Multispecific antibodies targeting il-13 and il-18

Abstract

To provide a multispecific antibody targeting IL-13 and IL-18, a method for producing the multispecific antibody, a method for simultaneously inhibiting IL-13 and IL-18 with the multispecific antibody, and a method for treating an IL-13 / IL-18-mediated disorder such as atopic dermatitis by administering the multispecific antibody.SOLUTION: A first moiety comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), wherein the first moiety specifically binds to interleukin-18 (IL-18), and b. a second moiety comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2), wherein the second moiety specifically binds to interleukin-13 (IL-13).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of immunology. Specifically, the present invention relates to interleukin-13 (IL-13). Multispecific antibodies targeting interleukin 13 (IL-13) and interleukin 18 (IL-18), and It relates to methods for making and using the same. [Background technology]

[0002] Atopic dermatitis (AD) is a condition characterized by a range of symptoms, including intense pruritus (e.g., severe itching), It is a chronic / recurring inflammatory skin disease characterized by scaly, dry eczematous lesions on the skin. The disease is extremely disruptive to daily life due to significant psychological problems, significant sleep deprivation and reduced quality of life. The pathophysiology of AD can be complicated by: Immunoglobulin E (IgE)-mediated sensitization and the complex interrelationship between the immune system and environmental factors The primary skin abnormality is an immunological disturbance that leads to IgE-mediated sensitization. The epithelial barrier dysfunction can be the result of both genetic mutations and local inflammation. AD often begins in childhood, before age 5, and can continue into adulthood.

[0003] Typical treatments for AD include topical lotions and moisturizers, topical corticosteroids, These include ointments, creams, or injections. However, most treatment options offer This only provides temporary and incomplete relief of symptoms. Furthermore, many patients with moderate to severe AD It becomes resistant to treatment with topical corticosteroids or calcineurin inhibitors. Therefore, there is a need in the art for novel targeted therapies for the treatment and / or prevention of AD. There are.

[0004] The pathogenesis of AD is multifactorial, and immune-mediated mechanisms include type 2 T helper cell (Th) 2) and type 2 innate lymphoid cells (ILC2s), which are characterized by inappropriate activation of these cells and are involved in inflammatory processes. This is accompanied by increased expression of interleukins, particularly interleukin 4 and IL-13 (Moyle et al.(2019)Exp Dermatol.28(7):756-768;R oediger et al. (2013) Nat Immunol.14(6):56 IL-13 plays a key role in the development and maintenance of inflammatory processes and epidermal barrier dysfunction. Through its prominent role in the pathophysiology of AD, it has emerged as one of the key cytokines. (Tsoi et al. (2019) J Invest Dermatol. 1 39(7):1480-1489).

[0005] Dupilumab, an anti-IL4Ra antibody, is currently approved by the U.S. Food and Drug Administration and the European Medicines Agency. It is approved by the US Food and Drug Administration for the treatment of moderate to severe forms of AD. Antibodies that specifically target IL-13, such as tralokinumab and tralokinumab, are also being developed.

[0006] IL-18 induces super Th1 cells that produce and secrete IFN-γ and IL-13. It is believed to be involved in the pathogenesis of AD (Terada et al. (2006)Proc Natl Acad Sci USA.103:8816-88 21) IL-18 is released by keratinocytes and inflammatory dendritic cells and is a key regulator of AD patients. It has been shown that serum IL-18 levels in AD patients significantly correlate with the skin lesion score. (Ikezawa et al. (2010) Allergy, Asthma & I Immunology Research 2(4):235-246). In vivo administration also induces Th2 differentiation, and in mice, CD4+ T cells-, IL-4-, and and STAT6-dependent IgE production (Yoshimoto et al. (2016) 00) Nat Immunol 1:132-137;Hoshino et al.( 2000) Eur J Immunol 30:1998-2006).

[0007] IL-13 and IL-18 affect many different cell types associated with AD Simultaneous blockade of IL-13 and IL-18 signaling as they are pro-inflammatory cytokines There remains a need for effective treatments that achieve both IL-13 and IL-18. Multispecific antibodies (e.g., bispecific antibodies) targeting these proteins may play a key role in this chronic inflammatory disease. This may address an unmet medical need.

[0008] For the production of bispecific IgG (BsIgG) by co-expressing two different antibodies One of the most common problems in ribosomal DNA synthesis is the unwanted homodimerization and undesired synthesis of the component heavy chains. Figure 1 shows a possible erroneous assembly, which is the unwanted pairing of a component light chain with a positive heavy chain. To overcome this problem of heavy chain homodimerization, a previously identified We used engineered disulfide bonds in combination with the "knob-into-hole" mutations One of the mutations, S354C, can be used to remodel the heavy chain for heterodimerization. :T366W / Y349'C:T366'S:L368'A:Y407V is almost quantitative This can result in efficient (approximately 95%) heterodimerization (Merchant et al., 1999). 98) However, this near-quantitative heterodimerization does not solve the problem of light chain pairing. Therefore, random light chain pairing is assumed, and among the antibodies produced, the desired bispecific Further improving both heavy chain heterodimerization and light chain pairing and thereby improving the purity, yield and quality of bispecific antibodies. This is what is required. Summary of the Invention

[0009] The present disclosure provides a method for treating an autoimmune or inflammatory disorder, comprising administering to a subject an inhibitor of IL13 and an IL The present invention provides a method for treating a rheumatoid arthritis, comprising administering 18 inhibitors simultaneously or sequentially. Although it is not our intention to do so, the present inventors have demonstrated that simultaneous blockade of IL13 and IL18 suppresses IL- unexpectedly superior in the treatment of autoimmune or inflammatory disorders compared with blockade of IL-13 or IL-18 alone We hypothesize that simultaneous blockade may result in improved efficacy. In some cases, simultaneous administration of an IL-13 antagonist and an IL-18 antagonist is also possible. Blocking may be achieved by using multispecific (e.g., bispecific) antibodies that bind to both IL-18 and IL-13. This includes administering antagonists that inhibit both IL-13 and IL-18, such as antibodies.

[0010] The present invention provides a multi-specific inhibitor targeting both IL-13 and IL-18 for the treatment of AD. To provide specific antibodies or fragments thereof and to advance clinical development and commercial manufacturing at reasonable cost. To ensure sufficient overall yield, purity and quality of the product.

[0011] In some embodiments, the multispecific antibody is a bispecific antibody. The antibody comprises: a) a first light chain variable domain that specifically binds interleukin-18 (IL-18); a) a first portion comprising a first heavy chain variable domain (VL1) and a first heavy chain variable domain (VH1); The second light chain variable domain (VL) specifically binds to interleukin-13 (IL-13). 2) and a second portion comprising a second heavy chain variable domain (VH2). .

[0012] In some embodiments, engineered multispecific antibodies (e.g., bispecific antibodies) or The fragments may be human or humanized (e.g., CDR-grafted) IgG (e.g., Ig IgG1, IgG2, IgG3 or IgG4) antibodies. In some cases, modified multiple Specific antibodies (e.g., bispecific antibodies) may be human or humanized (e.g., CDR-grafted). ) IgG1 antibody.

[0013] Undesirable Fc interactions with Fcγ receptors and the complement receptor C1q increase serum persistence. This can be separated from binding to the neonatal Fc receptor (FcRn), which can be conferred by FcRn. The observed in vivo serum persistence can be tuned by mutations in the IgG Fc. It has been shown that Fc modification can enhance the activity of endosomal proteins in acidic pH conditions. Improving Fc affinity to FcRn in the IgG antibody prolongs the pharmacokinetics of monoclonal antibodies. This is an effective approach to improve the quality of the YTE mutation set (Maeda, 2017). (M252Y, S254T, T256E according to EU numbering) or LS mutation set The nucleotide sequence (M428L, N434S according to EU numbering) in the Fc CH2 domain An example of such a developed mutation set is shown in Figure 1.

[0014] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segment is M252Y / S254T / T256E(YTE) according to EU numbering. In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or its The fragment contains M428L, N434S(LS) according to EU numbering.

[0015] Chain pairing mutations enhance complementarity at the CH3-CH3 interface of bispecific or multispecific antibodies. The introduction of α-glucan has been shown to be efficient in promoting chain heterodimerization. Many sets of chain-pairing mutations have been used in the generation of multispecific antibodies. Increase / decrease in side chain volume (T366W / S354C-T366S / L368A / Y40 7V / Y349C, knob-into-hole) (Ridgway, 1996), charge reversal (K409D / K392D-D399K / E356K, electrostatic steering)(Gunas ekaran, 2010) or multiple IgA substitution (SEEDbody) (Davis, 20 10).

[0016] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The fragment may be, for example, a strand-pairing fragment in combination with a silencing and / or half-life extending mutation. In some cases, the chain-pairing amino acid substitutions are knob-into-hole (KiH) mutations, e.g., engineered multispecific antibodies (e.g., bispecific antibodies). The antibody or a fragment thereof comprises a first constant heavy chain having an amino acid substitution of T366W and a second constant heavy chain having an amino acid substitution of T366W. a second constant heavy chain having amino acid substitutions of 66S, L368A and Y407V, Amino acid residues are numbered according to EU numbering.

[0017] In another embodiment, the chain-paired amino acid substitutions are knob-into-hole (KiH) ambulations. The first constant heavy chain has the amino acid substitutions S354C and T366W, and the second constant heavy chain has the amino acid substitutions Y34 a second constant heavy chain with amino acid substitutions of 9C, T366S, L368A, and Y407V; The amino acid residues are numbered according to the EU numbering system.

[0018] In further embodiments, engineered multispecific antibodies (e.g., bispecific antibodies) or The fragment is T366W / S354C-T366S / L368A / Y407V / Y Includes both 349C (KiH) and M252Y / S254T / T256E (YTE), The amino acid residues are numbered according to the EU numbering system.

[0019] Without being bound by theory, in some embodiments, multispecific antibodies ( For example, a bispecific antibody) or fragment thereof may be used in conjunction with hFc to mediate, for example, ADCC and / or contain one or more mutations to suppress CDC effector function. A set of mutations is, for example, LALA (L234A, L235A according to EU numbering). (Wines et al., 2000) or DAPA (D265 according to EU numbering) A, P329A) (Genentech, U.S. Pat. No. 6,737,056) Some researchers have used cloning agents to reduce effector function. We adopted the cross-subclass approach. , IgG2 variants were generated by point mutations from IgG4 (i.e., EU numbering H268Q, V309L, A330S, P331S) (An et al., 20 9) Another silent IgG1 antibody is the aglycosylated / non-glycosylated antibody. It contains the N297A mutation (Strohl et al., 2009). The mutation set used combines previously described techniques to remove some or all of the effects. achieve higher levels of silencing, up to complete loss of activator function. Examples are (D265A, N297A, P329A) (International Publication No. 2019068632 (Issue brochure Janssen) Manipulation of important residues in the Fc region responsible for effector function Other alternative approaches for the preparation or mutation of P CT International Publication No. 2009 / 100309 Brochure (Medimmune), International Publication Patent Application Publication No. 2006 / 076594 (Xencor), U.S. Patent Application Publication No. 06 / 0134709 (Macrogenics), U.S. Pat. No. 56 (Genentech), U.S. Patent Application Publication No. 2010 / 0166740 See specification (Roche).

[0020] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segment contains D265A / P329A (DAPA), and this amino acid residue is are numbered according to the numbering system.

[0021] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segment contains L234A / L235A (LALA), and these amino acid residues are located in the EU are numbered according to the numbering system.

[0022] In another embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The fragment is selected from the group consisting of positions 234, 235, 236, 297 and 299. and one or more cysteine ​​substitutions at amino acid residues 1 and 2 according to the EU numbering system. It is numbered.

[0023] In further embodiments, engineered multispecific antibodies (e.g., bispecific antibodies) or one or more cysteine ​​substitutions of the fragment are selected from positions 234, 235 and 236 In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) comprises In another embodiment, the modified multispecific antibody (e.g., In another embodiment, the modified The resulting multispecific antibody (e.g., bispecific antibody) contains a cysteine ​​substitution at position 236. nothing.

[0024] In some embodiments, engineered multispecific antibodies (e.g., bispecific antibodies) or The fragment may contain one or more amino acid substitutions that reduce or enhance Fc effector function. and engineered multispecific antibodies (e.g., bispecific antibodies) or The fragments may contain one or more amino acid substitutions and / or correct chain modifications that enhance the half-life of the fragments. Contains one or more amino acid substitutions that promote pairing.

[0025] Thus, for example, in some embodiments, engineered multispecific antibodies (e.g., bispecific antibody) or a fragment thereof is T366W / S35 according to EU numbering 4C-T366S / L368A / Y407V / Y349C(KiH) and YTE(M25 LS (M428L, N434S) and LS (M428L, N434S) In some embodiments, the modified multispecific antibody comprises a half-life extending mutation selected from the group consisting of: The antibody (e.g., bispecific antibody) or fragment thereof is designated T36 according to EU numbering. 6W / S354C-T366S / L368A / Y407V / Y349C(KiH) and L ALA (L234A, L235A), DAPA (D265A, P329A) and N297 In some embodiments, the Fc silencing mutation is selected from the group consisting of: The engineered multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be used in combination with the EU T366W / S354C-T366S / L368A / Y407V / Y3 by combining 49C (KiH), YTE (M252Y, S254T, T256E) and LS (M428 L, N434S) and LALA (L234 A, L235A), DAPA (D265A, P329A) and N297 The Fc-silencing mutations are selected.

[0026] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segments are L234A / L235A (LALA) and M252Y / S254T / T25 6E(YTE), the amino acid residues of which are numbered according to the EU numbering system. There are.

[0027] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segments include L234C and M252Y / S254T / T256E(YTE), The amino acid residues are numbered according to EU numbering. The modified multispecific antibody (e.g., bispecific antibody) or fragment thereof may be L235 C and M252Y / S254T / T256E (YTE), and these amino acid residues are In another embodiment, the modified multispecific The antibody (e.g., bispecific antibody) or fragment thereof may comprise G236C and M252Y / It contains S254T / T256E (YTE), and these amino acid residues are They are numbered as follows.

[0028] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segments are L234A / L235A(LALA), M252Y / S254T / T256 E(YTE) and T366W / S354C-T366S / L368A / Y407V / Y3 49C(KiH), and this amino acid residue is numbered according to the EU numbering system. are.

[0029] In one embodiment, an engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is The segments are L234C, M252Y / S254T / T256E(YTE) and T366 Including W / S354C-T366S / L368A / Y407V / Y349C(KiH), The amino acid residues are numbered according to EU numbering. The engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof may be L23 5C, M252Y / S254T / T256E(YTE) and T366W / S354C-T 366S / L368A / Y407V / Y349C(KiH), and these amino acid residues are , numbered according to EU numbering. In another embodiment, modified multiple specific The heterospecific antibody (e.g., bispecific antibody) or fragment thereof may have the following amino acid sequence: G236C, M252Y / S254T / T256E(YTE) and T366W / S354C-T366S / L36 8A / Y407V / Y349C (KiH), and these amino acid residues are represented by the EU numbering system. They are numbered according to the tag.

[0030] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The VH1 and VH2 domains of the nucleotide sequence are composed of complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3. 2, HCDR3, and VL1 and VL2 are LCDR1, LCDR2, LCDR3 Including, a. The VH1 domain comprises (e.g., in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 32, the amino acid sequence SEQ ID NO: 33 and the HCDR3 having the amino acid sequence SEQ ID NO: 34, or ii. The HCDR1 having the amino acid sequence SEQ ID NO: 35, the amino acid sequence SEQ ID NO: 36 and the HCDR3 having the amino acid sequence SEQ ID NO: 37, or iii. The HCDR1 having the amino acid sequence SEQ ID NO: 38, the amino acid sequence SEQ ID NO: 3 9 and the HCDR3 having the amino acid sequence SEQ ID NO: 40 and b. The VL1 domain is (e.g., in order): i. the LCDR1 having the amino acid sequence SEQ ID NO:4, the LCDR1 having the amino acid sequence SEQ ID NO:5 and the LCDR3 having the amino acid sequence SEQ ID NO:6, or ii. the LCDR1 having the amino acid sequence SEQ ID NO: 7, the LCDR1 having the amino acid sequence SEQ ID NO: 8 and the LCDR3 having the amino acid sequence SEQ ID NO: 9, or iii. The LCDR1 having the amino acid sequence SEQ ID NO: 10, the amino acid sequence SEQ ID NO: 1 the LCDR2 having the amino acid sequence SEQ ID NO: 1 and the LCDR3 having the amino acid sequence SEQ ID NO: 12 and c. The VH2 domain is (e.g., in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 46, the amino acid sequence SEQ ID NO: 47 and the HCDR3 having the amino acid sequence SEQ ID NO: 48, or ii. The HCDR1 having the amino acid sequence SEQ ID NO: 49, the amino acid sequence SEQ ID NO: 50 and the HCDR3 having the amino acid sequence SEQ ID NO: 51, or iii. The HCDR1 having the amino acid sequence SEQ ID NO: 52, the amino acid sequence SEQ ID NO: 5 3 and the HCDR3 having the amino acid sequence SEQ ID NO: 54 and d. The VL2 domain may be (e.g., in order): i. the LCDR1 having the amino acid sequence SEQ ID NO: 18, the LCDR1 having the amino acid sequence SEQ ID NO: 19 and the LCDR3 having the amino acid sequence SEQ ID NO: 20, or ii. The LCDR1 having the amino acid sequence SEQ ID NO: 21, the amino acid sequence SEQ ID NO: 22 and the LCDR3 having the amino acid sequence SEQ ID NO: 23, or iii. The LCDR1 having the amino acid sequence SEQ ID NO: 24, the amino acid sequence SEQ ID NO: 2 5 and the LCDR3 having the amino acid sequence SEQ ID NO:26 Includes.

[0031] In some embodiments, the first light chain is of the lambda type and the second light chain is of the kappa type. It is a type.

[0032] In some embodiments, the first light chain is lambda 1 type and the second light chain is kappa type. It is type 4.

[0033] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The ment comprises a VL1 domain comprising the amino acid sequence SEQ ID NO: 13 and a VL2 domain comprising the amino acid sequence SEQ ID NO: 2 7, containing the VL2 domain.

[0034] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The ment comprises a VH1 domain comprising the amino acid sequence SEQ ID NO: 41, a VH2 domain comprising the amino acid sequence SEQ ID NO: 13 a VL1 domain comprising the amino acid sequence SEQ ID NO:55; a VH2 domain comprising the amino acid sequence SEQ ID NO:56; The VL2 domain comprises SEQ ID NO: 27.

[0035] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The ment comprises a first light chain comprising the amino acid sequence shown in SEQ ID NO: 14 and a second light chain comprising the amino acid sequence shown in SEQ ID NO: 28. and a second light chain comprising an amino acid sequence selected from the group consisting of:

[0036] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The ment comprises a first heavy chain containing a heterodimerization modification and a second heavy chain containing a heterodimerization modification of the first heavy chain. and a second heavy chain comprising a heterodimerization modification complementary to the modification.

