Treatment methods for dry eye disease using TNFα antagonists

Abstract

This invention provides a composition for treating dry eye disease (DED). [Solution] A composition for the treatment of a patient having dry eye disease (DED), comprising a TNFα antagonist, is provided, characterized in that a therapeutically effective amount of the TNFα antagonist is administered to the patient based on the patient having a DED response marker, and the DED response marker is the rs1800693 response allele.

Description

[Technical Field]

[0001] This disclosure covers prediction methods, personalized therapies, kits, information in a transmissible form, and dry eye disease. Regarding treatment methods for patients with the condition. [Background technology]

[0002] Dry eye disease (DED) is caused by inflammation of the ocular surface and an increase in the osmolality of the tear film. Tears and O It is a common multifactorial disease of the crystalline surface (Lienert JP, Tarko L, Uchino M, Christen WG, Schaumberg DA.(20 16).Long-term Natural History of Dry Eye Disease from the Patient's Perspective. Ophthalmology. 123(2):425-33). The only available pharmacological The treatment is topical cyclosporine, which is an anti-inflammatory agent approved for increasing tear production. Steroids are also used to treat DED, but long-term use is contraindicated due to side effects. For more severe forms of DED, serum eye drops and scleral contact lenses are recommended. However, none of these treatments adequately address the underlying cause of DED. No (Marshall LL, Roach JM. (2016). Treatment) of Dry Eye Disease.Consult Pharm.2016;3 1(2):96-106).

[0003] Dry eye, also known as keratoconjunctivitis sicca, is a common eye condition affecting millions of people every year. is a disorder. This condition is particularly prevalent in postmenopausal women, especially due to hormonal changes after the loss of fertility. Dry eye can vary in severity among affected individuals. In mild cases, patients may experience aching pain, a dry sensation, and often a persistent irritation, as if caused by a small foreign body trapped between the eyelid and the ocular surface. In severe cases, vision can be substantially impaired. Other diseases, such as Sjögren's syndrome and cicatricial pemphigoid, can also lead to the dry eye condition. Transient dry eye symptoms associated with refractive corrective surgery have been reported to last from 6 weeks to over 6 months postoperatively in some cases.

[0004] Dry eye is thought to be caused by several unrelated etiologies, but the manifestation of complications all share a common effect, namely, the disruption of the precorneal tear film, resulting in exposure of the ocular surface, water loss, and cytokine production, which cause many of the symptoms outlined above (Lemp, Report of the National Eye In stitute / Industry Workshop on Clinical Tr ials in Dry Eyes, The CLAO Journal, volume [[ID=Z7]] 21, number 4, pages 221 - 231 (1995)).

[0005] Physicians employ several approaches to treat dry eye. One common approach is to replenish and stabilize the eye's tear film by instilling so - called artificial tears throughout the day. Other approaches include the use of intraocular inserts (such as punctal plugs) to promote tear retention or the stimulation of endogenous tear production.

[0006] One example of a tear substitute technique is to make the solution highly viscous, thereby making it difficult to expel from the eye. It contains buffered isotonic saline and aqueous solutions containing polymers. It also contains phospholipids and oils, etc., as in tears. Attempts have also been made to stabilize the tear film by supplying one or more components of the film. The composition has been shown to be useful in the treatment of dry eye; for example, McCulle y and Shine, Tear film structure and dry eye, Contactologia, volume 20(4), pages 145 -49 (1998); and Shine and McCulley, Keratocon junctivitis sicca associated with meibom ian secretion polar lipid abnormality,Ar Chives of Ophthalmology,volume 116(7),pa See ges 849-52 (1998).

[0007] Another approach involves providing a lubricant instead of artificial tears. For example, in the United States. Japanese Patent No. 4,818,537 (Guo) describes a lubricating liposome-based composition. The U.S. Patent No. 5,800,807 (Hu et al.) indicates that dryers The present invention discloses a composition containing therapeutic glycerin and propylene glycol.

[0008] These methods have achieved some success, but despite that, they are still ineffective in treating dry eye. Problems remain, as the use of tear substitutes is only temporarily effective. However, this is because it generally needs to be applied repeatedly while the patient is awake. It is not uncommon for patients to have to apply artificial tear solution 10 to 20 times a day. This can be not only troublesome and time-consuming, but also potentially very costly.

[0009] In addition to the attempts described above, which are primarily aimed at symptomatic relief of symptoms associated with dry eye, Methods and compositions aimed at treating the physiological conditions that cause these symptoms are also being explored. For example, U.S. Patent No. 5,041,434 (Lubkin) describes combined est Using sex steroids such as chlorogenic compounds to treat dry eye conditions in postmenopausal women Disclosed; U.S. Patent No. 5,290,572 (MacKeen) describes the process of micronizing The present invention discloses stimulating anterior segment tear film production using a calcium ion composition.

[0010] Such attempts to treat the root cause of dry eye often involve inflammation of the related eye tissues. The focus has been on treating meibomian gland dysfunction. The use of various drugs, including steroids (e.g., U.S. Patent No. 5,958,912; Marsh et al., Topical nonpreserved methyl prednisolone therapy for keratoconjuncti vitis sicca in Sjoegren's syndrome,Ophth almology, 106(4):811-816(1999); and Pflugfel der et al., U.S. Pat. No. 6,153,607), cytokine release inhibition Harmful drugs (Yanni, JM; et al. International Publication No. 00 / 03705 A1 pamphlet) Lett), Cyclosporine A (Tauber, J. Adv. Exp. Med. Biol.) 1998,438(Lacrimal Gland,Tear Film,and Dr. Eye Syndromes 2),969), and mucosal secretion stimulants (mucose cretatogue), for example, 15-HETE (Yanni et.al., US Patent) This is disclosed, including in Specification No. 5,696,166.

[0011] Many studies have shown that inflammatory sites, including tumor necrosis factor α (TNF-α), in affected tissues An increase in Cain has been reported (Massingale ML, Li X, Vallab hajosyula M, Chen D, Wei Y, et al.(2009).An alysis of inflammatory cytokines in the tears of dry eye patients.Cornea.28(9):1 023-7;Chen Y,Zhang X,Yang L,Li M,Li B,et al.(2014).Decreased PPAR-γ expression i n the conjunctiva and increased expressi on of TNF-α and IL-1β in the conjunctiva and tear fluid of dry eye mice.Mol Med Rep.9(5):2015-23). ​​TNFα levels and DE in tear film or conjunctival tissue. A correlation between D and clinical severity has also been observed (Lee SY, Han SJ, N am SM, Yoon SC, Ahn JM, et al. (2013).Analys is of tear cytokines and clinical correl ations in Sjoegren syndrome dry eye pati ents and non-Sjoegren syndrome dry eye p patients.Am J Ophthalmol.156(2):247-253). TNF-α is a pleomorphic cytokine, and its cell transport upon binding to its receptor is It is involved in regulation, activation, and host defense against various pathogens. Anti-TNF agents are used in rheumatoid arthritis It has demonstrated clinical efficacy in the treatment of human autoimmune diseases, including malaise and Crohn's disease. However, local anti-TNF therapy for DED is related to the involvement of TNF-α in DED. Despite the existence of evidence, it has not been evaluated (Ji YW, Byun YJ, Ch oi W, Jeong E, Kim JS, et al.(2013).Neutral ization of ocular surface TNF-α reduces ocular surface and lacrimal gland inflammation mation induced by in vivo dry eye.Invest Ophthalmol Vis Sci.54(12):7557-66).

[0012] Recent research by Hallak et al. suggests that the Va gene of brain-derived neurotrophic factor (BDNF) l66Met and two SNPs in the vitamin D receptor (VDR) gene, Fokl and Ap It has been shown that al may be related to DED (Hallak et al. l.,Investigative Ophthalmology & Visual Science September 2015,Vol.56,5990-5996) However, DED patients who are most likely to benefit from TNFα antagonism are There are no known SNPs that can be identified. [Overview of the project] [Means for solving the problem]

[0013] This specification describes the use of TNFα antagonists in patients with dry eye disease (DED). By identifying patients who are likely to respond well before treatment, T can be applied to these populations. Novel prediction method and individual that maximize the benefits of NFα antagonism and minimize its risks Differential therapy is offered. This finding is partly based on selection from the rs1800693 response allele. DED patients who carry the DED response markers are compared to DED patients who do not carry this allele. This decision is based on demonstrating an improvement in the response to the LME636 compared to the previous version.

[0014] Therefore, the inventors believe that there is a possibility of responding to TNFα antagonism. In various pharmaceutical products and methods, including the identification of patients with high DED risk, and TNFα antagonists Whether to prescribe antidote (e.g., LME636) to those patients, or alternative therapy regimens In assisting physicians in deciding whether or not to prescribe, the target is rs180069 It is intended that testing for the presence of three response alleles may be useful.

[0015] Therefore, one purpose of this disclosure is based on certain aspects of the patient's biochemical profile. , administer a therapeutically effective dose of a TNFα antagonist, such as a TNFα antibody like LME636, to the patient. The objective is to provide a method for treating patients with DED. Another objective of this disclosure is to provide a method for treating patients with DED. The target is based on certain aspects of the patient's biochemical profile, and TNFα antagonists, for example, L Patients with DED who are likely to respond to treatment with TNFα antibodies such as ME636 The objective is to provide a method for identification. Another objective of this disclosure is to provide a method for identifying the biochemical profile of a patient. Based on certain aspects, patients with DED may be given TNFα antagonists, such as LME636. This is a method for determining which TNFα antibody treatment is likely to be effective.

[0016] This specification discloses various methods for selectively treating patients with DED. In embodiments, these methods involve taking a biological sample from a patient and applying the DED response marker of the present disclosure. Assay the following: and if the patient has a response marker, then the treatment is effective. A small amount of TNFα antagonist, such as a TNFα antibody like LME636, is selectively administered to the patient. This includes the following.

[0017] This specification also states that patients with DED may use TNFα antagonists such as LME636. Various methods for predicting the likelihood of responding to TNFα antibody therapy are also disclosed. In some embodiments, these methods involve the DED of the present disclosure in a biological sample from a patient. This includes detecting response markers, where the presence of response markers indicates that the patient has TNFα antagonism. This indicates an increased likelihood of responding to drug treatment.

[0018] In a preferred embodiment, the TNFα antagonist is a TNFα-binding molecule, preferably an antibody or The antigen-binding portion is most preferably LME636. The D response marker is at least one DED response marker as shown in Table 1. .

[0019] Additional methods, uses, and kits are provided in the following description and attached claims. Those skilled in the art will be able to see further features, advantages and benefits of this disclosure from the following description and the attached claims. The details will become clear. [Brief explanation of the drawing]

[0020] [Figure 1] This shows the symptomatic changes from baseline to day 43 in 12 patients with the rs1800693 CC genotype (4 patients treated with LME636 and 8 patients treated with the medium). [Figure 2] This waterfall plot shows the change from baseline in the overall ocular discomfort score at day 26 for all patients treated with LME636 or the media, allowing for the visualization of symptomatic changes by treatment and rs1800693 genotype. [Figure 3] This waterfall plot shows the change from baseline in the overall ocular discomfort score at day 27 for all patients treated with LME636 or the media, allowing for the visualization of symptomatic changes by treatment and rs1800693 genotype. [Figure 4] This waterfall plot shows the change from baseline in the overall ocular discomfort score at day 28 for all patients treated with LME636 or the media, allowing for the visualization of symptomatic changes by treatment and rs1800693 genotype. [Figure 5] This waterfall plot shows the change from baseline in the overall ocular discomfort score at day 29 for all patients treated with LME636 or the media, allowing for the visualization of symptomatic changes by treatment and rs1800693 genotype. [Modes for carrying out the invention]

[0021] The term "comprising" is equivalent to "including". Furthermore, it includes "consisting of ~", for example X "including (comp The composition may consist of X alone, or something additional, such as X + Y. It may be included.

[0022] Regarding the numerical value x, the term "approximately" means ±10% unless otherwise specified in the context.

[0023] As used herein, the terms "subject" and "patient" refer to any human or non-human animal. This includes things. The term "non-human animals" includes all vertebrates, for example, non-human primates, etc. Mammals and non-mammals such as rhododendrons, dogs, cats, horses, cows, chickens, amphibians, and reptiles. It includes.

[0024] The term "assay" means to identify, screen, probe, test, and measure. It is used to refer to the act of doing or deciding, and this act is carried out by any conventional means. It may also be used to determine the presence of a specific gene or protein marker in the sample. SA assay, Northern blotting, imaging, serotype typing, cell typing, Gene sequencing, phenotyping, haplotyping, immunohistochemistry, Western It can be assayed using methods such as blotting and mass spectrometry. (The term "detect" is not used in this context.) (d) refers to the act of extracting specific information from a given source, and this is even if it is direct. Or it may be indirect. In some embodiments of the prediction methods disclosed herein, given The presence of such things (e.g., alleles, protein levels, etc.) indirectly affects biological samples, for example It is detected by querying the database. The terms "assay" and "determine" are used. "To do" refers to the transformation of a substance, for example, a biological sample, such as a blood sample or other tissue sample, into a certain state. The aim is to transform the sample from one state to another by subjecting it to physical testing.

[0025] The term "to obtain" means to acquire, for example, by some means, for example, through physical intervention (for example) If a biopsy, blood sampling, or non-physical intervention (e.g., transmission of information via a server) is performed on the property It means to acquire it as such.

[0026] The phrase "assaying a biological sample..." implies the presence of a given DED response marker in the sample. It is used to mean that it can be tested (directly or indirectly) with respect to any of the following. The existence of matter represents one probability, and the non-existence of matter represents another probability (probabilistic In situations where ty means, the presence or absence of such substance is a guideline for treatment decisions. It will be understood that it can be used in this way. For example, if a patient has a DED response marker The decision of whether or not to do so depends on determining whether a specific response allele actually exists in the patient. This is obtained by determining that the patient does not have a specific response allele. In either case, it is determined whether the patient has the presence of a DED response marker. The method disclosed herein determines, in particular, whether a particular individual has a DED response marker. This includes determining whether the patient has the DED response markers listed in Table 1. This is done by determining. Each of these decisions (i.e., existence or non-existence) remains as is. This provides the patient's allergy status, and therefore these determinations Each similarly provides an indicator of whether a particular individual responds better to TNFα antagonism. do.

