mediated or autoimmune disease or disorder or diagnosing or screening a subject to determine the subject's risk of developing an immune-mediated or autoimmune disease or disorder;

wherein the disease is associated with expression, increased and/or adverse IL-32 activity of IL-32 in the patient.

In this regard, several routes of administration may be used. In one embodiment of the invention, there is provided an antibody mixture as described above, which exhibits the characteristics of the antibodies of the invention when combined with the above-described antibodies or antigen binding fragments, anti-idiotypic antibodies or peptides or peptide-based compounds and/or compounds, all designed for intravenous, intramuscular, subcutaneous, intraperitoneal, intranasal, parenteral or as an aerosol administration.

As mentioned above, due to their binding specificity, the molecules of the invention, such as antibodies and fragments thereof, may preferably be used in the above-defined methods of treating, alleviating, diagnosing and/or screening for immune-mediated or autoimmune diseases or disorders associated with and/or caused by IL-32 expression, elevated and/or adverse IL-32 activity. For example, expressed, elevated and/or adverse IL-32 activity has been observed in Rheumatoid Arthritis (RA) synovial tissue biopsies, where the level of IL-32 expression is positively correlated with the severity of inflammation (Alsaleh et al, (2010), supra; Cagnard et al, (2005), supra). In addition to Rheumatoid Arthritis (RA), IL-32 has been found to be functionally associated with several other diseases, such as ankylosing spondylitis (Ciccia et al, (2012), supra), Inflammatory Bowel Disease (IBD), Myasthenia Gravis (MG), Chronic Obstructive Pulmonary Disease (COPD), asthma, Crohn's disease, psoriasis, vascular inflammation and atherosclerosis (Kobayashi et al, (2010)), atopic dermatitis and cancer (Alsaleh et al, (2010); Breenan and Beech, (2007); Asquith and Mclnnes (2007); Dinarello and Kim, (2006); Fantini et al, (2007); all supra). IL-32 may also play a role in the immune response to tuberculosis (Kundu and Basu, (2006); Neetea et al, (2006); supra). Moreover, increased transcription of IL-32 was observed following infection with bacteria and viruses such as mycobacterium tuberculosis (Netea et al, (2006), supra) or influenza a (Li et al, (2008), supra), suggesting that it may play a role in host defense.

Accordingly, in one embodiment, there is provided an anti-IL-32 antibody or IL-32 binding fragment thereof or composition as defined above for use in the above methods, wherein the disease is an autoimmune disease, preferably selected from the group consisting of Rheumatoid Arthritis (RA), ankylosing spondylitis and other forms of spondyloarthritis including but not limited to psoriatic arthritis (psoriatic arthritis), inflammatory bowel disease (IBD; including Crohn's disease, ulcerative colitis and Celiac disease), psoriasis, Myasthenia Gravis (MG), Chronic Obstructive Pulmonary Disease (COPD), asthma, tuberculosis, vascular inflammation and atherosclerosis, atopic dermatitis, tuberculosis and cancer including leukemia.

Due to the large number of molecules provided herein that are suitable for treating diseases associated with e.g. inflammation, the present invention also relates to methods of treating, diagnosing and/or prognosing the possible course and outcome of such diseases, and uses of the molecules of the invention, preferably wherein an immune-mediated or autoimmune disease or disorder is associated with expression, elevated and/or adverse activity of IL-32. In one embodiment, a method for treating such a disease is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above-described antibody or antigen-binding fragment, antibody cocktail exhibiting the characteristics of the antibodies of the invention in combination, anti-idiotypic antibody or peptide-based compound.

Furthermore, in one embodiment, the invention relates to a method of treating an immune-mediated or autoimmune disease or disorder associated with expression, elevated and/or adverse activity of IL-32 comprising administering to a subject a therapeutically effective dose of a ligand binding molecule comprising:

(i) at least one CDR of an anti-IL-32 antibody or IL-32 binding fragment thereof of the invention; or

(ii) At least one anti-idiotype antibody and/or peptide-based compound as defined above.

Therapeutic approaches based on the use of monoclonal antibodies that are specific for only one epitope of a particular antigen associated with or causing a disease may suffer from several disadvantages. For example, the difficulty of treatment and possible inefficiencies may arise from the diversity of pathogenesis that leads to a particular disease requiring the simultaneous targeting of several antigens. Furthermore, the inherent diversity of the patient population must be taken into account, involving for example polymorphisms, glycosylation heterogeneity or slight denaturation of a given antigen in different patients or in one patient, which may at least cause a reduction in the binding efficiency of the monoclonal antibodies used. Some of these drawbacks can be circumvented by, for example, performing a pretreatment screen to determine whether the antigen is immunologically relevant to the patient intended for treatment and to determine whether there are any epitope changes in a particular patient. However, such screening is often omitted due to treatment urgency or cost limitations. The invention therefore also relates to a method based on the application of more than one type of binding molecule to a patient at a time, i.e. to the application of a mixture of binding molecules. These binding molecules can specifically bind to one IL-32 isoform under different epitopes, each binding molecule applied can specifically bind to another IL-32 isoform, or several binding molecules are used to bind to several epitopes of more than one IL-32 isoform. In case the binding molecules of the invention are directed against (specifically bind to) one IL-32 isoform as antigen, their binding specificities are directed against different epitopes of said antigen. The use of such mixtures is particularly envisaged for the treatment of patients with autoimmune diseases such as APS1 who often cannot be monotherapied with one specific antibody due to the presence of autoantibodies against about 3000 endogenous antigens. In such cases, it is desirable that at least some relief from the symptoms can be achieved using a combination therapy of two or more monoclonal antibodies and/or peptides and peptide-based compounds of the invention having the same or different antigen specificities.

Thus, in one embodiment, there is provided another method of treating a disease, the method comprising administering to a subject a therapeutically effective dose of a mixture consisting essentially of at least two, three, four, five, and more components selected from the group consisting of:

-an antibody or antigen-binding fragment thereof of the invention that specifically binds the IL-32 isoform as defined above; and/or

-an anti-idiotype antibody of the invention and/or a peptide or peptide-based compound of the invention comprising an epitope specifically recognized by an antibody of the invention or an antigen-binding fragment thereof.

The invention naturally also extends to diagnostic and prognostic methods directed to diagnosing immune-mediated or autoimmune disorders and diseases associated with expression, elevated and/or adverse activity of one or more IL-32 isoforms, preferably IL-32 γ, and/or prognosing the development of a disease, i.e. its progression, response to treatment or recovery. Accordingly, in one embodiment, the invention relates to a method of diagnosing an immune-mediated or autoimmune disease or disorder associated with IL-32 expression, elevated and/or adverse activity in a subject, the method comprising contacting a biological sample of the subject with an anti-IL-32 antibody, or IL-32 binding fragment thereof, of the invention, and detecting the presence of IL-32. In a preferred embodiment, the IL-32 isoform detected is IL-32. gamma. Furthermore, in one embodiment, the invention relates to a method for detecting or determining IL-32 in an isolated biological sample, the method comprising: mixing the sample with an anti-IL-32 antibody of the invention, allowing the antibody to form a complex with any IL-32 isotype present in the mixture, and detecting the complex present in the mixture, preferably wherein IL-32 is IL-32 γ.

As already mentioned above, in one embodiment the invention relates to a kit for diagnosing an immune-mediated or autoimmune disease or disorder associated with IL-32 expression, said kit comprising an antibody or antigen-binding fragment, an anti-idiotype antibody or peptide-based compound, polynucleotide, vector or cell as described above, optionally comprising reagents and/or instructions for use. In connection with the kits of the invention, for example, within the container comprising the kit, may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Additionally or alternatively, the kit includes reagents and/or instructions for use in an appropriate diagnostic assay. The compositions, i.e. the kits of the invention, are of course particularly suitable for the diagnosis, prevention and treatment of diseases or disorders associated with the expression of IL-32, in particular for the treatment of diseases as described above. In a particularly preferred embodiment, the disease is associated with the expression of one or more IL-32 isoforms.

In another embodiment, the invention relates to a diagnostic composition comprising any of the binding molecules, antibodies, antigen-binding fragments, peptides or peptide-based compounds, polynucleotides, vectors or cells of the invention as described above, and optionally suitable means for detection, such as reagents customary in immunological or nucleic acid-based diagnostic methods. For example, the antibodies of the invention are suitable for use in immunoassays, wherein the antibodies of the invention can be used in liquid phase or can be bound to a solid support. Examples of immunoassays which can utilize the antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are Radioimmunoassays (RIA), sandwich (sandwich) (immunoassays), flow cytometry and western blot assays. The antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells to which they specifically bind. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier is soluble or insoluble. There are many different labels and labeling methods known to those skilled in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed above.

In this case, the invention also relates to a tool specifically designed for this purpose. For example, protein or antibody based arrays, e.g., loaded with any one of the antigens derived from one or more IL-32 isoforms and comprising disease-associated antigens, may be used to detect autoantibodies that may be present in patients with autoimmune diseases (particularly RA, IBD or APECED/APS1), or with antibodies or equivalent antigen binding molecules of the invention that specifically recognize any of those inflammation-associated antigens. The design of microarray immunoassays is outlined in Kusnezow et al, mol. cell Proteomics 5(2006), 1681-1696. The invention therefore also relates to a microarray loaded with binding molecules or antigens recognized according to the invention.

Definitions and embodiments

Unless otherwise indicated, the terms and embodiments used herein are given the definitions as provided and used in international applications WO2013/098419a1 and WO2013/098420 a 1. Additionally, the general terms used herein are given the definitions as provided in revision 2000 and reissue 2003, International Standard Book Number (ISBN) 0198506732, Oxford dictionary Biochemistry and Molecular Biology, Oxford university press, 1997.

It is noted that the terms "a" or "an" entity refer to one or more, or one or more, of the entities; for example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The terms "neutralizing" and "neutralizing antibody" are used as is common in the art, respectively, i.e., such an antibody refers to a reduction or elimination of at least some biological activity of an antigen or living microorganism. For example, the invention of the isotype-specific anti IL-32 antibody is a neutralizing antibody, such as in the example described in the determination of the amount, if in sufficient amount, the antibody eliminates or reduces one or more corresponding IL-32 isotype activity. Neutralization is generally defined by the 50% inhibitory concentration (IC50), and it can be statistically assessed based on the area under the neutralization titration curve (AUC). IC50 values for exemplary anti-IL-32 antibodies of the invention are described and shown herein, e.g., exemplary antibody 2C2 has an IL-32 γ IC50 value of 300 ng/ml.