[0037] In some embodiments, the first and second constant heavy chains comprise a heterodimerization modification. IgG1, and a) The heterodimerization modification of the first immunoglobulin heavy chain is a serine at position 366, a alanine at position 368 and valine at position 407, and a heterodimer of the second immunoglobulin heavy chain The dimerization modification includes tryptophan at position 366, or b) The heterodimerization modification of the second immunoglobulin heavy chain is a serine at position 366, a nucleotide at position 444, a nucleotide at position 446, a nucleotide at position 448, a nucleotide at position 449, a nucleotide at position 450, a nucleotide at position 451, a nucleotide at position 452, a nucleotide at position 453, a nucleotide at position 454, a nucleotide at position 455, containing an alanine at position 368 and a valine at position 407, and a heterodimer of the first immunoglobulin heavy chain. The dimerization modification involves tryptophan at position 366, The amino acid residues are numbered according to the EU numbering system.

[0038] In some embodiments, the multispecific antibody exhibits dual specificity through enhanced FcRn binding. It is a bispecific antibody containing mutations that enhance the half-life of the bispecific antibody.

[0039] In some embodiments, the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E(YTE), and these amino acid residues correspond to They are numbered accordingly.

[0040] In some embodiments, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:42. , and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:56.

[0041] In some embodiments, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57. , and the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO:58.

[0042] In some embodiments, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:42. , and the first light chain comprises the amino acid sequence set forth in SEQ ID NO: 14, and the second heavy chain comprises , the second light chain comprises the amino acid sequence set forth in SEQ ID NO: 56, and the second light chain comprises the amino acid sequence set forth in SEQ ID NO: 28. It includes the amino acid sequence.

[0043] In some embodiments, the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO:57. , and the first light chain comprises the amino acid sequence set forth in SEQ ID NO: 14, and the second heavy chain comprises , the second light chain comprises the amino acid sequence set forth in SEQ ID NO: 58, and the second light chain comprises the amino acid sequence set forth in SEQ ID NO: 28. It includes the amino acid sequence.

[0044] The multiple compounds of the present disclosure in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. Pharmaceutical compositions comprising specific antibodies (e.g., bispecific antibodies) or fragments thereof are also described herein. It is revealed in the book.

[0045] In some embodiments, the pharmaceutical composition further comprises one or more additional active agents.

[0046] Also disclosed herein are isolated nucleic acid molecules encoding the multispecific antibodies of the present disclosure.

[0047] Cloning or expression vectors containing one or more nucleic acid sequences as outlined above are also contemplated herein. The vectors disclosed herein are suitable for recombinant production of the multispecific antibodies of the present disclosure. In some embodiments, a set of two cloning or expression vectors is provided as described herein. The first vector contains a full-length heavy chain including a constant domain and a variable domain, and A heavy chain encoded by a first vector encodes a full-length light chain including constant and variable domains. The IL-18 and light chains combine to form the anti-IL-18 arm of the bispecific IgG antibody. and the second vector can contain a full-length heavy chain containing a constant domain and a variable domain. and a full-length light chain comprising constant and variable domains, encoded by a second vector. The heavy and light chains used are the anti-IL-13 chains of the bispecific IgG antibody as described herein. In some embodiments, the first and the second vector is an expression vector, and the first and second vectors in a common host cell Co-expression of vectors produces anti-IL-18 / IL-13 bispecific antibodies with high yield, purity, and activity IgG-like antibodies are provided.

[0048] Host cells containing one or more cloning or expression vectors as outlined above are also contemplated herein. Disclosed in the specification.

[0049] Also disclosed herein is a process for the production of the multispecific antibodies of the present disclosure, which Cultivating host cells as outlined above under conditions sufficient to express specific antibodies. and then purifying and recovering the multispecific antibody from the host cell culture. include.

[0050] Kits containing one or more cloning and / or expression vectors of the disclosure are also disclosed herein. The kit is shown to be capable of detecting the multispecific (e.g., bispecific) antibodies disclosed herein. Further included are instructions for making.

[0051] Also disclosed herein are kits comprising a multispecific antibody of the disclosure or a pharmaceutical composition of the disclosure, The kit may include instructions for use and a method for administering the multispecific antibody or pharmaceutical agent to a subject in need thereof. and a means for administering the composition.

[0052] In some embodiments, the means for administration is a syringe, an autoinjector, an injection pen, Includes vials and syringes, infusion pumps, patches or infusion bags and needles.

[0053] A method for simultaneously binding IL-13 and IL-18, comprising: Also disclosed herein are methods comprising contacting a subject with an effective amount of a multispecific antibody of the present disclosure. In some cases, the contacting is performed in vitro. In some cases, the contacting is performed ex vivo. In some cases, the contacting requires IL-13 and IL-18 inhibition. It is carried out in a subject, such as a human patient.

[0054] A method for simultaneously inhibiting the activity of IL-13 and IL-18, comprising administering to a plurality of mammalian cells with an effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. Also disclosed herein. In some cases, the contacting is performed in an in vitro or ex vivo culture. In some cases, the contacting is with a non-human animal, such as a non-human primate. In this case, contact is required for patients with IL-18 and IL-13 inhibition, such as those with atopic dermatitis. In some cases, IL-13 and IL-18 activity is measured by At least 10%, at least 25%, at least 50%, at least 75% or less In some cases, the reduction in IL-13 activity is due to the suppression of STAT-6 signaling. In some cases, the reduction in IL-18 activity is measured by a decrease in LPS / I signaling. IFNγ production, such as that induced by IgE-12. In some cases, the reduction in IL-18 and / or IL-13 activity is achieved by the methods described herein. and increasing the levels of IL-18 or IL-13, respectively, bound to the multispecific antibody. is measured by a decrease in the levels of free IL-18 or IL-13, respectively.

[0055] A method for simultaneously inhibiting the activity of IL-13 and IL-18 in a subject, comprising administering to a subject a therapeutically effective amount of IL-13 and IL-18. a method comprising administering to a subject an effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. Also disclosed herein are: IL- Simultaneous blockade of IL-13 and IL-18 was shown to be superior to blockade of IL-18 or to blockade of IL-13. It is hypothesized that these compounds may have complementary (e.g., synergistic) effects compared to one another. In some embodiments, simultaneous blockade of IL-13 and IL-18 compared to blockade of IL-18 may have complementary (eg, synergistic) effects.

[0056] A method of treating an IL-13 and / or IL-18 mediated disorder in a subject, comprising: administering to a subject a therapeutically effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. Also disclosed herein are methods. In some embodiments, the methods comprise administering a monospecific anti-IL-1 In some embodiments, the treatment is improved compared to treatment with a 3 antagonist. This method is an improved treatment compared to treatment with a monospecific anti-IL-18 antagonist. In some embodiments, the method comprises administering an anti-IL-13 or anti-IL-18 antagonist In some embodiments, the improvement is an improvement in treatment compared to treatment with Better Eczema Area and Severity Index (EASI) scores after 16, 24, 36 or 52 weeks of Core, better Physician Global Assessment (IGA) score, better pruritus numerical rating by scale scores and / or better dermatology-related quality of life index scores In some embodiments, the method comprises administering an anti-IL-13 or anti-IL-18 antagonist to a subject. This improvement was observed at 16, 24, and 36 minutes after treatment compared with the non-steroidal antihistamine treatment. or a lower atopic dermatitis severity scoring (SCORAD) score after 52 weeks Denoted by the core.

[0057] In some embodiments, the methods of the disclosure involve simultaneous blockade of IL13 and IL18 (e.g., , at a level prior to treatment with a multispecific antibody (e.g., a bispecific antibody) or fragment thereof. compared with control groups, the incidence of one or more AD-related biomarkers, particularly CCL17 / TARC and IgE (e.g., serum IgE), CCL26 / eotaxin-3, CCL22 / MDC, hsC RP, CD40, IL-13, IL-24, IL-22, IL-18 (e.g., serum IL -18, serum-free IL-18 (bioactive)) and IL-18BP (e.g., serum IL- The expression levels of one or more AD-related biomarkers selected from the list consisting of 18BP This reduces the

[0058] A method for inhibiting IgE antibody production in a subject, comprising administering to a subject (e.g., an effective amount of any of the methods of the present disclosure) IL-13 and IL-18 are administered simultaneously or sequentially to the subject (by administering a polyspecific antibody to the subject). Also disclosed herein are methods that include inhibiting the expression of the protein by

[0059] A method for inhibiting IFN-γ production in a subject, comprising administering to a subject an effective amount of any of the compounds of the present disclosure. IL-13 and IL-18 are administered simultaneously or sequentially to the subject (by administering a polyspecific antibody to the subject). Also disclosed herein are methods that include inhibiting the expression of the protein by

[0060] 1. A method for treating and / or preventing an inflammatory or immune condition, comprising administering to a subject (e.g., a subject) a therapeutically effective amount of administering the multispecific antibodies of the present disclosure to a subject in need of treatment and / or prevention of an inflammatory or immune condition The present invention includes simultaneous or sequential inhibition of IL13 and IL18 (by administering the same to the same patient). Also disclosed herein are methods of treating the inflammatory or immune condition. In a preferred embodiment, the inflammatory or immune condition is a skin condition. In a preferred embodiment, the skin condition is atopic dermatitis. In some cases, atopic dermatitis is moderate to severe atopic dermatitis. In some cases, atopic dermatitis is moderate to severe atopic dermatitis. Topical dermatitis, as determined by the Rajka / Langeland criteria score, Moderate to severe, with a Rajka / Langeland score of 4.5 In some embodiments, the method comprises administering one or more topical corticosteroids. In some embodiments, the atopic dermatitis further comprises administration of one or more steroids. It is poorly controlled by administration of topical corticosteroids.

[0061] In a further aspect, the multispecific antibodies (e.g., Disclosed herein are the use of antibodies (e.g., bispecific antibodies) or fragments thereof.

[0062] In a further aspect, a method for the manufacture of a medicament for the treatment and / or prevention of AD, comprising administering to a subject a compound of the present disclosure. The use of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof is disclosed herein. is shown.

[0063] Further details and embodiments are provided in the following sections. [Brief explanation of the drawings]

[0064] [Figure 1]The following illustrates the possible products when expressing two KiH-modified mAbs in the same host cell line: LHHL: light chain-heavy chain-heavy chain-light chain, this fraction contains the final bispecific; HHL: heavy chain-heavy chain-light chain; HL: heavy chain-light chain = half mAb; H: heavy chain; L light chain; LL: light chain hetero- and homodimers. [Figure 2] Schematic diagrams of plasmids A to D for expressing IL-13 and IL-18. Plasmids A and C encode the expression of anti-IL13 kappa LC and anti-IL13 knob HC, while plasmids B and D encode the expression of anti-IL18 lambda LC and anti-IL18 hole HC. [Figure 3] Schematic diagram of Furin-2A peptide (F2A) plasmids E and F. F2A technology allows combinatorial expression of multiple protein chains from a single promoter. In plasmids E and F, the first expression cassette encodes anti-IL18 lambda LC and anti-IL18 hole HC, and the second expression cassette encodes anti-IL13 kappa LC and anti-IL13 knob HC. [Figure 4] Schematic diagram of compatible plasmids G and H. Plasmid G encodes the expression of anti-IL13 kappa LC and anti-IL13 knob HC, while plasmid H encodes the expression of anti-IL18 lambda LC and anti-IL18 hole HC. [Figure 5] Schematic diagram of Furin-2A peptide (F2A) plasmid I with different combinations of protein chains in the expression cassettes compared to plasmids E and F. On plasmid I, the first expression cassette encodes anti-IL18 lambda LC, anti-IL18 hole HC, and anti-IL13 knob HC, and the second expression cassette encodes anti-IL13 kappa LC. [Figure 6-1] Figure 6: Illustrative melting curves of IL-13 / IL-18 bispecific antibodies. Figure 6A is the melting curve for bbmAb1. Figure 6B is the melting curve for bbmAb2. Figure 6C is the melting curve for bbmAb5. Figure 6D is the melting curve for bbmAb4. Figure 6E is the melting curve for bbmAb3. [Figure 6-2](As mentioned above.) [Figure 6-3] (As mentioned above.) [Figure 6-4] (As mentioned above.) [Figure 6-5] (As mentioned above.) [Figure 7] A comparison of the pharmacokinetic profiles in Tg276 mice following administration of bbmAb1 and bbmAb2 and Fc-silenced variants bbmAb6, bbmAb7, bbmAb8 and bbmAb9 is shown (pooled serum samples plotted by sampling time). [Figure 8] This figure illustrates the results of gene set variation analysis (GSVA) ​​of differentially expressed genes between control samples (skin biopsies) not treated with a cytokine cocktail that induces an atopic dermatitis (AD)-like transcriptome (control), samples treated with a cytokine cocktail that induces an atopic dermatitis (AD)-like transcriptome (induced samples), and samples co-incubated with an isotype control antibody (AD + isotype). Induced samples co-incubated with an anti-IL-18 antibody (AD + anti-IL18), an anti-IL-13 antibody (AD + anti-IL13), and a bispecific antibody that simultaneously inhibits IL-13 and IL-18 activity (AD + bbmAb1) show various degrees of inhibition. Data are shown for cells from five different donor samples. [Figure 9] 1 illustrates the results of t-distributed stochastic neighbor embedding (t-SNE) analysis (at perplexity 5) based on in vitro disease transcriptomes (AD+ isotype vs. control) of 507 upregulated genes as described in Example 8. [Figure 10] 1 illustrates the results of t-SNE analysis (at perplexity 5) based on in vitro disease transcriptomes (AD+ isotypes vs. controls) of 1485 differentially expressed genes as described in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0065] In order that the present disclosure may be more readily understood, specific terms are used throughout the detailed description. Unless otherwise defined, all scientific and technical terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the disclosure pertains.

[0066] definition Interleukin (IL)-18 (referred to herein simply as "IL-18") is a Produced primarily by macrophages and T cells as a precursor protein (pro-IL-18) After cleavage by caspase-1, it is secreted as an active protein (Din arello CA et al (1999) J Allergy Clin Immu nol;103:11-24). Under normal physiological conditions, IL-18 interacts with IL-12. Induction of cell-mediated immunity after infection with microbial products such as lipopolysaccharide (LPS) in a synergistic effect Related to induction (Sareneva T et al (2000) J Immunol;1 65(4):1933-8). After stimulation with IL-18, natural killer (NK) cells and and T cells produce cytokines, which play an important role in the activation of macrophages and other cells. IL-18 releases interferon gamma (INF-γ). In addition to its ability to induce NF-κB, it also has a variety of other functions. B activation, Fas ligand expression, induction of both CC and CXC chemokines, and competition This includes improved production of human immunodeficiency virus.

[0067] The term "IL-18" refers to interleukin-18 polypeptide, an IFN-γ-inducing factor. IL-, also known as interferon-gamma-inducing factor or INF-γ-inducing factor Throughout this specification, the term IL-18 refers to the pro- or mature form. Unless otherwise indicated, pro-IL-18 (mature IL-18 before protease cleavage) is used. The term "IL-18" interchangeably encompasses both the precursor of IL-18 and mature IL-18 (after protease cleavage).

[0068] Interleukin (IL)-13 (referred to herein simply as "IL-13") is a It is a pleiotropic cytokine produced mainly by Th2 cells and ILC2, but relatively moderately To a lesser extent, mast cells, basophils, eosinophils, natural killer cells, macrophages, and dendritic cells Free IL-13 is also produced by IL-1 in all cells of the human body. It binds to the a1 subunit of the IL-13 receptor (IL-13Ra1), particularly in monocytes and B cells. In a cascade reaction, this binding is catalyzed by dimerization of Janus kinase 1. signal transducer and activator of JAK1 and tyrosine kinase 2 (TYK2) It supports the recruitment of IL-4Ra, which induces the formation of IL-4 receptors and signal transducers of transcription. phosphorylation of transcription activating factor 6 (STAT6), a transcription factor that promotes TH2 differentiation, and IgE This leads to class switching (Silverberg et al. (2017) Derm atol Clin.35(3):327-334;Goenka et al(201 1) Immunol Res. 50(1):87-96). The term "IL-13" refers to IL It is a synonym for -13 polypeptide or interleukin-13 polypeptide.

[0069] In all cases, the terms "comprises" and "comprises" "s)," "comprising," and the like are used in reference to sequences (e.g., amino acid sequences) and The array "consists of," "is made up of," or "is not made up of" It should be understood that the term "contains" or "is" may also be used to describe the invention. When used, the phrase "consisting essentially of" refers to the active pharmaceutical ingredient included in a method or composition. Any excipients that are inactive for the intended purpose of the drug genus or species and method or composition. In some embodiments, the phrase "consisting essentially of" refers to the vehicle of the multispecific antibody of the present disclosure. The phrase "antibody" expressly excludes the inclusion of one or more additional active agents other than the body. "Consisting essentially of" means that the multispecific antibody of the present disclosure is a multispecific antibody. The inclusion of two co-administered agents is expressly excluded.

[0070] The term "antibody" as used herein refers to an antibody that binds to an antigen non-covalently and reversibly. A polypeptide of the immunoglobulin family (or a polypeptide of For example, natural IgG-type antibodies are made up of molecules interconnected by disulfide bonds. It is a tetramer containing at least two heavy (H) chains and two light (L) chains connected together. Each heavy chain is composed of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain. The heavy chain constant domain is composed of three domains, CH1, CH2 and CH3. The chain comprises a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (abbreviated herein as C The VH and VL regions are called complementarity-determining regions (CDRs). These regions can be further divided into regions of hypervariability and more conserved regions called framework regions (FRs). Each VH and VL has the following sequences from the amino terminus to the carboxy terminus: Three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR The variable regions of the heavy and light chains are composed of CDR3, CDR4, and FR4. The antibody contains a binding domain, which may be referred to herein as an antigen-binding domain. The body's constant regions are responsible for the expression of various cells of the immune system (e.g., effector cells) and the classical complement system. Contains the first component (C1q), which may mediate the binding of immunoglobulins to host tissues or factors .

[0071] The term "antibody" includes monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, and the like. Antibodies, including but not limited to, antibodies, bispecific or multispecific antibodies and anti-idiotypic (anti-Id) antibodies The antibody may be of any isotype / class (e.g., IgG, IgE, Ig M, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3 , IgG4, IgA1 and IgA2).

[0072] Both the light and heavy chains are divided into regions of structural and functional homology. and "variable" are used functionally. In this respect, the light (VL) and heavy (VH) chain segments It is recognized that both the variable domains of the light chain determine antigen recognition and specificity. The constant domains of the heavy chain (CL) and heavy chain (CH1, CH2 or CH3) are involved in secretion, transplacental transfer, and These confer important biological properties such as motility, Fc receptor binding, and complement fixation. The numbering of the region domains is such that the more distant they are from the antigen binding site or amino terminus of the antibody, the greater the number of the region domains. The N-terminus of the molecule contains the variable region and the C-terminus contains the constant region; The CH3 and CL domains comprise the carboxy termini of the heavy and light chains, respectively.