[0027] [Table 1]

[0028] Table 1 shows the various response alleles of this disclosure. The first column is the gene in which the SNP in the second column is located. The third column provides the approximate location of the SNP in the gene.

[0029] The SNPs listed in Table 1 are those that occur when any of the given response alleles are present (i.e., affected (Whether the person is homozygous or heterozygous with respect to a given response allele) TNF While it has predictive value regarding α-antagonism, as will be discussed in the following examples, the CC gene Patients with this genotype tend to show greater improvement in response to LME636 compared to patients with the CT or TT genotype. There was a direction.

[0030] The term "dry eye" is also known as keratoconjunctivitis sicca or keratoconjunctivitis sicca, and refers to the anterior tear film. A common ophthalmic disorder involving the breakdown of the eye's outer surface and the resulting loss of moisture from the exposed outer surface. In certain embodiments, "dry eye" is characterized as moderate to severe. The severity is determined by a person skilled in the art using a general ocular discomfort score, for example, as described herein. The determination is based on the following. The method for determining the severity of dry eye is also, for example, the 2007 International Dry Eye Research Group (2007 International Dry Eye Work) DEWS Definition and Classification Guidelines by shop ("The Ocular Surf") See ace, April 2007, Vol. 5, No. 2, pages 75-92. It is also described in Sullivan et al. (Investigative Ophthalmology & Visual Science,December This method is described in (2010, Vol. 51, pp. 6125-6130).

[0031] The term "TNFα" refers to tumor necrosis factor alpha (also known as kaketin), and This involves endotoxins or other stimuli from numerous cell types, including monocytes and macrophages. TNFα is a naturally occurring mammalian cytokine produced in response to inflammatory responses. It is a major mediator of immunological and pathophysiological responses (Grell, M ., et al. (1995) Cell, 83:793-802). "TNFα" contains, Wild-type TNFα and polymorphic variations of TNFα from various species (e.g., humans, mice, and monkeys) This includes the body and functional equivalents of TNFα. The functional equivalents of TNFα relating to this disclosure are Preferably, wild-type TNFα (e.g., human TNFα) and at least about 85%, 95%, and 96%. It has overall sequence identity of %, 97%, 98%, or even 99%, and is inflammatory and immune. It substantially retains the ability to mediate physiological and pathophysiological responses.

[0032] When used herein, "TNFα antagonist" refers to TNFα function, expression and / or cyanotype. Antagonizing Gunar signaling (for example, by blocking the binding of TNFα to TNFα receptors) This refers to molecules that have the ability to reduce, inhibit, decrease, or delay something. Non-exclusive examples of TNFα antagonists include TNFα-binding molecules and TNFα-receptor-binding molecules. Molecules are mentioned. Part of the methods, regimens, kits, processes, uses, and compositions disclosed herein. In this embodiment, a TNFα antagonist is used.

[0033] A "TNFα-binding molecule" is a molecule that binds to the human TNFα antigen, either alone or in combination with other molecules. This refers to any molecule that has the ability to bind. A binding reaction is, for example, the receptor of TNFα. This includes binding assays, competition assays, or bioassays to determine inhibition of binding to the body. By alternative methods (qualitative assays) or any type of binding assay, irrelevant specificity, however Ideally, a negative control test should be performed using an antibody of the same isotype, such as an anti-CD25 antibody. This may be shown as a standard. Non-limiting examples of TNFα-binding molecules include small molecules and TNFα receptors. It binds to TNFα as a decoy and as produced by B cells or hybridomas. Antibodies and chimeras, CDR grafts, or human antibodies or any fragment thereof, for example, F(ab ')2 and Fab fragments, as well as single-chain or single-domain antibodies are included. Preferably T NFα-binding molecules antagonize TNFα function, expression, and / or signaling (for example, (to reduce, inhibit, decrease, delay). Methods, regimens, kits, and procedures disclosed herein. In some embodiments of the process, use, and composition, TNFα-binding molecules are used.

[0034] A "TNFα receptor binding molecule" is a molecule that binds to the human TNFα receptor, either alone or in combination with other molecules. This refers to any molecule that has the ability to attach and bind. A binding reaction is, for example, a TNFα receptor. Binding assays, competition assays, or bioassets to determine inhibition of the binding of the substance to TNFα Using a standard method (qualitative assay) including (i) or any type of binding assay, unrelated Specificity, but ideally based on a negative control test using the same isotype antibody. This may be shown. Non-limiting examples of TNFα receptor binding molecules include small molecules, TNFα decoys. , and the anti-TNFα receptor as produced by B cells or hybridomas Body and chimeras, CDR grafts, or human antibodies or any fragment thereof, e.g., F(ab')2 The Fab fragment and single-chain or single-domain antibodies are included. Preferably TNFα Receptor-binding molecules antagonize TNFα function, expression, and / or signaling (for example, (to reduce, inhibit, decrease, delay). Methods, regimens, kits, and procedures disclosed herein. In some embodiments of the process, use, and composition, a TNFα receptor-binding molecule is used.

[0035] The term "antibody," as used herein, refers to all antibodies and any antigen-binding antibodies thereof. It may contain a portion or a single chain. Naturally occurring "antibodies" are linked to each other by disulfide bonds. It is a glycoprotein containing at least two linked heavy (H) chains and two light (L) chains. Each heavy chain has a heavy chain variable region (V in this specification). H (abbreviated as) and includes the heavy chain constant region. The constant region contains three domains, CH1, CH2, and CH3. Each light chain has a variable light chain region ( The light chain includes a constant region (VL, as abbreviated herein) and a constant region of the light chain. The constant region of the light chain is one domain , including CL. V H and V L The domain is called the Framework Domain (FR) and is even more advanced. Hypervariability, also known as the supervariable region or complementarity-determining region (CDR), in which conserved regions are scattered. It can be further divided into the following regions. Each V H and V L The amino terminology is carboxyl The order towards the end is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, It contains three CDRs and four FRs arranged in FR4. The variable regions of the heavy and light chains are related to the antigen and It contains interacting binding domains. The constant region of the antibody is connected to various cells of the immune system (e.g., EFF). It can mediate the binding of immune globulins to host tissues or factors, including effector cells and the first component of the classical complement system (C1q). In some embodiments of the methods, regimens, kits, processes, uses, and compositions of the present disclosure, an antibody against TNFα or a TNFα receptor, preferably an antibody against TNFα, such as LME636, is used. The term "antigen-binding portion" of an antibody, as used herein, refers to a fragment of an antibody that retains the ability to specifically bind to an antigen (e.g., TNFα). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the scope of the term "antigen-binding portion" of an antibody include a Fab fragment, which is a monovalent fragment consisting of the V , V , CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; a Fd fragment consisting of V

[0036] and the CH1 domain; a Fv fragment consisting of the V and V The antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments included within the scope of the term "antigen-binding portion" of an antibody include a Fab fragment, which is a monovalent fragment consisting of the V , V L H H domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; a Fd fragment consisting of V and the CH1 domain; a Fv fragment consisting of the V [[ID=2出来る。抗体の「抗原結合部分」という用語の範囲内に含まれる結合断片の例としては、V<及びCH1ドメインからなるFd断片;抗体の単一のアームのV L 及びV H ドメインからなるFv断片;V H ドメインからなるdAb断片(Ward et al., 1989 Nature 341 :544 - 546);及び単離CDRが挙げられる。例示的抗原結合部位は、配列番号1 ~6(表2)に示されるとおりのLME636のCDR、好ましくは重鎖CDR3を含む <及びV L H は別個の遺伝子によりコードされるが、組換え方法を用いて合成リンカーによってつなぎ合わせて、それらが単一のタンパク質 L To make the chain, where V H D [[ID=4出ることができ、ここではV [[ID=及びV 、組換え方法を用いて合成リンカーによってつなぎ合わせて、それらが単一のタンパク質 鎖として作られることを可能にすることができ、ここではV L 及びVH The domains form pairs They form monovalent molecules (known as single-chain Fv(scFv); for example, Bird et al. al., 1988 Science 242:423-426; and Huston et al. al.,1988 Proc.Natl.Acad.Sci.85:5879-588 See 3). Such single-chain antibodies are also included within the scope of the term "antibody." As shown in the diagram, the single-chain antibody and antigen-binding moiety are obtained using prior art known to those skilled in the art. In some embodiments of the methods, regimens, kits, processes, uses, and compositions disclosed herein, T Single-chain antibodies or antibodies against NFα (e.g., LME636) or TNFα receptors The antigen-binding portion is used.

[0037] When used herein, "isolated antibody" refers to other antibodies with different antigen specificities. This refers to antibodies that do not qualitatively contain TNFα (for example, an isolated antibody that specifically binds to TNFα). (Substantially contains no antibodies that specifically bind to antigens other than the one specified.) Term: "Monoclonal antibody" Alternatively, "monoclonal antibody composition" as used herein refers to an antibody with a single molecular composition. Refers to molecular preparations. The term "human antibody" is used herein within the framework. The antibody contains variable regions in both the region and the CDR region that are derived from human sequences. This is intended. "Human antibodies" do not need to be produced from humans, human tissue, or human cells. No. The human antibodies of this disclosure do not contain amino acid residues (e.g., in) that are not encoded by the human sequence. In vitro random or site-directed mutagenesis, or in vivo recombination of antibody genes This occurs either through N-nucleotide addition at junctions or in vivo in somatic cell apoptosis. This disclosure may include mutations introduced by mutations. Methods, regimens, kits, and products In some embodiments of the process, use, and composition, the TNFα antagonist is a human antibody, isolated antibody, and / or monoclonal antibodies. Methods, regimens, kits, processes, and uses of the disclosed herein. In other embodiments of the use and composition, the TNFα antagonist is a recombinant single-chain (scFv) antibody. .

[0038] The term “K D This term is intended to refer to the dissociation rate of a specific antibody-antigen interaction. "K D When used herein, " is K d vs K a The ratio (i.e., K d / K a ) requested It is intended to refer to the dissociation constant, which is expressed as the molar concentration (M) of the antibody. D The value can be determined using methods that are well established in the relevant art. K D The method for determining this is publicly known in the relevant technical field, for example, Biacore (registered trademark) A biosensor system such as a system is used. In some embodiments, a TNFα antagonist is used. Antidrugs, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding portions, e.g., LM) E636) or a TNFα receptor binding molecule (e.g., a TNFα receptor antibody or its antigen-binding portion) (minutes) contains approximately 5-250 pM of K in human TNFα D They are joined together.

[0039] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigen site. Within the range of each antigen site, the variable region of the antibody "arm" is bound by numerous weak non-covalent forces. It interacts with the antigen at this site; the more interactions there are, the stronger the affinity. Various species of TNF Standard assays for determining the binding affinity of antibodies to α are known in the art. Examples include ELISA, Western blotting, and RIA. Antibody binding reaction rate (For example, binding affinity) is also publicly known in the relevant field, such as through Biacore analysis. It can also be evaluated using standard assays of knowledge.

[0040] As determined in accordance with the methodologies known in the art and described herein. These TNFα functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological) Antibodies that "inhibit" one or more of the physical activity (such as physical activity) will not function in the absence of the antibody (or in the absence of any unrelated specific characteristics). A statistically significant reduction in specific activity compared to that observed when a control antibody of the opposite sex is present. It will be understood that this is related to the small. Antibodies that inhibit TNFα activity are, for example, measured At least about 10%, at least 50%, 80%, or 90% of the parameters are statistically measured. This significantly affects the reduction and the characteristics of the methods, uses, processes, kits and compositions of this disclosure. In a specific embodiment, the TNFα antibody used is a TNFα antibody with a TNFα enzyme concentration of 95%, 98%, or more than 99%. Potential activity may be inhibited.

[0041] The term "derivative" means, unless otherwise specified, a specified sequence (e.g., variable domain) The TNFα antagonist relating to this disclosure, for example, a TNFα binding molecule (for example, a TNFα antibody or The antigen-binding portion, for example, LME636, or the TNFα receptor-binding molecule (for example, TNFα Amino acid sequence variants of receptor antibodies or their antigen-binding portions, and covalent modifications (e.g., pe The term "machine" is used to define and use processes such as hydroxylation, deamidation, hydroxylation, phosphorylation, and methylation. "Active derivatives" are qualitative derivatives that are common to the TNFα antagonists of this disclosure, such as TNFα-binding molecules. It contains molecules having physical activity. Functional derivatives include TN as disclosed herein. This includes fragments and peptide analogs of Fα antagonists. The fragments include, for example, the full-length of a specified sequence. Includes a region within the sequence of the polypeptide shown. TNFα antagonists disclosed herein Functional derivatives (e.g., functional derivatives of LME636) are preferably disclosed herein. The V of the TNFα-binding molecule H and / or V L Array (for example, V in Table 2) H and / or V L Distribution (columns) and at least approximately 65%, 75%, 85%, 95%, 96%, 97%, 98%, or more V has 99% overall sequence identity. H and / or V L Includes domain and human T It effectively retains its ability to bind to NFα.

[0042] The phrase "substantially identical" refers to related amino acid or nucleotide sequences (e.g., V H also is V L The domain is identical to a specific reference sequence or (e.g., a conserved amino This means that there will be only a negligible difference (due to acid substitution). The difference lies in the specified area (e.g., V H or V L One or two of the five amino acid sequences of the domain Substitutions (e.g., conservative substitutions such as swapping serine with threonine, or antibody activity Minor amino acid changes (such as substitutions at positions not involved in structural integrity or complement binding) This includes. In the case of antibodies, the second antibody has the same specificity and its affinity is at least It has 50%. It is substantially identical to the sequence disclosed herein (e.g., at least about 85%). The sequence (of which the sequence identity is %) is also part of this disclosure. In some embodiments, derivatives TNFα antibodies (e.g., derivatives of LME636, e.g., LME636 biosimilar antibodies) The sequence identity is approximately 90% or more based on the sequence of this disclosure, for example, 90%, 91%, 92%. Even if it is %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher good.