Central and peripheral tolerance

Self-tolerance is the process by which the immune system does not respond to antigens of components of the body. Self-tolerance is accomplished by the death or inactivation of autoreactive T and B cells, which can occur as part of central tolerance in central (reproductive) immune organs (thymus or bone marrow) or as peripheral tolerance in tissues most often considered secondary immunity (e.g., spleen, lymph nodes, intestine). Self-tolerance is an important feature of the normal immune system. Failure to establish and/or maintain self-tolerance results in autoimmunity that can cause autoimmune diseases with serious health effects on the host organism.

T cells and B cells can develop central tolerance to those antigens present in the reproductive immune organs. In bone marrow, B cells develop tolerance to widely expressed bone marrow-specific antigens and antigens introduced through the blood circulation. In the thymus, thymic medullary epithelial cells can express hundreds of autoantigens that are presented to developing T cells. The gene responsible for the widespread expression of autoantigens in thymic medullary epithelial cells is AIRE (autoimmune regulator). AIRE activates a variety of tissue-specific genes that are normally expressed only in specific peripheral organs (e.g., insulin in pancreatic islets). In the absence of a functional AIRE gene, no antigen is presented, T cells are not inactivated, and autoimmunity to self-antigens develops, leading to pathological phenomena in APECED patients and AIRE-deficient mice.

Another important gene in tolerance induction is foxp 3. This gene encodes a transcription factor that regulates fate inducing immunosuppression in T lymphocytes that attack (engage) self-antigens in the thymus and possibly also in the periphery. The inability to encode a functional FOXP3 protein is a characteristic of IPEX patients, who therefore also have a generalized autoimmune disease.

The central and peripheral tolerance are described in more detail in the corresponding sections on pages 62-63 of international application WO2013/098419a1, the disclosure of which is incorporated herein by reference.

Peptides and polypeptides:

the term "peptide" is understood to include the terms "polypeptide" and "protein" (which may sometimes be used interchangeably herein) and any amino acid sequence within the meaning of the present invention, such as those of the heavy and light chain variable and constant regions of the invention. Likewise, fragments of proteins and polypeptides are also contemplated, which may be referred to herein as "peptides". However, the term "peptide" preferably denotes an amino acid polymer comprising at least 5 consecutive amino acids, preferably at least 10 consecutive amino acids, more preferably at least 15 consecutive amino acids, even more preferably at least 20 consecutive amino acids and especially preferably at least 25 consecutive amino acids. In addition, the peptides according to the invention typically have no more than 100 consecutive amino acids, preferably less than 80 consecutive amino acids and more preferably less than 50 consecutive amino acids.

As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide", such as the antibodies of the present invention, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a product of a particular length. Thus, "peptide," "dipeptide," "tripeptide," "oligopeptide," "protein," "amino acid chain," or other terms used to refer to one or more chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be substituted or used interchangeably with any of these terms.

The term "polypeptide" also means the product of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or may be produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. The polypeptide may be produced by any means, including by chemical synthesis.

The polypeptides of the invention may have a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. However, the term "polypeptide" preferably denotes an amino acid polymer comprising at least 100 amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, whereas polypeptides having no defined three-dimensional structure can adopt a large number of different conformations and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety attached to the protein by an oxygen-containing or nitrogen-containing side chain of an amino acid residue (e.g., a serine residue or an asparagine residue).

An "isolated" polypeptide or fragment, variant, or derivative thereof means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide may be removed from its native or native environment. For the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as native or recombinant polypeptides that have been isolated, fractionated or partially or substantially purified by any suitable technique.

"recombinant peptide, polypeptide or protein" refers to a peptide, polypeptide or protein produced by recombinant DNA techniques (i.e., produced by a cell, microorganism or mammal transformed with an exogenous recombinant DNA expression construct encoding a fusion protein that includes the desired peptide). Proteins or peptides expressed in most bacterial cultures are generally devoid of polysaccharides. The protein or polypeptide expressed in yeast may have a glycosylation pattern that is different from the glycosylation pattern expressed in mammalian cells.

Also included as polypeptides of the invention are fragments, derivatives, analogs and variants of the above polypeptides and any combinations thereof. The terms "fragment," "variant," "derivative," and "analog" include peptides and polypeptides having an amino acid sequence that is substantially similar to the amino acid sequence of a native peptide. The term "sufficiently similar" means that a first amino acid sequence comprises a sufficient or minimal number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common domain and/or common functional activity. For example, amino acid sequences comprising a common domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical are defined herein as substantially similar. Preferably, the variant will be substantially similar to the amino acid sequence of a preferred peptide of the invention, in particular to an antibody or antibody fragment, or to a synthetic peptide or peptide-based compound comprising an epitope recognized by an antibody of the invention or a fragment, variant, derivative or analogue of any of them. Such variants typically retain the functional activity of the peptides of the invention, i.e., are bound by the antibodies of the invention. Variants include peptides that differ in amino acid sequence from the native and wt peptides, respectively, by one or more amino acid deletions, additions and/or substitutions. They may be naturally occurring variants or artificially designed variants.

When referring to an antibody or antibody polypeptide of the invention, the terms "fragment," "variant," "derivative," and "analog" include any polypeptide that retains at least some of the antigen-binding properties of the corresponding native binding molecule, antibody, or polypeptide. Fragments of the polypeptides of the invention include proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein. Variants of the antibodies and antibody polypeptides of the invention include fragments as described above, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced by using mutagenesis techniques known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of the binding molecules of the invention, such as the antibodies and antibody polypeptides of the invention, are polypeptides that have been altered to exhibit additional characteristics not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a binding molecule or fragment thereof, an antibody, or a "derivative" of an antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may replace proline; 5-hydroxy lysine can be substituted for lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine may replace lysine.

Anti-idiotypic antibodies：

When referring to antibodies or other binding molecules, the term "anti-idiotype antibody" includes molecules that bind to a unique antigenic peptide sequence on the variable region of an antibody at or near the antigen binding site, thereby suppressing a particular immune response that would otherwise be elicited by a given autoantibody. In a similar manner, synthetic peptides or peptide-based compounds comprising an epitope specifically recognized by an antibody of the invention may be used.

Anti-idiotype antibodies can be obtained in a similar manner to other antibodies. The detection of a specific anti-idiotype antibody is performed by any type of cross-linking, either by agglutination (in nephelometry or turbidimetry), precipitation (radioimmunodiffusion) or sandwich immunoassays, such as ELISA. U.S. patent application No. 20020142356 provides a method for obtaining a population of anti-idiotypic monoclonal antibodies directed against a high concentration of high molecular weight antibodies specific for a target antigen, wherein the population of anti-idiotypic monoclonal antibodies has a wide range of binding affinities for selected antibodies specific for the target antigen, and wherein a subset of the population of anti-idiotypic antibodies can be selected that have the desired affinity for a particular application.

Us patent application No. 20020142356 describes a competitive immunoassay for antigens using antibodies as coating (coat) and anti-idiotype antibodies as detection, or vice versa. Other references disclosing the use of anti-idiotypic antibodies as alternative antigens include Losman et al, Cancer Research,55(1995) (23 support.S): S5978-S5982; becker et al, J.of Immunol. methods 192(1996), 73-85; baral et al, International j. of cancer,92(2001), 88-95; and Kohen et al, Food and Agriculture Immunology,12(2000), 193-. The use of anti-idiotype antibodies in the treatment of disease or against parasites is known in the art; see, e.g., Sacks et al, J.Exper. medicine,155(1982), 1108-.

Determination of similarity and/or identity of molecules：

The "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide with the sequence of a second peptide. If identical or conservative amino acid substitutions are present, the amino acid of one peptide is similar to the corresponding amino acid of a second peptide. Conservative substitutions include those described in Dayhoff, M.O. eds, The Atlas of Protein sequences and Structure5, National biological Research Foundation, Washington, D.C. (1978), and Argos, EMBO J.8(1989), 779-. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, gin, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and-Asp, Glu.

Preferably, the percent identity or similarity between two sequences is accomplished using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90:5873-Property of (2)And (4) determining. At NCBI: (http:// www.ncbi.nlm.nih.gov/blast/Blast.cge) Such algorithms are incorporated in the BLASTn and BLASTp programs of Altschul et al (1990) J.mol.biol.215: 403-410.

Percent identity or similarity is performed using the standard parameters of the BLASTn and BLASTp programsProperty of (2)And (4) determining.

BLAST polynucleotide search was performed using the BLASTn program.

With respect to the conventional parameters, the "maximum target sequence" box may be set to 100, the "short query string" box may be checked, the "expected threshold" box may be set to 10, and the "font size" box may be set to 28. Regarding the scoring parameters, the "match/mismatch score" can be set to 1, -2, and the "gap penalty (cost)" box can be set to linear. With respect to the filter and masking parameters, the "low complexity region" box may not be checked, the "species specific repeat sequence" box may not be checked, the "masking for look-up table only" box may be checked, and the "masking lower case" box may not be checked.

BLAST protein search was performed using the BLASTp program. With respect to the conventional parameters, the "maximum target sequence" box may be set to 100, the "short query string" box may be outlined, the "expected threshold" box may be set to 10, and the "font size" box may be set to "3". With respect to the scoring parameters, the "matrix" box may be set to "BLOSUM 62", and the "gap penalty" box may be set to "Presence: 11, extension: 1 ", the" composition adjustment "box may be set to" conditional composition score matrix adjustment ". With respect to the filter and masking parameters, the "low complexity region" box may not be checked, the "mask for look-up table only" box may not be checked, and the "mask lower case" box may not be checked.