[0073] The phrase "antibody fragment" as used herein refers to one or more portions of an antibody. In some embodiments, these portions are parts of the constant domain of an antibody, e.g., Examples include the crystallizable fragment (Fc), the constant (C) domain, etc. In embodiments, these moieties bind non-covalently, reversibly, and specifically to antigens. an antigen-binding fragment that retains the ability to bind to an antigen, referred to herein as an antigen-binding domain The term "antigen-binding fragment" as used herein refers to an antigen-binding fragment that binds to an epitope of an antigen. specifically interact with the target (e.g., binding, steric hindrance, stabilizing / destabilizing, spatial separation) A binding fragment refers to one or more portions of an antibody that retain their ability to bind to the antibody. Examples of binding fragments include , single-chain Fv (scFv) (with or without internal cysteine ​​bridges), disulfide-linked Fv (sdFv), F(ab)2 fragment, Fab fragment, F(ab')2, F(ab') fragment, consisting of VL, VH, CL and CH1 domains two Fab fragments linked by a disulfide bridge in the hinge region a bivalent fragment containing the VL and VH domains of a single arm of an antibody; Fv Fragment;V H dAb fragments consisting of domains (Ward et al., ( 1989) Nature, 341:544-546); and isolated complementarity determination of antibodies These include, but are not limited to, CDRs or other epitope-binding fragments.

[0074] As used herein, "Fc" or "Fc region" refers to a region comprising CH2 and CH3, Optionally, the Fc region includes any portion of an antibody hinge region. Each half antibody of the present disclosure is composed of two polypeptide chains that dimerize to form a single antibody. For example, a half antibody having an IL-18 scFv contains an Fc polypeptide chain. This half antibody contains an IL-18 scFv linked to a polypeptide chain. and two Fc polypeptide chains dimerize into the Fc region of the multispecific antibody of the present disclosure. Like all polypeptide chains, the Fc polypeptide chain has an N-terminal and a C-terminal and ends, each of which contains an antigen-binding domain (e.g., an IL-18 binding domain or IL-13 binding domain).

[0075] Antibody fragments include single domain antibodies, maxibodies, minibodies, and intrabodies. , diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (e.g. , Hollinger and Hudson, (2005) Nature Biote (See, Technology 23:1126-1136).

[0076] An antibody fragment comprises a complementary light chain polypeptide (e.g., VL-CL-VL-CL) and Together, the tandem Fv segments form a pair of antigen-binding regions (e.g., VH-CH1- The VH-CH1 pair can be assembled into a single chain molecule (Zapata et al., 1995) Protein Eng., 8:1057-1062; and U.S. Pat. Nos. 5, 6 41,870).

[0077] The term "half antibody" refers to an antibody molecule, antibody fragment, or antibody-like molecule that contains a single antigen-binding domain. A half antibody refers to a molecule or a portion of a polyspecific binding molecule. In one embodiment, a half antibody is, for example, an IgG A half antibody refers to a pair of heavy and light chains of an antibody. In one embodiment, a half antibody comprises a VL domain and a CL domain. A polypeptide comprising a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a second polypeptide comprising a CH3 domain (i.e., Fd and Fc), The VL and VH domains comprise an antigen-binding domain. refers to a polypeptide comprising an Fv domain and an Fc polypeptide chain (including a CH2 domain and a C H3 domain and optionally a hinge region). Either the first half antibody, the second half antibody, or both the first and second half antibodies may contain an additional antibody. In some polyspecific binding molecule embodiments, the first half-antibody may comprise a first half-antibody binding domain. The antibody associates, e.g., heterodimerizes, with a second half antibody. In the construct, the first half antibody is covalently linked to the second half antibody.

[0078] The term "monospecific molecule" as used herein refers to a molecule that binds to one epitope on a target antigen. In some embodiments, the monospecific molecule of the present disclosure is a monospecific molecule. In some embodiments, the monospecific molecules of the present disclosure are monospecific antibody-like molecules. The term "bispecific molecule" refers to a binding molecule that binds to two different antigens. The term "trispecific molecule" refers to a multispecific Fc that binds to a target antigen via three different binding moieties. It refers to an Fc that contains a multispecific binding molecule that binds to three different antigens. In embodiments, the bispecific molecules of the present disclosure are bispecific antibody-like molecules. In embodiments, the polyspecific binding molecules of the present disclosure are multispecific antibody-like molecules.

[0079] The term "multispecific antibody" refers to an antibody that binds to more than one epitope of an antigen or more than one antigen. The recognition of each antigen is generally achieved via an "antigen-binding domain." In particular, bispecific antibodies bind to two different antigens, either on the same antigen or on different antigens. All bispecific IgG molecules, i.e., natural immunoglobulins, recognize the same epitope. Bispecific antibodies, which are indistinguishable in their composition, are bivalent and contain at least two different F It maintains an asymmetric structure due to the presence of the v region. Depending on the method of preparation and the origin of the heavy and light chains, These may further differ in the constant regions of the heavy or light chains (Brinkmann and and Kontermann, 2017).

[0080] Bispecific antibodies are "heterodimers," meaning that one moiety targets a first target. a portion derived from a first antibody specific for the target and another portion derived from a second antibody specific for a second target "Heterodimerization modification" aims to promote such formation. Modifications to one or both portions of the antibody resulting in the formation of a heterodimeric bispecific antibody. The Fc domains of two IgG1 parts of an antibody are used to form a bispecific antibody. An example of a heterodimerization modification is the modification of a bulky amino acid (aa) side chain in the first heavy chain with a "knob" (S354C, T366W) with a small aa side chain and a "hole" (Y349C, T366S, L368A, Y407V) were introduced into the second heavy chain, and both An additional disulfide bridge in the CH3 region linking the heavy chains was introduced (Merch ant et al., Nat.Biotechnol.,16:677-681(19 98), p. 678, Table 1).

[0081] The terms "mismatch" or "mismatch formation" or "misassembled" refer to a duplex. Conjugate binding is targeted to different parts of the protein complex of interest, such as specific antibodies. This means that the protein complex does not look or behave as intended. An example of a mismatch in the context of a bispecific antibody is shown in Figure 1.

[0082] The terms "recognize" or "bind" as used herein mean that the epitope Whether linear, punctuated, or conformational, the epitope is A binding molecule, antibody or antigen-binding fragment thereof that detects and interacts with (e.g., binds to or recognizes) The term "epitope" refers to a segment to which an antibody or antigen-binding fragment of the present disclosure is specific. An epitope is a site on an antigen that specifically binds to a target molecule. It can be formed from both non-adjacent amino acids that are aligned by folding. Epitopes that are expressed are generally retained upon exposure to denaturing solvents, whereas epitopes that are expressed by tertiary folding are not. Epitopes formed by the cleavage of a protein are generally lost upon treatment with denaturing solvents. Typically, at least 3, 4, 5, 6, 7, 8, 9, 10, 11 unique spatial conformations 12, 13, 14, or 15 amino acids. Methods for this include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996) or electron microscopy. It is the part of an antibody that recognizes an epitope.

[0083] The phrases "specifically bind" or "selectively bind" refer to binding to an antigen (e.g., a protein). Used in the context of describing the interaction between an antibody, antibody fragment, or antibody-derived binding agent When used in a heterogeneous population of proteins and other biological materials, e.g., a biological sample, e.g., A binding reaction that is determinative of the presence of an antigen, for example in a blood, serum, plasma, or tissue sample. Therefore, under specific immunoassay conditions, a specific binding specificity is The antibody or binding agent binds to the specific antigen at least twice as much as background and In one aspect, the designated immune antigen does not bind substantially to any significant amount to other antigens present in the antibody. Under assay conditions, antibodies or binders with a particular binding specificity will exhibit a high level of activity over background binds to a specific antigen at least 10 times more than the amount of other antigens present in the sample Specific binding to an antibody or binding agent under such conditions does not result in qualitative binding to the antibody or binding agent. It may be necessary for the agent to be selected for its specificity for a particular protein. If desired or appropriate, this selection may be extended to include other species (e.g., mouse or rat). ) or other subtypes by subtracting antibodies that cross-react with the molecule. Alternatively, in some aspects, antibodies or antibody fragments that cross-react with a particular desired molecule are used. The account is selected.

[0084] The term "antigen-binding site" refers to the determinants that form the interface that binds to an antigen or its epitope. The term "antigen-binding site" refers to the portion of an antibody that contains the antigen. It can be used interchangeably with binding moiety. The original binding site generally forms an interface that binds to the antigenic polypeptide (at least 4 The antibody molecule generally comprises one or more loops (single amino acid or amino acid mimics). An antigen-binding site may comprise at least one or two CDRs and / or hypervariable loops or more generally Typically, it comprises at least 3, 4, 5 or 6 CDRs and / or hypervariable loops.

[0085] The term "complementarity determining region" or "CDR" as used herein refers to a region that is responsible for antigen specificity and It generally refers to the sequence of amino acids in the antibody variable region that confers binding affinity. The heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3) and each The light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The positions of the framework regions are defined according to various known definitions in the art, for example, Kab can be determined using the at, Chothia, IMGT, AbM and combined definitions (See, for example, Kabat et al., (1991) “Sequences of Pr oteins of Immunological Interest,”5th Ed .Public Health Service,National Institute tes of Health, Bethesda, MD; Johnson et al. ,(2001)Nucleic Acids Res.,29:205-206;Cho thia&Lesk,(1987)J.Mol.Biol.,196:901-917; Chothia et al.,(1989)Nature,342:877-883; Chothia et al.,(1992)J.Mol.Biol.,227:799 -817;Lefranc MP (2001) Nucleic Acids Res., 29:207-209;Al-Lazikani et al.,(1997)J.Mo (See I. Biol., 273:927-748.) The antigen-binding site is defined as follows: Also described in the literature: Ruiz et al., (2000) Nucleic Acids ds Res.,28:219-221;MacCallum et al.,(199 6) J. Mol. Biol., 262:732-745; and Martin et al. .,(1989)PNAS.USA,86:9268-9272;Martin et. al.,(1991)Methods Enzymol.,203:121-153; and Rees et al., In Sternberg MJE (ed.), Pr. otein Structure Prediction,Oxford Univer City Press, Oxford, 141-172 (1996). Kabat numbered. Under the scheme, in some embodiments, The DR amino acid residues are 31-35 (HCDR1), 50-65 (HCDR2), and 95- 102 (HCDR3); CDR amino acid sequence in the light chain variable domain (VL) The acid residues are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). DR3). Under the numbering scheme, in some embodiments The CDR amino acids in VH are 26 to 32 (HCDR1), 52 to 56 (HCDR2), ) and 95 to 102 (HCDR3); the CDR amino acid residues in VL are , 26~32(LCDR1), 50~52(LCDR2) and 91~96(LCDR3) The Kabat and Chothia combination numbering scheme In some embodiments, the CDRs are Kabat CDRs, Chothia CDRs, corresponding to amino acid residues that are part of the DR or both. For example, in some embodiments, The CDRs are amino acid residues 26 to 35 (e.g., ... HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and human VL, e.g., amino acid residues 24 to 34 (LCDR1) in a mammalian VL, e.g., a human VL; It corresponds to 50-56 (LCDR2) and 89-97 (LCDR3). Under IMGT, The CDR amino acid residues in VH are approximately 26-35 (CDR1), 51-57 (CDR2), ) and 93 to 102 (CDR3), and the CDR amino acid residues in VL are Approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the C The DR region can be determined using the program IMGT / DomainGapAlign. do.

[0086] The term "humanized" forms of non-human (e.g., murine) antibodies refer to antibodies derived from non-human immunoglobulins. For the most part, humanized antibodies are chimeric antibodies that contain minimal sequence. Residues from the hypervariable regions of mouse, rat, or mouse antibodies with the desired specificity, affinity, and potency are selected. Substituted by residues from the hypervariable region (donor antibody) of a non-human species such as rabbit or non-human primate. In some cases, the human immunoglobulin (recipient antibody) is replaced by a human immunoglobulin (recipient antibody). Replacing immunoglobulin framework region (FR) residues with corresponding non-human residues Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies , comprising substantially all of at least one, and generally two, variable domains, All or substantially all of the fragments correspond to those of a non-human immunoglobulin, and all or substantially all of the fragments correspond to those of a non-human immunoglobulin. Qualitatively, all are of human immunoglobulin lo sequence. Humanized antibodies are optionally , at least part of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525(1986);Riechmann et al.,Natur e 332:323-329 (1988); and Presta, Curr. Op. Str. See the following review articles and See also Vaswani and Hamil and the references cited therein. ton,Ann.Allergy,Asthma&Immunol.1:105-115 (1998);Harris,Biochem.Soc.Transactions 2 3:1035-1038(1995);Hurle and Gross,Curr.O p.Biotech.5:428-433(1994).

[0087] As used herein, the term "human antibody" refers to an antibody in which both the framework and CDR regions are The present invention also includes antibodies having variable regions derived from sequences of human origin. In this case, the constant region may also be, for example, a human germline sequence or a variant of a human germline sequence. Mutated versions or, for example, Knappik, et al. (2000. J Mol Biol ol 296,57-86) derived from human framework sequence analysis. Antibodies contain consensus framework sequences derived from human sequences.

[0088] The human antibodies of the invention may omit amino acid residues that are not encoded by human sequences (e.g., in vivo). Random or site-directed mutagenesis in vitro or somatic mutation in vivo These may include mutations introduced by the nucleotide sequence or conservative substitutions that promote stability or production. However, the term "human antibody" as used herein refers to antibodies derived from other mammalian species, such as mice. It includes antibodies in which germline-derived CDR sequences have been grafted onto human framework sequences. Not intended.

[0089] As used herein, a "modification" or "mutation" of an amino acid residue / position refers to a modification of the original amino acid. refers to a change in the primary amino acid sequence compared to the amino acid sequence of the original, and the change is For example, typical modifications result from the substitution of a residue by another amino acid (or substitution (e.g., conservative or non-conservative substitution) at said position, "Amino acid substitution" or "deletion" includes the insertion of one or more amino acids at said residues / positions. The mutation of a given amino acid sequence by a different amino acid residue It refers to the replacement of an existing amino acid residue. Generally, and preferably, the modification is a replacement of the original (or "wild" amino acid residue). at least one of the variant polypeptides compared to a polypeptide comprising the amino acid sequence For example, in the case of antibodies, the physico-biochemical activities to be altered are The property can be binding affinity, binding capacity and / or binding efficacy to a target molecule.

[0090] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants are those that contain the same or essentially the same amino acid sequence. A nucleic acid encoding an amino acid sequence or an essentially equivalent nucleic acid if the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids can be found in any given For example, the codons GCA, GCC, GCG, and GCU all encode proteins. It encodes the amino acid alanine, so that at all positions where alanine is specified by a codon In the position, the codons can be changed to any of the corresponding codons described, and the encoded Such nucleic acid variations are "silent variations" and do not alter the polypeptide produced. This is one type of conservatively modified variation. Every nucleic acid sequence also describes every possible silent variation of the nucleic acid. Each codon in the acid sequence (AUG, which is usually the only codon for methionine, and AUG, which is usually the only codon for tryptophan) that all codons except the one that is TGG can be modified to yield a functionally identical molecule Accordingly, each silent variation of a nucleic acid which encodes a polypeptide , implicit within each sequence described.

[0091] With respect to polypeptide sequences, "conservatively modified variants" refer to chemically similar amino acids. Substitution of an amino acid with a non-naturally occurring acid, including individual substitutions, deletions, or additions to a polypeptide sequence. Conservative substitution tables providing functionally similar amino acids are known in the art. Such conservatively modified variants include variants, interspecies homologs and alleles of the polymorphisms of the invention. The following eight groups are conservative substitutions for each other: Contains amino acids: 1) alanine (A), glycine (G); 2) aspartic acid (D) , glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine ( R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), Phosphorus (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7 ) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (e.g. See, for example, Creighton, Proteins (1984). In embodiments, the phrase "conservative sequence modifications" refers to modifications of an amino acid sequence of an antibody or antibody-like molecule. It refers to amino acid modifications that do not significantly affect or alter binding properties. It is used for this purpose.

[0092] In the context of two or more nucleic acid or polypeptide sequences, the terms "percent identical" or " "Percent identity" refers to two or more sequences or subsequences that are the same. Two sequences are Use one of the following sequence comparison algorithms over a comparison window or designated region: for maximum agreement, measured by manual alignment and visual inspection. When compared and aligned, a specific percentage of amino acid residues or nucleotides are the same (i.e., 60% across a specified region or, if not specified, across the entire sequence) , optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity). Optionally, identity may be at least A region that is at least about 50 nucleotides (or 10 amino acids), more preferably at least 10 0 to 500 nucleotides or 1000 nucleotides or more (or 20, 50, 200 amino acids) It exists over a range of acids.

[0093] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. A sequence comparison algorithm is used to compare test and reference sequences input into a computer. Sub-array coordinates are specified as needed, and the array algorithm program parameters are used. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm is then run based on the program parameters. The percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0094] As used herein, the term "comparison window" refers to the region over which two sequences are optimally aligned. The sequence can then be compared to a reference sequence at the same number of flanking positions, typically between 20 and 600, but usually about 50 to 600. about 200, more usually about 100 to about 150. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison is known in the art. and Waterman (1970) Adv. Appl. Math. 2:482c Local homology algorithm, Needleman and Wunsch (1970) J. Pearson homology alignment algorithm in Mol. Biol. 48:443 and Lipman,(1988)Proc.Nat'l.Acad.Sci.US A 85:2444 similarity search method, computerized implementation of these algorithms Implementation (Wisconsin Genetics Software P ackage,Genetics Computer Group,575 Scien GAP, BESTFIT, FASTA and and TFASTA) or manual alignment and visual inspection (e.g., Brent e t al.,(2003)Current Protocols in Molecule This can be done by the methods described in the literature (see, for example, J. Immunol. Biology).

[0095] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are: There are the BLAST algorithm and the BLAST 2.0 algorithm, Altschul et al. (1977) Nuc. Acids Res. 2 5:3389-3402; and Altschul et al., (1990) J.Mo Biol. 215:403-410. The software for this is provided by the National Center for Biotechnology Information. This algorithm is publicly available from the University of California, San Diego. High-scoring sequence pairs (HSPs) are then identified by identifying short words of length W in the query sequence. This includes identifying by specifying a word of the same length in a database sequence. When aligned with the T is called the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits find longer HSPs containing them. The word hits act as seeds for initiating searches for the cumulative alignments. The core is extended in both directions along each sequence for as far as it can grow. The cumulative score is calculated by multiplying the nucleotides For a given sequence, the parameter M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always <0) For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction stops when: If the investment score falls by an amount X from its maximum achieved value; if the cumulative score is one or more negative values If the scoring of residues becomes zero or less due to the accumulation of alignments; or The BLAST algorithm parameters W, T, and X are Determine the sensitivity and speed of the alignment. BLASTN program (nucleotide sequence For , the default is Word Length (W) of 11, Expectation (E) of 10, M=5, N=- For amino acid sequences, the BLASTP program uses the data A word length of 3 and an expectation (E) of 10 as faults and BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) Using alignment (B), 10 predictions (E), M=5, N=-4 and comparison of both strands .

[0096] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., , Karlin and Altschul (1993) Proc. Natl. Acad. (See, e.g., J. Sci. USA 90:5873-5787). BLAST algorithm One measure of similarity provided by the system is the minimum sum probability (P(N)), which is the sum of two An index of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, the nucleic acid may be one that has a minimum sum probability of about 0.5 in a comparison of the test nucleic acid to the reference nucleic acid. less than 2, more preferably less than about 0.01, and most preferably less than about 0.001; Considered similar to the reference sequence.