[0043] With respect to natural polypeptides and their functional derivatives, "identity" in this specification means the maximum Percent identity is achieved, and any conservative substitution is not part of sequence identity. Align the array without considering the following, and then, if necessary, introduce gaps. The percentage of amino acid residues in the candidate sequence that are identical to the residues of the natural polypeptide, and It is defined as follows. Extension or insertion of the N-terminus or C-terminus is not interpreted as diminishing identity. Alignment methods and computer programs are well known. Percent Identity can be verified using standard alignment algorithms, e.g., Altshul et al. ((1990) J.Mol.Biol.,215:403 410) Basic Local Alignment Search Tool (BLAST: Basic L ocal Alignment Search Tool);Needleman et al. ((1970) J.Mol.Biol., 48:444 453) algorithm M; or Meyers et al. (1988) Comput.Appl.Bios It can be determined by the algorithm in ci., 4:11 17). Parameter 1 The formula is a Blosum 62 scoring matrix with a gap penalty of 12 and gap extension. Penalty 4 and frame shift gap penalty 5 may also apply. Two amino acids Percent identity between acid or nucleotide sequences is also determined by the ALIGN program (version E. Meyers and W. Miller (198) are incorporated into (2.0) 9) Using the CABIOS, 4:11-17) algorithm, PAM120 weighted remaining This is determined using the base table, gap length penalty 12, and gap penalty 4. It can also be done this way.

[0044] "Amino acids" refers to all naturally occurring L-α-amino acids, for example, and D-α-amino acids. Contains amino acids. The term "amino acid sequence variant" refers to the amino acid sequence variant when compared with the sequence relating to this disclosure. This refers to molecules that have some difference in their amino acid sequence. For example, the poly(P) of the specified sequence according to this disclosure. The amino acid sequence variants of the peptide still retain the ability to bind to human TNFα. No acid sequence variants include substitution variants (at least one in the polypeptide relating to this disclosure) This refers to a compound in which an amino acid residue has been removed and replaced with another amino acid in the same position. ), insertion mutants (directly adjacent to an amino acid at a specific position in the polypeptide relating to this disclosure) (Those in which one or more amino acids are inserted adjacent to each other) and deletion mutants (polypepto) This includes (in which one or more amino acids have been removed in the form of butylene).

[0045] The term "pharmaceutically acceptable" does not imply interference with the efficacy of the biological activity of one or more active ingredients. It means a non-toxic substance that does not cause harm.

[0046] With respect to a compound, for example, a TNFα-binding molecule or another drug, the term "administer" refers to the compound. It is used to refer to delivering goods to a patient via any route.

[0047] As used herein, "therapeutic dose" refers to a single or multiple dose administered to a patient (such as a human). When administered, it treats, prevents, prevents, cures, or delays the onset of disorders or recurrent disorders. To reduce its severity and improve at least one of its symptoms, or TNFα is effective in extending patient survival beyond the expected survival rate when no other treatment is available. Antagonists, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding moieties, for example, L ME636) or TNFα receptor binding molecules (e.g., TNFα receptor antibodies or their antigen binding) This refers to the amount of a portion of an active ingredient administered alone (e.g., a TNFα antagonist, e.g., L). When applied to ME636), this term refers to the component alone. When applied to combinations... This term refers to the therapeutic effect, regardless of whether the drugs are administered in combination, sequentially, or simultaneously. This refers to the total amount of active ingredients added.

[0048] The terms "treatment" or "to treat" refer to a situation where a person is at risk of contracting a disease or is suspected of having a disease. Treatment of patients who have been diagnosed with an illness or disease or medical condition. This includes both preventive or protective treatment (as applicable) and curative or disease-modifying treatment. and control clinical relapses. The treatment aims to prevent, cure, and treat the disorder or relapsing disorder. To delay the onset, reduce its severity, or improve one or more of its symptoms, or To extend a patient's survival beyond what would be expected without medical treatment, It may be administered to patients who have or are likely to acquire the disorder later in life.

[0049] The phrase "responds to treatment" refers to a specific treatment, such as a TNFα-binding molecule (e.g., LME63). 6) Used to mean that the patient demonstrates a clinically meaningful benefit from the treatment upon delivery. In the case of DED, such criteria include, for example, improvement in the overall ocular discomfort score. All of these criteria are acceptable measures of whether the patient is responding to a given treatment. The phrase "responds to treatment" is interpreted relatively rather than as an absolute response. This is intended. For example, a DED patient who has a DED response marker, DED response marker Compared to DED patients without the condition, those who receive more benefits from TNFα antagonist treatment are considered to have a greater effect. It is predicted that carriers of these DED response markers will be even better treated with TNFα antagonists. In response to this, it can simply be said that the patient "responds to treatment" with TNFα antagonists.

[0050] The phrase "receive data" can mean, for example, verbally, electronically (for example, by email, etc.) Encoded on a disc or other medium, in writing or by any other available means. Therefore, it is used to mean acquiring information as possession.

[0051] When used herein, “select” and “choose” in relation to a patient refer to a specific patient. From a larger patient population, a particular patient meets a certain criterion, for example, Being specially selected based on (or resulting from) having a DED response marker It is used to mean that. Similarly, "to treat selectively" means to treat patients with DED. This refers to providing, where the patient in question is selected from a larger patient population, and the specific patient is selected from a larger patient population. They are specially selected based on meeting certain criteria, for example, DED patients are selected if they meet the criteria for DED. It is specifically selected for treatment because it has a response marker. Similarly, "it is selectively administered." "From a larger patient population, if that particular patient meets a predetermined criterion, for example, a specific gene mark" Specially selected based on (or resulting from) having Kerr or other biological markers This refers to administering drugs to a patient. It also means to select, treat selectively, and administer selectively. This means that standard treatment regimens are not delivered based solely on the fact that the patient has DED. Rather, it means that personalized therapy is delivered to patients based on their specific biological characteristics. When used in the specification, the selection of a treatment method refers to a method having a DED response marker. Rather than referring to the accidental treatment of a patient, it refers to the patient having DEDI response markers. This refers to the careful selection of administering TNFα antagonists to patients based on the following criteria. Therefore, selective treatment is the careful selection of TNFα antagonists. This differs from standard treatment, which involves delivering a specific drug to all patients regardless of their allergy status.

[0052] As used herein, "predict" means that an individual having DED has a TNFα-binding molecule The possibility of responding to treatment by or better than by medical Information to enable healthcare providers to make decisions is provided in the manner described herein. This indicates that the response can be predicted with 100% accuracy. Instead, those skilled in the art will understand that this refers to an increase in probability.

[0053] When used herein, "possibility" and "potential" mean that an event may occur. It is a measure of how likely something is to happen. It can be used synonymously with "probability." Possibility refers to a probability that exceeds speculation but does not reach the level of certainty. Therefore, common sense, training, or experience A rational person using experience can conclude that a certain event is likely, given the circumstances. In some cases, an event is possible. In some embodiments, once the possibility is confirmed, the patient The patient may be treated with a TNFα-binding molecule (or treatment may be continued, or treatment may be increased in dosage). (to be moved to the additional stage), or the patient does not need to be treated (or treatment is interrupted, or treatment is more (Transition to a lower dose.)

[0054] The phrase "increased probability" refers to an increase in the likelihood that a certain event will occur. For example, Some methods described herein may indicate that patients may respond to treatment with TNFα-binding molecules. An increase in TNFα-binding molecules compared to DED patients without DED response markers or an increase in TNFα-binding molecules It is possible to predict whether there will be an increased likelihood of a better response to treatment.

[0055] As used herein, "SNP" refers to "single nucleotide polymorphism." Single nucleotide polymorphisms are genomes. A single nucleotide in (or other shared sequences) between members of a biological species or individuals These are DNA sequence mutations that occur when there is a difference between the paired chromosomes in a DNA sequence. Most SN P has only two alleles, and usually one is more common than the other in the population. SNPs are genes Exons or introns, upstream or downstream untranslated regions of genes, or simply genomic locations ( It can exist in the coding region of a gene (i.e., it is not transcribed). This can be silent due to redundancy in the genetic code (i.e., synonymous polymorphism), and SNPs can also cause changes in the sequence of the encoded polypeptide (i.e., non-synonymous). (Polymorphism). In this disclosure, SNPs are referred to by their single nucleotide polymorphism database (dbSNP) rs number, e.g. For example, it is identified by "rs1800693". dbSNPs are identified by the National Biotechnology Institute. - Information Center (NCBI: National Center for Biotech) (Natural Information) National Human Genome Institute (NHGRI:Nat ional Human Genome Research Institute) In the free public archives of various intraspecific and interspecific genetic variations developed and operated by the same organization, be.

[0056] Polymorphic regions such as SNPs usually have conserved sequences before and after them in the genome of the target population. Therefore, the location of polymorphic sites is often, in the case of SNPs, generally "SNP context arrangement". The consensus nucleic acid sequence (for example, 30-60 nucleos) surrounding the polymorphic region is called a "sequence." It can be determined based on the context sequence of the SNP disclosed herein. Therefore, the NCBI is available at www.ncbi.nlm.nih.gov / snp. You can refer to an SNP database. Alternatively, the location of polymorphism sites can be found in genes, mRNA transcripts. , relative to the origin of the BAC clone, or even to the start codon (ATG) of protein translation It can also be identified by its position on the corresponding reference sequence (e.g., GeneBank deposit). Those skilled in the art will know that the location of a specific polymorphic site in each individual within the target population corresponds to the consensus sequence. Or the presence of one or more insertions or deletions in the genome of the individual when compared to a reference sequence. This may result in the reference sequence or context sequence not being located at the exact same position. To understand the identity of the selective allele at the polymorphic site to be detected, and its A person skilled in the art is provided with either or both of the reference sequence and / or context sequence in which the polymorphic region exists. When this is done, robust for detecting selective alleles of polymorphic sites in any given individual. Designing specific and accurate assays is routine for those skilled in the art. Therefore Those skilled in the art can determine the relationship between the reference sequence or context sequence and the start codon of such sequence. (By referring to a specific location, the location of any polymorphic region described herein is specified The determination is merely for convenience, and the literal, arbitrary, specifically named nucleos The cydoid position is known in the gene typing method described herein or in the art. The gene markers of the present invention were tested using any of the other gene typing methods. Any nucleotide position in which the same polymorphic region is actually located at the same gene locus in any individual They will understand that this also includes placement.

[0057] In addition to SNPs, gene polymorphisms include gene enhancers, exons, introns, and protons. This includes translocations, insertions, substitutions, and deletions occurring in motors, 5'UTR, 3'UTR, etc.

[0058] When used herein, "rs1800693" refers to tumor necrosis factor receptor 1 (TNF The human tumor necrosis factor receptor superfamily, also known as R1, member 1A(T The sixth of the NFRSF1A) gene (GenBank accession number NM_001065.3) This refers to T / C SNPs located within the intron region. The TNFRSF1A protein is the major one. It is one of the TNFα receptors, is involved in the NF-κB pathway, and mediates apoptosis. It also regulates inflammation. The rs1800693 polymorphism site is located on chromosome 12:6330843. The term "rs1800693 response allele" is used herein, and when used in this specification, it refers to rs18 The "C" allele (or "G" allele in the case of a non-coding chain) or "T" allele located at the 00693 polymorphism site. (In the case of a non-coding strand, this refers to the A allele.) Methods, uses, and embodiments of the kits disclosed herein In this state, the patient has at least one rs1800693 response allele.

[0059] The aforementioned response alleles are useful for predicting the response of DED patients to TNFα antagonism. In some embodiments, DED patients with CC, CT, or TT genotypes are TN It may respond to treatment with Fα antagonists, such as TNFα antibodies like LME636. It is considered to be.

[0060] As those skilled in the art will recognize, nucleic acid samples containing a specific SNP are complementary double-stranded molecules, Often, therefore, references to specific sites on the sense strand are similar to references on the complementary antisense strand. This refers to the region in question. Similarly, for SNPs on both copies of one strand of a chromosome. References to specific genotypes obtained are obtained for the same SNP on both copies of the other strand. It is equivalent to the complementary genotype. Therefore, for example, rs1800693 on the coding strand The T / C genotype for the polymorphic region is determined by the A / G genotype for the polymorphic region on the non-coding strand. It is equivalent to the gene type.

[0061] As used herein, “genome sequence” refers to the DNA sequence present in the genome. A region within a rell, the allele itself, or a chromosome containing the allele of interest. ) contains a larger DNA sequence.

[0062] The products of DED response markers include nucleic acid products and polypeptide products. "Petido products" are polypeptides encoded by DED response markers (markes). This refers to the DNA of the DED response marker (e.g., geno). (e.g., pre-mRNA, mRNA, miRNA, etc.) It refers to an object or a fragment thereof.

[0063] "Equivalent gene markers" refer to gene markers that are interrelated with the target allele. For example, it may exhibit linkage disequilibrium (LD) with the target allele, or it may be gene-linked. The determination of whether a patient has a DED response marker involves examining a biological sample from the patient for alleles. Rather than directly investigating the gene itself, equivalent genetic markers may be used. There are various programs that assist in determining LDs for SNPs, such as HaploB. lock(bioinfo.cs.technion.ac.il / haplobloc (Available in k / ), HapMap, and WGA Viewer are available.

[0064] The term "probe" refers to a substance that specifically detects another substance, such as a substance related to a DED response marker. This refers to any substance composition useful for detection. A probe is a DED response marker, or D Oligonucleotides that specifically hybridize to the genome sequence of nucleic acid products of ED response markers It may also be a cytoplasm (including conjugated oligonucleotides). Gonucleotides are highly specific to receptor molecules (for example, antibodies specific to their antigens). Oligonucleotides covalently bound to a chromophore or molecule containing a specific ligand (e.g., an antigen). It refers to Otid. The probe is also used to amplify specific regions within the DED response marker. For example, it may be a PCR primer together with another primer. Furthermore, the probe is These may be antibodies that specifically bind to the polypeptide products of these alleles. Furthermore, pro The marker detects an equivalent genetic marker to the DED response marker (e.g., one that binds to or harbors a marker that binds to it). It may be any material composition having the ability to bleed. In a preferred embodiment, In this case, the probe specifically detects the nucleic acid sequence (preferably genomic DNA) of the target allele. It either breeds or specifically binds to polypeptide sequences.