A polynucleotide:

the term "polynucleotide" is intended to encompass both the singular and the plural, and refers to an isolated nucleic acid molecule or construct, for example, messenger rna (mrna) or plasmid dna (pdna). Polynucleotides may include conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide means a nucleic acid molecule, DNA or RNA that has been removed from its native environment. For example, for the purposes of the present invention, a recombinant polynucleotide encoding an antibody contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the invention. Isolated polynucleotides or nucleic acids according to the invention also include those molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or may include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region, but any flanking sequence (e.g., promoter, ribosome binding site, transcription terminator, intron, etc.) is not part of the coding region. The two or more coding regions of the invention may be present in a single (single ) polynucleotide construct, e.g. on a single vector, or may be present in separate polynucleotide constructs, e.g. on separate (different) vectors. In addition, any vector may contain a single coding region, or may include two or more coding regions, e.g., a single vector may encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, respectively. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, fused or unfused to nucleic acids encoding binding molecules, antibodies or fragments, variants or derivatives thereof. Heterologous coding regions include, but are not limited to, specialized elements (specialized elements) or motifs (motifs), such as secretion signal peptides or heterologous functional domains.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide may typically include a promoter and/or other transcriptional or translational control elements operatively associated with one or more coding regions. The operable association is: in this case, the coding region for the gene product, e.g., a polypeptide, is associated with one or more regulatory sequences, which direct the expression of the gene product under the influence or control of the one or more regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" if the promoter function is induced to cause transcription of an mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or with the ability of the DNA template to be transcribed. Thus, a promoter region may be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of affecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. Other transcriptional control elements besides promoters, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.

Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (immediate early promoter, with intron-a attached), simian virus 40 (early promoter), and retroviruses (e.g., rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbit β -globulin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, various translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses, particularly internal ribosome entry sites or IRES, also known as CITE sequences.

In other embodiments, the polynucleotide of the invention is RNA, for example, in the form of messenger RNA (mrna), small hairpin RNA (shrna), small interfering RNA (sirna), or any other RNA product.

The polynucleotide and nucleic acid coding regions of the invention may be associated with other coding regions encoding secretion or signal peptides, which direct the secretion of the polypeptide encoded by the polynucleotide of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the growing protein chain has begun to be excreted across the rough endoplasmic reticulum. One of ordinary skill in the art knows that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the intact or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of a sequence that retains the ability to direct secretion of a polypeptide with which it is operatively associated is used. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase. However, it is also possible to produce the polypeptides of the invention, in particular immunoglobulins and fragments thereof, intracellularly.

Expression of：

The term "expression" as used herein refers to a process by which a gene produces a biochemical, such as an RNA or polypeptide. The process includes any manifestation of the functional presence of genes within the cell, including but not limited to gene knockouts as well as transient and stable expression. Expression includes, but is not limited to: transcribing the gene into messenger RNA (mrna), transfer RNA (trna), small hairpin RNA (shrna), small interfering RNA (sirna), or any other RNA product; and translating such mRNA into one or more polypeptides. If the final desired product is a biochemical, expression includes generating the biochemical and any precursors. Expression of a gene results in a "gene product". As used herein, a gene product can be a nucleic acid (e.g., a small interfering RNA (sirna), messenger RNA, produced by transcription of a gene) or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids that are post-transcriptionally modified (e.g., polyadenylation), or polypeptides that are post-translationally modified (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.).

Various expression vector/host systems can be utilized to contain and express the polynucleotide sequences. These expression vector/host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage, plasmid or cosmid DNA expression vectors; yeast transformed with a yeast expression vector; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

For expression of a peptide, polypeptide or fusion protein (hereinafter referred to as "product") in a host cell, a procedure such as that described below can be used. The restriction fragment containing the DNA sequence encoding the product may be cloned into a suitable recombinant plasmid containing an origin of replication acting in the host cell and a suitable selectable marker. Plasmids may include promoters for inducible expression of the product (e.g., pTrc (Amann et al, Gene 69(1988),301-315) and pETl Id (Studier et al, Gene expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), 60-89)). Recombinant plasmids can be introduced into host cells by, for example, electroporation, and cells containing the recombinant plasmid can be identified by selecting for markers on the plasmid. Assays specific for the product can be used to induce and detect expression of the product in the host cell.

In some embodiments, the DNA encoding the product/peptide may be optimized for expression in a host cell. For example, the DNA may include codons for one or more amino acids that are predominant in the host cell relative to other codons for the same amino acid.

Alternatively, expression of the product may be performed by in vitro synthesis of the protein in a cell-free extract which is also particularly suitable for incorporation of modified or non-natural amino acids for functional studies, see also below. When the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein passesIntracellular proteases undergo rapid protein degradation, and the use of in vitro translation systems is advantageous over in vivo gene expression. The most commonly used cell-free translation system consists of extracts from rabbit reticulocytes, wheat germ and E.coli. The total extract was prepared as a crude extract containing all macromolecular components (70S or 80S ribosomes, tRNA' S, aminoacyltRNA synthetases, initiation, elongation and termination factors, etc.) required for heterologous RNA translation. To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regeneration systems (phosphocreatine and creatine phosphokinase for eukaryotic systems, and phosphoenolpyruvate and pyruvate kinase for e.coli lysates), and other cofactors known in the art (Mg)^{2 +}，K⁺Etc.). Suitable transcription/translation Systems are commercially available, for example from Promega, Roche Diagnostics, and Ambion, Applied Biosystems (Anderson, C. et al, meth.Enzymol.101(1983),635 & 644; Arduengo, M. et al (2007), The Role of Cell-Free Rabbitexpression Systems in Functional Proteomics in Kudlick, Katzen and Bennett eds., Cell-Free Expression Vol.2007.Austin, Tx: Landes biosciences, 2006.1-18; Chen & Zubay, meth.Enzymol.101(1983), 674-90; Ere et al, Biotechnol.prog.22 (1570), 1570).

Host cell：

With respect to the present invention, the host cell may be any prokaryotic or eukaryotic cell, such as a bacterial cell, an insect cell, a fungal cell, a plant cell, an animal cell or a human cell. Preferred fungal cells are, for example, those of the genus saccharomyces, in particular those of the species saccharomyces cerevisiae (s. The term "prokaryotic" is intended to include all bacteria that can be transformed or transfected with a DNA or RNA molecule for expressing an antibody or corresponding immunoglobulin chain of the present invention. Prokaryotic hosts may include gram-negative and gram-positive bacteria such as, for example, escherichia coli, salmonella typhimurium (s.typhimurium), Serratia marcescens (Serratia marcocens), and bacillus subtilis. The term "eukaryotic" is intended to include yeast cells, higher plant cells, insect cells and preferably mammalian cells, most preferably HEK293 cells, NSO cells, CSO cells and CHO cells. Depending on the host used in the recombinant production process, the antibody or immunoglobulin chain encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated. The antibodies of the invention or corresponding immunoglobulin chains may also include an initial methionine amino acid residue. Any technique commonly known to those of ordinary skill in the art can be used to transform or transfect a host with a polynucleotide of the present invention. In addition, methods for preparing fused, operably linked genes and expressing them in, for example, mammalian cells and bacteria are known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructs and methods described therein can be used to express the antibodies of the invention or the corresponding immunoglobulin chains in eukaryotic or prokaryotic hosts. Typically, expression vectors containing promoter sequences that promote efficient transcription of the inserted polynucleotide are used in conjunction with the host. Expression vectors typically contain an origin of replication, a promoter and a terminator, as well as specific genes that provide phenotypic selection of transformed cells. Suitable source cells for DNA sequences and host cells for immunoglobulin expression and secretion ("Catalogue of Cell Lines and hybrids," fine edition (1985) Rockville, Maryland, u.s.a., which is incorporated herein by reference) may be obtained from a variety of sources, such as the American Type Culture Collection (American Type Culture Collection). Furthermore, transgenic animals (preferably mammals) comprising cells of the invention can be used for large scale production of antibodies of the invention.

The transformed host may be grown in a fermentor and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the whole antibodies (the whole antibodies), dimers, individual light and heavy chains thereof, or other immunoglobulin forms of the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like; see Scopes, "Protein Purification", Springer Verlag, n.y. (1982). The antibody of the invention, or its corresponding immunoglobulin chain(s), can then be isolated from the growth medium, cell lysate, or cell membrane fraction (fraction). For example, the isolation and purification of the recombinantly expressed antibodies or immunoglobulin chains of the invention can be any conventional method, such as preparative chromatographic separation and immunological separation, such as, for example, those separations comprising the use of monoclonal or polyclonal antibodies directed against, for example, the constant regions of the antibodies of the invention. It will be apparent to those skilled in the art that the antibodies of the invention may be further conjugated to other moieties for use in, for example, drug targeting and imaging applications. Expression of the antibody or antigen may be followed by such conjugation to the attachment site chemically, or the conjugation product may be engineered at the DNA level to the antibody or antigen of the invention. The DNA is then expressed in a suitable host system and, if necessary, the expressed protein is collected and renatured.

For pharmaceutical use, substantially pure immunoglobulins having at least about 90% to 95% homology are preferred, and 98% to 99% or more homology are most preferred. Once purified or partially purified to the desired homology, the antibodies can be used therapeutically (including in vitro) or to develop and perform assay procedures.

The invention also relates to a method for producing a cell capable of expressing an antibody of the invention or one or more immunoglobulin chains corresponding thereto, the method generally comprising genetically engineering the cell with a polynucleotide or vector of the invention. The cells obtainable by the method of the invention can be used, for example, to test the interaction of an antibody of the invention with its antigen.

ELISA-determination:

enzyme-linked immunosorbent assays (ELISA) for various antigens include those based on colorimetric, chemiluminescent and fluorimetric assays. ELISA has been successfully applied to determine low amounts of drugs and other antigenic components in plasma and urine samples, does not include an extraction step and is easy to perform. ELISAs for detecting antibodies against (anti, to) protein antigens often utilize direct binding of short synthetic peptides to the plastic surface of microtiter plates. Due to the synthetic nature of the peptide and the efficient purification method using high performance liquid chromatography, the peptide is generally very pure. Short peptides have the disadvantage that they generally represent linear epitopes, not conformational or discontinuous epitopes. To present conformational epitopes, long peptides or intact native proteins are used. Direct binding of protein antigens to the hydrophobic polystyrene carrier (support) of the plate can cause partial or complete denaturation of the bound protein and loss of conformational epitopes. This effect can be avoided by coating the plate with antibodies that mediate the immobilization of the antigen (capture ELISA).