[0097] The percent identity between two amino acid sequences is calculated using the method of E. Meyers and W. Mil ler (Comput. Appl. Biosci. 4:11-17 (1988)) algorithm (which is incorporated into the ALIGN program (version 2.0)) PAM120 weight residue table, 12 and a gap penalty of 4. The percent identity between two amino acid sequences is calculated using the method of Needleman and Wunsc h (J. Mol. Biol. 48: 444-453 (1970)) algorithm (This is done with the GCG software package, available at www.gcg.com) P program), Blossom62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and 1, 2, Length weights of 3, 4, 5 or 6 may be used to determine the likelihood of a stray mark.

[0098] Two nucleic acid sequences or polypeptides other than the percentage of sequence identity noted above As another indication that the polypeptides encoded by the first nucleic acid are substantially the same, the peptide is immunologically directed against a polypeptide encoded by a second nucleic acid, as described below. Therefore, the polypeptides may be cross-reactive with antibodies raised against the For example, a peptide may be a peptide having a sequence similar to that of a second polypeptide, such as when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is As described below, two molecules or their complements can hybridize to each other under stringent conditions. Further indication that two nucleic acid sequences are substantially identical. In some cases, the same primers can be used to amplify the sequence.

[0099] The term "nucleic acid" or "polynucleotide" refers to a nucleic acid or polynucleotide in either single-stranded or double-stranded form. It refers to ribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. Unless otherwise specified, the term refers to a nucleic acid that has similar binding properties to the reference nucleic acid and is identical to naturally occurring nucleotides. Nucleic acids containing known analogues of natural nucleotides that are metabolized similarly are also included unless otherwise specified. Unless otherwise specified, a particular nucleic acid sequence implicitly includes conservatively modified variants thereof (e.g., degenerate sequences). Don substitutions), alleles, orthologs, SNPs and complementary sequences and sequences explicitly indicated Specifically, degenerate codon substitutions are those that substitute one or more selected (or all) codons. The third position of the amino acid sequence is substituted with mixed base and / or deoxyinosine residues. This can be achieved by activating the ATP-dependent ATPase (Batzer et al., Nucleic Acid cid Res.19:5081(1991);Ohtsuka et al.,JB iol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0100] The nucleotides in "polynucleotide" or "nucleic acid" are bromouridine and inosidine. base modifications such as thiazolinone derivatives, phosphorothioates, phosphorodithioates, phosphorose phosphorodiselenoate, phosphoroanilothioate, phosphoroanilader The amino acid sequence may include modifications such as ribose modifications such as ribose and phosphoramidate.

[0101] The term "vector" refers to a vector suitable for transformation or transfection of a host cell. and (in conjunction with a host cell) driving the expression of one or more heterologous coding regions operably linked thereto. Any molecule or entity (e.g., nucleic acid, platform) containing a nucleic acid sequence that regulates and / or modulates means a microorganism (e.g., a microorganism ...

[0102] As used herein, the term "operably linked" or "functionally linked" refers to two This refers to the functional relationship between the above polynucleotide (e.g., DNA) segments. The term "transcriptional regulatory sequence" refers to the functional relationship of a transcriptional regulatory sequence to a sequence to be transcribed. For example, a promoter or Enhancer sequences stimulate or regulate the transcription of a coding sequence in an appropriate host cell or other expression system. When a sequence is transcribed, it is operably linked to a coding sequence. The promoter transcriptional regulatory sequences linked to the promoter are all physically adjacent to the sequence to be transcribed. However, some transcriptional regulatory sequences, such as enhancers, It does not need to be physically adjacent to the coding sequence whose transcription it enhances, nor does it need to be positioned in close proximity to it. There's no need.

[0103] The term "co-expression" refers to the expression of different polypeptides in a single host in which all polypeptides are common. Co-expression of bispecific antibodies means that they are expressed together in a cell. This means that the different parts that form the target antibody are expressed in a single common host cell. Several expression vectors, such as one for each half of the polyspecific antibody, are introduced into the expression host cell. Alternatively, one expression vector may be constructed that encodes all parts of the bispecific antibody. Co-expression can be achieved by incorporating the C-terminus. , the carboxyl-terminal amino acid of a polypeptide chain having a free carboxyl group (-COOH) As used herein, "N-terminus" refers to a molecule having a free amine group (-NH2). The amino acid refers to the amino-terminal amino acid of a polypeptide chain.

[0104] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues. The terms are used interchangeably herein. The term refers to a nucleotide sequence in which one or more amino acid residues are present in a corresponding naturally occurring nucleotide. Amino acid polymers are artificial chemical mimics of existing amino acids, and naturally occurring amino acids It also applies to amino acid polymers and non-naturally occurring amino acid polymers unless otherwise specified. To the extent possible, a particular polypeptide sequence also implicitly embraces conservatively modified variants thereof.

[0105] The term "in vivo half-life" as used herein refers to the time a compound circulates in the blood of a mammal. The half-life of a molecule of interest or a variant thereof is referred to as the half-life of the molecule of interest or a variant thereof.

[0106] Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be produced by human bone marrow It can be produced by the hybridoma method using a mouse-human heteromyeloma cell line. (Kozbor, J Immunol; (1984) 133:3001; r, Monoclonal Isolated Antibody Productio n Techniques and Applications,pp51-63,Ma (Rcel Dekker Inc, 1987). An alternative method is to use both human variable regions. including the use of phage libraries or transgenic mice that utilize repertoires (Winter G;(1994)Annu Rev Immunol 12:433- 455, Green LL, (1999) J Immunol Methods 231 :11-23).

[0107] Several strains of transgenic mice are currently available, and their mouse immunological properties are The immunoglobulin locus has been replaced with a human immunoglobulin gene segment (T omizuka K,(2000)Proc Natl Acad Sci,97:7 22-727;Fishwild DM(1996)Nature Biotechno l 14:845-851;Mendez MJ,(1997)Nature Gene Upon antigen challenge, these mice express the antibody of interest. It is possible to generate a repertoire of human antibodies from which antibodies can be selected. Of particular note is the ability to generate human antibodies. The Trimera™ system (Eren R et al.) in which lymphocytes are transplanted into irradiated mice al., (1988) Immunology 93:154-161), humans (or other The large number of pooled lymphocytes effectively achieves the in vitro isolation antibody production procedure, followed by Selective Lymphocyte Isolation Antibody System (SLAM, Babcook et al, Proc Natl Acad Sci (1996)93:7843-78 48), limiting dilution and selection procedure and Xenomouse™ (Abgenix Inc.) An alternative approach is to use Morphodoma™ technology to Available from rphotek Inc.

[0108] Phage display technology can be used to generate human antibodies and fragments thereof. (McCafferty; (1990) Nature, 348:552-553 and G riffiths AD et al(1994)EMBO 13:3245-3260 This technique involves the production of protein genes from filamentous bacteriophages such as M13 or fd. The isolated antibody variable domain gene is inserted into either the major or minor coat of the offspring. cloned in frame and presented as functional isolated antibody fragments on the surface of phage particles. Selection based on the functional properties of the isolated antibodies. As a result, genes encoding isolated antibodies exhibiting these properties are selected. The Ray technology is directed at individuals suffering from a disease or disorder or alternatively at non-immunized humans. Antigen-specific antibodies are selected from a library made from human B cells collected from donors. (Marks; J Mol Bio (1991) 222:581- 591). If an intact human isolated antibody containing an Fc domain is desired, the desired constant Establish a stable cell line by displaying the phage in a mammalian expression vector containing the region. The resulting fragments must be re-cloned.

[0109] Affinity maturation techniques (Marks; Biotechnol (1992) 10:779-7 83) can be used to provide binding affinity, and natural variants of the heavy and light chain variable regions by successively replacing and selecting based on improved binding affinity of the primary human isolate The affinity of the antibody is improved. Variations of this technique, such as "epitope imprinting" A variety of forms are now available (WO 93 / 06213; Waterhouse et al., 2004). use; Nucl Acids Res (1993)21:2265-2266).

[0110] The term "pure" when used in the context of purified bispecific antibodies refers to intact UPLC- Using an MS mass screening approach, cells express bispecific antibodies of different bispecific antibodies after co-expression in selected cells and Protein-A purification in vitro. It relates to the purity and identity of the combinations and constructs. Purity or purity refers to the heterologous and The method of the present invention refers to the relative quantification of intact and homodimeric bbmAbs. Correctly formed heterodimers with a relative purity of >85% based on the mass signal intensity Bispecific antibodies can be observed.

[0111] The terms "therapeutically acceptable amount," "therapeutically effective amount," or "therapeutically effective dose" are and interchangeably refer to an amount sufficient to produce a desired result (i.e., a decrease in disease activity, a decrease in disease activity, In some embodiments, the therapeutic effect is therapeutically acceptable. An acceptable amount is one that does not induce or cause undesirable side effects. The amount that can be achieved is determined by administering a low dose initially and then gradually increasing the dose until the desired effect is achieved. "Prophylactically effective doses" and "therapeutically effective doses" of the molecules of the present disclosure can be determined by The "effective dose" refers to the amount of IL-13 and IL-18 that is effective in treating various diseases, including those associated with IL-13 and IL-18 activity. The onset of symptoms may be prevented or the severity of disease symptoms may be reduced as a result.

[0112] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g. For example, mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except where indicated, the terms "patient" or "subject" are used throughout this specification. Used interchangeably in writing.

[0113] As used herein, a "patient in need of treatment" or a "subject in need of treatment" and the like refer to molecules or pharmaceuticals of the present disclosure used for detection, diagnostic procedures and / or treatment, etc. This includes subjects, such as mammalian subjects, who may benefit from administration of the composition.

[0114] The terms "treat," "treating," "treatment," "prevent," "preventing," or "Prevention" includes therapeutic treatment, prophylactic treatment, and reducing a subject's risk of developing a disorder or other risk. Treatment does not require complete cure of the disorder, but rather may involve the treatment of symptoms or underlying conditions. As used herein, a human antibody or fragment thereof includes the reduction of risk factors associated with the The variable region or full-length chain of the antibody is derived from a human germline immunoglobulin gene. If obtained from a line that does, it is a "product" of or "derived from" a particular germline sequence. Such systems include heavy or light chain variable regions or full-length heavy or light chains. Transgenic mice carrying human immunoglobulin genes were injected with target antigens. Immunization or human immunoglobulins displayed on phage with an antigen of interest. This involves screening a library of human germline immunoglobulin genes. A human antibody or fragment thereof that is the "product" of or "derived from" a sequence The amino acid sequence of the antibody was compared with that of human germline immunoglobulins to identify human antibodies. The human germline immunoglobulin with the closest sequence (i.e., highest percent identity) to the human sequence Specific human germline immunoglobulins can be identified by, for example, selecting sequences. Human antibodies that are the "product" of or "derived from" sequences, e.g., naturally occurring somatic clones, Amino acid sequence changes compared to the germline sequence due to the deliberate introduction of mutations or site-directed mutations However, the human antibodies selected will generally contain human germline immunoglobulins. The amino acid sequence is slightly different from the amino acid sequence encoded by the globulin gene. and at least 90% identical to the germline immunoglobulin amino acid sequence of another species (e.g., Amino acid residues that identify a human antibody as human compared to the mouse germline sequence In certain cases, human antibodies are encoded by germline immunoglobulin genes. At least 60%, 70%, or 80% of the amino acid sequence , 90% or at least 95% or even at least 96%, 97%, 98% or 99% Generally, a human antibody derived from a particular human germline sequence can be identical to a human germline antibody. The amino acid sequence encoded by immunoglobulin genes of the cytoplasm and up to 10 amino acids In certain cases, human antibodies are derived from germline immunoglobulin genes The encoded amino acid sequence and up to five or even up to four, three, two or one amino acid residues. The difference in acidity may be indicated.

[0115] Various aspects of the invention are described in further detail in the sections and subsections that follow.

[0116] Multispecific antibodies that bind to IL-13 and IL-18 I. IL-18 Binding Domain The present disclosure provides multispecific antibodies (e.g., bispecific antibodies) engineered to bind human IL-18. The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are provided. Recognition of IL-18 by IL-18 specific antibodies occurs via the "IL-18 antigen binding domain." This is interchangeably referred to as the "IL-18 binding domain."

[0117] In a preferred embodiment, a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof The antibody contains one IL-18 binding domain and is a multispecific binding molecule with respect to IL-18 binding. In another preferred embodiment, the polyspecific binding molecule comprises multiple ILs. a multispecific binding molecule comprising two IL-18 binding domains, e.g., two IL-18 binding domains; The molecule is multivalent with respect to binding IL-18, preferably bivalent with respect to binding IL-18.

[0118] In some embodiments, the IL-18 binding domain of the multispecific antibody (e.g., bispecific antibody) The antibody or a fragment thereof is preferably IL-18 scFv or IL-18 Fab. These include IL-18 Fab.

[0119] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags ment for IL-18 was 10 -4 M~10 -8 M, e.g. 10 -5 M~10-7 M, for example , 10 -6 M or 10 -7 Binding affinity K of M D The IL-18 binding domain has the following structure:

[0120] In a preferred embodiment, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof are In another preferred embodiment, the antibody comprises one anti-IL-18 Fab. The combined domain contains two IL-18 Fabs. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof are also contemplated herein. .

[0121] In some examples, Fabs can be prepared according to methods known in the art. The immunoglobulin monomer is cleaved to produce two Fab fragments and an Fc fragment. The enzyme pepsin can be used to cleave below the hinge region to produce F(ab')2 The F(ab')2 fragment is formed by It can be separated into two Fab' fragments by mild reduction. Fab fragment is the non-covalent and CH interactions that occur across the large interface between the heavy and light chain polypeptides. It is highly stable due to the presence of a stabilizing disulfide bond between the I and CL domains. (Glover & Humphreys, Chapter 2, Antibodies es,Vol 1:Production and Purification,Klu wer Academic / Plenum Publishers,New York (See, e.g., 2004, edited by G Subramanian). Linker orientation and For example sizes, see, e.g., Hollinger et al. (1993) PNAS USA90:6444-6448, Patent Application Publication No. 2005 / 0100543, U.S. Patent Application Publication No. 2005 / 017 5606, U.S. Patent Application Publication No. 2007 / 0014794 and International Publication No. Pamphlet No. 2006 / 020258 and International Publication No. 2007 / 024715 Please refer to the fret.

[0122] The phrases "Fab that binds human IL-18" and "IL-18 Fab" refer to a Fab that binds human IL-18. In one embodiment, IL-18 Fab has equivalent binding affinity to IL-18. In another embodiment, the antibody binds to IL-18 with efficacy comparable to that of the full-length antibody. The IL-18 Fab has a lower binding affinity, e.g., lower than the full-length antibody. Although the antibody binds to IL-18 with a binding affinity that is not incompatible with the biological activity described herein, provide a relevant response.

[0123] In the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof A preferred IL-18Fab for use comprises the amino acid sequence set forth in SEQ ID NO:41 It comprises VH and VL comprising the amino acid sequence shown in SEQ ID NO:13.

[0124] In one aspect, the present disclosure provides a method for the production of an IL-18 antibody comprising an IL-18 binding domain, e.g., an IL-18 Fab, Polynucleotides encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof The present disclosure also provides isolated nucleic acid molecules encoding these Fabs.

[0125] VH having the amino acid sequence shown in SEQ ID NO: 41, and a multispecific antibody (e.g., a VL having an IL-18 binding domain) comprising an IL-18 binding domain, the VL having an amino acid sequence Disclosed herein are isolated nucleic acid molecules encoding the IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG8, IgG9, IgG1, IgG1, IgG4, IgG1, IgG2, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG4, IgG1, IgG2, IgG4, IgG4, IgG5, IgG6, IgG1, will be done.

[0126] In one embodiment, the IL of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof The IL-18 binding domain (e.g., IL-18 Fab) is expressed in mammalian cells. In one aspect, the sequence of the present disclosure is encoded by a transgene that is codon optimized. The whole construct of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof can be administered to a mammalian Encoded by a transgene whose entire sequence is codon-optimized for expression in animal cells Codon optimization is the process of optimizing the number of synonymous codons (i.e., codons that encode the same amino acid) in the coding DNA. This refers to the discovery that the frequency of occurrence of such codons (which encode the nucleotide sequence) is biased in different species. Due to degeneracy, identical polypeptides may be coded for by a variety of nucleotide sequences. Various codon optimization methods are known in the art, for example, At least U.S. Patent Nos. 5,786,464 and 6,114,148 This includes the methods disclosed in the document.

[0127] II. IL-13 Binding Domain The present disclosure provides multispecific antibodies (e.g., bispecific antibodies) engineered to bind to human IL-13. The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are provided. Recognition of IL-13 by IL-13 specific antibodies occurs via the "IL-13 antigen binding domain." This is interchangeably referred to as the "IL-13 binding domain."

[0128] In a preferred embodiment, a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof The polyspecific binding molecule comprises one IL-13 binding domain and binds to bound IL-13. In another preferred embodiment, the polyspecific binding molecule is a polyvalent binding molecule having multiple ILs. a multispecific binding molecule comprising an IL-13 binding domain, e.g., two IL-18 binding domains, is multivalent with respect to binding IL-18 and preferably bivalent with respect to binding IL-13 become.

[0129] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof The IL-13 binding domain of the antibody is preferably an IL-13 scFv or an IL-13 Fab. These antibodies include IL-13 Fab.

[0130] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags ment for IL-13 is 10 -4 M~10 -8 M, e.g. 10 -5 M~10 -7 M, for example 10 -6 M or 10 -7 Binding affinity K of M D The IL-13 binding domain comprises the following:

[0131] In a preferred embodiment, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof are In another preferred embodiment, the antibody comprises one IL-13 Fab. The binding domain contains two IL-13 Fabs. The binding domain contains three or more IL-13 Fabs. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof are also contemplated herein. do.

[0132] The phrases "Fab that binds human IL-13" and "IL-13 Fab" refer to a Fab that binds human IL-13. In one embodiment, the IL-13 Fab has a similar binding affinity to the IL-13 Fab. In other embodiments, the antibody binds to IL-13 with potency comparable to that of a full-length antibody. The -18 Fab has a lower binding affinity, e.g., binds to IL-1 with lower binding affinity than the full-length antibody. -13, yet still provide the biological responses described herein.

[0133] Uses with the Disclosed Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof A preferred IL-13 Fab for H and VL comprising the amino acid sequence shown in SEQ ID NO:27.

[0134] Uses with the Disclosed Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof Another preferred IL-13 Fab for and VL comprising the amino acid sequence shown in SEQ ID NO: 71.

[0135] In one aspect, the present disclosure provides a multimeric antibody comprising an IL-13 binding domain, e.g., an IL-13Fab. Polynucleotides encoding specific antibodies (e.g., bispecific antibodies) or fragments thereof The present disclosure also provides isolated nucleic acid molecules encoding these Fabs.

[0136] VH having the amino acid sequence shown in SEQ ID NO: 55, A multispecific antibody comprising an IL-13 binding domain comprising a VL having the amino acid sequence Disclosed herein are isolated nucleic acid molecules encoding the IgG1-binding fragments (bispecific antibodies) or fragments thereof. can be.

[0137] VH having the amino acid sequence shown in SEQ ID NO: 85, and A multispecific antibody comprising an IL-13 binding domain comprising a VL having the amino acid sequence Also disclosed herein are isolated nucleic acid molecules encoding the bispecific antibody or fragments thereof. do.