[0065] The phrase "specifically hybridizes" refers to stringent hybridization. It is used to refer to hybridization under certain conditions. The triggering conditions are known to those skilled in the art, and Current Protocols i n Molecular Biology,John Wiley & Sons,N. See Y. (1989), 6.3.1-6.3.6. This document contains aqueous solutions. A non-aqueous method is also described, and either can be used. An example of an ibridization condition is 6 × sodium chloride / sodium citrate (SSC Hybridization at approximately 45°C, followed by 0.2×SSC and 5 in 0.1% SDS. At least one wash at 0°C. Stringent hybridization conditions. The second example involves hybridization in 6×SSC at approximately 45°C, followed by 0.2×SS C, at least one wash at 55°C in 0.1% SDS. Stringent high Another example of hybridization conditions is hybridization in 6×SSC at approximately 45°C. This is followed by at least one wash in 0.2×SSC and 0.1%SDS at 60°C. Further examples of trigger hybridization conditions include those in 6×SSC at approximately 45°C. Hybridization followed by 0.2×SSC, at least in 0.1% SDS at 65°C. This is a single wash. For high-stringent conditions, use 0.5M sodium phosphate, 7% Hybridization in SDS at 65°C, followed by 0.2×SSC in 1% SDS at 65°C This includes at least one wash at °C.

[0066] The term "region of nucleic acid" is used to refer to a small sequence within a larger nucleic acid sequence. For example, a gene is a region of a chromosome, and an exon is a region of a gene.

[0067] In the context of polypeptides, the term "specifically binds" means that the probe binds to an undesirable polypeptide. Rather than randomly binding to cydops, it targets a given polypeptide (e.g., DED response). It is used to mean binding to the polypeptide product of the marker. However, "Specifically binding" means that the probe provides a useful measurement of the presence of a given polypeptide target. The ability to cross-react with undesirable polypeptides is not hindered by the cross-reactivity in question. Do not exclude reactivity.

[0068] The term "to have the ability" is used to mean that a given result is achievable, for example. A probe that has the ability to detect the presence of a specific substance is one that uses that probe to identify This means that the substance can be detected.

[0069] "Oligonucleotides (oliogonucelotide)" are short, for example, 2~ This refers to a nucleotide sequence of 100 base pairs.

[0070] The term "biological sample" as used herein refers to identification, diagnosis, prediction, or monitoring. This refers to patient samples that can be used for hysterosalpingography. Preferred samples include synovial fluid, blood, and blood-derived materials. Products (buffy coat, serum, and plasma, etc.), lymph, urine, tears, saliva, hair follicle cells, Cerebrospinal fluid, buccal swab, feces, synovial fluid, synovial cells, sputum, or tissue sample (e.g., cartilage sample) Examples include: In addition, those skilled in the art will know that some samples, after fractionation or purification procedures, are derived from, for example, whole blood. You will understand that the analysis can be much easier once the DNA has been isolated.

[0071] TNFα antagonists The various pharmaceutical compositions, regimens, processes, uses, methods, and kits disclosed are TNF Alpha-antagonists, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding moieties, for example) LME636) or a TNFα receptor binding molecule (e.g., a TNFα receptor antibody or its antigen binding molecule) Use the combined portion.

[0072] In one embodiment, a TNFα antagonist is used, for example, a TNFα-binding molecule (e.g., a TNFα antibody). Or its antigen-binding portion, for example LME636), is the hypervariable region CDRH1, CDRH2 and and at least one heavy chain variable domain (V) containing CDRH3 H ) including the CDRH1 The first amino acid sequence has sequence number 1, and the second amino acid sequence has sequence number 2. , and the CDRH3 has the amino acid sequence SEQ ID NO: 3. In one embodiment, TN Fα antagonists, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding portions, e.g., LME636) includes at least the ultra-variable regions CDRL1, CDRL2 and CDRL3. Another light chain variable domain (VL ) includes, and the CDRL1 is the amino acid sequence SEQ ID NO: 4 CDRL2 has the amino acid sequence SEQ ID NO: 5, and CDRL3 has the amino acid sequence SEQ ID NO: 5. It has the no-acid sequence, sequence number 6.

[0073] In one embodiment, a TNFα antagonist is used, for example, a TNFα-binding molecule (e.g., a TNFα antibody). Or its antigen-binding portion, for example, LME636) is V H Domain and V L Including the domain, Here: a)V H The domain is (for example in the sequence): i) hypervariable region CDRH1, CDRH It comprises 2 and CDRH3, wherein CDRH1 has the amino acid sequence SEQ ID NO: 1, and the CD RH2 has amino acid sequence number 2, and the aforementioned CDRH3 has amino acid sequence number 2 Item 3 has; and b) V L The domain is the hypervariable region CDRL1, CDR (for example, within the sequence). It includes L2 and CDRL3, and the CDRL1 has the amino acid sequence SEQ ID NO: 4, and the C DRL2 has amino acid sequence sequence number 5, and CDRL3 has amino acid sequence sequence It has the number 6.

[0074] In one embodiment, a TNFα antagonist is used, for example, a TNFα-binding molecule (e.g., a TNFα antibody). Or its antigen-binding portion, for example LME636), is a) an amino acid shown as SEQ ID NO: 8. Acid sequence-containing heavy chain variable domain (V H );b) Amino acid sequence shown as Sequence ID No. 10 Light chain variable domain (V L );c)V containing the amino acid sequence shown as Sequence ID No. 8 H V containing the domain and the amino acid sequence shown as SEQ ID NO: 10 L Domain; d) Array V includes the hypervariable region indicated as number 1, sequence number 2, and sequence number 3. H domain; e ) V including the hypervariable region shown as Sequence ID 4, Sequence ID 5 and Sequence ID 6 L domain ; or f) V including the hypervariable region shown as Sequence ID 1, Sequence ID 2, and Sequence ID 3 H Includes a domain and a hypervariable region indicated as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. V L Includes the domain.

[0075] For easier reference, the amino acid sequence of the hypervariable region of the LME636 scFv antibody is as follows: Provided in Table 2.

[0076] [Table 2]

[0077] In some embodiments, a TNFα antagonist is used, for example, a TNFα-binding molecule (e.g., TNFα The antibody or its antigen-binding moiety (e.g., LME636) contains the light chain of SEQ ID NO: 7. Other implementations Morphologically, the TNFα antagonist contains the heavy chain of SEQ ID NO: 8. In other embodiments, TN The Fα7 antagonist comprises the light chain of SEQ ID NO: 7 and the heavy chain of SEQ ID NO: 8. In some embodiments, In other embodiments, the TNFα antagonist contains three CDRs of SEQ ID NO: 7. The α-antagonist contains three CDRs of SEQ ID NO: 8. In other embodiments, the TNFα antagonist is Includes three CD-Rs with sequence number 7 and three CD-Rs with sequence number 8. Sequence number 7 and sequence number The CDR for No. 8 is shown in Table 2. In other embodiments, the TNFα antagonist is the sequence of SEQ ID NO: 9. Includes: [ka]

[0078] The hypervariable domain can be associated with any kind of framework domain, but preferably of human origin. The preferred framework area is as described above by Kabat EA et al. It is included. The preferred heavy chain framework is LME63 as shown in Sequence ID No. 10. This is a heavy chain framework of 6 antibodies: [ka]

[0079] The preferred light chain framework is the LME636 antibody as shown in SEQ ID NO: 11. It is a light chain framework: [ka]

[0080] When used in the sequence of sequence numbers 10 and 11, (X) n=3-50 is CD Represents R

[0081] In one embodiment, a TNFα antagonist is used, for example, a TNFα-binding molecule (e.g., a TNFα antibody). Or its antigen-binding portion, for example LME636), a) has a hypervariable region CDRH1 in its sequence, The first domain includes CDRH2 and CDRH3 (the aforementioned CDRH1 is an amino acid sequence) CDRH2 has the number 1, and CDRH3 has the amino acid sequence SEQ ID NO: 2, and CDRH3 (b) has the amino acid sequence sequence number 3); and (b) hypervariable regions CDRL1, CDRL2 and The second domain containing CDRL3 (the CDRL1 has amino acid sequence number 4) CDRL2 has the amino acid sequence SEQ ID NO: 5, and CDRL3 has the amino acid sequence SEQ ID NO: 5 having column array number 6); and c) either the N-terminal end of the first domain and the C-terminal end of the second domain or the C-terminal end of the first domain and the N-terminal end of the second domain selected from single-chain binding molecules comprising an antigen-binding site comprising a peptide linker that binds thereto.

[0082] Alternatively, the TNFα antagonist used in the method of the present disclosure, such as a TNFα binding molecule (e.g., a TNFα antibody or an antigen-binding portion thereof), may comprise a derivative of the TNFα binding molecule shown by the sequence (sequnence) herein (e.g., a pegylated version of LME636). Alternatively, the TNFα antagonist used in the method of the present disclosure, such as a TNFα binding molecule (e.g., a TNFα antibody or an antigen-binding portion thereof), may have a V H or V L domain that is substantially identical to the V shown herein or V H or V L domain (e.g., those shown in SEQ ID NO: 8 and 7). The anti-TNFα antibody disclosed herein may have a V H or V L domain that is substantially identical thereto. The anti-TNFα antibody disclosed herein may comprise a heavy chain that is substantially identical to that shown as SEQ ID NO: 8 and / or a light chain that is substantially identical to that shown as SEQ ID NO: 7. The anti-TNFα antibody disclosed herein may comprise a heavy chain comprising SEQ ID NO: 8 and a light chain comprising SEQ ID NO: 7. The anti-TNFα antibody disclosed herein may comprise: a) one heavy chain comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO: 8 and a constant portion of a human heavy chain; and b) one light chain comprising a variable domain having an amino acid sequence substantially identical to that shown in SEQ ID NO: 7 and a constant portion of a human light chain. Alternatively, the TNFα antagonist used in the method of the present disclosure, such as e.g., or, may comprise For example, a TNFα-binding molecule (e.g., a TNFα antibody or its antigen-binding portion) is as shown herein. These may also be amino acid sequence variants of the reference TNFα-binding molecule. Such derivatives and mutations In all cases, TNFα antagonists were present at approximately 50 nM or less, approximately 20 nM or less, and approximately 10 nM. The following applies to concentrations of the aforementioned molecules of approximately 5 nM or less, or more preferably approximately 3 nM or less, at a concentration of approximately 1 nM (= It has the ability to inhibit the activity of human TNFα at 30 ng / ml by 50%, and the said inhibitory activity is For example, Chiu et al.,2011,PLoS ONE,Vol 6,issu As described in e 1,e16373, it is involved in neutralizing TNFα cytotoxicity of L929 cells. It is measured by performing an assay.

[0083] This disclosure also includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CD. RL3, or one or more amino acid residues of the framework, typically just a few (e.g.) For example, 1 to 4 mutations in the corresponding DNA sequence, such as site-directed mutagenesis. TNFα antagonists that are altered by the development of TNFα, for example, TNFα-binding molecules (e.g., TNFα antibodies) Or it may also include its antigen-binding moiety, e.g., LME636). This disclosure relates to such altered T It contains DNA sequences that encode NFα antagonists.

[0084] This disclosure also relates to TNFα antagonists that have binding specificity to human TNFα, such as TN Fα-binding molecule (e.g., TNFα antibody or its antigen-binding portion, e.g., LME636), details It contains a TNFα antibody that has the ability to inhibit the binding of TNFα to its receptor, and approximately 50 nM The following are approximately 20 nM or less, approximately 10 nM or less, approximately 5 nM or less, or more preferably approximately 3 nM or less. The following molecules inhibit the activity of 1 nM (=30 ng / ml) human TNFα by 50% at the concentration of the aforementioned molecule. A TNFα antibody that has the ability to do so (the aforementioned inhibitory activity is among the TNFα cytotoxicity of L929 cells) This also includes (measured by assaying the sum).

[0085] In a preferred embodiment, the anti-TNFα anti-TNFα used in the methods, uses, kits, etc. of the disclosed herein The substance is LME636, which contains the sequence of sequence number 9. LME636 is human TNF It inhibits α and is recombinantly produced in Escherichia coli (E. coli) using standard expression techniques. Humanized monoclonal scFv consisting of 254 amino acids (molecular mass: 26.7 kDa) This is an antibody fragment. This molecule is formed by a mobile amino acid sequence consisting of glycine and serine. Variable light and heavy chains of covalently linked monoclonal rabbit anti-human TNFα antibodies. Complementarity-determining regions (CDRs) and specific framework residues from the region sequence are derived from the human light chain and It was genetically engineered by grafting it onto a heavy chain variable region framework.

[0086] In one embodiment, methionine derived from the start codon in the expression vector is If it is not cleaved after translation, it is present in the final protein. In that case, LME 636 has the sequence of sequence number 12: [ka]

[0087] The methods, kits, and other preferred TNFα antibodies used in the methods, kits, and other uses disclosed herein are internationally published. Pamphlet No. 2009 / 155723 and International Publication Pamphlet No. 2012 / 051734 This is what is shown in Let.

[0088] Assay techniques, diagnostic methods, and methods for creating information in a transmissible form The methods of the present disclosure are useful for predicting the responsiveness of DED patients to treatment or improvement, and to treatment with TNFα antagonists, such as LME636 These methods are particularly useful for determining whether a patient has a DED response marker in a sample from the patient using .

[0089] Biological samples from patients may be assayed for the presence of DED response markers by any applicable conventional means, and the means will be selected according to whether the particular marker is within the range of any of exons, introns, non-coding portions of mRNA or non-coding genomic sequences .

[0090] Numerous biological samples, such as blood, synovial fluid, buffy coat, serum, plasma, lymph, feces , urine, tears, saliva, cerebrospinal fluid, buccal swabs, sputum, or tissue, are used to identify the presence of allergens or proteins, the expression levels of genes or proteins, and the activities of proteins . In the methods of the present disclosure, various sources within the biological sample can be used, for example the genomic DNA obtained from the biological sample may be assayed to detect DED response markers , or the products of DED response markers obtained from the biological sample, such as nucleic acid products (e.g., DNA , pre-mRNA, mRNA, microRNA, etc.) and polypeptide products (e.g., expressed [[ID=3�]]proteins) may be assayed .

[0091] The inventors have determined that various SNP alleles in Table 1 are useful for predicting the responsiveness of a particular patient to treatment with TNFα antagonism (e.g using LME636 ​In a preferred embodiment, to determine whether the subject has a DED response marker Therefore, the genomic sequence of the DED response marker is analyzed.

[0092] As described in the examples, our latest findings are in SNP rs1800693. The presence of related genotypes affects TNFα antagonism against DED (e.g., LME636). This leads to the conclusion that DED response markers may be useful in predicting improvements in response. Its presence can be detected by various gene typing techniques. Typically, such gene typing The Iping technique involves identifying the region containing the target polymorphic site (e.g., SNP), or the region adjacent to it. One or more oligonucleotides complementary to the target are used. The inheritance of a specific polymorphic region is targeted. The sequence of oligonucleotides used for subtyping is typically a context sequence or It is designed based on the reference array.