However, overexpressed recombinant proteins are often insoluble and require purification and renaturation under denaturing conditions when antibodies to conformational epitopes are to be analyzed. See, for example, U.S. patent application No. 20030044870 for a general ELISA using a recombinant fusion protein as a coat protein.

Binding molecules：

"binding molecules" as used in the context of the present invention mainly relates to antibodies and fragments thereof, but may also refer to other non-antibody molecules that bind to the "molecule of interest" of the present invention, wherein the molecule of interest is a protein of the glycoprotein class known as cytokines, in particular an interleukin selected from the group of different IL-32 isoforms. In a particularly preferred embodiment, the molecule of interest is IL-32 γ. The molecules of interest of the present invention are defined in further detail in the description of the specific embodiments above and below of the invention. Binding molecules of the invention include, but are not limited to, hormones, receptors, ligands, Major Histocompatibility Complex (MHC) molecules, chaperones such as Heat Shock Proteins (HSPs), and cell-cell adhesion molecules such as members of the cadherins, integrins, C-type lectins, and immunoglobulin (Ig) superfamily. Thus, for clarity only, and without limiting the scope of the invention, most of the following embodiments are discussed with respect to antibodies and antibody-like molecules that represent preferred binding molecules for the development of therapeutic and diagnostic agents.

Antibody:

the terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin is a molecule that binds to a molecule of interest of the invention as defined above and below, which molecule comprises at least the variable domain of a heavy chain, and typically at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are well understood; see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual (Cold spring harbor Laboratory Press,2nd ed.1988). The terms "binding" and "recognition" are used interchangeably with respect to the binding affinity of a binding molecule, e.g., an antibody, of the invention.

As defined above and below, any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to a molecule of interest is interchangeably referred to herein as a "binding molecule", "binding fragment", or "immunospecific fragment".

Antibodies of the invention or antigen binding fragments, immunospecific fragments, variants or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, murinized or chimeric antibodies, single chain antibodies, epitope binding fragments, e.g., Fab ', and F (ab')₂Fd, Fvs, single-chain Fvs (scFv), single-chain antibody, disulfide-linked Fvs (sdFv), antibody comprising V_LOr V_HFragments of the domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the antibodies disclosed herein). scFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5892019. In this regard, the antigen-binding fragment of an antibody can also be a domain antibody (dAB), also known as a single domain antibody (sdAB) or a nanobody^TM(nanobodies^TM) (Ablynx, Inc., Gente, Belgium), see, e.g., De Haard et al, J.Bacteriol.187(2005), 4531-; holt et al, Trends Biotechnol.21(2003), 484-490. As will be discussed in further detail below, the term "immunoglobulin" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon) and whereinSome subclasses (e.g., γ 1 to γ 4). It is the nature of the chain that determines the "class" of an antibody as IgG, IgE, IgM, IgD, IgA and IgY, respectively. The immunoglobulin subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional specialization. The immunoglobulin or antibody molecules of the invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.) or subclass of immunoglobulin molecule. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art in view of this disclosure and, thus, fall within the scope of the present invention. While all immunoglobulin classes are clearly within the scope of the present invention, the following discussion is generally directed to the IgG class of the immunoglobulin molecule. With respect to IgG, standard immunoglobulin molecules include two identical light chain polypeptides having a molecular weight of about 23000 daltons and two identical heavy chain polypeptides having a molecular weight of 53000-70000. The four chains are typically connected by disulfide bonds in the "Y" configuration, where the light chain bracketing (blacket) begins at the mouth of the "Y" and continues through the heavy chain of the variable region.

As is evident from the classification of the exemplary anti-IL-32 antibodies of the invention listed in table 1 above, the exemplary antibodies of the invention are of the IgG3 or IgG1 class, and may be involved in the regulatory T cell response and/or epithelial cell priming in these AIRE deficient states. By

Et al, in clin. exp. immunol. (2012); these findings were confirmed by the classification of the corresponding autoantibodies found in AIRE-deficient mice as described in doi:10.1111/cei.12024, the disclosure of which is incorporated herein by reference. Thus, in a preferred embodiment of the invention, the antibody of the invention is of the IgG type, even more preferably IgG3 or IgG 1.

IgG structure:

light chains are classified as either kappa or lambda (kappa, lambda). Each heavy chain species may be associated with a kappa or lambda light chain. In general, the light and heavy chains are covalently bound to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence continues from the N-terminus at the fork end of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the light chain (V)_L) Part and heavy chain (V)_H) The variable domains of the moieties determine the recognition and specificity of the antigen. In contrast, the constant domains of the light Chain (CL) and heavy chains (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they are further away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually include the carboxy-termini of the heavy and light chains, respectively.

As described above, the variable regions allow the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, V of the antibody_LDomains and V_HThe domains or subsets of Complementarity Determining Regions (CDRs) combine to form variable regions that define a three-dimensional antigen binding site. The tetrabasic antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, by V_HAnd V_LThree CDRs on each of the chains define an antigen binding site. Any antibody or immunoglobulin fragment that contains sufficient structure to specifically bind to a molecule of interest of the present invention is interchangeably referred to herein as a "binding fragment" or an "immunospecific fragment".

In naturally occurring antibodies, the antibody includes six hypervariable regions, sometimes referred to as "complementarity determining regions" or "CDRs," present in each antigen-binding domain, which are short, non-contiguous sequences of amino acids specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional conformation in a liquid (aqueous) environment. The "CDRs" are flanked by four relatively conserved "framework" regions or "FRs" that exhibit little intermolecular variability. The framework regions predominantly adopt a β -sheet conformation, and the CDRs form loops that connect, or in some cases form part of, the β -sheet conformation. Thus, the framework regions serve to form a scaffold that positions the CDRs in the correct orientation by interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. The complementary surface facilitates non-covalent binding of the antibody to its cognate epitope(s). For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify amino acids that include the CDR and framework regions, respectively, because they have been precisely defined; see "Sequences of Proteins of Immunological Interest," Kabat, E.et al, U.S. department of Health and Human Services, (1983); and Chothia and Lesk, J.mol.biol.196(1987),901-917, the entire contents of which are incorporated herein by reference.

Where there are two or more definitions of a term used and/or accepted in the art, the definition of the term as used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, "Sequences of proteins of Immunological Interest" (1983) and Chothia and Lesk, J.mol.biol.196(1987),901-917, which are incorporated herein by reference, wherein the definition includes overlapping or subsets of amino acid residues when compared to each other. However, the use of either definition to refer to the CDRs of an antibody or variant thereof is intended to fall within the scope of the terms as defined and used herein. Suitable amino acid residues comprising the CDRs defined by each of the above-cited references are listed in table 2 below for comparison. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular hypervariable region or CDR of the human IgG subtype of an antibody given the amino acid sequence of the variable region of that antibody.

Table 2: CDR definition¹

	Kabat	Chothia
			VH CDR1	31-35	26-32
VH CDR2	50-65	52-58
			VH CDR3	95-102	95-102
VL CDR1	24-34	26-32
			VL CDR2	50-56	50-52
VL CDR3	89-97	91-96

¹The numbering of all CDR definitions in Table 2 is according to the numbering convention set forth by Kabat et al (see below).

Kabat et al also define a numbering system for variable domain sequences that can be applied to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al in U.S. Dept. of Health and Human Services, "Sequence of Proteins of immunological Interest" (1983). Unless otherwise indicated, reference to numbering of particular amino acid residue positions in an antibody of the invention, or antigen-binding fragment, variant or derivative thereof, is according to the Kabat numbering system, however the Kabat numbering system is theoretical and may not apply equally to each antibody of the invention. For example, depending on the position of the first CDR, the following CDR may move in either direction.

In one embodiment, the antibody of the invention is not an IgM or a derivative thereof having a pentavalent structure. In particular, IgM is less useful than IgG and other bivalent antibodies or corresponding binding molecules in particular applications of the present invention, particularly in therapeutic applications, because IgM often exhibits non-specific cross-reactivity and very low affinity due to its pentavalent structure and insufficient affinity maturation.

In a particularly preferred embodiment, the antibody of the invention is not a polyclonal antibody, i.e. it essentially consists of one specific antibody species, rather than a mixture obtained from a plasma immunoglobulin sample.

Antibody fragment, animalization:

antibody fragments, including single chain antibodies, may include one or more variable regions, alone or in combination with all or a portion of: a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain. The invention also includes fragments that bind to the molecules of interest of the invention, the fragments comprising any combination of one or more variable regions and the hinge, CH1, CH2 and CH3 domains. Antibodies of the invention or immunospecific fragments thereof identical to the monoclonal antibodies isolated according to the methods of the invention, particularly to the human monoclonal antibodies, may be from any animal source including birds and mammals. Preferably, the antibody is a human antibody, a murine antibody, a donkey antibody, a rabbit antibody, a goat antibody, a guinea pig antibody, a camel antibody, a llama (llama) antibody, an equine antibody or a chicken antibody. In another embodiment, the variable region may be of cartilaginous fish (condricthoid) in the source (e.g. from sharks).

In a particularly preferred embodiment of the invention, the antibody is a naturally occurring human monoclonal antibody, or binding fragment, derivative and variant thereof, cloned from a human subject, which specifically binds to a specific IL-32 isotype of the invention, preferably IL-32 γ, as defined in detail above and below, e.g., in table 1, the figures (in particular fig. 1 to 4) and the examples (e.g., in examples 2 and 6).

Optionally aligning and employing the framework regions of the human antibodies according to the relevant human germline variable region sequences in the database; see, e.g., Vbase (http:// Vbase. MRC-cpe. cam. ac. uk /) hosted by the MRC protein engineering center (Cambridge university, UK). For example, amino acids that are considered likely to be derived from a true germline sequence deviation may be due to PCR primer sequences introduced during cloning. In contrast to artificially generated human-like antibodies, such as single chain antibody fragments (scfvs) from phage display antibody libraries or heterologous mice, the human monoclonal antibodies of the invention are characterized by: (i) obtained using a human immune response rather than an animal surrogate, i.e., the production of antibodies in humans in response to the native IL-32 isotype in the relevant conformation; (ii) protecting an individual from, or at least significantly minimizing, the presence of symptoms of a disease, such as SLE; and (iii) because the antibody is of human origin, the risk of cross-reactivity to self-antigens is minimized. Thus, according to the present invention, the terms "human monoclonal antibody", "human monoclonal autoantibody", "human antibody" and the like are used to denote an IL-32 binding molecule of human origin having the specified isotype specificity for IL-32, i.e., the binding molecule has been isolated from a human cell (such as a B cell) or a hybridoma cell thereof, or the cDNA of the binding molecule has been cloned directly from the mRNA of a human cell (e.g., a human memory B cell). Human antibodies are still considered "human", even if amino acid substitutions are made in the antibody, for example to improve its binding properties.