[0138] Constructs (e.g., clones) comprising one or more of the foregoing isolated nucleic acid molecules and polynucleotides. The foregoing constructs (e.g., cloning or expression vectors) are also included in the present disclosure. Host cells containing one or more of the expression vectors are also included in the disclosure.

[0139] In one aspect, the present disclosure provides a multispecific antibody (e.g., a bispecific antibody) disclosed herein. and a recombinant nucleic acid construct comprising a polynucleotide encoding the polypeptide (or a fragment thereof). The polynucleotide encodes an IL-13 binding domain or a fragment thereof. It includes a nucleic acid sequence that

[0140] III. Domains and Antibodies of Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof and region connection and orientation As used herein, the term "linked" or "linking" refers to multispecific One portion of an antibody (e.g., a bispecific antibody) or fragment thereof may be attached to another portion of the molecule. It refers to being directly or indirectly connected. Direct connection is one form of connection, and In the text, this is referred to as "fused" or "fusion." Using a molecule with the form ABC as an example, Thus: Part A is directly linked to part B and indirectly linked to part C (part A is directly linked to part B) (This may also be referred to as being fused to the VH-internal linker-VL.) Using scFv, VH is indirectly linked to VL and directly linked to an internal linker (The linker may also be described as being fused to both the VL and VH).

[0141] In some embodiments, multispecific antibodies (e.g., bispecific antibodies) or their flags The IL-18 binding domain and the IL-13 binding domain of the cleavage membrane are linked. In embodiments, the linkage is a direct linkage, such that the regions are fused to one another. In some embodiments, the IL-18 binding domain and / or the IL-13 binding domain is F c polypeptide chain. In some embodiments, the IL-13 binding domain (e.g., For example, an IL-13 Fab is linked to an IL-18 binding domain, e.g., via a polypeptide linker. In some embodiments, the IL-13 binding domain (e.g., For example, an IL-13 Fab) is linked, e.g., fused, to the N-terminus of the IL-18 binding domain. do.

[0142] IV. Multispecific Antibody Formats In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies are multivalent, e.g., bivalent with respect to one antigen and monovalent with respect to another antigen. Exemplary bispecific antibodies may be bivalent. a first antigen-binding domain (e.g., a first VL and a VH of one of the antigens (e.g., a VH of one of the antigens) and a VH of a second antigen or epitope (e.g., IL-13) In some embodiments, the second antigen-binding domain has a different structure. The first and second epitopes may be located on the same antigen, e.g., the same protein (or multimeric protein). In some embodiments, the first and second epitopes are located on the nucleotide sequence of on different antigens, e.g., on two proteins (or subunits of a multimeric protein). In an embodiment of the present disclosure, the bispecific antibody is a first epitope or antigen (e.g., I Heavy chain variable domain sequences and light chain variable domain sequences having binding specificity for α- and β-actin (L-18) and further having binding specificity for a second epitope or antigen (e.g., IL-13). The binding domain comprises:

[0143] Protocols for making bispecific or heterodimeric antibodies are known in the art. For example, the "knob-in" method described in U.S. Pat. No. 5,731,168 is well known. -hole approach; see, for example, International Publication No. WO 2009 / 089004, International Publication No. 2006 / 106905 and International Publication No. 2010 / 129304 Electrostatic steering Fc pairing as described in the pamphlet; e.g., WO 2007 / 1 Strand exchange engineering domain (SEED) heterodimers as described in brochure 10205 -Formation; see, for example, International Publication No. 2008 / 119353, International Publication No. 2011 / 131746 brochure and International Publication No. 2013 / 060867 brochure Fab arm exchange as described, for example, in U.S. Pat. No. 4,433,059 Heterobifunctional reagents with amine- and sulfhydryl-reactive groups such as Double antibody conjugates, e.g., by antibody cross-linking to generate specific structures; see, e.g., U.S. Pat. Reduction of disulfide bonds between two heavy chains as described in US Pat. No. 4,444,878 Half-antibodies (heavy chain-light chain pairs or Bispecific antibodies or antibody-like molecular determinants produced by recombining Fab; e.g. For example, a trifunctional antibody, e.g., three Fab' flags cross-linked through sulfhydryl reactive groups. ment, such as those described in U.S. Pat. No. 5,273,743; biosynthetic binding proteins Preferably disulfide or amine reactive chemical crosslinking through the protein, e.g., the C-terminal tail. Pairs of scFvs bridged via a bifunctional antibodies, e.g., through leucine zippers replacing the constant domains; Fab fragments with different binding specificities dimerized with other Fab fragments, for example, U.S. Pat. Such as those described in US Pat. No. 5,582,996; bispecific and oligospecific monovalent and and oligovalent receptors, e.g., the CH1 region of one antibody, typically along with the associated light chain. and the VH region of another antibody via a polypeptide spacer ( the VH-CH1 regions of two Fab fragments, e.g., those described in U.S. Pat. No. 5,591,828 Bispecific DNA-antibody conjugates, such as those described in Cross-linking of antibodies or Fab fragments via cleavage, e.g., U.S. Pat. No. 5,635,602. as described in the specification; bispecific fusion proteins, e.g., hydrophilic Expression constructs containing two scFvs together with a heterocyclic helical peptide linker, e.g., Such as those described in US Patent No. 5,637,481; multivalent and multispecific binding proteins Proteins, such as the Ig heavy chain variable region binding domain, commonly referred to as diabodies, A polypeptide having one domain and a second domain containing the binding region of an Ig light chain variable region. Dimers of amides (creating higher order structures for bispecific, trispecific or tetraspecific molecules) For example, as described in U.S. Pat. No. 5,837,242, bispecific / multispecific antibodies, such as those described in U.S. Pat. No. 5,837,821; The peptide sequence is a sequence of linked VL and VH chains that can be dimerized to form a 2-valent molecule. a minibody construct further linked to an antibody hinge region and CH3 region with a pacer; short in either orientation that can dimerize to form bispecific diabodies linked with a peptide linker (e.g., 5 or 10 amino acids) or no linker at all VL and VH domains; e.g., those described in U.S. Pat. No. 5,844,094. polymers and tetramers; e.g., as described in U.S. Pat. No. 5,864,019 C-terminal, which is further associated with a VL domain to form a series of Fvs (or scFvs). VH domains (or family members) linked by peptide bonds with crosslinkable groups at a string of VL domains in a VL domain member; see WO 2011 / 028952 As described in the pamphlet No. 1, one of the antigens binds monovalently and one of the antigens binds bivalently. VL and VH domains, scFv or Fab; and VL and VL, as described, for example, in U.S. Pat. No. 5,869,620. A single-chain binding polypeptide in which both VH domains are linked through a peptide linker through non-covalent or chemical cross-linking using scFv or diabody-type formats can be combined into multivalent structures, for example to form homodivalent, heterodivalent, trivalent and tetravalent structures. Further exemplary multispecific and bispecific molecules include, but are not limited to, and methods for making same are described, for example, in U.S. Pat. No. 5,910,573, U.S. Pat. No. 5,932,448, U.S. Pat. No. 5,959,083, U.S. Pat. ,989,830, U.S. Pat. No. 6,005,079, U.S. Pat. No. 6,2 39,259, U.S. Pat. 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The contents of the above-referenced application are incorporated herein by reference in their entirety. Thus, in some embodiments, the IL-13 / IL-18 multispecific antibodies of the present disclosure (e.g., Multispecific or bispecific antibodies (e.g., bispecific antibodies) are known in the art and are described above. IL-13 binding domain and IL-18 binding domain in one of the different formats Preferred formats for the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure include: The set is described in more detail below.

[0144] V. Representative Anti-IL-13 / IL-18 Bispecific Antibodies The amino acid sequences in Table 1 are examples of IL-13 / IL-18 bispecific antibodies and portions thereof. do.

[0145] [Table 1]

[0146] [Table 2]

[0147] [Table 3]

[0148] [Table 4]

[0149] [Table 5]

[0150] [Table 6]

[0151] [Table 7]

[0152] [Table 8]

[0153] [Table 9]

[0154] Table 10

[0155] Table 11

[0156] Table 12

[0157] Table 13

[0158] Table 14

[0159] Table 15

[0160] Table 16

[0161] Table 17

[0162] Table 18

[0163] Table 19

[0164] [Table 20]

[0165] [Table 21]

[0166] In some embodiments, the multispecific antibodies described herein have a sequence similar to that of Table 1, compared to the sequences of Table 1. A bispecific antibody containing an amino acid sequence with one, two, or three substitutions, deletions, or insertions. .

[0167] Representative IL-13 / IL-18 bispecific antibody formats are illustrated in Table 2. All bbmAbs combine anti-IL13 and anti-IL-18 binding domains and bind to human Ig Based on the G1 format, all contain YTE half-life extension mutations, while two are L Further containing an ALA silencing mutation. More particularly, bbmAb2, bbmA b1 and bbmAb3 contain the YTE half-life extending mutation in the Fc. and bbmAb5 contain both LALA silencing and YTE half-life extending mutations. bbmAb2, bbmAb1, bbmAb4 and bbmAb5 are the same as mAb1 and mAb5. bbmAb3 combines the variable domains of mAb1 and mAb3, while bbmAb4 combines the variable domains of mAb1 and mAb2. Combine domains.

[0168] bbmAb2, bbmAb4, and bbmAb3 bind to the anti-IL-18 heavy chain Fc and the KiH knob. While carrying heterodimerization mutations, the KiH hole mutations are anti-IL-13 heavy chain bbmAb2, bbmAb4, and bbmAb3 are located in the anti-IL-18 heavy chain Fc. Carrying a KiH knob mutation and a KiH hole mutation in the anti-IL-13 heavy chain Fc .

[0169] [Table 22]

[0170] VI. Modifications of Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof of the Disclosure The present application provides antibodies having various modifications in the binding domain, variable domain and / or constant region. Variants of the molecules and / or fragments thereof described herein and fusions of the disclosed molecules For example, the disclosed multispecific antibodies (e.g., bispecific antibodies) or The Fc region of the fragment can be wild-type, or it can be a fragment that can be used to induce various outcomes. Preferred modifications to the Fc include " LS mutation (M428L, N434S, (EU numbering)) and YTE mutation Mutations (M252Y, S254T, T256E (EU numbering)), effector silencing "DAPA" mutations for cytotoxicity (D265A, P329A (EU numbering)) and knob-in-hole mutations that promote proper strand pairing (e.g., knob S354C, T 366W; Hall Y349C, T366S, L368A, Y407V (EU numbering )) is included.

[0171] A. Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof with Variable Region Modifications to The VH and VH of a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure Each of the VL domains contains hypervariable region CDR1, CDR2 and CDR3 sequences. In certain embodiments, one or more of these CDR sequences have conservative modifications of the amino acid sequence. The resulting modified molecule retains or has enhanced binding properties compared to the parent antibody.

[0172] Furthermore, in certain instances, the framework region may be modified to maintain or enhance the antigen-binding ability of the antibody. Mutating residues within the region has proven beneficial (e.g., Quee U.S. Pat. No. 5,530,101 to n et al.; U.S. Pat. No. 5,58 No. 5,089; U.S. Pat. No. 5,693,762 and U.S. Pat. No. 6,180 (See, e.g., U.S. Pat. No. 3,370). Such mutations can be used to improve binding properties. by incorporating the variable region framework of the molecule of the disclosure (e.g., antibody or antibody fragments) It can modify the nucleotide sequence of a molecule (such as a nucleotide sequence).

[0173] Another type of variable region modification is to modify the VH and / or VL CDR1, CDR2 and / or CDR3. Mutating amino acid residues within the DR3 domain, thereby modifying one or more of the antibodies of interest improving the binding properties (e.g., affinity) of a compound, known as "affinity mutation." To introduce the mutations, site-directed mutagenesis or PCR-mediated mutagenesis can be used. The effects on antibody binding or other functional properties of interest can be determined by the methods described herein. These can be evaluated in in vitro or in vivo assays such as those provided in the Examples. Modifications (as discussed above) can be introduced. Mutations can include amino acid substitutions, additions or Deletions may also be made, generally of up to 1, 2, 3, 4 or 5 residues within the CDR regions. , preferably one or two residues are changed.

[0174] by introducing appropriate nucleotide changes into the encoding DNA or by synthesis of the desired variant. , amino acid sequence mutations of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof Such variants can be prepared, for example, by substitution of residues within the amino acid sequence of the molecule. Any combination of deletion, insertion, and substitution may be used to construct the final construct. This is done to arrive at a product that retains the desired antigen-binding characteristics. Acid changes may also alter post-translational processing of the molecule, such as changing the number or position of glycosylation sites. obtain.

[0175] The present application provides multispecific antibodies with conservative amino acid modifications in the variable and / or constant regions. Includes variants of antibodies (e.g., bispecific antibodies) or fragments thereof.

[0176] B. Multispecific Antibodies (e.g., Bispecific Antibodies) with Enhanced Heterodimerization or Their Fragment Incorrect heterodimerization of two antibody heavy chain domains can prevent the formation of desired multispecific antibodies (e.g., This may be an obstacle to improving the yield of purified antibodies (e.g., bispecific antibodies) or fragments thereof. EP 1870459 A1; U.S. Pat. No. 5,555,623 No. 82,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910 ,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,4 41; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 20062 04493A1 specification; and WO 2009 / 089004A1 pamphlet As disclosed, the two heavy chain domains of a bispecific or multispecific antibody or antibody-like molecule A variety of approaches are available for promoting in-dimerization.

[0177] The present disclosure provides a method for the dimerization (heterodimerization) of two interacting heterologous polypeptides. Promoting and / or reducing the dimerization of two identical polypeptides (homodimerization) Generally, each of the two interacting polypeptides is a C of an antibody. The Fc region contains H2 and CH3 domains. The CH3 domain is the most abundant Fc region in all isotypes. type, class or subclass, and preferably IgG (IgG1, IgG2, IgG3 and The constant region is derived from an antibody of the IgG4 class, most preferably IgG1.

[0178] Generally, the polypeptides of the present disclosure comprise a CH1 domain, a CH2 domain, a hinge domain, VH domains, VL domains, CDRs, etc., and / or antigen binding domains as described herein fragments, such as scFv and / or Fab, which contain, in addition to the CH3 domain, other antigens These antibody fragments include antibody fragments of the various types described herein. Antibodies, such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human Antibodies, bispecific or multispecific antibodies, camelized antibodies, anti-idiotypic (anti-Id) antibodies and antibody complexes. Heterodimerization of two different heavy chains at the CH3 domain While homodimerization of identical heavy chains results in the desired antibody or antibody-like molecule, In an exemplary embodiment, two or more heteropolypeptides are used, resulting in a reduced yield of the desired antibody or molecule. The peptide chains comprise a CH3 domain and are included in the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure. It comprises two chains that form a molecule in either the antibody (specific antibody) or fragment format. In one embodiment, the two heteropolypeptide chains comprising a CH3 domain are unmodified chains. The modified strands include modifications that favor heterodimeric association of the polypeptides compared to the modified strands. Various examples of latex are provided below.

[0179] Knob-in-Hole (KIH) (also known as "Key-in-Hole") The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure may contain constant domains. one or more mutations, e.g., multiple mutations, to one or more of the domains, e.g., the CH3 domain In one example, a multispecific antibody (e.g., a bispecific antibody) of the present disclosure or its The fragments each comprise a heavy chain Fc or constant domain of an antibody, e.g., CH2 or CH3 In one example, two heavy chain constant domains, e.g., CH2 or CH3 domains of a multispecific antibody (e.g., bispecific antibody) or fragment thereof The main chain contains one or more mutations that allow heterodimeric association between the two chains. In embodiments, one or more mutations are present in a multispecific antibody (e.g., a bispecific antibody) or its In one embodiment, one or more of the fragments are located on the CH2 domains of the two heavy chains of the fragment. Natural mutations may be present in at least one of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof. In one embodiment, the heavy chain constant domains are located on the CH3 domains of the two polypeptides. a first polypeptide of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof comprising One or more mutations to the peptide generate a "knob" and multiple domains comprising the heavy chain constant domain. a specific antibody (e.g., a bispecific antibody) or a fragment thereof directed against a second polypeptide; One or more mutations that affect the heavy chain constant domain form a "hole" and allow the multispecific antibody to bind to the heavy chain constant domain. Heterodimerization of polypeptides of the antibody (e.g., bispecific antibody) or fragments thereof connects the "holes" and "knobs" (e.g., interacts with, e.g., a second polypeptide) The CH2 domain of the first polypeptide or the CH2 domain of the second polypeptide interacts with the CH2 domain of the second polypeptide. the CH3 domain of the first polypeptide interacting with the CH3 domain of the second polypeptide). As the term is used herein, a "knob" refers to a multispecific polypeptide comprising, for example, a heavy chain constant domain. the interface of the first polypeptide of a specific antibody (e.g., a bispecific antibody) or fragment thereof The heavy chain constant domains are attached to the heteromultimers so that the heavy chain constant domains project from the heteromultimers, thus stabilizing the heteromultimers. a second polypeptide of a polyspecific antibody (e.g., a bispecific antibody) or fragment thereof; At least one amino acid that is capable of occupying a compensatory "hole" at the interface of refers to the acid side chain, which favors heteromultimerization over homomultimerization. The fragment may be present at the original interface or may be present at the interface (e.g., by altering the nucleic acid encoding the interface). Preferred import residues for the formation of the knob are generally and natural amino acid residues, preferably arginine (R), phenylalanine (F), It is selected from tyrosine (Y) and tryptophan (W). Most preferred is tryptophan. In a preferred embodiment, the original residues for the formation of the overhang are: Alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine It has a small side chain volume.

[0180] "Whole" refers to a multispecific antibody (e.g., a bispecific antibody) or antibody containing a heavy chain constant domain. at least one of the fragments is recessed from the interface of the second polypeptide refers to a single amino acid side chain and therefore is not a multispecific antibody (e.g., corresponding polypeptides on the adjacent interface of the first polypeptide of a polypeptide chain (e.g., a bispecific antibody) or fragment thereof. The holes can be present at the original interface or can be synthetically introduced. (e.g., by altering the nucleic acid encoding the interface). The preferred import residue is typically a natural amino acid residue, preferably alanine (A). , serine (S), threonine (T) and valine (V). Most preferred are , serine, alanine or threonine. In a preferred embodiment, The original residues of the Contains tryptophan and others.

[0181] In one embodiment, the first CH3 domain is either a "knob" or a "hole" (see above). To generate sequences of Proteins of immunological interest,5th ed., Vol. 1 (1991; NIH, Bethesda, Md.), pp. 688- 696) with mutations at residues 366, 405, or 407 according to the EU numbering scheme. and a second CH3 domain that heterodimerizes with the first CH3 domain. residue 407 if there is a mutation at residue 366 in the H3 domain, the first CH3 domain If there is a mutation at residue 405 in the first CH3 domain, there is a mutation at residue 394 or at residue 40 in the first CH3 domain. If there is a mutation at 7, there is a mutation at residue 366 (EU numbering), and the first Generate a "hole" or "knob" complementary to the "knob" or "hole" of the CH3 domain. do.