[0093] Numerous methods and devices are available for identifying the presence of DED response markers. Methods well known in the technical field, such as phenol / chloroform extraction, GentAS S Purification of PUREGENE DNA (registered trademark) from ystems (Qiagen, CA) The system extracts DNA (genomic DNA and cDNA) for SNP detection from biological samples. It can be manufactured. For DNA sequence detection, the sense strand or antisense strand within the region is used. This may involve examining one or more nucleotides located in any of the nucleotide chains. The presence of polymorphism in this region indicates that a sequence-specific probe, such as a hydrolysis probe from Taqman, is needed. Beams, beacons, scorpions; or high-speed devices for detecting markers or polymorphisms. DNA obtained by PCR using a bridization probe (genomic DNA or It may be detected from cDNA. For the detection of polymorphisms, sequence-specific probes are used, as they are intended. It specifically hybridizes with the allele's genomic DNA, or, in some cases, the target RNA. Primers and probes for polymorphic sites (e.g., SNPs) can be found at www. NCBI SNP database available at ncbi.nlm.nih.gov / snp These probes may be designed based on the context sequences listed in [the relevant document]. Secondary detectable molecules that may be labeled for contact detection or that specifically bind to the probe. Contact may be made. PCR products can also be detected by DNA binding agents. Next The PCR product is then subjected to any DNA sequencing method available in the art. This allows for sequencing. Alternatively, the presence of alleles is not limited, Sanger method-based sequencing, pyrosequencing, or next-generation sequencing It can be detected by sequencing using any sequencing method (Sh endure J.and Ji,H.,Nature Biotechnology( 1998), Vol.26, Nr 10, pages 1135-1145). SNP The optimized allele identification assay is from Applied Biosystems (Fost You can also purchase it from (Er City, California, USA).

[0094] To investigate specific polymorphisms (e.g., SNPs), for example, hybridization-based Methods, for example, dynamic allele-specific hybridization (DASH) gene typing PING, detection of polymorphic sites (e.g., SNPs) by molecular beacons (Abravaya K., et al.(2003)Clin Chem Lab Med.41:468-474 ), Luminex xMAP technology (registered trademark), Illumina Golden G ate® technology and commercially available high-density oligonucleotide SNP arrays (e.g., A ffymetrix Human SNP5.0 GeneChip (registered trademark) is 5 Genome-wide typing capable of gene typing over 00,000 human SNPs (Implementing the Sei), BeadChip (registered trademark) kit from Illumina, for example Human660W-Quad and Human 1.2M-Duo); enzyme-based Methods such as restriction fragment length polymorphism (RFLP), PCR-based methods (e.g., tetraprimer) ARMS-PCR), Invader assay (Olivier M. (2005) Mut at Res.573(1-2):103-10), various primer elongation assays (test Output formats, e.g., MALDI-TOF mass spectrometry, electrophoresis, blotting. , and incorporated into ELISA-like methods), TaqMan® assay, and Ligonucleotide ligase assays; and other post-amplification methods, e.g., single-strand conformation Polymorphism analysis (Costabile et al. (2006) Hum. Mutat.27 (12):1163-73), temperature gradient gel electrophoresis (TGGE) denture gel electrophoresis, modified high-performance liquid chloroform Mathematics, high-resolution melting analysis, DNA mismatch binding protein assays (e.g.) MutS protein from Thermus aquaticus It binds to different single nucleotide mismatches with different affinities, and capillary electrophoresis (When used, it can distinguish all 6 mismatch sets), SNPLex ( (Registered trademark) (Exclusive SNP detection system available from Applied Biosystems) Tem), capillary electrophoresis, mass spectrometry, and various sequencing methods, for example By applying various techniques, including pyrosequencing and next-generation sequencing, Yes, it is possible. A commercially available kit for SNP gene typing is, for example, Fluidigm D. Dynamic Array (registered trademark) IFC (Fluidigm), TaqMan (registered trademark) (Registered Trademark) SNP Gene Typing Assay (Applied Biosystems) MassARRAY(registered trademark) iPLEX Gold (Sequenom), Type Examples include the -it Fast(registered trademark) SNP probe PCR kit (Quiagen). It is possible.

[0095] In some embodiments, the presence of polymorphic sites (e.g., SNPs) in the patient is a hybrid It is detected using a dilation assay. In a hybridization assay, The presence of a genetic marker indicates that the nucleic acid from the sample is complemented by a nucleic acid molecule, such as an oligonucleotide progenitor. Determined based on the ability to hybridize with various hybridization processes. A sample is available. In some cases, the hybridization of the probe to the target sequence is performed. The bound probe can be visualized, for example, and directly detected by Northern or Southern assays. These assays isolate either DNA (Southern) or RNA (Northern). Next, DNA or RNA that is frequently cleaved in the genome and then assayed as a marker. It is then cut by a series of restriction enzymes that do not cleave in any of the vicinity of the DNA or RNA. For example, it is separated on an agarose gel and transferred to a membrane. One or more labeled probes, for example radionucleotides or binders (e.g., SYBR) (Registered Trademark) Green) is used to create low, medium, or high strings. The membrane is brought into contact under gentle conditions. Unbound probes are removed, and the labeled probes become visible. The presence of binding is detected by chemical reaction. In some embodiments, an array, such as MassARR, is used. AY (registered trademark) system (Sequenom, San Diego, California) The subject may be genetically typed using ia (USA).

[0096] Even if conventional gene typing methods are modified for use in gene typing, Such conventional methods include, for example, DNA amplification techniques such as PCR and its variations, and dyes. Rectosequencing combined with Luminex xMAP® technology Examples include O hybridization, SSP typing, and SBT.

[0097] Sequence-Specific Oligonucleotides (SSO) igonucleotide typing involves PCR target amplification and immobilization onto beads. Hybridization of PCR products into a panel of sequence-specific oligonucleotides, Detection of probe-coupled amplification products by color development and subsequent data analysis are used. Those skilled in the art will understand this. The sequence-specific oligonucleotide (SSO) hybridization described is One Provided by Lambda, Inc. (Canoga Park, CA) or Lifecodes HLA Typing Kit (Tepnel Life Science) Luminex (registered trademark) technology (Luminex, Corp.) It can also be implemented using various commercially available kits, such as in combination with ation (TX). You will understand that. LABType(registered trademark)SSO is a sequence-specific oligonucleotide. Identify HLA alleles using creotide (SSO) probes and color-coded microspheres. This is a reverse SSO (rSSO) DNA typing solution. The target DNA is polymerized. The reaction is amplified by a enzyme chain reaction (PCR), and then hybridized with a bead probe array. This assay is performed in a single well of a 96-well PCR plate; therefore, It can process 96 samples at once.

[0098] Sequence-Specific Primers (SSPs) s) Typing is a PCR-based technique, unlike DNA-based typing which uses sequence-specific techniques. Use primers. The SSP method, under controlled PCR conditions, completely matches the target sequence. This is based on the principle that only primers with matching sequences will produce amplification products. Sequence-specific primer pairs selectively target a target sequence specific to a single allele or allele population. It is designed for amplification. The PCR product can be visualized on an agarose gel. A control primer pair matching a non-allelic sequence present in all samples improves PCR amplification efficiency. It serves as an internal PCR standard for verification. Those skilled in the art will understand the sequence-specific plastic described. Low, moderate, and high resolution gene typing by imer typing, Oler up SSP(trademark) kit (Olerup, PA) or (Invitrogen) or Allset and(TM) Gold DQA1 Low Resolution SSP (Invitro You will understand that this can be done using various commercially available kits, such as gen. .

[0099] Sequence-based typing (SBT) is, This method is based on PCR target amplification, followed by sequencing and data analysis of the PCR product.

[0100] In some cases, RNA, such as mature mRNA and premRNA, also exhibits the presence of specific polymorphisms. It can be used to determine (see Table 1). mR transcribed from a given gene Sequence analysis of NA is not limited to Northern blot analysis, nuclease protection analysis, etc. NPA (Polymerase-Assisted Aspiration), In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction ( RT-PCR, RT-PCR ELISA, TaqMan-based quantitative RT-PCR (Probe-based quantitative RT-PCR) and SYBR green-based quantitative RT-PCR This can be carried out using any known method in the relevant technical field, including CR. In one example, mRNA level detection involves isolated mRNA and a DED response marker. Therefore, oligonucleotides that can hybridize to the encoded mRNA This involves contact. Nucleic acid probes are typically, for example, full-length cDNA, or Oligonucleotides with a length of at least 7, 15, 30, 50, or 100 nucleotides, etc. under stringent conditions, it is sufficient to specifically hybridize to mRNA. It may be partial. Hybridization of mRNA and probe is the marker in question. This indicates that - is being expressed. In one format, RNA is immobilized on a solid surface, for example For example, isolated RNA is flowed onto an agarose gel, and the mRNA is removed from the gel by a membrane such as nitrocellulose. By transcribing, the probe is brought into contact with the gene. The amplification primer is the 5' or Annealing to the 3' region (positive and negative chains, or vice versa), with short regions in between. It is defined as a pair of nucleic acid molecules that can contain a region. Generally, amplification primers are approximately It is 10 to 30 nucleotides long and adjacent to a region of approximately 50 to 200 nucleotides long. Under appropriate conditions and with suitable reagents, such a primer will produce a nucleus adjacent to the primer. It is possible to amplify nucleic acid molecules containing the creotide sequence. PCR products are not limited to However, gel electrophoresis and staining with DNA-specific stains or hybridization to labeled probes are not performed. Detection can be performed by any preferred method, including dilation.

[0101] In some cases, the presence of polymorphism in patients affects the polypeptide products of DED response markers. This can be determined by analysis (see Table 1). Detection of polypeptide products is limited. Although not performed, immunocytochemical staining, ELISA, flow cytometry, and Western blot are used. Any method in the art, including lot analysis, spectroscopy, HPLC, and mass spectrometry. This can be carried out using publicly known methods.

[0102] One method for detecting polypeptide products in a sample involves specifically comparing them with marker proteins. This method uses a probe, which is a binding protein (e.g., an antibody) that has the ability to interact with the target. Preferably, a labeled antibody, its binding site, or other binding partner may be used. The body may be of monoclonal or polyclonal origin, or may be biosynthetically produced. The binding partner may also be a naturally occurring molecule or a synthetically produced molecule. This may be done. The amount of complexed protein is the standard amount described in the art. Determined using a protein detection method. Immunological assay design, theory and protocol For a detailed review, see Practical Immunology, Butt, Including WR, ed., Marcel Dekker, New York, 1984, Numerous texts in this technical field can be referenced. Proteins by labeled antibodies Various assays are available for detecting the substance. Direct labeling involves attaching an antibody to the firefly. This includes light or luminescent tags, metals, dyes, radionuclides, etc. Indirect labeling can be performed using alkaline phosphatase, hydrogen peroxidase, etc., as described in the technology. It contains various enzymes that are well known in the field. In a one-step assay, if present, polyps The butyl product is immobilized and incubated with the labeled antibody. The antibody binds to the sample. After washing to remove unbound molecules, the sample is assayed for labeling. It can be done.

[0103] The use of immobilized antibodies specific to proteins or polypeptides is also intended by this disclosure. The antibody is then used to detect magnetic or chromatographic matrix particles, or the surface of the assay site (matrix). Microtiter wells, etc., solid substrate material pieces (plastic, nylon, paper, etc.) It can be immobilized on various solid supports. An antibody or multiple antibodies can be immobilized on a solid support. Assay strips can be prepared by coating them in a layered manner. This strip is then immersed in the test sample and quickly processed through the washing and detection steps. This can generate measurable signals, such as colored spots.

[0104] In the two-step assay, the polypeptide product of the immobilized DED response marker was found to be unlabeled. The body may be incubated with the antibody. Then, if present, the unlabeled antibody complex will be incubated with the unlabeled antibody. It binds to a secondary labeled antibody that is specific to the labeled antibody. The sample is washed, and the presence of the label is checked. The choice of marker used for antibody labeling will vary depending on the application. However, the selection of the marker is easily determined by those skilled in the art. Antibodies are radioactive. It may be labeled with a sex atom, enzyme, chromophore or fluorescent moiety, or a colorimetric tag. Tag-adding label The choice will also depend on the desired detection limit. Enzyme assays (ELISA) are Typically, these are formed by the interaction between the enzyme-tagged complex and the enzyme substrate. This enables the detection of colored products. Some examples of radioactive atoms include: 32 P, 125 I, 3 H , and 14 P is one example. Some examples of enzymes include horseradish peroxidase, and A Lucalyphosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase Examples include enzymes. Examples of chromophore components include fluorescein and rhodamine. For example, antibodies are conjugated to these labels by methods known in the art. They may be used. For example, the enzyme and chromophore molecules may be dialdehyde, carbodiimide, di The antibody may be conjugated using a coupling agent such as maleimide. Alternatively, Conjugation may be carried out by ligand-receptor pairs. Some suitable ligands Examples of receptor pairs include biotin-avidin or biotin-streptavidin, and antibodies. - Antigens are mentioned.

[0105] In one embodiment, the present disclosure relates to a sandwich technique for detecting polypeptide products in biological samples. The technique involves the use of two antibodies that have the ability to bind to the target protein. For example, one is immobilized on a solid support and the other is free in a solution. It is labeled with some easily detectable chemical compound. Examples of scientific labels include, but are not limited to, radioisotopes, fluorescent compounds, and enzymes or similar substances. Other products that change color or become electrochemically active upon exposure to the reactant or enzyme substrate. The molecules that are produced are listed below. When a sample containing polypeptide products is placed in this system... The polypeptide product binds to both the immobilized antibody and the labeled antibody. This result is reflected in the support surface. This is a "sandwich" immune complex on the surface. Unbound sample components and excess labeled antibodies are washed away. By running the sample and measuring the amount of labeled antibody complexed with proteins on the support surface, multiple Combined proteins are detected. Sandwich immunoassays are highly specific and extremely... It shall be highly sensitive, provided that appropriate detection limit labels are used.

[0106] Preferably, the presence of polypeptide products in the sample is detected by radioimmunoassay or enzyme-linked immunoassay. Standard methods, competitive enzyme-linked immunosorbent assay, dot blot, Western blot, chromatography Graphing, preferably high-performance liquid chromatography (HPLC), or in the art It is detected by other known assays. Characteristics of antibodies against proteins or polypeptides Heteroimmunological binding can be detected directly or indirectly.