Antibodies derived from a human immunoglobulin repertoire or from animals transgenic for one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described below and, for example, by Kucherlapati et al in U.S. patent No. 5939598, represent human-like antibodies to distinguish them from truly human antibodies of the invention.

For example, the pairing of the heavy and light chains of a human-like antibody (such as synthetic and semi-synthetic antibodies typically isolated from phage display) does not necessarily reflect the original pairing as it occurs in the original human B cell. Thus, Fab and scFv fragments obtained from recombinant expression libraries commonly used in the art can be considered artificial, with all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides affinity matured antibodies isolated from selected human subjects that are characterized by their therapeutic efficacy.

Grafted antibodies (equivalents)

The present invention also relates to grafted antibodies (interchangeably referred to as equivalents) containing CDRs respectively derived from an antibody of the invention, such as an IL-32 antibody. Such grafted CDRs include humanized antibodies in which CDRs from an antibody of the present invention have been grafted or in which CDRs containing one or more amino acid substitutions have been grafted. The CDRs can be grafted directly into the human framework as described above or into the framework of antibodies from animal sources. Framework changes can also be incorporated by generating a framework library, if desired. As described in more detail below, optimization of the CDR and/or framework sequences may be performed separately and combined in sequence, or may be performed simultaneously.

To generate a grafted antibody, the donor CDRs of an antibody of the invention are grafted onto an antibody acceptor variable region framework. Methods for grafting antibodies and generating CDR variants to optimize activity have been described previously (see, e.g., International patent applications WO 98/33919; WO 00/78815; WO 01/27160). This procedure can be performed to achieve grafting and affinity reacquisition of the donor CDR simultaneously. Likewise, the method can be used alone or in combination with CDR grafting to improve or optimize the binding affinity of the variable region. The method for conferring donor CDR binding affinity onto an acceptor variable region is applicable to both heavy and light chain variable regions, and thus can be used to simultaneously graft and optimize the binding affinity of antibody variable regions.

The donor CDR can be altered to comprise a plurality of different amino acid residue changes at all or selected positions within the donor CDR. For example, a random or biased (biased) pool or preselected subset of twenty naturally occurring amino acid residues can be introduced into a donor CDR to produce a different population of CDR species. Inclusion of CDR variant species into different populations of variable regions allows for generation of variant species that exhibit optimized binding affinity for a predetermined antigen. A range of possible changes can be made at the donor CDR positions. Some or all of the above possible changes that can be selected for alteration can be introduced into the transplant donor CDR population. Individual positions in the CDRs can be selected to introduce changes, or various positions with altered amino acids can be combined and screened for activity.

One approach is to change all amino acid positions along the CDR by replacing at each position with, for example, all twenty naturally occurring amino acids. Substitutions at each position can occur in the context of other donor CDR amino acid positions, such that a significant portion of the CDRs retain the true donor CDR sequences and thus the binding affinity of the donor CDRs. For example, an acceptor variable region framework, whether native or altered, can be grafted with a population of CDRs that contain a single positional substitution at each position within the CDR. Likewise, the acceptor variable region framework can be targeted for grafting with a population of CDRs comprising more than one changed position to incorporate all 20 amino acid residues or amino acid subsets. One or more amino acid positions within a CDR or set of CDRs to be grafted can be altered and grafted into the recipient variable region framework to generate a population of grafted antibodies. It is to be understood that a CDR having one or more relocation positions may be combined with one or more other CDRs having one or more relocation positions, if desired.

A population of CDR variant species with one or more altered positions can be combined with any or all of the CDRs that make up the binding pocket (binding pocket) of the variable region. Thus, an acceptor variable region framework can be targeted for simultaneous incorporation of a donor CDR variant population at one, two, or all three acceptor CDR positions in either the heavy or light chain. The choice of which CDR or the number of CDRs to target with an amino acid position change will depend on, for example, whether the entire CDR needs to be grafted into a recipient, or whether the method is performed for the purpose of optimizing affinity.

Another method for selecting donor CDR amino acids to be altered for use in conferring donor CDR binding affinity onto an antibody acceptor variable region framework is to select highly variable known or readily identifiable CDR positions. For example, the variable region CDR3 is typically highly variable. Thus, this region can be selectively targeted for amino acid position changes in transplantation procedures to ensure binding affinity reacquisition or augmentation, either alone or in conjunction with relevant receptor variable framework changes.

Murine antibody:

an example of an antibody produced by transplantation as described above is a murine antibody. As used herein, the term "murine antibody" or "murine immunoglobulin" refers to an antibody that includes one or more CDRs from a human antibody of the invention; and a human framework region comprising amino acid substitutions and/or deletions and/or insertions based on the mouse antibody sequence. The human immunoglobulin providing the CDRs is referred to as the "parent" or "acceptor", while the mouse antibody providing the framework alterations is referred to as the "donor". The constant region need not be present, but if present, is typically substantially identical to a mouse antibody constant region, i.e., at least about 85-90%, preferably about 95% or more identical. Thus, in some embodiments, a full-length murinized human heavy or light chain immunoglobulin comprises a mouse constant region, human CDRs, and a substantially human framework having a plurality of "murinized" amino acid substitutions. Typically, a "murinized antibody" is an antibody comprising a murinized variable light chain and/or a murinized variable heavy chain. For example, a murine antibody does not encompass, for example, a typical chimeric antibody, for example because the entire variable region of the chimeric antibody is non-mouse. A modified antibody that has been "murinized" by a "murinization" process binds to the same antigen as the parent antibody that provides the CDRs and is generally less immunogenic in mice than the parent antibody.

Antibody fragments：

As used herein, the term "heavy chain portion" includes amino acid sequences derived from immunoglobulin heavy chains. The polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide used in the present invention can include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, a binding polypeptide used in the invention may lack at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). As described above, one of ordinary skill in the art will appreciate that these domains (e.g., heavy chain portions) can be modified such that their amino acid sequences differ from naturally occurring immunoglobulin molecules.

In certain antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, the heavy chain portion of one polypeptide chain of a multimer is identical to the heavy chain portion of a second polypeptide chain of the multimer. Alternatively, the heavy chain moiety-containing monomers of the invention are not identical. For example, each monomer may comprise a different target binding site, thereby forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies disclosed herein, or antigen-binding fragments, variants or derivatives thereof, consist of a single polypeptide chain (such as an scFv) and are expressed intracellularly (intrabodies) for potential in vivo therapeutic and diagnostic applications.

The binding polypeptide heavy chain portions disclosed herein for use in diagnostic and therapeutic methods may be derived from different immunoglobulin molecules. For example, the heavy chain portion of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain portion may include a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

Thus, as also exemplified in the examples, in one embodiment the constant region or a part thereof of the antibody of the invention, in particular the CH2 domain and/or the CH3 domain, but optionally also the CH1 domain, is heterologous to the variable region of the native human monoclonal antibody isolated according to the method of the invention. In this case, one or more of the heterologous constant regions is preferably of human origin in the case of therapeutic use of the antibodies of the invention, but may also be of rodent origin, for example, in the case of animal studies; see also the examples.

As used herein, the term "light chain portion" includes amino acid sequences derived from immunoglobulin light chains. Preferably, the light chain moiety comprises V_LDomain or C_LAt least one of a domain.

As previously indicated, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "V_HThe "domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, while the term "CH 1 domain" includes the first (predominantly amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to V_HA domain, and is the amino terminus of the hinge region of an immunoglobulin heavy chain molecule.

As used herein, the term "CH 2 domain" includes that portion of the heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system, see Kabat EA et al, supra). The CH2 domain is unique in that it is not closely paired with another domain. In contrast, two N-linked branched sugar chains were inserted between the two CH2 domains of the intact native IgG molecule. It is well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains about 108 residues.

As used herein, the term "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. The hinge region comprises about 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains; see Roux et al, j. immunol.161(1998), 4083.

As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 region and the CL region are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 (positions 226 or 229, EU numbering system) using the Kabat numbering system.

As used herein, the terms "linked," "fused," or "fused" are used interchangeably. These terms mean that two or more elements (elements) or components are joined together by any means, including chemical coupling or recombinant means. By "in-frame fusion" is meant that the Open Reading Frames (ORFs) of two or more polynucleotides are joined (in a manner that maintains the correct translational reading frame of the original ORF) to form a continuous, longer ORF. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (which segments are not typically so joined in practice). Although the reading frame is thus formed continuously throughout the fused segment, the segments may be physically or spatially separated by, for example, in-frame linking sequences. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused in-frame, but separated by polynucleotides encoding at least one immunoglobulin framework region or other CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide. Thus, in one embodiment, the polynucleotide is a cDNA encoding the variable region and at least a portion of the constant domain. In one embodiment, the polynucleotide is a cDNA encoding the variable and constant domains of the antibodies of the invention as defined herein.

Epitope:

the minimum size of a peptide or polypeptide epitope of an antibody is considered to be about four to five amino acids. The peptide or polypeptide epitope preferably comprises at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Because a CDR can recognize its tertiary form of an antigenic peptide or polypeptide, the amino acids comprising the epitope need not be contiguous and, in some cases, may not even be on the same peptide chain. In the present invention, the peptide or polypeptide epitope recognized by the antibody of the invention comprises a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 and about 30 or between about 30 and about 50 contiguous or non-contiguous amino acids (i.e., at least one IL-32 isoform) of the molecule of interest of the invention, or other IL-32 isoform homologous sequences in the case where the antibody recognizes more than one isoform.