[0182] In another embodiment, the first CH3 domain may be either the "knob" or the "hole" (top (as described above) and a second CH3 domain that heterodimerizes with the first CH3 domain. To achieve this, a mutation was made at residue 366 (EU numbering) to alter the first CH3 domain. To create "holes" or "knobs" that are complementary to the "knobs" or "holes" of the input. Mutations were made at residues 366, 368 and / or 407 (EU numbering). In this form, the mutation to the first CH3 domain is a tyrosine (Y) residue at position 366. In one embodiment, the first CH3 mutation is T366Y. In embodiments, the mutation to the first CH3 domain is a tryptophan at position 366 ( In one embodiment, the first CH3 mutation is T366W. In embodiments, the heterodimer is formed with a first CH3 domain having a mutation at position 366. Mutations to the second CH3 domain (e.g., tyrosine introduced at position 366) that enhance and having tryptophan (Y) or tryptophan (W), e.g., mutation T366Y or T366W. (including mutations at position 366, 368, and 407) (EU numbering). In embodiments, the mutation at position 366 is a serine (S) residue. The mutation at position 368 introduced an alanine (A), and the mutation at position 407 introduced a valence amino acid (V). In an embodiment, the mutations are T366S, L368A, and Y40 In one embodiment, a multispecific antibody (e.g., a bispecific antibody) or fragment thereof The first CH3 domain of the segment contains the mutation T366Y, and the first CH3 domain The second CH3 domain, which heterodimerizes with the nucleotides T366S, L368A, and Y407V or vice versa. In one embodiment, a multispecific antibody (e.g., a bispecific the first CH3 domain of said antibody or fragment thereof comprises the mutation T366W; The second CH3 domain, which heterodimerizes with the first CH3 domain, contains the mutation T36 6S, L368A and Y407V or vice versa.

[0183] Any of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure Further knob-in-hole mutation pairs suitable for use in See WO 1996 / 027011, which is incorporated herein in its entirety. and Merchant et al., (1998) Nat. Biotechnol. , 16:677-681.

[0184] In any of the embodiments described herein, the CH3 domain contains a pair of cysteine ​​residues. Without being bound by theory, the The introduction of a pair of cysteine ​​residues capable of forming sulfide bonds allows the formation of heterodimerized antibodies. In an embodiment, the first CH3 domain has a residue at position 354 ( (EU numbering) contains a cysteine ​​and heterodimerizes with the first CH3 domain The second CH3 domain contains a cysteine ​​at position 349 (EU numbering). In some embodiments, the first CH of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof The 3 domain contains a cysteine ​​at position 354 (e.g., containing the mutation S354C) and tyrosine (Y) at 366 (e.g., containing the mutation T366Y) and The second CH3 domain, which heterodimerizes with the main domain, has a cysteine ​​at position 349 (e.g. , containing the mutation Y349C), serine at position 366 (e.g., containing the mutation T366S), alanine at position 368 (e.g., containing the mutation L368A) and ba In embodiments, the antibody may be a multispecific antibody (e.g., a nucleotide sequence encoding a nucleotide sequence of interest) or a nucleotide sequence encoding a nucleotide sequence of interest. The first CH3 domain of the antibody or fragment thereof has a CDR at position 354. Cysteine ​​(e.g., containing mutation S354C) and tryptophan at position 366 (W ) (e.g., containing the mutation T366W), and a heterodimer with the first CH3 domain. The second CH3 domain, which binds to the cysteine ​​at position 349 (e.g., mutation Y349 C), serine at position 366 (e.g., containing the mutation T366S), alanine (e.g., containing the mutation L368A) and valine at position 407 (e.g., containing the mutation containing the mutation Y407V).

[0185] IgG heterodimerization In one embodiment, the polynucleotides of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof Heterodimerization of peptide chains (e.g., half antibodies) is the process of synthesising C IgG1 antibodies. In one embodiment, the H3 domain is increased by introducing one or more mutations into the H3 domain. The mutation is a F405L mutation in the second CH3 domain (EU numbering scheme). It contains a K409R mutation in one CH3 domain that pairs with the ATP-binding domain. Similarly or alternatively, positions 366, 368, 370, 399, 405, 407 and 4 09 (EU numbering). Preferably, the polypeptide containing such a mutation Heterodimerization of tides is achieved under reducing conditions, e.g., 10–100 mM 2-MEA (e.g., , 25, 50 or 100 mM 2-MEA) at 25 to 37°C, e.g., 25°C or 37°C. This is achieved in 1 to 10 hours, for example, 1.5 to 5 hours, for example, 5 hours.

[0186] Using techniques known in the art, the amino acid substitutions described herein may be used to identify the CH3 domain. Usually, the DNA encoding the heavy chain is introduced into the The oligonucleotides are genetically modified using techniques described in the Clinical Approach. Nucleotide-mediated mutagenesis replaces the DNA encoding the two hybrid heavy chains. This is the preferred method for preparing mutants. This technique is described by Adelman et al. ., (1983) DNA, 2:183, and are well known in the art.

[0187] Suitable IgG heterodimerization strategies are described, for example, in US Pat. No. 6,213,999, the contents of which are incorporated herein by reference in their entirety. WO 2008 / 119353, ... Publication No. 2011 / 131746 and Publication No. 2013 / 060867 It is stated in the pamphlet.

[0188] In any of the embodiments described herein, the CH3 domain contains a pair of cysteine ​​residues. Without being bound by theory, the The introduction of a pair of cysteine ​​residues capable of forming sulfide bonds allows the formation of heterodimerized multispecific and providing stability to heterologous antibodies (e.g., bispecific antibodies) or fragments thereof. In an embodiment, the first CH3 domain contains a cysteine ​​(EUNA) at position 354. The first CH3 domain contains a second CH3 domain that heterodimerizes with the first CH3 domain. The amino acid contains a cysteine ​​at position 349 (EU numbering).

[0189] polar crosslinking In one embodiment, the polynucleotides of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof Heterodimerization of peptide chains (e.g., half-antibodies) is based on rational "polar-bridge" This is increased by introducing mutations in the heterodimer configuration, which results in similar Residues with (or complementary) physical properties, while differing in homodimer configuration By interacting with residues of physical properties and residues at the binding interface between the two polypeptide chains, In particular, these mutations prevent polar residues from interacting with polar residues in heterodimer formation. In contrast, homodimers are designed to interact with other hydrophobic residues. In the formation of dimers, residues are mutated so that polar residues interact with hydrophobic residues. Favorable interactions in heterodimer configurations and unfavorable interactions in homodimer configurations The optimal interaction is that the CH3 domain prefers to form heterodimers rather than homodimers. These work together to increase the likelihood of forming a

[0190] In an exemplary embodiment, the above mutations are at residues 364, 368, 39 of the CH3 domain. It is made at one or more of positions 9, 405, 409 and 411 (EU numbering).

[0191] In one embodiment, Ser364Leu, Thr366Val, Leu368Gln, Asp 399Lys, Phe405Ser, Lys409Phe and Thr411Lys One or more mutations selected from the group consisting of: For example, Ser364Leu: the original serine residue at position 364 is replaced with leucine; Thr 366Val: The original residue of threonine at position 366 is replaced with valine; Leu368Gln : The original residue of leucine at position 368 is replaced with glutamine; Asp399Lys: position 39 The original residue aspartic acid at position 9 is replaced by lysine; Phe405Ser: The original residue at position 405 The residue phenylalanine is replaced by serine; Lys409Phe: the original residue lysine at position 409 is replaced by phenylalanine; Thr411Lys: the original residue of threonine at position 411 is It is substituted with lysine.

[0192] In another embodiment, Tyr407Phe, Lys409Gln and Thr411Asp Other CH3s may be introduced (e.g., , Tyr407Phe: the original residue tyrosine at position 407 is replaced with phenylalanine; Ly s409Glu: The original lysine residue at position 409 is replaced with glutamic acid; Thr411As p: the original residue of threonine at position 411 is replaced by aspartic acid).

[0193] In a further embodiment, one CH3 domain contains Ser364Leu, Thr366Va l, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Ph and Thr411Ly, while having one or more mutations selected from the group consisting of The CH3 domain of The mutant has one or more mutations selected from the group consisting of:

[0194] In one exemplary embodiment, the original residue of threonine at position 366 of one CH3 domain is is replaced by valine, while the original residue of tyrosine at position 407 in the other CH3 domain is , is replaced by phenylalanine.

[0195] In another exemplary embodiment, the original residue of the serine at position 364 in one CH3 domain is , is replaced by leucine, while the original residue of leucine at position 368 in the same CH3 domain is , is replaced by glutamine.

[0196] In yet another exemplary embodiment, a phenylalanine amino acid at position 405 of one CH3 domain is The original residue of lysine at position 409 of this CH3 domain was replaced with serine. The residue is replaced by a phenylalanine, while the lysine at position 409 of the other CH3 domain The original residue is replaced with glutamine.

[0197] In yet another exemplary embodiment, one CH3 domain has an aspartic acid at position 399. The original residue of is replaced by lysine, and the original threonine at position 411 of the same CH3 domain is The residue is replaced by a lysine, while the original threonine at position 411 in the other CH3 domain is The residue is substituted with aspartic acid.

[0198] Using techniques known in the art, the amino acid substitutions described herein may be used to identify the CH3 domain. Typically, the DNA encoding the heavy chain is introduced into a The oligonucleotides are genetically modified using techniques described in the Practical Approach. Nucleotide-mediated mutagenesis generates substitution mutations in the DNA encoding the two hybrid heavy chains. This is the preferred method for preparing isomers. This technique was developed by Adelman et al. , (1983) DNA, 2:183, and are well known in the art.

[0199] Polar cross-linking strategies are described, for example, in US Pat. No. 6,49 ... the contents of which are incorporated herein by reference in their entirety. International Publication No. 2006 / 106905 and International Publication No. 2009 / 08900 No. 4 Pamphlet and Gunasekaran K et al., (2010) JB Biol Chem., 285:19637-19646.

[0200] In any of the embodiments described herein, to introduce a pair of cysteine ​​residues, The H3 domain may be further mutated. The introduction of a pair of cysteine ​​residues capable of forming sulfide bonds allows the formation of heterodimerized multispecific It is believed that the addition of the hydroxyl group provides stability to heterologous antibodies (e.g., bispecific antibodies). The first CH3 domain contains a cysteine ​​at position 354 (EU numbering) and the first C The second CH3 domain, which heterodimerizes with the H3 domain, is located at position 349 (EU numbering The ring contains cysteine.

[0201] C. Multispecific Antibodies (e.g., Bispecific Antibodies) with Extended In Vivo Half-Life or Their Fragments Fragment The present multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be used in vivo. It may be further modified to extend its half-life.

[0202] Various strategies are available, for example, polyethylene glycol (PEG), reCODE PEG, antibody scaffold, polysialic acid (PSA), hydroxyethyl starch (HES), Chemical linkage to albumin-binding ligands and carbohydrate shields; albumin, IgG, Fc Genetic fusion to proteins that bind to serum proteins, such as Rn and transferrin Nanobodies, Fabs, DARPins, avimers, affibodies and anticalins Coupling (genetic or chemical) to other binding moieties that bind to serum proteins, such as rPEG, albumin, albumin domains, albumin-binding proteins, and Fc or by incorporation into nanocarriers, slow-release formulations or medical devices. and extending the half-life of the indicated multispecific antibodies (e.g., bispecific antibodies) or fragments thereof. It can be used to

[0203] One or more amino acid modifications (i.e., substitutions, insertions, or deletions) may be made to the IgG constant domain or These FcRn-binding fragments (preferably Fc regions or fragments thereof) and administering to the subject a multispecific antibody (e.g., a bispecific antibody) of the present disclosure that has an extended half-life in vivo. For example, the method described in WO 1998 / 023444 can be used to prepare a human antibody (specific antibody) or a fragment thereof. No. 23289, International Publication No. 1997 / 34631 and U.S. Pat. See U.S. Pat. No. 6,277,375. The disclosed multispecific antibodies (e.g., Preferred modifications to the Fc of the antibody (bispecific antibody) or fragment thereof are to increase half-life. For this purpose, the "LS" mutation (M428L, N434S (EU numbering)) and the "YTE " mutations (M252Y, S254T, T256E (EU numbering)).

[0204] Additionally, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may comprise one or more can be conjugated or fused to the above human serum albumin (HSA) polypeptide or a portion thereof making the molecule more stable in vivo or have a longer half-life in vivo This technique is known in the art and is described, for example, in WO 02 / 04499. Pamphlet No. 1993 / 15199, Pamphlet No. WO 1993 / 15200 and WO 2001 / 77137; and EP 413622 See the specification. Use of N-terminal fragments of HSA for fusion to polypeptides The use of the gene has also been proposed (for example, European Patent No. 399666). stabilized by mechanically or chemically fusing or conjugating molecules to albumin; The shelf life may be extended and / or in solution in vitro and / or in vivo. The activity of the molecule is maintained for an extended period of time. Additional methods for HSA fusion include, for example: WO 2001 / 077137 and WO 2001 / 077137, which are incorporated herein by reference. and WO 2003 / 06007. In certain embodiments, In this case, expression of the fusion protein is carried out in a mammalian cell line, such as a CHO cell line.

[0205] D.Fc silenced In embodiments of the disclosure that incorporate one or more constant domains, e.g., heavy chain constant regions, hF one to silence, for example, ADCC and / or CDC effector function within The inclusion of these mutations may be beneficial. Activation of immune cells may increase the cross-linking of target cells. However, human Fc binds to both high and low affinity FcR gamma receptors. Thus, cross-linking of receptors (e.g., CD3) on immune cells and subsequent activation Gonism can occur upon binding in the absence of tumor targeting. Cross-linking of the human Fc can induce antibody-dependent cellular cytotoxicity (ADCC). When complexed with α- and β-actin, it also binds to complement proteins and induces complement-dependent cytotoxicity (CDC). Therefore, residues in Fc that reduce or abrogate these interactions Mutations to the α-terminal β ... It can focus on the effects of children.

[0206] In embodiments, the multiple binding sites of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof are One or more, for example all, of the chain constant region domains may contain DAPA mutations (e.g., EU numbering For example, the contents of each as a whole include Shields RL et al., (2001) J Bi, incorporated by reference herein. ol Chem.,276(9):6591-604; U.S. Patent Application Publication No. 2015 / 0 See specification 320880A1.

[0207] In embodiments, the multiple binding sites of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof are One or more, e.g., all, of the amino acid sequence constant region domains may be mutated with a LALA mutation (e.g., EU numbering For example, the contents of each as a whole include Hezareh M et al., (2001) Journal of Clinical Nutrition, vol. 1, pp. 111-114, 2001, incorporated by reference. al of Virology,75(24):12161-12168;Shield s RL et al., (2001) supra.

[0208] In embodiments, the multiple binding sites of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof are One or more, for example all, of the amino acid sequence constant region domains contain the N279A mutation (EU numbering by Tao), the contents of each of which are incorporated by reference in their entirety. MH&Morrison SL(1989)J Immunol.143(8):259 5-601; Shields RL et al., (2001) see above). .

[0209] Further Fc mutations to provide silenced effector functions are The disclosure of International Publication No. WO 2014 / 145806 is incorporated herein by reference. The silent IgG1 antibody is described in WO 2014 / 023146 (e.g., Figure 7). Examples from the 145806 brochure include E233P, L234V, L235A and S Contains a 267K mutation and a deletion of G236 (G236del). Another example from International Publication No. 2014 / 145806 is E233P, L Contains the 234V and L235A mutations and the deletion of G236 (G236del). Another example from WO 2014 / 145806 of a resistant IgG1 antibody is Contains the S267K mutation.

[0210] E. Compounds The present disclosure provides a method for the preparation of a fusion protein comprising the addition of a heterologous protein or polypeptide (or The fragments thereof, preferably at least 10, at least 20, at least 30, At least 40, at least 50, at least 60, at least 70, at least 80, recombinantly fused to a polypeptide of at least 90 or at least 100 amino acids Polyspecific antibodies that are covalently or chemically conjugated (including both covalent and non-covalent conjugation) antibody or antibody fragment thereof. Methods for fusing or conjugating proteins, polypeptides or peptides to , are known in the art. See, e.g., U.S. Pat. No. 5,336,603, U.S. Pat. No. 5,622,929, U.S. Pat. No. 5,359,046, U.S. Pat. ,349,053, U.S. Pat. No. 5,447,851 and U.S. Pat. 112,946; EP 307434 and EP 367166 International Publication No. 1996 / 04388 and International Publication No. 1991 / 0 Brochure No. 6570; Ashkenazi et al., (1991) PNAS. USA 88:10535-10539;Zheng et al.,(1995)J. Immunol.154:5590-5600; and Vil et al., (1992 ) PNAS.USA 89:11337-11341.

[0211] Further fusion proteins have been developed using gene shuffling, motif shuffling, and exon shuffling. nucleotide shuffling and / or codon shuffling (collectively referred to as "DNA shuffling") DNA shuffling can be achieved through the use of multispecific antibodies ( can be used to alter the activity of a specific antibody (e.g., a bispecific antibody) or fragment thereof. Generally, U.S. Patent No. 5,605,793, U.S. Patent No. 5,811,238 Specification, U.S. Patent No. 5,830,721, U.S. Patent No. 5,834,252 and U.S. Patent No. National Patent No. 5,837,458; Patten et al., (1997) Cu rr.Opinion Biotechnol.8:724-33;Harayama( 1998)Trends Biotechnol.16(2):76-82;Hanss on et al., (1999) J.Mol.Biol.287:265-76; and Lorenzo&Blasco(1998)Biotechniques,24(2): 308-313 (each of these patents and publications is incorporated herein by reference in its entirety). Multispecific antibodies (e.g., bispecific antibodies) or their The fragments may be subjected to error-prone PCR, random nucleotide insertion, or other The fragments of this molecule can be altered by subjecting them to random mutagenesis by the method The polynucleotide encoding the fragment may be one or more components of one or more heterologous molecules, It can be recombined with chief, section, part, domain, fragment, etc.

[0212] Additionally, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be purified. In a preferred embodiment, the nucleic acid sequence may be fused to a marker sequence, such as a peptide to facilitate transcription. The marker amino acid sequence may be a hexa-histidine peptide, for example, one of many commercially available. However, the tag provided by the pQE vector (QIAGEN, Inc., 925 9 Eton Avenue, Chatsworth, CA, 91311). Gentz et al., (1989) PNAS.USA 86:821-824 For example, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags that can be used include the influenza hemagglutinin protein (Wilson et al., 2001). n et al., (1984) Cell 37:767) and the "flag" tag. Examples of suitable tags include, but are not limited to, a hemagglutinin ("HA") tag corresponding to an epitope associated with a nucleotide sequence. .

[0213] In other embodiments, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof The molecules are conjugated to diagnostic or detectable agents. Such molecules can be used to determine the effectiveness of a particular treatment. As part of a clinical testing procedure, such as determining the onset, development, progression and / or progression of a disease or disorder Such diagnosis and detection may be useful for monitoring the severity or for prognosis. , horseradish peroxidase, alkaline phosphatase, beta-galactosidase prosthetic groups, such as but not limited to strontium phosphate; Fluorescent materials, such as but not limited to streptavidin / biotin and avidin / biotin; Igaumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythritol luminescent materials, such as but not limited to luminol; bioluminescent materials, such as but not limited to radioactive materials, including but not limited to luciferase, luciferin, and aequorin; However, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur Yellow (35S), tritium (3H), indium (115In, 113In, 112In and 111In,), technetium (99Tc), thallium (201Ti), gallium ( 68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), Fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm , 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P , 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn and 117Ti n, etc.; and various positron-emitting metals and non-emitting metals using positron emission tomography Detection methods include, but are not limited to, various enzymes, radioactive paramagnetic metal ions, etc. This can be achieved by coupling the molecule to a releasable substance.