[0107] Dot blotting uses antibodies as probes to detect desired proteins. Therefore, it is routinely done by those skilled in the art (Promega Protocols a nd Applications Guide,Second Edition,199 1, Page 263, Promega Corporation). Dot blot binding. The sample is applied to the membrane using a device. The labeled probe is incubated with the membrane, and then the tan The presence of protein was detected.

[0108] Western blot analysis is well known to those skilled in the art (Sambrook et al., M Olecular Cloning,A Laboratory Manual,198 9,Vol.3,Chapter 18,Cold Spring Harbor La (boratory). In Western blotting, the sample is separated by SDS-PAGE. The gel is transferred to a membrane. The membrane is incubated with the labeled antibody to extract the desired protein. A substance was detected.

[0109] The assays described above are not limited to immunoblotting and immunodiffusion methods. This includes steps such as immunoelectrophoresis or immunoprecipitation. In some embodiments, automated analysis is performed. The presence of DED response markers is determined using a device.

[0110] This specification describes the presence or absence of DED response markers in biological samples from patients. Patients with DED, including detection, will respond to treatment with TNFα antagonists. A method for predicting the likelihood of this is disclosed, where: a) the presence of a DED response marker indicates that the patient is a) Increased likelihood of responding to treatment with TNFα antagonists; and b) DED response The absence of the marker reduces the likelihood that the patient will respond to treatment with TNFα antagonists. This indicates.

[0111] In some embodiments, the method further includes the step of obtaining a biological sample from a patient. The steps described here are performed before the assay steps.

[0112] In some embodiments, the DED response marker is used to analyze the biological sample and the nucleus of the DED response marker. Acid products, polypeptide products of DED response markers, or equivalent genes of DED response markers It is detected by assaying with respect to the child marker. In some embodiments, DE D response markers are used to assay biological samples for the genomic sequence of DED response markers. It is detected by the following. In some embodiments, the biological sample is synovial fluid, blood, serum, feces, The sample is selected from the group consisting of plasma, urine, tears, saliva, cerebrospinal fluid, leukocyte samples, and tissue samples.

[0113] In some embodiments, the presence of a DED response marker is measured by Northern blot analysis, poly Polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), TaqM AN-based assays, direct sequencing, dynamic allele-specific hybridization Dysization, high-density oligonucleotide SNP arrays, restriction fragment length polymorphism (RFLP) Assays, primer extension assays, oligonucleotide ligase assays, single-strand con Emission polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denatured high-performance liquid chromatography Raffy, High-Resolution Melting Analysis, DNA Mismatch Binding Protein Assay, SNPLex (Registered Trademark) Capillary electrophoresis, Southern blotting, immunoassay, immunohistochemistry Biochemistry, ELISA, flow cytometry, Western blotting, HPLC, and mass spectrometry It is detected by a technique selected from a group of methods.

[0114] In some embodiments of the methods and uses described herein, the TNFα antagonist is a TNFα-binding molecule or a TNFα receptor binding molecule. In some embodiments, a TNFα binding molecule or T The NFα receptor binding molecule is the TNFα binding molecule. In some embodiments, TNFα binding The compound molecule is either a TNFα antibody or its antigen-binding portion.

[0115] In some embodiments of the methods and uses described herein, the TNFα antibody is a recombinant humanized antibody. Yes. In some embodiments of the methods and uses of this disclosure, recombinant humanized TNFα antibody is L It's the ME636.

[0116] Treatment methods and use of TNFα antagonists According to the method of this disclosure, clinicians can provide personalized therapy to DED patients. In other words, according to this method, patients are selectively treated with TNFα antagonists (e.g., LME636). Should it be done, or should the patient be selectively treated with commercially available treatments or topical cyclosporine? It is possible to determine whether or not a patient has DED. To maximize the benefits of TNFα antagnosing in the entire population, This risk can be minimized. TNFα antagonists, for example, TNFα binding molecules ( For example, a TNFα antibody or its antigen-binding moiety (e.g., LME636) or a TNFα receptor binding moiety. Synthetic molecules (e.g., TNFα receptor antibodies or their antigen-binding moieties) are particularly used as DED response markers. In patients having DED, treatment, prevention, or reduction as disclosed herein ( It is understood that it is useful for improving signs and symptoms, structural changes, and eye discomfort, for example. It is likely.

[0117] TNFα antagonists, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding portions). , for example LME636) or TNFα receptor binding molecules (for example TNFα receptor antibodies or so The antigen-binding portion is used in vitro, ex vivo, or incorporated into pharmaceutical compositions. When administered in vivo to an individual (e.g., a human patient), for example, a DED response marker can be detected. In patients having -, DED can be treated, alleviated, or prevented. The pharmaceutical composition is intended to The formulation will be adapted to the illustrated route of administration (for example, oral compositions are generally (including inert diluents or food carriers). Other non-limiting examples of routes of administration include parenteral administration. For example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (for example, inhalation), transdermal administration (topical administration), transmucosal administration, and rectal administration. Examples include: Pharmaceutical compositions suitable for each intended route are well known in the art. be.

[0118] TNFα antagonists, for example, TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding portions). , for example LME636) or TNFα receptor binding molecules (for example TNFα receptor antibodies or so The antigen-binding portion (of the substance) is used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. This is possible. Such compositions may include, in addition to TNFα antagonists, carriers, various diluents, fillers, and salts. It may contain buffers, stabilizers, solubilizers, and other materials well known in the art. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition used in the method disclosed herein is Furthermore, additional therapeutic agents for treating specific targeted disorders may also be included. For example, the pharmaceutical composition It may also include other anti-inflammatory agents. Such additional factors and / or agents may include TNFα-binding molecules. To produce a synergistic effect with, or with TNFα antagonists, such as TNFα-binding molecules (for example) TNFα antibody or its antigen-binding moiety (e.g., LME636) or TNFα receptor-binding molecule The most common side effects are those caused by (for example, TNFα receptor antibodies or their antigen-binding moieties). It may be included in this pharmaceutical composition in order to keep its amount to a minimum.

[0119] The pharmaceutical composition used in the method disclosed herein may be manufactured by conventional methods. In one embodiment... The pharmaceutical composition is provided in a lyophilized form. For immediate administration, the pharmaceutical composition is suitable for aqueous solutions. It is dissolved in a carrier, such as sterile water for injection or sterile buffered saline. When it is considered desirable to prepare a large volume of solution for administration by injection, The process involves mixing human serum albumin or the patient's own heparinized blood with physiological saline. It can be advantageous. If such physiologically inactive proteins are present in excess, the injection solution and The loss of antibodies due to adsorption to the walls of containers and tubes used together is prevented. Albumin When used, the preferred concentration is 0.5-4.5% by weight of physiological saline. Other products The preparations include liquid or lyophilized formulations.

[0120] Antibodies, such as antibodies against TNFα, are typically available in an aqueous form that can be administered immediately as a parenteral agent. It is formulated as a lyophilized product, or as a lyophilized product that is reconstituted with a suitable diluent before administration. It is either one of the following. In some embodiments of the methods and uses of this disclosure, a TNFα antagonist For example, TNFα antibodies, such as LME636, are formulated as lyophilized products. The dried formulation can be reconstituted into a small liquid volume (e.g., 2 ml or less) to enable subcutaneous administration. This allows for the provision of a solution with a low antibody aggregation level.

[0121] The appropriate dosage naturally depends on, for example, the specific TNFα antagonist being used, such as TN Fα-binding molecule (e.g., TNFα antibody or its antigen-binding moiety, e.g., LME636) or T NFα receptor binding molecule (e.g., TNFα receptor antibody or its antigen-binding portion), host, administration Depending on the form, the nature and severity of the condition under treatment, and the nature of the prior treatment the patient received It will depend on the individual patient and will vary. Ultimately, TNFα antagonists to treat each individual patient. The amount will be determined by the healthcare provider in charge. In some embodiments, the healthcare provider in charge The provider may administer a low dose of a TNFα antagonist and observe the patient's response. Other embodiments In this case, the initial dose of one or more TNFα antagonists administered to the patient was high, followed by relapse. The dose is gradually reduced until the signs of [symptoms] appear. Higher doses of TNFα antagonists may be administered, here Once the optimal therapeutic effect for that patient is achieved, the dosage is generally not increased further. do not have.

[0122] In the implementation of a part of the treatment method or use disclosed herein, a therapeutically effective amount of a TNFα antagonist, for example TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding moieties, e.g., LME636) Or a TNFα receptor-binding molecule (e.g., a TNFα antibody or its antigen-binding portion) in a patient, for example It is administered to mammals (e.g., humans). The method disclosed herein depends on the presence of a DED response marker. It is understood that selective treatment should be provided for patients who have the condition (i.e., patients with DED). This is because if the patient is ultimately treated with a TNFα antagonist, such TNFα antagonist therapy The law does not necessarily exclude monotherapy. In fact, patients with TNFα antagonists When selected for treatment, the method described herein allows a TNFα antagonist (e.g., LME636) to be administered. Whether administered alone or in combination with other therapeutic drugs for the treatment of the patient's DED, Either is acceptable. When administered in combination with one or more additional therapeutic agents, TNFα antagonists are used in combination with other therapeutic agents. It may be administered simultaneously with the drug or sequentially. Sequential administration is acceptable. If so, the appropriate administration sequence of TNFα antagonists and other therapeutic agents used in combination, as well as co-delivery, should be considered. The appropriate dosage will be determined by the attending physician.

[0123] TNFα antagonists are conveniently administered parenterally, intravenously, for example, in the precubital vein or other peripheral veins. It can be administered intravenously, intramuscularly, or subcutaneously. The duration of intravenous (IV) therapy depends on the severity of the disease being treated and the individual patient's condition. And will vary depending on individual responses. Also, subcutaneous use of the pharmaceutical composition of this disclosure (SC) therapy is also planned. IV or SC therapy using the pharmaceutical composition of this disclosure The appropriate duration of the law and the timing of therapy administration will be determined by the healthcare provider.

[0124] In certain embodiments, a TNFα antagonist, such as LME636, is used in the periorbital, conjunctival, Intra-Tenon's capsule, intra-anterior chamber, intravitreous, intraocular, subretinal, subconjunctival, retrobulbar, or intratubular injection, etc. By tissue injection; via catheter or other porous, non-porous, or gelatinous material. Eyes with implantable devices such as retinal pellets, intraocular implants, suppositories, or implants. By direct application to the conjunctival sac; by topical eye drops or ointments; or by application to the conjunctival sac or adjacent to the sclera By using a sustained-release device implanted (transscleral), in the sclera (intrascleral), or within the eye. It can be delivered directly to the eye. Intratubular injection is performed by draining Schlemm's canal from the venous collecting ducts. It may be inside or within Schlemm's tube.

[0125] For ocular delivery, the antibody of the present invention is delivered with an ophthalmologically acceptable preservative, co-solvent, surfactant, and viscosity Combined with accelerators, penetration enhancers, buffers, sodium chloride, or water, for aqueous sterilization of ophthalmology. A suspension or solution may be formed. Topical ophthalmic preparations may be packaged, for example, in a multi-dose form. It may be used. Therefore, a preservative may be necessary to prevent microbial contamination during use. Suitable preservative Preservatives include chlorobutanol, methylparaben, propylparaben, and phenylethyl Alcohol, disodium edetate, sorbic acid, polyquaternium-1, or to those skilled in the art Other known preservatives include those typically present in concentrations of 0.001–1.0% w / v. It is used at this level. The unit dose composition of the present invention may be sterile, but is typically treated with preservatives. It is not treated. Therefore, such compositions generally do not contain preservatives.

[0126] In certain embodiments, compositions intended for topical administration to the eye are provided as eye drops or eye ointments. It is then formulated, and the total amount of antibody can be approximately 0.1-10.0% (w / w). The amount of TNFα antagonist, such as LME636, is approximately 5.0 to 10.0% (w / w). The most preferred concentration is approximately 6.0% (w / w).

[0127] In specific situations, the composition of the present invention will be administered as a topical solution. The ease of formulation and the fact that the patient can administer 1-2 drops of the solution to the affected eye are important factors. Aqueous solutions are generally preferred because they allow for easy administration of the composition. However, This composition may also be used as a suspension, a viscous or semi-viscous gel, or other type of solid or semi-solid composition. That's fine.

[0128] The therapeutically effective amount of antibodies present in the formulation is, for example, the desired dose volume and one or more doses The dosage is determined by considering the administration method. Approximately 1.0 mg / ml to approximately 100 mg / ml, preferred. The appropriate dosage range is approximately 5.0 mg / ml to approximately 80 mg / ml, and most preferably approximately 10.0 mg / ml. An exemplary antibody concentration in the formulation is approximately 1-60 mg / ml.

[0129] As a general suggestion for systemic administration, the TNFα antagonist to be administered, for example, LME636 The effective dose, whether administered as a single dose or more, is approximately 0.1 to approximately 100 mg / The range may be within the patient's weight in kg, and a typical range for the antibodies used is, for example, approximately 0. Approximately 3 to 20 mg / kg, more preferably approximately 0.3 to 15 mg / kg, is administered daily. However, other dosage regimens may be useful. Progress in this therapy is ongoing. It can be easily monitored by the next technology.

[0130] This specification includes a) the therapeutic effective dose based on the patient having a DED response marker. Patients with DED, including selective administration of TNFα antagonists to patients. A selective treatment method is disclosed, where the DED response marker is rs1800693 response. It is an allergen.

[0131] In some embodiments, the method further includes the step of obtaining a biological sample from a patient. The steps described here are performed before the assay steps.

[0132] In some embodiments of the methods and uses of this disclosure, the DED response marker is used to respond to a biological sample. Nucleic acid products of DED response markers, polypeptide products of DED response markers, or DED response It is detected by performing an assay against an equivalent genetic marker to the response marker.

[0133] In some embodiments of the methods and uses of this disclosure, the DED response marker is used to respond to a biological sample. It is detected by assaying the genomic sequence of the DED response marker.

[0134] In some embodiments of the methods and uses described herein, the biological sample may be synovial fluid, blood, serum, or feces. A sample is selected from the group consisting of stool, plasma, urine, tears, saliva, cerebrospinal fluid, leukocyte samples, and tissue samples. ru.