Binding characteristics:

"binding" or "identifying" as used interchangeably herein generally refers to: a binding molecule (e.g., an antibody) binds to a predetermined epitope through its antigen binding domain; and that the binding causes some complementarity between the antigen binding domain and the epitope. According to this definition, it is believed that when an antibody binds to an epitope, it "specifically binds" to the epitope through its antigen binding domain more readily than it binds to a randomly unrelated epitope. The term "specificity"Used herein to define the relative affinity of binding to an epitope by an antibody. For example, antibody "a" can be said to have a higher specificity for a given epitope than antibody "B", or antibody "a" can be said to bind to epitope "C" with a higher specificity than its specificity for the relevant epitope "D". An unrelated epitope is typically part of a non-specific antigen (e.g., BSA, casein, or any other designated polypeptide) that can be used to assess the binding specificity of a given binding molecule. In this regard, the term "specifically binds" refers to K that an antibody binds to a non-specific antigen in a specific manner_DAt least two times lower K_DBinding to a predetermined antigen. The term "highly specific" binding as used herein refers to the relative K of an antibody to a specific target antigen_DK binding to other ligands than the antibody_DAt least ten times lower.

The term "immunoglobulin binding property" or other binding property, if present, for all grammatical forms of antibodies and antigens refers to the specificity, affinity, cross-reactivity, and other binding properties of an antibody.

By "preferentially binds" is meant that a binding molecule, such as an antibody, specifically binds to an epitope more readily than the antibody binds to a related, similar, homologous, or similar epitope. Thus, an antibody that "preferentially binds" to a given epitope is more likely to bind to the given epitope than to a related epitope, even though such an antibody may cross-react with the related epitope. With respect to a particular antigen, such as a specific IL-32 isotype, the term "preferentially binds" means that a binding molecule, e.g., an antibody, specifically binds to an IL-32 isotype more readily than the antibody binds to a related, similar, homologous, or similar IL-32 isotype.

By way of non-limiting example, if a binding molecule such as an antibody has a dissociation constant (K) for a second epitope that is greater than that of the antibody_D) Low K_DBinding to a first epitope, it can be considered that the binding molecule preferentially binds to said first epitope. In another non-limiting example, if the antibody is to a second epitope K than the antibody_DAn affinity that is at least one order of magnitude lower than the affinity for the first epitope, then it can be considered that the antibody preferentially binds toThe first epitope. In another non-limiting example, if the antibody is to a second epitope K than the antibody_DAn affinity that is at least two orders of magnitude lower binds to a first epitope, and the antibody can be considered to preferentially bind to the first epitope.

In another non-limiting example, a binding molecule, such as an antibody, can be considered to preferentially bind a first epitope if it binds said first epitope with a lower k (off) than the antibody's rate of dissociation (k (off)) for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it binds said first epitope with an affinity that is at least one order of magnitude lower than the antibody's k (off) for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind a first epitope if it binds the first epitope with an affinity that is at least two orders of magnitude lower than the antibody's k (off) for a second epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives, disclosed herein can be considered to be less than or equal to 5 x 10^-2Second of^-1、10^-2Second of^-1、5×l0^-3Second of^-1Or l0^-3Second of^-1The off-rate (k (off)) of (a) binds to the molecule of interest, fragment or variant thereof of the invention. More preferably, the antibodies of the invention can be considered to be at less than or equal to 5 × 10^-4Second of^-1、10^-4Second of^-1、5×10^-5Second of^-1Or 10^-5Second of^-1、5×10^-6Second of^-1、10^-6Second of^-1、5×10^-7Second of^-1Or 10^-7Second of^-1The off-rate (k (off)) of (a) binds to the molecule of interest, fragment or variant thereof of the invention.

Binding molecules, e.g., antibodies or antigen binding fragments, variants, or derivatives, disclosed herein can be considered to be greater than or equal to 10³M^-1Second of^-1、5×10³M^-1Second of^-1、10⁴M^-1Second of^-1Or 5X 10⁴M^-1Second of^-1Binding ratio (o)n rate) (k (on)) binds to a molecule of interest, fragment or variant thereof of the invention. More preferably, the antibodies of the invention can be considered to be greater than or equal to 10⁵M^-1Second of^-1、5×10⁵M^-1Second of^-1、10⁶M^-1Second of^-1Or 5X 10⁶M^-1Second of^-1Or 10⁷M^-1Second of^-1The binding rate (k (on)) of (a) binds to the molecule of interest, fragment or variant thereof of the invention.

An antibody is considered to competitively inhibit binding of a reference antibody to a given epitope if the binding molecule, e.g., antibody, preferentially binds to the epitope to an extent that prevents binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art (e.g., a competitive ELISA assay). An antibody can be considered to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term "affinity" refers to a measure of the strength of binding of a single epitope to the CDRs of a binding molecule (e.g., an immunoglobulin molecule); see, e.g., Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the strength of the functional combination of the immunoglobulin mixture with the antigen; see, e.g., Harlow, pages 29-34. Avidity is related to the affinity of each immunoglobulin molecule in the population for a specific epitope as well as the valency of the immunoglobulin and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure (such as a polymer) may be one of high avidity. The affinity or avidity of an antibody for an antigen may be determined experimentally using any suitable method; see, for example, Berzofsky et al, "Antibody-Antibody Interactions" InFundamental Immunology, Paul, W.E. eds., Raven Press New York, N Y (1984) and Kuby, Janisimmunology, W.H.Freeman and Company New York, N Y (1992)And the method described therein. Conventional techniques for measuring the affinity of an antibody for an antigen include ELISA, RIA and surface plasmon resonance methods. If the measurements are performed under different conditions (e.g., salt concentration, pH), the affinity of the particular antibody-antigen interaction measured may vary. Therefore, standard solutions of antibodies and antigens, as well as standard buffers, are preferably used for affinity and other antigen binding parameters (e.g., K)_D、IC₅₀) The measurement of (2).

Binding molecules of the invention, such as antibodies or antigen-binding fragments, variants or derivatives thereof, may also be described or embodied in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; i.e. a measure of the correlation between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope different from the epitope that induced its formation. Cross-reactive epitopes usually contain many of the same complementary structural features as the inducing epitope and, in some cases, may actually be more suitable than the original.

For example, certain antibodies have a degree of cross-reactivity in that they bind related but non-identical epitopes, e.g., epitopes that are at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identical to a reference epitope (as calculated using methods known in the art and described herein). An antibody is considered to have little or no cross-reactivity if it does not bind to an epitope that has less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity to a reference epitope (as calculated using methods known in the art and described herein). An antibody is considered "highly specific" for an epitope if it does not bind to any other analog, ortholog, or homolog of the epitope.

According to the inventionBinding molecules, such as antibodies or antigen-binding fragments, variants or derivatives thereof, may also be described or embodied in terms of their binding affinity to a molecule of interest of the present invention. Preferred binding affinities include dissociation constants or Ks_DLess than 5X 10^-2M、10^-2M、5×10^-3M、10^-3M、5×10^-4M、10^-4M、5×10^-5M、10^-5M、5×10^-6M、10^-6M、5×10^-7M、10^-7M、5×10^-8M、10^-8M、5×10^-9M、10^-9M、5×10^-10M、10^-10M、5×10^-11M、10^-11M、5×10^{- 12}M、10^-12M、5×10^-13M、10^-13M、5×10^-14M、10^-14M、5×10^-15M or 10^-15Those binding affinities of M. Typically, the antibody is present in 10^-7Dissociation constant (K) of M or less_D) Binding to its predetermined antigen. Preferably, the antibody is present in 10^-9Dissociation constant (K) of M or less_D) And still more preferably at 10^-11Dissociation constant (K) of M or less_D) Binding to its cognate antigen.

Modification of antibodies：

Can be further processedDecorationImmunoglobulin or a cDNA encoding the same. Thus, in yet another embodiment, the method of the invention comprises any of the steps of one or more steps of producing a chimeric antibody, a humanized antibody, a single chain antibody, a Fab fragment, a diabody, a fusion antibody, a labeled antibody, or an analog of any of these. Corresponding methods are known to the person skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. When derivatives of the antibodies are obtained by phage display techniques, the surface plasmon resonance method employed in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to the same epitope as any one of the antibodies provided by the invention (Schier, Human antibodies hybrids 7(1996), 97-105; Malmborg, J.Immunol.methods 183 (1995)),7-13). The production of chimeric antibodies is described, for example, in international application WO 89/09622. For example, methods for producing humanized antibodies are described in European application EP-A10239400 and in International application WO 90/07861. Other sources of antibodies to be utilized according to the present invention are so-called xenogeneic antibodies. For example, the general principles for the production of xenogenous antibodies (e.g., human antibodies) in mice are described in International applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As noted above, in addition to complete antibodies, the antibodies of the invention may exist in a variety of forms, including, for example, Fv, Fab and F (ab)₂And single strands; see, for example, International application WO 88/09344.

The antibodies of the invention, or one or more immunoglobulin chains corresponding thereto, may be further modified using conventional techniques known in the art (e.g., by using one or more deletions, one or more insertions, one or more substitutions, one or more additions and/or one or more recombinations and/or one or more other modifications of amino acids known in the art, either alone or in combination). Methods for introducing such modifications in the DNA sequence on which the amino acid sequence of an immunoglobulin chain is based are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Modifications of the antibodies of the invention include chemical and/or enzymatic derivatization at one or more constituent amino acids, including: side chain modification; main chain modification; and N-and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and attachment of carbohydrate or lipid moieties, cofactors, and the like. Likewise, the invention includes the production of chimeric proteins comprising the described antibodies or some fragment thereof fused at the amino terminus to a heterologous molecule (such as a marker or drug). The antigen binding molecules generated in this way can be used to target drugs to cells expressing the appropriate surface structures of diseased cells and tissues, respectively. Such targeting and binding to cells can facilitate delivery of therapeutically or diagnostically active agents as well as gene therapy/gene delivery. Molecules/particles with the antibodies of the invention will specifically bind to cells/tissues expressing a particular antigen of interest and may therefore have diagnostic and therapeutic uses.