[0214] The present application provides multispecific antibodies (e.g., bispecific antibodies) conjugated to a therapeutic moiety or their The molecules of the present disclosure or fragments thereof may be used as cytotoxins, therapeutic agents, such as cytostatic or cytocidal agents, or radioactive metal ions, e.g. It can be conjugated to an alpha-emitter therapeutic moiety. This includes any drug that is harmful to humans.

[0215] Furthermore, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be used in certain The therapeutic moiety or drug moiety may be conjugated to a therapeutic moiety or drug moiety that modifies the biological response of the The term should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be , may be a protein, peptide, or polypeptide that possesses a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, and Pseudomonas aeruginosa exotoxin. proteins, such as tumor necrosis factor, α-interleukin, -interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasma angiogenic agent; or a biological response modifier, e.g., a ribosomal protein; Examples include fluoxetine and the like.

[0216] For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to molecules, see For more information, see Saito et al., (2003) Adv. Drug Deliv. R ev.55:199-215;Trail et al.,(2003)Cancer Immunol.Immunother.52:328-337;Payne(2003 )Cancer Cell 3:207-212;Allen(2002)Nat.Re v.Cancer,2:750-763;Pastan and Kreitman(2 002)Curr.Opin.Investig.Drugs,3:1089-1091 ;Senter&Springer(2001)Adv.Drug Deliv.Rev See also .53:247-264.

[0217] Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be used as radioimmunoconjugates. They are also conjugated to radioisotopes to make cytotoxic radiopharmaceuticals, also called Examples of radioisotopes that can be conjugated to molecules for diagnostic or therapeutic use include: These include iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art, for example, but not limited to, , Denardo et al., (199 8)Clin Cancer Res.4(10):2483-90;Peterson et al.,(1999)Bioconjug.Chem.10(4):553-7 ; and Zimmerman et al., (1999) Nucl.Med.Biol. See 26(8):943-50.

[0218] Techniques for conjugating therapeutic moieties to antibodies are known, see, for example, Arnon et al. al.,“Monoclonal Antibodies For Immunota rgeting Of Drugs In Cancer Therapy”,in M onoclonal antibodies and cancer therapy, Reisfeld et al.(eds.),pp.243-56(Alan RL iss, Inc. 1985); Hellstrom et al., “Antibodi es For Drug Delivery”,in Controlled Drug Delivery(2nd Ed.), Robinson et al.(eds.) ,pp.623-53(Marcel Dekker,Inc.1987);Thorp e,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review”,in Monoclon al Antibodies 84:Biological And Clinical Applications,Pinchera et al.(eds.),pp.4 75-506(1985);“Analysis,Results,And Futur e Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” ,in Monoclonal Antibodies For Cancer Det ection And Therapy,Baldwin et al.(eds.), pp.303-16 (Academic Press 1985) and Thorpe e See t al., (1982) Immunol. Rev. 62:119-58 .

[0219] The multispecific antibody (e.g., bispecific antibody) or fragment thereof may also be attached to a solid support. This is particularly useful for immunoassays or purification of target antigens. Suitable solid supports include glass, cellulose, polyacrylamide, nylon, and polystyrene. Examples of suitable resins include, but are not limited to, polyethylene, polyvinyl chloride, or polypropylene.

[0220] VII. Methods of Producing Antibodies of the Invention Polypeptides of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure When the peptides are cross-linked, these functional linkages can be achieved using methods known in the art. A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of agents include protein A, carbodiimide, and N-succinimidyl-S-acetyl -thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTN B), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridinyl) lysyldithio)propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimide) imidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (e.g., Karpovsky et al.,(1984)J.Exp.Med.160:168 6; Liu et al. (1985) PNAS.USA 82:8648 Another method is the Behring In (1985) method. s.Mitt.No.78:118-132;Brennan et al.,(198 5) Science 229:81-83) and Glennie et al., (19 87) J. Immunol. 139:2367-2375). The complexing agents were SATA and sulfo-SMCC, both from Pierce Chemistry. and is available from Cal Co. (Rockford, IL).

[0221] Alternatively, the present multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may be A DNA construct encoding a desired molecule is introduced into an expression vector, and the desired molecule is expressed in the same host cell. The vector can be recombinantly produced by expressing and assembling the vector.

[0222] A. Preparation of Polypeptide Chains Polypeptides and antibodies and fragments thereof (e.g., half antibodies) are not limited to conventional monoclonal antibodies. Clonal antibody techniques, e.g., Kohler and Milstein, (1975) Nature 2 56:495 produced by a variety of techniques, including the standard somatic cell hybridization technique There are many techniques for producing monoclonal antibodies, such as viral or Oncogenic transformation, may be used.

[0223] The animal system for preparing hybridomas is the murine system. Doma production is a well-established procedure. Immunization Protocol and Fusion Immunization Techniques for isolation of spleen cells are known in the art. , mouse myeloma cells) and fusion procedures are also known.

[0224] Chimeric or humanized antibodies used in this disclosure may be derived from mouse monoclonal antibodies prepared as described above. The D-sequence encoding the heavy and light immunoglobulin chains can be prepared based on the sequence of a single clonal antibody. NA can be obtained from the mouse hybridoma of interest and purified using standard molecular biology techniques. The immunoglobulin sequences may be modified to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to generate chimeric antibodies, murine variable fragments can be synthesized using methods known in the art. The region can be linked to a human constant region (see, e.g., US Pat. No. 6,233,199, to Cabilly et al.). (See Patent No. 4,816,567.) The murine CDR regions can be inserted into a human framework using methods known in the art. See, for example, U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. No. 5,225,539 to Queen. U.S. Patent No. 5,530,101 to et al.; U.S. Patent No. 5,585, 089; U.S. Patent No. 5,693,762 and U.S. Patent No. 6,180,3 See specification No. 70.

[0225] In certain embodiments, the antibodies or antibody-like molecules of the present disclosure are human monoclonal antibodies. Such human monoclonal antibodies possess parts of the human immune system rather than the mouse system. These can be generated using transgenic or transchromosomic mice. Transgenic and transchromosomic mice are referred to herein as HUm, respectively. These mice include those referred to as Ab mice and KM mice, collectively referred to herein as "human Ig mice." It is called "su".

[0226] HUmAb mice (Medarex, Inc.) have inactivated endogenous μ and κ chain loci. Unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulins, along with targeted mutations that enhance immune responses. containing a human immunoglobulin gene minilocus encoding globulin sequences (e.g. , Lonberg, et al., (1994) Nature 368(6474):8 56-859). Therefore, this mouse expresses mouse IgM or κ. In response to immunization, the introduced human heavy and light chain transgenes exhibited class II phenotype. undergoing cross-replication and somatic mutation to generate a high-affinity human IgGκ monoclonal ( Lonberg et al., (1994) above; Lonberg, (1994) Ha ndbook of Experimental Pharmacology 113: 49-101; Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13:65-93 and Harding and Lonberg, (1 995)Reviewed in Ann.NYAcad.Sci.764:536-546. The preparation and use of Ab mice and the genomic modifications carried by such mice are described in detail in Tay lor et al., (1992) Nucleic Acids Research 20:6287-6295;Chen et al.,(1993)Internati onal Immunology 5:647-656;Tuaillon et al .,(1993)PNAS USA 94:3720-3724;Choi et al. .,(1993)Nature Genetics 4:117-123;Chen e t al.,(1993)EMBO J.12:821-830;Tuaillon e t al.,(1994)J.Immunol.152:2912-2920;Tayl or et al.,(1994)International Immunology 579-591; and Fishwild et al., (1996) Nature Biotechnology 14:845-851, all of which are further described in The contents of which are specifically incorporated herein by reference in their entirety. U.S. Pat. No. 5,545,806 to Rg and Kay; U.S. Pat. No. 5,569 ,825; U.S. Patent No. 5,625,126; U.S. Patent No. 5,633,4 25; U.S. Patent No. 5,789,650; U.S. Patent No. 5,877,397 No. 5,661,016; U.S. Pat. No. 5,814,318 U.S. Patent No. 5,874,299; and U.S. Patent No. 5,770,429 U.S. Patent No. 5,545,807 to Surani et al.; WO 1992 / 103918 to Lonberg and Kay International Publication No. 1993 / 12227, International Publication No. 1994 / 25585 Pamphlet No. 1997113852 Pamphlet No. 1998 / 24884 and WO 1999 / 45962; and See WO 2001 / 14424 to Korman et al. Please refer to.

[0227] In another embodiment, the human antibodies used in the present disclosure contain a human heavy chain transgene and a human light chain transgene. In transgenes and transchromosomes, such as mice with transchromosomes, These may be produced using mice carrying human immunoglobulin sequences in the mouse. These mice, called "M mice," were developed in response to an international public inquiry into Ishida et al. This is described in detail in brochure No. 2002 / 43478.

[0228] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are , which are available in the art and can be used to generate human antibodies used in the present disclosure. For example, an alternative called Xenomouse (Abgenix, Inc.) Transgenic systems can be used. Such mice are described, for example, in Kucherla et al. U.S. Pati et al., U.S. Pat. No. 5,939,598; U.S. Pat. No. 6 ,075,181; U.S. Pat. No. 6,114,598; U.S. Pat. Nos. 50,584 and 6,162,963.

[0229] Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are , which are available in the art and can be used to generate the human antibodies used in this disclosure. For example, a human heavy chain transchromosome and a human light chain transchromosome called "TC mouse" can be used. Mice carrying both transchromosomes can be used; such mice are Mizuka et al., (2000) PNAS USA 97:722-727 Furthermore, cattle carrying human heavy and light chain transchromosomes have been reported in the art. It has been described in the field of surgery (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894), which produces the human antibodies used in this application. It can be used to make

[0230] Human monoclonal antibodies screened from a library of human immunoglobulin genes Human antibodies can also be prepared using phage display methods for isolating human antibodies. Such phage display methods are well established in the art or are described below. See, for example, U.S. Patent No. 5,222,232 to Ladner et al. 3,409; U.S. Pat. No. 5,403,484; and U.S. Pat. No. 5,577 No. 1,698; U.S. Pat. No. 5,427,908 to Dower et al. No. 5,580,717; McCafferty et al. U.S. Patent No. 5,969,108 and U.S. Patent No. 6,172,197 to I. No. 5,885,799 to Griffiths et al. No. 3; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731 Specification; U.S. Patent No. 6,555,313 Specification; U.S. Patent No. 6,582,915 Specification See U.S. Patent No. 6,593,081.

[0231] Human monoclonal antibodies used in this disclosure are capable of generating a human antibody response upon immunization. Alternatively, the antibody can be prepared using SCID mice in which human immune cells have been reconstituted. Such mice are described, for example, in U.S. Pat. No. 5,476,999 to Wilson et al. 96 and U.S. Pat. No. 5,698,767.

[0232] Methods for making bispecific antibodies are known in the art and are discussed herein.

[0233] B. Methods for Producing Recombinant Molecules In one embodiment, the present application provides a multispecific antibody (e.g., a bispecific antibody) or fragment thereof. The present invention provides a method for recombinantly producing one or more major polypeptide chains of a menthol-containing polypeptide, comprising: 1) one or more nucleic acid molecules encoding each of the polypeptide chains of the polyspecific binding molecule. 2) creating a DNA construct; and 2) introducing said DNA construct into one or more expression vectors. 3) co-transfecting said expression vectors in one or more host cells. and 4) expressing and assembling the molecule in a host cell or in solution.

[0234] In this regard, the present disclosure provides multispecific antibodies (e.g., bispecific antibodies) described herein. or fragments thereof, such as the IL-13 binding domain and and an IL-18 binding domain. In embodiments, the isolated nucleic acid is a single contiguous polynucleotide. In other embodiments, the isolated polynucleotide comprises two or more nucleotides. are arranged on a contiguous nucleic acid sequence.

[0235] In one embodiment, the isolated nucleic acid comprises a sequence encoding an IL-13 binding domain or a fragment thereof. In one embodiment, the sequence includes a sequence encoding an IL-18 binding domain or fragment thereof. a sequence encoding an IL-13 binding domain or a fragment thereof and an IL-18 binding domain The sequences encoding the polypeptides are located on separate polynucleotides, which are referred to as "nucleic acid molecules" It is also called a "set of

[0236] In one embodiment, a sequence encoding an IL-13 binding domain or a fragment thereof and an I The sequences encoding the L-18 binding domains are arranged on a single polynucleotide.

[0237] In an exemplary embodiment, the DNA sequence encoding the light chain of the antibody and the heavy chain of the first half antibody The DNA sequences encoding the vectors are placed in separate expression vectors. The expression vectors are co-transfected into host cells in a ratio that results in The corresponding heavy and light chains are expressed in a host cell and assemble into functional molecules.

[0238] In another exemplary embodiment, the DNA sequence encoding the light chain of the antibody and the heavy chain of the first half antibody are The DNA sequences encoding the chains are placed in an expression vector. The expression vector is then transferred to a host The encoded heavy and light chains can be expressed in the host cell, Assembled into functional molecules.

[0239] Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof as described herein Cloning and expression of one or more nucleic acid molecules or a set of nucleic acid molecules encoding the compound Vectors are provided herein, which vectors are useful for the recombinant production of polyspecific binding molecules. Multispecific antibodies (e.g., bispecific antibodies) as described herein or their A process for the production of fragments of the present invention is provided herein, which process comprises multiple specific Conditions sufficient to express a specific antibody (e.g., a bispecific antibody) or fragment thereof. The method further comprises culturing the host cells disclosed herein and then producing multispecific antibodies. Purifying and recovering the antibody (e.g., bispecific antibody) or fragment thereof from the host cell culture. This includes:

[0240] The variable or constant regions of the molecules described herein, such as to promote heterodimerization Any desired mutations above may be introduced at this stage as described herein.

[0241] The DNA sequences encoding the heavy or light chains of the molecule are prepared by de novo solid phase DNA synthesis or Created by PCR mutagenesis of an existing sequence (e.g., a sequence as described in the Examples below). Narang et al., (1979) Meth. Enzymol. 6 8:90 phosphotriester method; Brown et al., (1979) Meth. Enzymol.68:1 09 phosphodiester method; Beaucage et al .,(1981) Tetra.Lett.,22:1859 diethyl phosphoramidite and the solid support method of U.S. Pat. No. 4,458,066. Direct chemical synthesis of nucleic acids can be achieved by methods such as PCR Technology. : Principles and Applications for DNA Amplif ication, H.A. Erlich (Ed.), Freeman Press, NY ,NY,1992;PCR Protocols: A Guide to Method s and Applications, Innis et al. (Ed.), Aca. demic Press, San Diego, CA, 1990; Mattila et al. al., (1991) Nucleic Acids Res.19:967; and Ec kert et al.,(1991) PCR Methods and Application Mutations to polynucleotide sequences by PCR, as described in Refs. 1:17. Different introductions can be performed.

[0242] Expression vectors and host cells for producing the above molecules are also provided in the present disclosure. A "vector" is suitable for transformation or transfection of a host cell, (in conjunction with) directing the expression of one or more heterologous coding regions operably linked thereto and / or or any molecule or entity containing a regulatory nucleic acid sequence (e.g., nucleic acid, plasmid, A variety of expression vectors can be used to express molecules. Polynucleotides encoding the chains or binding domains can be expressed. Both viral and non-viral expression vectors are used to produce antibodies in mammalian host cells. Non-viral vectors and systems can be used to generate vectors containing plasmids or Episomal vectors (typically containing expression cassettes for protein or RNA expression) ) and human artificial chromosomes (see, e.g., Harrington et al. (1997) Nat Genet 15:345). For example, mammals Non-viral vectors useful for expressing polynucleotides and polypeptides in (e.g., human) cells The vectors used were pThioHis A, B & C, pcDNA3.1 / His, and pE BVHis A, B&C, (Invitrogen, San Diego, CA), MP SV vectors and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, and vectors based on rhesus, adeno-associated virus, herpesvirus, SV40, papillomavirus Virus, HBP Epstein-Barr virus-based vector, vaccinia virus vector Examples include the phage virus and Semliki Forest virus (SFV). Brent et al., (1995) supra; Smith, Annu.Rev.Microbiol.49:807 and Rosenfeld et al., (1992) Cell 68:143. I want to be done that.

[0243] The choice of expression vector will depend on the intended host cell in which the vector will be expressed. Typically, the expression vector contains a polynucleotide encoding the antibody chain or fragment. promoter and other regulatory sequences (e.g., enhancers) operably linked to the In some embodiments, an inducible promoter is used to express the inserted sequence. Inducible promoters include, for example, arabinose, lac Z, metallothionein promoter, or heat shock promoter. Cultures of the resulting organisms are grown under non-inducing conditions to allow the population to grow until the expression product is fully expressed by the host cells. The promoter can be expanded without bias towards better tolerated coding sequences. In addition, other regulatory elements may also be incorporated into multispecific antibodies (e.g., bispecific antibodies) or their fragments. These may be required or desired for efficient expression of the heavy and light chains of the fragment. Elements typically include an ATG initiation codon and an adjacent ribosome binding site or other sequence. In addition, the efficiency of expression can be improved by the inclusion of an enhancer appropriate for the cell system used. (e.g., Scharf et al., (1994) Resul ts Probl. Cell Differ. 20:125; and Bittner et. al., (1987) Meth. Enzymol., 153:516. For example, the SV40 enhancer or CMV enhancer can be used to enhance expression in mammalian host cells. It is possible to increase expression in

[0244] The expression vectors can be prepared by inserting the above sequences for the heavy and / or light chains or fragments thereof. A secretory signal sequence is located to form a fusion protein with the encoded polypeptide. More often, the inserted antibody or antibody-like molecule sequence is contained in a vector. The sequences encoding the light and heavy chain variable domains are linked to a signal sequence before being inserted. The vector used to insert the gene sometimes also encodes a constant region or a portion thereof. Such vectors express the variable regions as fusion proteins with the constant regions, thereby allowing the expression of intact Typically, such constant regions are those found in humans. be.

[0245] Host cells for harboring and expressing the molecules can be either prokaryotic or eukaryotic. E. coli can be used to clone and express the polynucleotides of the present disclosure. Other microbial hosts suitable for use include Bacillus, e.g., Bacillus subtilis, Bacillus spp. For example, Bacillus subtilis and other Enterobacteriaceae obacteriaceae, e.g., Salmonella, Serratia Serratia and various Pseudomonas species In these prokaryotic hosts, those skilled in the art can construct expression vectors. It is also possible to use vectors containing expression control sequences compatible with the host cell (e.g., an origin of replication, In addition, there are any number of different known promoters, e.g. For example, lactose promoter system, tryptophan (trp) promoter system, beta-lactosine promoter system, The promoter is either the tamase promoter system or a promoter system derived from phage lambda. , typically controlling expression, optionally with an operator sequence, and a ribosome binding site It has a sequence of transcription and translation, and initiates and completes transcription and translation. The antibodies of the present disclosure can also be expressed in combination with a baculovirus vector. Insect cells can also be used.