[0135] In some embodiments of the methods and uses of this disclosure, the DED response marker is Northern Lot analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-P) CR), TaqMan-based assays, direct sequencing, dynamic sequencing Segment-specific hybridization, high-density oligonucleotide SNP array, restriction fragment length Polymorphism (RFLP) assay, primer extension assay, oligonucleotide ligase assay SAY, single-strand conformational polymorphism analysis, temperature gradient gel electrophoresis (TGGE), denaturation high Fast liquid chromatography, high-resolution melting analysis, DNA mismatch binding protein assay I, SNPLex (registered trademark), capillary electrophoresis, Southern blotting, immunoassay SEI, immunohistochemistry, ELISA, flow cytometry, Western blot, HPL It is detected by a technique selected from the group consisting of C and mass spectrometry.

[0136] kit The present invention also provides a method for detecting DED response markers in biological samples (test samples) from patients. It also includes kits. Using such kits, patients with DED can receive TNFα antagonists, for example TNFα-binding molecules (e.g., TNFα antibodies or their antigen-binding moieties, e.g., LME636) Alternatively, treatment with a TNFα receptor binding molecule (e.g., a TNFα antibody or its antigen-binding portion) It is possible to predict whether it will respond (or has a higher responsiveness). For example, this kit contains DED response markers in biological samples, their allele products and / or probes capable of detecting equivalent genetic markers of those alleles (e.g., Oligonucleotides, antibodies, labeled compounds, or other drugs This kit may include (agents). This kit also shows that the patient is responding to treatment with a TNFα antagonist. This may also include a description of the provision of predictions about possible outcomes.

[0137] The probe specifically hybridizes with genome sequences, nucleic acid products, or polypeptide products. It is possible. Exemplary probes are oligonucleotides that specifically hybridize to the response alleles in Table 1. Rheotides or conjugated oligonucleotides (e.g., DNA, cDNA, mRNA) (e.g.); primer extension oligonucleotides, allele-specific primers, allele-specific Target primers, allele-specific probes, and primer extension primers This includes combinations such as the following. Optionally, this kit includes probes that target an internal standard allele. This can include any allele exhibited by the general population. Internal standard allele The detection is designed to ensure the performance of the kit. The kit of this disclosure also includes, for example, Buffers, preservatives, or protein stabilizers may also be included. This kit is also detectable This kit may also include components necessary for the detection of effective drugs (e.g., enzymes or substrates). It can also be assayed and compared with a control sample or a series of samples that can be compared with the test sample included. A control sample may also be included. Each component of the kit is usually sealed in a separate container. All the various containers, along with the instruction manual, are included in a single package.

[0138] Such kits also include TNFα antagonists, such as TNFα-binding molecules (e.g., TNFα antibodies). or its antigen-binding portion, e.g., LME636) or a TNFα receptor-binding molecule (e.g., TN Fα antibody or its antigen-binding moiety) (e.g., in liquid or lyophilized form) or TNFα antagonist ( The kit may also include a pharmaceutical composition containing (as described above). Thus, such a kit may include TNF It is useful for the selective treatment of DED using alpha-adrenergic antagonists (e.g., LME636). Such kits include means of administering TNFα antagonists (e.g., syringes and vials, pre-filled containers). These kits may include a syringe, pre-filled pen, and instructions for use. For example, DED treatment delivered in combination with an enclosed TNFα antagonist, such as LME636. It may also include additional therapeutic agents (as listed above).

[0139] The term "means of administration" is not limited to pre-filled syringes, vials, and syringes. This includes administering drugs to patients using injection pens, automatic infusion devices, intravenous drip bags, pumps, etc. This term is used to refer to any available means of administration for systemic administration. In such cases, the patient may self-administer the medication (i.e., administer the medication themselves), or a physician may administer the medication. They may administer drugs.

[0140] overview The attached description above provides details of one or more embodiments of this disclosure. Alternatively, in the test, any method and materials similar to or equivalent to those described herein may be used. While other methods and materials may be used, preferred methods and materials are described here. Other features, purposes, and The advantages will become apparent from this description and the claims. In the claims, the singular form refers to the plural form unless otherwise specifically indicated by the context. Including elephants. Unless otherwise defined, all scientific and technical terms used herein are defined in this disclosure. It has the same meaning as generally understood by those skilled in the art to which it belongs. All patents and publications are incorporated by reference. The following embodiments are preferred examples of the present disclosure. Embodiments are provided to illustrate them in more detail. These examples are not included in the attached claims. The scope of the patentable subject matter of this disclosure is limited as defined by the scope of [the relevant provision]. It must not be interpreted as an object. [Examples]

[0141] Example 1-CLME636X2202A study: LME636 is used for the signs of dry eye disease and Improve symptoms CLME636X2202A is used in patients with severe dry eye disease (DED) to treat LME This was a proof-of-concept (PoC) study to determine the efficacy and safety of local ocular treatment with 636. The primary objective of this PoC study was determined by the overall ocular discomfort score on day 29 of treatment. The effectiveness of locally administered LME636 in reducing ocular symptoms compared to LME636 media. The objective was to demonstrate the efficacy. An important secondary objective was to improve the overall ocular discomfort score >20. The objective was to determine the percentage of patients (responder analysis). DNA samples were collected from patients who had given informed consent, and preliminary pharmacological genetic analysis was performed. By performing this procedure, we identified genetic factors that may influence the response to LME636 treatment. Physogenic analysis is described in Example 2.

[0142] During the treatment phase, 69 patients received LME636, and 65 received LME636 via Participants were randomized to receive a total dose. Six doses were administered via LME636 and LME636 media. Seven and 64 patients completed this study and were included in the perprotocol analysis. The academic characteristics and baseline characteristics are well equal between these two groups, including the following parameters. The balance was maintained (LME636 vs. LME636 media): average age 61.7 vs. 58.8 years; 61 female patients versus 54 female patients; mean overall ocular discomfort at baseline: 77.9 vs. 80.3.

[0143] Regarding the primary efficacy endpoint, the change from baseline in the overall ocular discomfort score was measured. The concentration (LME636 vs. LME636 medium) was -7.9 [1.45 SE] vs. - on day 29. 3.6 [1.49SE] (Main time points and per-protocol analysis set) ) and on day 43 -10.5[1.74SE] vs -5.4[1.77SE] (full analysis) This was a preliminary time point based on the lysis set. Baseline on day 29. The 90% confidence interval for the difference in change from was -7.7 to -0.8.

[0144] Regarding LME636 versus LME636 media, more than 20 units of general ocular discomfort occurred on day 29. The number of patients who achieved improvement from baseline was 12 (17.9%) versus 3 (4.7%). ) and the p-value based on the chi-squared test and Wald interval was 0.018 (responder (Analysis).

[0145] In conclusion, these results met the pre-specified criteria for the primary analysis and the responder analysis. This was further supported by the findings. The improvement lasted from day 29 to day 43.

[0146] Example 2: Materials and methods for pharmacogenetic (PG) analysis Example 2.1: Sample and Treatment A total of 127 patient samples were collected. Patients who met all of the following criteria were selected for this pharmacological genetic analysis. Incorporated into the analysis: • We submitted written informed consent for the pharmacological genetic study. • Successfully extracted DNA and performed gene typing. • Included in the overall analysis of the clinical trial.

[0147] Blood samples are collected from patients who have given their consent at individual testing facilities, and then processed at Pharmaceuti. cal Product Development(Wilmington, NC 28) Shipped to 401) Covance (Indianapolis, IN 46214) By doing so, each patient's genomic DNA is extracted from their blood, and Clinical Refer TaqMa Gene typing data was generated using n technology. Of the nine SNPs, eight showed coloration. This was successful, and one (rs115575857) developed color due to the complexity of the local DNA sequence. None. For these eight SNPs, genetics were found in 126 out of 127 DNA samples. Child typing was successful. One patient sample had problems with DNA quality, and gene typing The piping failed. Two duplicate samples were included as quality control. .

[0148] Being located at the drug target (TNF-α) or its receptor (TNFR1), or Sjögren Based on the fact that these are genetic mutations highly associated with the syndrome, several candidate genes were selected for PG analysis. We also selected single nucleotide polymorphisms (SNPs) (see Table 1).

[0149] [Table 3]

[0150] Example 2.2: Statistical Analysis Primary efficacy endpoint The primary efficacy endpoint is the base of the overall ocular discomfort score at day 29 of treatment. The change was measured from the baseline. A per-protocol set was used for efficacy analysis.

[0151] To test for genotype effects in patients treated with LME636: baseline general symptoms The items include pleasure score, genotype, treatment duration, interaction between genotype and treatment duration, age, and race. A repeated measures analysis of mixed models was performed to determine the baseline of overall discomfort among genotype groups. We present estimates of the difference in the mean change from the input and the associated 90% confidence intervals.

[0152] For testing the interaction between genotype and treatment on day 29 of treatment: ANCOVA model The study used a metric that included baseline global discomfort score, age, and race as covariates. Estimates of the difference in the mean change in overall discomfort from baseline between genetic groups and their associations. A 90% confidence interval was presented.

[0153] All statistical tests were two-tailed. Bonferroni correction was applied to adjust for multiple tests. For any genetic effect that achieved p ≤ 0.1 after adjusting for multiple testing, individual patient data A scatter plot or waterfall plot showing the data will be presented.

[0154] Important secondary efficacy endpoints An important secondary efficacy endpoint is overall eye health from baseline to day 29 of treatment. Percentage of patients (determined as responders) with an improvement in their discomfort score > 20. The efficacy analysis was performed using a per-protocol set.

[0155] Fisher's exact test was used. Genes associated with the primary endpoint were identified. Only the genotype was analyzed. Percentage of patients with an improvement in the overall ocular discomfort score >20 between genotype groups. He presented the vintage.

[0156] Example 3: PG Analysis Results Example 3.1: Comparison of PG and the entire test population We successfully performed gene typing on a total of 126 patient samples for eight SNPs. The two duplicates introduced for quality control had eight SNP genotypes. It showed 100% agreement. Of the 126 patients with genotype data, 88 were cured. They entered treatment. Of those 88 patients who had genotype data and entered treatment, 8 Six people were in the perprotocol analysis set. In contrast, all trials were perprotocol The col analysis set included 131 patients. Table 3 shows the PG population and the overall population, respectively. Demographic characteristics in the trial, baseline overall ocular discomfort scores, and treatment The response rate is summarized below. The PG population showed a high response rate to these variables, particularly overall ocular palsy at baseline. When determined by pleasure scores, it appeared similar to all other tests.

[0157] [Table 4]

[0158] Example 3.2: Analysis of primary efficacy endpoint - Initial association test Similar to comprehensive clinical trials, mixed model repeated measures analysis was used to analyze genotype and day 29 of treatment. We tested the association between the change from baseline in the overall ocular discomfort score and the patient's overall ocular discomfort score. Of the eight SNPs examined, only rs1800693 was related to the response to LME636. The effect was significant after Bonferroni correction (p<0.0001) (Table 4). SNP rs1800629 showed a nominal significant association with the clinical endpoint (p=0). (0.0159), this significance disappeared after Bonferroni correction (p=0.1272).

[0159] [Table 5]

[0160] As shown in Table 5, the genotype effect of rs1800693 on symptom improvement is LME The condition appeared only in patients treated with 636 and not in patients treated with the other medium. CC genotype Patients tended to show greater improvement than those with the CT or TT genotype.

[0161] [Table 6]

[0162] Example 3.3: Analysis of the primary efficacy endpoint - Interaction between treatment and genotype Next, the inventors conducted a covariance analysis to determine whether there is an interaction between the treatment and the genotype. The ANCOVA model was used for the test. Similar to the initial mixed model analysis, on day 29 of treatment... Only rs1800693 showed a significant interaction (p=0.0076) (Table 6). The effect remained significant even after Bonferroni correction (p=0.0608).

[0163] [Table 7]

[0164] [Table 8]

[0165] Interaction testing was performed only on day 29 of treatment. However, there were missing data on individual treatment days. There was a loss. To improve the understanding of the interaction between treatment and rs1800693, the inventor They further tested the interactions from day 23 to day 28. As shown in Table 7, 2 Similar trends in interactions were observed from days 3 to 29.

[0166] [Table 9]

[0167] [Table 10]

[0168] Example 3.4: Analysis of important secondary efficacy endpoints Secondary efficacy endpoint analysis focused on response rates between genotype groups. rs180 Since 0693 is the only SNP associated with the primary efficacy endpoint, rs18 Only 00693 was analyzed. As shown by Fisher's exact test, the response was The rate was much higher in patients with the CC genotype compared to patients with the CT or TT genotype (Table 8). These results support the analysis of the primary efficacy endpoint. However, Since the number of responders is small, it is advisable to interpret the results carefully.

[0169] [Table 11]

[0170] [Table 12]

[0171] Example 3.5: Visualization of individual patient data This pharmacological genetic analysis involved 12 patients with the CC genotype. The changes in symptoms were visualized. To compare between treatments, the overall ocular discomfort scores of these 12 CC genotype patients were... The change from baseline was plotted against the treatment day from baseline to day 43. As shown in Figure 1, 3 out of 4 patients treated with LME636 showed signs of aging over time. There was significant symptomatic improvement. In contrast, among the eight patients treated with the medium, there was a consistent 10% improvement. No patients showed symptom improvement higher than the rank indicated. LME636 and vegetative state on day 29 of treatment The difference in symptom improvement between the two groups was statistically significant according to ANCOVA analysis (p<0.0001). .

[0172] Waterfour measurement of the change from baseline in the overall ocular discomfort score for all patients Further visualization using plots made it possible to visualize symptom changes by treatment and genotype. (Figures 2-5). Interestingly, only minimal mediation responses were observed in patients with the CC genotype. On the other hand, within the CT and TT groups, a media response of >10 occurred. From this data, the CC gene Patients with this type of condition may have certain dry eye disorders that are unlikely to be alleviated by simply using a medium. This was suggested.