Sample/specimen:

as used herein, the term "sample" or "biological sample" refers to any biological material obtained from a subject or patient. In one aspect, the sample can include blood, cerebrospinal fluid ("CSF"), or urine. In other aspects, the sample can include whole blood, plasma, monocytes (PBMCs) enriched for peripheral blood (such as lymphocytes (i.e., T cells, NK cells, or B-cells), monocytes, macrophages, dendritic cells, and basophils), and cultured cells (e.g., B cells from a subject). The sample may also comprise a biopsy or tissue sample (including tumor tissue). In still other aspects, the sample can include whole cells and/or lysates of the cells. In one embodiment, the sample comprises Peripheral Blood Mononuclear Cells (PBMCs). The sample may be collected by methods known in the art.

Identification of anti-IL-32 antibodies, isolation of corresponding B cells, and recombinant expression of anti-IL-32 antibodiesThe identification of B cells specific for the anti-IL-32 antibodies of the invention (as listed in table 1 and as exemplified for the IL-32 γ isotype) and the molecular cloning of antibodies showing the specificity of interest and the recombinant expression and functional characteristics of the antibodies can be performed generally as described in international applications WO2013/098419a1 and WO2013/098420 a 1; see the examples section in the above international application, in particular examples 1 and 2 on pages 118-120 of WO2013/098419a1 and examples 1 to 4 on pages 27-31 of WO2013/098420 a1, the disclosures of which are incorporated herein by reference.

Briefly, in one embodiment of the invention, a culture of single or oligoclonal B cells is cultured and the supernatant of the culture containing antibodies produced by the B cells is screened for the presence and affinity of antibodies specific for one or more IL-32 isotypes, as described in this example. In another embodiment, patient sera are first screened for the presence of autoantibodies against the IL-32 isotype, and those with high titers (titer ) are then selected for peripheral blood mononuclear cell isolation; see example 2 of WO2013/098419a1, page 118-120, the disclosure of which is incorporated herein by reference. The screening process involves screening for fragments, peptides or derivatives that bind the same type of IL-32. Subsequently, isolating the antibody whose binding is detected or the cells producing said antibody; see example 3 on page 120 of WO2013/098419a1, the disclosure of which is incorporated herein by reference. Thus, a preliminary screen can be performed on a candidate donor panel using a sample comprising antibody secreting cells (e.g., total peripheral blood or serum). In particular, monocytes can be isolated from blood or lymphoid tissue using standard separation techniques for isolating Peripheral Blood Mononuclear Cells (PBMCs), such as gradient centrifugation. After and/or before this separation step, serum (or plasma), cell culture supernatant or samples of cells (obtained from different patients, different tissues and/or at different time points) can be pre-screened using standard techniques for detecting the presence of antibodies and antibody-secreting cells (e.g., ELISA, BIACORE, Western blot), FACS, SERPA, antigen array, neutralization of viral infection in cell culture systems, or ELISPOT assays). This document provides several examples showing such techniques as the use of ELISPOT for characterizing immune responses in vaccinated donors (Crotty et al, Immunol meth.286(2004), 111-.

After identifying a candidate anti-IL-32 antibody and a B cell secreting the antibody, respectively, a nucleic acid sequence encoding the antibody of interest is obtained, comprising the steps of: preparing a B cell, and obtaining/sequencing a nucleic acid encoding an antibody of interest from the B cell, and further inserting said nucleic acid into or using said nucleic acid to prepare an expression host capable of expressing the antibody of interest, culturing or subculturing the expression host with the antibody of interest expressed, and optionally purifying the antibody of interest. It goes without saying that the nucleic acid can be manipulated intermediately to introduce restriction sites, to change codon usage and/or to add or optimize transcriptional and/or translational regulatory sequences. These techniques are prior art and may be performed without undue burden by those skilled in the art. For example, the heavy chain constant region may be exchanged with or eliminated entirely from a different type of heavy chain constant region. The variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding capability to more than one target, or a chimeric heavy and light chain combination can be employed. Once genetic material is available, the design of analogs that retain their ability to bind to the desired target as described above is well established. Methods for cloning antibody variable regions and generating recombinant antibodies are known to those skilled in the art and are described, for example, in Gilliland et al, Tissue antibodies 47(1996), 1-20; doenecke et al, Leukemia 11(1997), 1787-. However, in a preferred embodiment of the invention, the B-cells and expression of the corresponding antibodies are obtained by the method described in international application WO2013/098420 a1, in particular example 3 on pages 28-30 thereof, the disclosure of which is incorporated herein by reference.

Diseases and disorders:

the terms "disorder" and "disease" are used interchangeably herein, unless otherwise indicated. The term "autoimmune disease" as used herein is a disease or disorder arising from and directed to an individual's own tissue or organ or co-segregation or manifestation thereof or causing a condition therefrom. Autoimmune diseases are mainly caused by abnormal regulation of the adaptive immune response and the formation of autoantibodies or autoreactive T cells against self-structures. Almost all autoimmune diseases have an inflammatory component. Autoinflammatory diseases are primarily inflammatory, and some classical autoinflammatory diseases are caused by genetic defects in the innate inflammatory pathway. In auto-inflammatory diseases, no autoreactive T cells or autoantibodies are found. In many of these autoimmune and autoinflammatory diseases, there may be many clinical and laboratory indications, including but not limited to, hypercholesterolaemia, high autoantibody levels, antigen-antibody complex deposition in tissues, benefits from corticosteroid or immunosuppressive therapy, and lymphoid cell aggregation in infected tissues. Without being limited by theory on B cell mediated autoimmune diseases, it is believed that B cells demonstrate a pathogenic role in human autoimmune diseases through a variety of mechanical pathways including autoantibody production, immune complex formation, dendritic cell and T cell activation, cytokine synthesis, direct chemokine release, and providing foci for ectopic new lymph fluid production. Each of these pathways may be involved in the pathology of autoimmune diseases to varying degrees.

As used herein, an "autoimmune disease" can be an organ-specific disease (i.e., an immune response specifically directed against an organ system, such as the endocrine system, hematopoietic system, skin, cardiopulmonary system, gastrointestinal and hepatic systems, renal system, thyroid, ear, neuromuscular system, central nervous system, etc.) or a systemic disease that may affect multiple organ systems (including but not limited to Systemic Lupus Erythematosus (SLE), rheumatoid arthritis, polymyositis, autoimmune polyendocrinopathy syndrome type 1 (APS 1)/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apected)), or the like. Preferred such diseases include, but are not limited to, Multiple Sclerosis (MS), various forms of autoimmune rheumatic diseases including, but not limited to, rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, sjogren's syndrome, scleroderma, lupus (including, but not limited to, SLE and lupus nephritis), polymyositis/dermatomyositis and psoriatic arthritis, autoimmune skin diseases including, but not limited to, psoriasis, pemphigus group diseases, bullous pemphigoid diseases and cutaneous lupus erythematosus, and autoimmune endocrine disorders including, but not limited to, diabetes-related autoimmune diseases such as type 1 or insulin-dependent diabetes mellitus (T1DM or IDDM), autoimmune thyroid diseases including, but not limited to, graves' disease and thyroiditis, and diseases affecting autoimmune production including, but not limited to, autoimmune polyendocrine syndrome type 1 (APS 1)/autoimmune polyendocrine-candidiasis-candidia Mycosis-ectoderm dystrophy (apected), myasthenia gravis (MG/thymoma)).

Preferred diseases include, for example, SLE, RA, spondyloarthritis, psoriatic arthritis, T1DM, MS, psoriasis, sjogren's syndrome, graves' disease, thyroiditis and glomerulonephritis and APS 1. Still more preferred are RA, SLE and MS, and most preferred is SLE.

Labeling and diagnosis:

the labeling agent may be coupled directly or indirectly to the antibody or antigen of the invention. One example of indirect coupling is the use of spacer (spacer) moieties. In addition, the antibodies of the invention may include other domains, which are linked by covalent or non-covalent bonds. The ligation may be based on gene fusion according to methods known in the art and described above, or may be performed by chemical cross-linking as described, for example, in international application WO 94/04686. The other domains present in the fusion protein comprising the antibody of the invention may preferably be linked by a flexible linker (linker), advantageously by a polypeptide linker, wherein said polypeptide linker comprises a plurality of hydrophilic peptide-bonded amino acids, of sufficient length to span the distance between the C-terminal end of said other domains and the N-terminal end of the antibody of the invention (or vice versa). A therapeutically or diagnostically active agent can be coupled to an antibody or antigen-binding fragment thereof of the invention by a variety of means. This includes, for example, single chain fusion proteins comprising the antibody variable regions of the invention coupled to a therapeutically or diagnostically active agent by covalent means such as peptide bonds. Other examples include molecules comprising at least one antigen-binding fragment covalently or non-covalently coupled to other molecules, including those in the following non-limiting illustrative list. Traunecker, int.J. cancer surp.SuDP 7(1992),51-52 describes the bispecific agent janusin in which the Fv region against CD3 is coupled to soluble CD4 or other ligands such as OVCA and IL-7. Likewise, the variable regions of the antibodies of the invention can be constructed as Fv molecules and coupled to replacement ligands (such as those shown in the cited references). Higgins, j.infect. disease 166(1992),198-202 describes heteroconjugated antibodies consisting of OKT3 cross-linked to an antibody directed against a specific sequence in the GP 120V 3 region (heteroconjugated antibodies). Such heteroconjugate antibodies can also be constructed using at least the variable regions comprised in the antibodies of the methods of the invention. Other examples of specific antibodies include those described in Fanger, Cancer treat. Res.68(1993),181-194 and Fanger, Crit. Rev. Immunol.12(1992), 101-124. Conjugates, i.e. immunotoxins comprising conventional antibodies, have been widely described in the art. The toxin may be coupled to the antibody by conventional coupling techniques, or an immunotoxin containing a protein toxin moiety may be produced as a fusion protein. The antibodies of the invention can be used in a corresponding manner to obtain such immunotoxins. Examples of such immunotoxins are those described in Byers, SeminarsCell. biol.2(1991),59-70 and Fanger, Immunol. today 12(1991), 51-54.