[0173] conclusion This preliminary pharmacogenetic analysis uses candidate gene techniques to investigate the mechanism of action of LME636. We focused on genes associated with [specific condition] and genes linked to Sjögren's syndrome. Due to the small sample size, only eight SNPs were included in the analysis to reduce the burden of adjusting for multiple tests. It was alleviated. Three SNPs in the TNF-α gene are found in patients with autoimmune diseases such as rheumatoid arthritis. It has been reported to be related to the response to anti-TNF agents in (Julia A, Ferna ndez-Nebro A, Blanco F, Ortiz A, Canyete JD ,et al.(2016).A genome-wide association study identifies a new locus associated with the response to anti-TNF therapy in rheumatoid arthritis.Pharmacogenomics J .16(2):147-50). The TNFR1 gene is mainly associated with multiple sclerosis (MS). One SNP causes exon 6 skipping, and soluble TNFR1 (sTNF) (R1) results in the production of (De Jager PL, Jia X, Wang J, de Bakker PI, Ottoboni L, et al.(2009).Meta-a analysis of genome scans and replication identify CD6,IRF8 and TNFRSF1A as new mu ltiple sclerosis susceptibility loci.Nat Genet.41(7):776-82;Gregory AP,Dendrou C A, Attfield KE, Haghikia A, Xifara DK, et al (2012).TNF receptor 1 genetic risk mirr ors outcome of anti-TNF therapy in multi ple sclerosis.Nature.23;488(7412):508-11 ). In DED patients, the Sjögren's syndrome risk allele affects the response to LME636. To investigate the potential impact, four SNPs strongly associated with Sjögren's syndrome were analyzed in this study. (Lessard CJ, Li H, Adrianto I, Ice JA, R asmussen A, Grundahl KM, et al. (2013).Vari ants at multiple loci implicated in both innate and adaptive immune responses ar e associated with Sjoegren's syndrome.Na t Genet.45(11):1284-92).

[0174] sTNFR1 is constitutively released from the cell membrane by TNF-α-converting enzyme, and sjög Its levels increase during the course of various human diseases, including Lennill syndrome, uveitis, and glaucoma. (Touchard E, Bloquel C, Bigey P, Kowalczu k L,Jonet L,et al.(2009).Local ocular im munomodulation resulting from electrotra nsfer of plasmid encoding soluble TNF re ceptors in the ciliary muscle.Gene Ther. 16(7):862-73; Sakimoto T, Yamada A, Sawa M. (2009).Release of soluble tumor necrosis factor receptor 1 from corneal epithelium um by TNF-alpha-converting enzyme-depend ent ectodomain shedding.Invest Ophthalmo l Vis Sci.50(10):4618-21;Sakimoto T, Ohni Shi T, Ishimori A. (2014). Significance of ectodomain shedding of TNF receptor 1 in ocular surface.Invest Ophthalmol Vis Sc i.55(4):2419-23). ​​In contrast, sudden disruption of the sTNFR1 production process The mutation causes periodic syndromes, which are autoinflammatory disorders (Magnotti F, Vi). tale A, Rigante D, Lucherini OM, Cimaz R, et al.(2013).The most recent advances in p. athophysiology and management of tumors necrosis factor receptor-associated peri odic syndrome(TRAPS):personal experience and literature review.Clin Exp Rheumatoid l.31(3 Suppl 77):141-9). Therefore, there is an imbalance in sTNFR1 production. This may contribute to the pathogenesis of human diseases. Some mechanistic studies suggest that sTNFR1 is linked to TNF-α activity. It may act as a physiological attenuator or a buffer that enhances the effects of TNF-α. It was suggested that it could function as a system (Aderka D, Engelmann). H, Maor Y, Brakebusch C, Wallach D. (1992).S. tabilization of the bioactivity of tumor necrosis factor by its soluble receptor sJ Exp Med.175(2):323-9;Gregory AP,Dend rou CA,Attfield KE,Haghikia A,Xifara DK, et al.(2012).TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis.Nature.23;488(7412):5 (08-11). rs1800693 is considered to be a novel mechanism for sTNFR1 production. The study found that the rs1800693 CC genotype is associated with increased signaling to TNF-α. Regarding this matter, and that this change in signal transduction is due to a change in the localization of sTNFR1 within the cell, It has been proven that this can be obtained (Housley WJ, Fernandez SD, Vera K,Murikinati SR,Grutzendler J,et al.(201 5).Genetic variants associated with auto immunity drive NFκB signaling and response ses to inflammatory stimuli.Sci Transl M ed.10;7(291):291ra93).

[0175] As reported in the 1000 Human Genome Database, the carrier rate of CC is higher in Caucasians and African Americans. In contrast, (In contract ), the carrier rate of CC was found in Caucasians and Africans in the LME636X2202 study. The percentages are 11.5% (11 / 96) and 12.7% (2 / 19), respectively. The literature mentions dryers. No genome-wide genetic association studies have been reported for this disease. rs180069 It is unclear whether factor 3 has any impact on the risk of dry eye disease.

[0176] The present inventors particularly focus on TNFα antagonistic activity in DED, for example, TNFα antibodies, for example We identified specific genetic variations that predict the response to LME636. The finding is that certain SNPs may be associated with an increased likelihood of patients developing DED. This could not have been predicted based on that. The findings disclosed herein are based on the fact that SNPs This could not have been predicted based solely on the specific genes that were studied. In fact, the tests Of the eight SNPs, four are known to be related to the TNFα pathway, and four are related to Sjögren's syndrome. Although it was known to be associated with the syndrome, rs180069 located in the TNFR1 gene Only group 3 showed a significant effect on the response to LME636 treatment. Patients with the CC genotype... Patients with the CT or TT genotype tend to show much greater improvement in symptoms compared to patients with the CT or TT genotype. Therefore, the extent to which a patient responds to medication depends on the patient's specific disease condition. Whether the patient carries related alleles, or whether the patient carries an SNP in a specific gene. It is not possible to make predictions based solely on this.

[0177] The present inventors believe that the prediction method and personalized therapy disclosed herein are such as LME636. By identifying patients who may respond before treatment with TNFα antagonists, DED To maximize the benefits of TNFα antagonism and minimize its risks in patients with [condition]. It is concluded that it is useful for obtaining.

[0178] Sequence List [ka] [ka] [ka] [ka] [ka] [ka]

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Claims

1. A method for selectively treating patients with dry eye disease (DED), wherein the patient Based on the presence of a DED response marker, a therapeutically effective dose of TNFα antagonist is selected for the patient. This includes administering the DED response marker as the rs1800693 response allele. A method.

2. This is a method of selectively treating patients with dry eye disease (DED) with TNFα antagonists. That is, a) Treatment with the TNFα antagonist based on the presence of the DED response marker in the patient Selecting the aforementioned patient for treatment; and b) Thereafter administering a therapeutically effective dose of the TNFα antagonist to the patient. Including; A method wherein the DED response marker is the rs1800693 response allele.

3. This is a method of selectively treating patients with dry eye disease (DED) with TNFα antagonists. That is, a) Assaying biological samples from the patient for DED response markers; and b) Subsequently, based on the fact that the biological sample from the patient has the DED response marker Selectively administer a therapeutically effective dose of TNFα antagonist to the patient. Including; A method wherein the DED response marker is the rs1800693 response allele.

4. This is a method of selectively treating patients with dry eye disease (DED) with TNFα antagonists. That is, a) Assaying biological samples from the patient for DED response markers; b) Subsequently, based on the fact that the biological sample from the patient has the DED response marker Selecting the patient for treatment with the TNFα antagonist; and c) Thereafter administering a therapeutically effective dose of the TNFα antagonist to the patient. Including; A method wherein the DED response marker is the rs1800693 response allele.

5. The assay step involves the biological sample being subjected to the nucleic acid product of the DED response marker, Polypeptide products of the DED response marker, or equivalent genes of the DED response marker. The method according to claim 3 or 4, comprising performing an assay with respect to a marker.

6. The assay step involves the biological sample being subjected to the genome sequence of the DED response marker. The method according to claim 5, comprising performing an assay relating to the present invention.

7. The aforementioned biological samples include synovial fluid, blood, serum, feces, plasma, urine, tears, saliva, cerebrospinal fluid, and white blood cells. A method according to any one of claims 3 to 6, selected from the group consisting of a sample and a tissue sample. 。

8. The assay steps include Northern blot analysis and polymerase chain reaction (PCR). ), reverse transcription polymerase chain reaction (RT-PCR), TaqMan-based assay, Electosequencing, dynamic allele-specific hybridization, high-density sequencing Rigonucleotide SNP arrays, restriction fragment length polymorphism (RFLP) assays, primer extension Assays, oligonucleotide ligase assays, single-strand conformational polymorphism analysis, temperature Gradient gel electrophoresis (TGGE), denatured high-performance liquid chromatography, high-resolution melting fraction Analysis, DNA mismatch binding protein assay, SNPLex®, capillary - Electrophoresis, Southern blotting, immunoassay, immunohistochemistry, ELISA, fluoroscopy Selected from the group consisting of itometry, Western blotting, HPLC, and mass spectrometry. The method according to any one of claims 3 to 7, including the technique of [doing something].

9. TNFα antagonists for use in the treatment of patients with dry eye disease (DED) Based on the fact that the patient has a DED response marker, the therapeutically effective amount of TNFα is administered. The antagonist will be administered to the patient, and the DED response marker will be rs180069 A TNFα antagonist characterized by being a tri-response allele.

10. TNFα antagonists for use in the treatment of patients with dry eye disease (DED) At a) Based on the presence of the DED response marker, the patient is determined to have the TNFα antagonist Treatment will be chosen as a drug treatment; and b) Subsequently, a therapeutically effective dose of the TNFα antagonist is administered to the patient. The DED response marker is characterized by being the rs1800693 response allele. NFα antagonist.

11. TNFα antagonists for use in the treatment of patients with dry eye disease (DED) At a) The biological sample from the patient will be assayed for DED response markers. and b) Treatment based on the fact that the biological sample from the patient has the DED response marker. An effective amount of the TNFα antagonist is selectively administered to the patient. The DED response marker is characterized by being the rs1800693 response allele. NFα antagonist.

12. TNFα antagonists for use in the treatment of patients with dry eye disease (DED) At a) The biological sample from the patient will be assayed for DED response markers. ； b) Based on the fact that the biological sample from the patient has the DED response marker, The patient was selected for treatment with a TNFα antagonist; and c) A therapeutically effective dose of the TNFα antagonist is selectively administered to the patient. The DED response marker is characterized by being the rs1800693 response allele. NFα antagonist.

13. Patients with dry eye disease (DED) will likely respond to treatment with TNFα antagonists. A method for predicting the possibility of the presence of a DED response marker in a biological sample from the patient. The assay includes determining the presence or absence of the DED response marker, and the presence of the DED response marker indicates the patient This indicates an increased likelihood that the person will respond to treatment with the TNFα antagonist, and the D A method in which the ED response marker is the rs1800693 response allele.

14. The process further includes the step of obtaining the biological sample from the patient, wherein the step of obtaining the sample is performed The method according to claim 13, which is performed before the step of dispensing.

15. The assay step involves the biological sample being subjected to the nucleic acid product of the DED response marker, Polypeptide products of the DED response marker, or equivalent genes of the DED response marker. The method according to any one of claims 11 to 13, which includes performing an assay with respect to the marker. Law.

16. The assay step involves the biological sample being subjected to the genome sequence of the DED response marker. The method according to claim 15, comprising performing an assay relating to the present invention.

17. The aforementioned biological samples include synovial fluid, blood, serum, feces, plasma, urine, tears, saliva, cerebrospinal fluid, and white blood cells. A selection from the group consisting of a sample and a tissue sample, according to any one of claims 11 to 16. method.

18. The assay steps include Northern blot analysis and polymerase chain reaction (PCR). ), reverse transcription polymerase chain reaction (RT-PCR), TaqMan-based assay, Electosequencing, dynamic allele-specific hybridization, high-density sequencing Rigonucleotide SNP arrays, restriction fragment length polymorphism (RFLP) assays, primer extension Assays, oligonucleotide ligase assays, single-strand conformational polymorphism analysis, temperature Gradient gel electrophoresis (TGGE), denatured high-performance liquid chromatography, high-resolution melting fraction Analysis, DNA mismatch binding protein assay, SNPLex®, capillary - Electrophoresis, Southern blotting, immunoassay, immunohistochemistry, ELISA, fluoroscopy Selected from the group consisting of itometry, Western blotting, HPLC, and mass spectrometry. The method according to any one of claims 11 to 17, including the technique of [doing something].

19. Predicting the response to TNFα antagonist treatment in patients with dry eye disease (DED). A method for creating information in a communicable form for the purpose of a) Based on the presence of a DED response marker in the biological sample from the patient, the patient is T Determining the increased likelihood of responding to treatment with an NFα antagonist, the DED response The response marker is the rs1800693 response allele; and b) The results of the steps to be determined, in tangible or intangible media for use in communication. Recording in form A method that includes this.

20. The method according to any one of claims 1 to 19, wherein the DED is moderate to severe. use.

21. Claims 1 to 20, wherein the DED response marker is rs1800693 C / C allele. The method or use described in any one of the items.

22. Claim 1, wherein the TNFα antagonist is a TNFα-binding molecule or a TNFα-receptor-binding molecule. The method or use described in any one of paragraphs 21 to 21.

23. The method or use according to claim 22, wherein the TNFα antagonist is a TNFα-binding molecule.

24. Claim 23, wherein the TNFα-binding molecule is a TNFα antibody or its antigen-binding portion. The method or use of.

25. a) Heavy chain variable domain (V) containing the amino acid sequence shown as Sequence ID No. 8 Ｈ ); b) Light chain variable domain (V) containing the amino acid sequence shown as Sequence ID No. 7 Ｌ ); c) V containing the amino acid sequence shown as Sequence ID No. 8 Ｈ Domain and Sequence ID 7 V containing the amino acid sequence shown Ｌ domain; d) V including the hypervariable region shown as Sequence ID 1, Sequence ID 2, and Sequence ID 3 Ｈ Do main; e) V including the hypervariable region shown as Sequence ID 4, Sequence ID 5 and Sequence ID 6 Ｌ Dome in; f) V including the hypervariable region shown as Sequence ID 1, Sequence ID 2, and Sequence ID 3 Ｈ Do V includes the main and the hypervariable region shown as Sequence ID 4, Sequence ID 5, and Sequence ID 6. Ｌ domain; and g) Sequence of sequence number 9 The method or use according to claim 24, wherein the TNFα antibody contains a TNFα antibody.

26. The method or use according to claim 25, wherein the TNFα antibody is a recombinant antibody.

27. The method or use according to claim 26, wherein the TNFα antibody is LME636.

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