The fusion protein may also include a cleavable linker or cleavage site for a protease. These spacer moieties may in turn be insoluble or soluble (Diener et al, Science 231(1986),148) and may be selected to enable release of the drug from the antigen at the target site. Examples of therapeutic agents that can be coupled to the antibodies and antigens of the invention for immunotherapy are chemokines, homing molecules, drugs, radioisotopes, lectins, and toxins. The drugs that can be conjugated to the antibodies and antigens of the invention depend on the disease condition for which the conjugated molecule is intended. For example, antibodies specific for targets useful in the treatment of neoplastic disease can be conjugated to compounds classically referred to as antineoplastic drugs (e.g., mitomycin C, daunorubicin, and vinblastine). In the case of using the radioisotope conjugated antibodies or antigens of the invention for e.g. tumor immunotherapy, certain isotopes may be preferred over others depending on such factors as leukocyte distribution and stability and emission. Depending on the autoimmune response, certain emitters may be preferred over others. In general, alpha and beta particles emitting radioisotopes are preferred in immunotherapy. Preference is given to short-range, high-energy alpha emitters, e.g.²¹²And (4) Bi. Examples of radioisotopes that may be bound to the antibodies or antigens of the invention for therapeutic purposes are¹²⁵I、¹³¹I、⁹⁰Y、⁶⁷Cu、²¹²Bi、²¹²At、²¹¹Pb、⁴⁷Sc、¹⁰⁹Pd and¹⁸⁸re. Other therapeutic agents that can be conjugated to the antibodies or antigens of the invention, as well as in vitro and in vivo therapeutic regimens, are known to, or can be readily determined by, one of ordinary skill in the art. Non-limiting examples of suitable radionuclides for labeling are¹⁹⁸Au、²¹²Bi、¹¹C、¹⁴C、⁵⁷Co、⁶⁷Cu、¹⁸F、⁶⁷Ga、⁶⁸Ga、³H、¹⁹⁷Hg、¹⁶⁶Ho、¹¹¹In、^113mIn、¹²³I、¹²⁵I、¹²⁷I、¹³¹I、¹¹¹In、¹⁷⁷Lu、¹⁵O、¹³N、³²P、³³P、²⁰³Pb、¹⁸⁶Re、¹⁸⁸Re、¹⁰⁵Rh、⁹⁷Ru、³⁵S、¹⁵³Sm and^99mtc. Other molecules suitable for labeling are fluorescent or luminescent dyes, magnetic particles, metals, and molecules that can be detected by secondary enzymatic or binding steps (such as enzymes or peptide tags). The eighth edition of the fluorescent Probes and Research Products manual (Handbook of fluorescent Probes and Research Products) lists commercially available fluorescent Probes suitable for use as labels in the present invention, the disclosure of which is incorporated herein by reference. Magnetic particles suitable for use in magnetic particle-based assays (MPA) may be selected from paramagnetic, diamagnetic, ferromagnetic and superparamagnetic materials.

General methods of Molecular and cellular biochemistry that can be used for diagnostic purposes can be found in standard textbooks such as the third edition of Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual) (Sambrook et al, Harbor Laboratory Press 2001), the fourth edition of Short Protocols in Molecular biology (compendium for Molecular biology) (eds.) (Ausubel et al, John Wiley & Sons 1999), protein methods (Bollag et al, John Wiley & Sons 1996). Reagents, detection devices (means, methods, means) and kits for diagnostic purposes are available from commercial suppliers such as Pharmacia Diagnostics, Amersham, BioRad, Stratagene, Invitrogen and Sigma-Aldrich as well as from sources given in any of the references cited herein, in particular the patent literature.

Treatments and drugs:

as used herein, the term "treatment" or "treatment" refers to both therapeutic treatment and prophylactic (preventative) or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of an autoimmune and/or autoinflammatory disease. Beneficial or desired clinical effects, whether detectable or undetectable, include, but are not limited to: alleviation of symptoms, diminishment of extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). "treatment" may also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include: those already having the condition or disorder, as well as those susceptible to the condition or disorder, or those in whom the manifestation of the condition or disorder is to be prevented.

The terms "drug," "drug/medicament," or "drug" are used interchangeably herein if not otherwise specified, and shall include, but are not limited to, all of the following: (A) articles, pharmaceuticals and formulations for internal or external use, and any substance or mixture of substances intended for the diagnosis, cure, mitigation, treatment or prevention of a disease in humans or other animals; and (B) articles, pharmaceuticals and formulations (except food products) intended to affect the structure or any function of the human or other animal body; and (C) an article intended to be a component of any of the articles specified in items (A) and (B). The term "drug", "medicine" or "drug/medicament" shall include a complete formulation intended for use in humans or other animals, including one or more "agents/agents", "compounds", "substances" or "(chemical) components/ingredients/compositions" and in some other cases also other pharmaceutically inactive excipients as fillers, disintegrants, lubricants, glidants, binders or to ensure ease of transport, disintegration, deaggregation, dissolution and bioavailability of the "drug", "medicine" or "drug/medicament" at the intended target site (e.g. skin, stomach or gut) within the body of the human or other animal. The terms "agent/medicament", "compound" or "substance" are used interchangeably herein and shall in a more specific case include, but are not limited to, all pharmacologically active agents, i.e., agents/medicaments that induce a desired biological or pharmacological effect or whose ability to induce such a possible pharmacological effect has been studied or tested by the methods of the present invention.

Examples of "antirheumatic" and immunosuppressive drugs include chloroquine, hydroxychloroquine, gold sodium thiobenzoate (myocrisin), auranofm (auranofm), sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate), adalimumab and the like, azathioprine, D-penicillamine, gold salts (oral), gold salts (intramuscular), minocycline, cyclosporines including cyclosporine a and exo cyclosporine, tacrolimus, mycophenolate mofetil, cyclophosphamide, staphylococcus aureus protein a (Goodyear and Silverman, j.exp.med., (2003),125-39), including salts and derivatives thereof, and the like.

Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen and sustained release ibuprofen (retard), fenoprofen, piroxicam, flurbiprofen, naproxen, ketoprofen, naproxen, tenoxicam, benorilate, diclofenac, naproxen, nabumetone, indomethacin (indomethacin), ketoprofen, mefenamic acid, diclofenac, fenbufen, apazone, acemetacin, tiaprofenic acid, indomethacin, sulindac, tolmetin, phenylbutazone, diclofenac and sustained release diclofenac, Cyclooxygenase (COX) -2 inhibitors (such as GR 253035, MK966, celecoxib(s) (diclofenac (R) and diclofenac

4- (5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl)), benzenesulfonamide and valdecoxibAnd meloxicam

Including salts and derivatives thereof, and the like. Preferably, the "non-steroidal anti-inflammatory drug" or "NSAID" is aspirin, naproxen, ibuprofen, indomethacin, or tolmetin. Such NSAIDs are optionally used with analgesics (such as codeine, tramadol, and/or dihydrocodeine) or narcotics (such as morphine).

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, e.g., a human patient, for which diagnosis, prognosis, prevention or treatment is desired.

A drug carrier:

pharmaceutically acceptable carriers and routes of administration can be obtained from the corresponding literature known to those skilled in the art. The pharmaceutical compositions of the present invention may be formulated according to methods well known in the art; see, for example, Remington at the University of Sciences in philiadelphia: the Science and practice of Pharmacy (2000), ISBN 0-683-306472, Vaccine protocols, second edition Robinson et al, Humana Press, Totowa, New Jersey, USA, 2003; second edition of Therapeutic Peptides and Proteins, edited by Taylor and Francis, Banga: Formulation, Processing, and Delivery Systems (2006), ISBN: 0-8493-1630-8. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (e.g., oil/water emulsions), various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers may be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to a subject in a suitable dosage. Administration of suitable compositions can be by different routes. Examples include administration of compositions containing pharmaceutically acceptable carriers by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations (such as nasal spray formulations) include purified aqueous or other solutions of active agents with preservatives and isotonicity agentsAnd (4) liquid. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Also contemplated in the present invention are pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., "Nanobodies)^TM") and the like. Such oral formulations may be in the form of tablets, capsules, powders, liquids or semi-solids. Tablets may include solid carriers such as gelatin or adjuvants. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers; see also O' Hagan et al, Nature Reviews, drug discovery 2(9) (2003), 727-735. Can be found in Remington's Pharmaceutical Sciences, MacePublizing Company, Philadelphia, PA,17^thOther guidance regarding formulations suitable for various types of administration is found in (1985) and corresponding newer versions thereof. For a brief review of methods for drug delivery, see Langer, Science 249(1990), 1527-.

Administration of drugsScheme(s)

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Typical doses may be, for example, in the range of 0.001 to 1000 μ g (or in the range of nucleic acid expression or inhibition of expression); however, doses below or above this exemplary range are also contemplated, particularly in view of the above factors. Generally, a conventional administration regimen for a pharmaceutical composition should be in the range of 1 μ g to 10mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1. mu.g to 10mg units per minute per kilogram of body weight, respectively. The progress may be monitored by periodic evaluation. Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils. Intravenous carriers include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention may contain other agents, such as antineoplastic agents and cytotoxic drugs, depending on the intended use of the pharmaceutical composition.

In addition, co-administration or sequential administration of other agents may be desirable. A therapeutically effective dose or amount refers to an amount of active ingredient sufficient to alleviate a symptom or condition. In cell cultures or experimental animals, the therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED 50.

Preferably, the therapeutic agent in the composition is present in an amount sufficient to prevent inflammation or suppress an immune response.

These and other embodiments are disclosed and encompassed by the specification and examples of the present invention. Other documents on any of the materials, methods, uses and compounds to be used in accordance with the present invention may be retrieved from public libraries and databases using, for example, electronic equipment. For example, the public database "Medline" hosted by the U.S. national center for biotechnology information and/or the national library of national institutes of health may be utilized. Other databases and web addresses, such as those of the European Bioinformatics Institute (EBI), a part of the European Molecular Biology Laboratory (EMBL), are known to those skilled in the art and can be obtained using an internet search engine. An overview of biotech patent information and a survey of relevant sources of patent information that can be used for backtracking searches and for new intelligence announcements is given in Berks, TIBTECH12(1994), 352-.

The foregoing disclosure generally describes the present invention. Several documents are cited throughout the specification text. A full and enabling disclosure of the literature is found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications cited throughout this application, including those disclosed in the background section, and manufacturer's specifications, instructions for use, etc.) are expressly incorporated herein by reference; however, there is no admission that any of the cited documents are indeed prior art with respect to the present invention.

A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.