[0246] In some preferred embodiments, mammalian host cells can be used to express the multispecific antibodies (e.g., The antibodies may be used to express and generate antibodies (e.g., bispecific antibodies) or fragments thereof. For example, mammalian host cells may be hybridoma cells that express endogenous immunoglobulin genes. The cell line may be a cell line (e.g., the 1D6.C9 myeloma hybridoma clone) or may be an exogenous The cell line may be a mammalian cell line (e.g., SP2 / 0 myeloma cells) carrying an expression vector for These include any mortal or immortal normal or abnormal animal cells or Examples of suitable cells include human cells capable of secreting intact immunoglobulins. Several host cell lines have been developed, including CHO cell lines, various Cos cell lines, and HeLa These include cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express peptides is generally described in, for example, Winnacke et al. r,FROM GENES TO CLONES,VCH Publishers,N. Expression vectors for mammalian host cells contain expression control sequences, e.g. For example, replication origins, promoters and enhancers (e.g., Queen et al., 1986) Immunol. Rev. 89:49-68) and essential processes Processing information sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites These expression vectors are generally used in mammalian Suitable promoters include those derived from genes or mammalian viruses. , may be constitutive, may be cell type specific, may be stage specific, and / or may be regulatable. Useful promoters include, but are not limited to, promoters of the genus Met. Tarothionein promoter, constitutive adenovirus major late promoter, dexamethasone Inducible MMTV promoter, SV40 promoter, MRP pol III promoter -, constitutive MPSV promoter, tetracycline-inducible CMV promoter (e.g., human immediate-early CMV promoter), constitutive CMV promoter and other promoters known in the art. Examples of promoter-enhancer combinations include:

[0247] The method for introducing an expression vector containing a polynucleotide sequence of interest is to For example, calcium chloride transfection is commonly used for prokaryotic cells. However, calcium phosphate treatment or electroporation may be used for other cell hosts. (See Ambrook, et al., supra.) Other methods include, for example, electrical Perforation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection tion, ballistic method, virosomes, immunoliposomes, polycation:nucleic acid conjugates , naked DNA, artificial virions, fusion to herpesvirus structural protein VP22 (El liot and O'Hare, (1997) Cell 88:223), drug-induced Enhanced DNA uptake and ex vivo transduction are often used for recombinant proteins. For long-term, high-yield production of high-quality antibodies, stable expression is desirable. The cell lines stably expressing the binding fragments are derived from viral origins of replication or endogenous expression. It can be prepared using the expression vector of the present disclosure containing the elements and a selectable marker gene. After introduction of the vector, cells are grown in rich media for 1-2 days and then switched to selective media. The purpose of a selectable marker is to confer resistance to selection; The presence of allows growth of cells that successfully express the introduced sequence in selective medium. Resistant, stably transfected cells can be cultured using tissue culture techniques appropriate to the cell type. It can be propagated by

[0248] The antibody or fragment thereof is generally released from the culture medium as a secreted polypeptide. However, if it is directly produced without a secretory signal, it can also be recovered from host cell lysates. If the molecule is membrane-bound, this can be achieved by eluting it with an appropriate detergent solution (e.g., Trito n-X100) can be used to release it from the membrane.

[0249] If the molecule is produced in a recombinant cell other than that of human origin, it may be a protein or However, the heteromultimer is substantially homogeneous and is completely free of polypeptides. Purifying molecules from recombinant cell proteins or polypeptides to obtain a preparation As a first step, the culture medium or lysate is usually centrifuged to remove certain cell debris. The produced molecules are separated by hydroxylapatite chromatography, gel electrophoresis, and It can be conveniently purified by electrophoresis, dialysis or affinity chromatography, and is preferably A common purification technique is affinity chromatography. Precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin Sepharose Chromatography, on anion or cation exchange resins (e.g., polyaspartic acid columns) Chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate Other techniques for protein purification are also available, such as fractionation by ammonium precipitation (mRNA ).

[0250] VIII. Uses of the Antibodies of the Invention A. Diagnostic and General Therapeutic Uses The antibodies of the present disclosure have many diagnostic and therapeutic applications. For example, they can be used in enzyme immunoassays. The arms can be used to bind specific epitopes on the enzyme and other parts of the molecule. The enzyme immunoassay using antibody-like molecules is et al.(Nolan et al.,(1990)Biochem.Bioph Acta. 1040:1-11). Multispecific antibodies are useful for treating various diseases. It can also be used for the diagnosis of diseases such as autoimmune diseases (Songsivilai et al. al., (1990) Clin. Exp. Immunol. 79:315). The antigen-binding domain of one of the molecules is expressed in tissue samples (in vitro, ex vivo, in vivo). and other binding sites may be detectable as described herein. The marker may be bound to a chelator that tightly binds to a radionuclide (Le Douss al et al.,(1992)Int.J.Cancer Suppl.7:58- 62;Le Doussal et al.,(1993)J.Nucl.Med.34 :1662-1671;Stickney et al.,(1995)Cancer Res.51:6650-6655).

[0251] The antibodies of the present disclosure have in vitro and in vivo diagnostic and therapeutic utility. These antibodies can be used in culture, e.g., in vitro, to treat, prevent, or diagnose various disorders. Alternatively, it may be administered to a cell in vivo or in a subject, for example, in vivo.

[0252] In one aspect, the molecules of the disclosure detect the presence of IL-13 and / or IL-18 in a biological sample. The term "detecting" as used herein is useful for detecting the presence of In certain embodiments, the biological sample is a sample of cells or tissue. In certain embodiments, such tissues contain higher levels of IL-1 as compared to other tissues. The present invention includes normal and / or cancerous tissues that express IL-13 and / or IL-18.

[0253] In one aspect, the present disclosure provides a method for detecting the presence of IL-13 and / or IL-18 in a biological sample. In certain embodiments, the method comprises: contacting the biological sample with the multispecific antibody of the present disclosure and forming a complex between the antibody and the antigen; The biological sample may include a urine or blood sample. It can be seen, but is not limited to.

[0254] Also included are methods for diagnosing disorders associated with IL-13 and / or IL-18 expression. In certain embodiments, the methods include contacting a test cell with a multispecific antibody of the disclosure; IL-13 and / or IL-13 in the test cells by detecting binding of the multispecific molecule Determining the expression level (either quantitatively or qualitatively) of IL-18; and control cells (e.g., normal cells of the same tissue origin as the test cells or non-virally infected cells) The expression levels of IL-13 and / or IL-18 in the test cells and IL-13 and / or IL- 18 expression levels in the test cells compared to the control cells, If IL-3 and / or IL-18 are present at higher levels, it is likely that IL-13 and / or IL-18 are present at higher levels. indicates the presence of an IL-18-associated disorder. In certain embodiments, the test cells The antibodies are obtained from individuals suspected of having a pathological disorder mediated by IL-13 and IL-18.

[0255] In certain embodiments, methods of diagnosis or detection, such as those described above, include, for example, "FACS" assays. The present invention also includes detecting binding of the multispecific molecules of the present disclosure using a ELISA.

[0256] Certain other methods can be used to detect binding of the multispecific antibodies of the present disclosure. Such methods include Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay assays, protein A immunoassays and immunohistochemistry (IHC), Examples of assays include, but are not limited to, antigen binding assays, which are well known in the art.

[0257] In certain aspects, the multispecific antibodies of the present disclosure are labeled. The label may be a label that can be directly detected. Labels or moieties (such as fluorescent, chromophoric, electron-dense, chemiluminescent and radioactive labels) and examples thereof For example, moieties such as enzymes or ligands that are indirectly detected through enzymatic reactions or molecular interactions. These include, but are not limited to, minutes.

[0258] B. Pharmaceutical Compositions and Dosage Forms Many of the disclosed compounds are useful in the uses and methods of the present disclosure in treating atopic dermatitis or related conditions. Pharmaceutical compositions comprising polyspecific antibodies (e.g., bispecific antibodies) or fragments thereof are disclosed herein. The compositions are provided herein and may comprise one or more pharmaceutically acceptable carriers and / or diluents. Further includes:

[0259] The phrase "pharmaceutically acceptable" refers to a compound that is suitable for use in animals, and more particularly in humans. approved by a federal or state regulatory agency or in compliance with the United States Pharmacopoeia or means listed in another generally recognized pharmacopoeia.

[0260] The term "pharmaceutical composition" refers to a pharmaceutical composition containing at least one active ingredient (e.g., an antibody or flagella of the present disclosure). ment) and at least one pharmaceutically acceptable excipient, diluent or carrier. Refers to...

[0261] Pharmaceutical compositions of therapeutic and diagnostic agents may be prepared, for example, as lyophilized powders, slurries, aqueous solutions, Mixed with physiologically acceptable carriers, excipients or stabilizers in the form of a lotion or suspension (See, for example, Hardman, et al. (2001) G odman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill,New Yo rk,NY;Gennaro(2000)Remington:The Scien ce and Practice of Pharmacy, Lippincott, W. illiams,and Wilkins,New York,NY;Avis,e t al.(eds.)(1993)Pharmaceutical Dosage F orms:eral Medications,Marcel Dekker,NY;L ieberman, et al. (eds.) (1990) Pharmaceutica l Dosage Forms:Tablets,Marcel Dekker,NY; Lieberman, et al. (eds.) (1990) Pharmaceutic al Dosage Forms:Disperse Systems,Marcel Dekker, NY; Weiner and Kotkoskie (2000) Exci (See Patient Toxicity and Safety.) The choice of dosage regimen depends on several factors, including serum or tissue turnover of the entity. The rate of response, the level of immunogenic symptoms of the entity, and the activity of target cells within the biological matrix. In certain embodiments, the administration regimen includes the availability of the therapeutic agent delivered to the patient. The amount of therapeutic agent delivered is therefore maximized for an acceptable level of side effects. The amount of biologic will depend in part on the particular entity and the severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available ( For example, Wawrzynczak (1996) Antibody Therapy, Bi os Scientific Pub.Ltd,Oxfordshire,UK;Kre sina(ed.)(1991) Monoclonal Antibodies, Cyt okines and Arthritis,Marcel Dekker,New Y ork, NY; Bach (ed.) (1993) Monoclonal Antib. odies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker,New York,NY;Bae rt,et al.(2003)New Engl.J.Med.348:601-60 8;Milgrom,et al.(1999)New Engl.J.Med.341 :1966-1973;Slamon,et al.(2001)New Engl.J .Med.344:783-792;Beniaminovitz,et al.(20 00)New Engl.J.Med.342:613-619;Ghosh,et a l.(2003)New Engl.J.Med.348:24-32;Lipsky, et al.(2000)New Engl.J.Med.343:1594-1602 (See Dekker, Inc., New York, NY) .

[0262] Determining the appropriate dose can be accomplished using, for example, methods known or inferred in the art that affect the treatment. Clinical trials are performed using parameters or factors that affect or are predicted to affect treatment. Usually, the dosage is started at a slightly lower amount than the optimum amount, and then increased to the desired amount. Increase in small increments for any negative side effects until optimal effect is achieved. Important diagnostic measures include, for example, measures of inflammatory symptoms or levels of inflammatory cytokines produced. Levels include:

[0263] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the particular patient, composition and administration. The dosage regimen is effective in achieving the desired therapeutic response without being toxic to the patient. The dosage level selected may be varied to obtain the desired amount of active ingredient. The activity of the particular composition or its esters, salts or amides, the route of administration, the time of administration, the The rate of excretion of the specific compound being used, the duration of treatment, and the specific composition used in combination other drugs, compounds and / or substances used in combination with the These factors include weight, condition, general health and previous medical history and similar factors known in the medical arts. The effect depends on a variety of pharmacokinetic factors.

[0264] Compositions comprising the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof The compound may be administered by continuous infusion or at intervals of, for example, one day, one week, or 1 to 7 times per week. It can be delivered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally or by inhalation. A dose may be provided.

[0265] The desired use of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure may be The amount is approximately the same as for antibodies or polypeptides on a moles / kg body weight basis. The dose administered to a subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 It can be 1 or 12 or more.

[0266] For multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure: The dosage administered to a patient is about 0.0001 mg / kg to about 100 mg / kg of patient body weight. For example, about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg of patient body weight The multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof may The unit dose is about 0.1 mg to 100 mg, for example, about 1 mg to 5 mg, about 5 mg to about 10 mg. g, about 10mg to about 25mg, about 25mg to about 50mg, about 50mg to about 100mg, about It can be from 100 mg to about 150 mg.

[0267] If a series of doses are administered, these may be administered, for example, about every day, about every week, or about every two weeks. Approximately every 3 weeks, approximately every 4 weeks (monthly), approximately every 2 months, approximately every 3 months (4 months a year) This dose may be administered approximately every six months (up to 10 times), for example, based on disease progression, adverse events, or medical history. Administration may be continued for other periods of time as determined by the physician, for example, about 2, 3, or 4 hours. The fixed dose may be administered from 1 to up to about 17 or more times.

[0268] The amount effective for a particular patient will depend on the condition being treated, the overall health of the patient, the method of administration, and the amount of The dosage may vary depending on factors such as the method, route, and dose, as well as the severity of side effects (see, e.g., Mayn ard,et al.(1996)A Handbook of SOPs for G ood Clinical Practice,Interpharm Press,B oca Raton,Fla.;Dent(2001)Good Laboratory and Good Clinical Practice, Urch Publ.,L (See London, UK).

[0269] Optionally, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof may be used. The agent contains a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the injection site. Further, the composition may be incorporated into a treatment with, for example, an inhalant or nebulizer and an aerosolizing agent. Pulmonary administration may also be used by use of a formulation such as those described in the patents cited herein, which are incorporated by reference in their entirety. Nos. 6,019,968 and 5,985, each of which is incorporated herein by reference. 320, U.S. Pat. No. 5,985,309, U.S. Pat. No. 5,934,27 No. 2, U.S. Patent No. 5,874,064, U.S. Patent No. 5,855,913 The specification, U.S. Patent No. 5,290,540 and U.S. Patent No. 4,880,078 and International Publication No. 1992 / 19244 pamphlet, International Publication No. 1997 / 32 Pamphlet No. 572, Pamphlet WO 1997 / 44013, Pamphlet WO 1 998 / 31346 and WO 1999 / 66903 Please refer to.

[0270] The multispecific antibodies of the present disclosure can be synthesized using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and The route and / or mode of administration for the antibody selected will vary depending on the desired results. administration routes, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes, e.g., injection Parenteral administration is generally administered by injection into the intestine and topically. may refer to modes of administration other than intravenous, intramuscular, intra-arterial, etc., including but not limited to: intravenous, intramuscular, intra-arterial , intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular These include intrathecal, intrathecal, epidural, and intrasternal injections and infusions. The compositions may be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasally, intravenously, or intramuscularly. Administration may be orally, vaginally, rectally, sublingually or topically.

[0271] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof In one aspect, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are administered by infusion. In one aspect, the multispecific antibodies of the present disclosure (or fragments thereof) are administered subcutaneously. The antibody (eg, bispecific antibody) or fragment thereof is administered intravenously.

[0272] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used in, for example, , injection devices, injection pens, vials and syringes, pre-filled syringes, auto-injectors, Infusion pumps, patch pumps, infusion bags and needles, etc. may be used to administer the drug via any of the above routes. The multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof may be administered in a single dose. If the compound is administered in a controlled or sustained release system, the compound may be administered in a controlled or sustained release system. Pumps can be used (Langer, supra; Sefton, 1987, CRC Cri t.Ref Biomed.Eng.14:20;Buchwald et al.,1 980,Surgery 88:507;Saudek et al.,1989,N. Engl. J. Med. 321:574). Controlled or sustained release of the therapeutic agent can be achieved (e.g., Medical Applications of Controlled Release,Lange r and Wise (eds.), CRC Pres., Boca Raton, Fl. a.(1974);Controlled Drug Bioavailability ,Drug Product Design and Performance,Smo Len and Ball (eds.), Wiley, New York (1984); Ranger and Peppas,1983,J.,Macromol.Sci.R See ev. Macromol. Chem. 23:61; Levy et al. .,1985,Science 228:190;During et al.,198 9,Ann.Neurol.25:351;Howard et al.,1989,J Neurosurg. 7 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. US Patent No. 5,916,597; US Patent No. 5,912,015; US Patent No. No. 5,989,463; U.S. Pat. No. 5,128,326; International Publication No. Pamphlet No. 1999 / 15154; and Pamphlet No. WO 1999 / 20253 Examples of polymers that may be used in sustained release formulations include, but are not limited to: However, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate) ), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid) , polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (P Examples include poly(lactide-co-glycolide) (PLGA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert and It is free of extractable impurities, stable on storage, sterile, and biodegradable. Release or sustained release systems can be placed in close proximity to the prophylactic or therapeutic target, thus allowing for minimal Requires only systemic doses (e.g., Goodson, in Medical Appli cations of Controlled Release, op. cit., vol. 2, p. (See p. 115-138 (1984)).

[0273] Controlled release systems are described in detail in Langer (1990, Science 249:1527-153 3). The use of one or more multispecific antibodies (e.g., bispecific antibodies) of the present disclosure is discussed in the review by

[0033] Methods known to those skilled in the art for preparing sustained release formulations containing polyspecific antibodies or fragments thereof are also known. Any of the techniques described in the above may be used, for example, the techniques described in the above, which are incorporated herein by reference in their entirety. U.S. Patent No. 4,526,938, International Publication No. 1991 / 05548, pamphlet International Publication No. 1996 / 20698, Ning et al., 199 6,”Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Su stained-Release Gel,”Radiotherapy&Oncolo gy 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circula ting Emulsions,”PDA Journal of Pharmaceu tical Science&Technology 50:372-397,Clee K et al., 1997, “Biodegradable Polymer C carriers for a bFGF Antibody for Cardiova scular Application,”Pro.Int'l.Symp.Contr ol.Rel.Bioact.Mater.24:853-854 and Lam et a l., 1997, “Microencapsulation of Recombina nt Humanized Monoclonal Antibody for Loc al Delivery,”Proc.Int'l.Symp.Control Rel See Bioact. Mater. 24:759-760.

[0274] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure may be administered locally. When used, they may be in the form of ointments, creams, transdermal patches, lotions, gels, shampoos, Formulated in the form of a spray, aerosol, solution, emulsion or other form known to those skilled in the art. For example, Remington's Pharmaceutical Sciences es and Introduction to Pharmaceutical Do sage Forms, 19th ed., Mack Pub.Co., Easton, See Pa. (1995). Non-sprayable topical dosage forms are compatible with topical application. A carrier or one or more excipients, which in some cases have a dynamic viscosity greater than that of water. Viscous semi-solid or solid forms are typically used. Suitable formulations include, but are not limited to: However, examples include liquids, suspensions, emulsions, creams, ointments, powders, liniments, and patches. They are then sterilized or modified to affect various properties, such as osmotic pressure, as required. The composition is mixed with auxiliary agents (such as preservatives, stabilizers, wetting agents, buffers or salts) to provide the desired effect. Other suitable topical dosage forms include, in some cases, a topical formulation in combination with a solid or liquid inert carrier. The active ingredient is in a mixture with a pressurized volatile substance (e.g., a gaseous propellant, e.g., Freon). or sprayable aerosol preparations packaged in squeeze bottles. Humectants or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are known in the art.

[0275] Compositions comprising the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof When the substance is administered intranasally, it may be in the form of an aerosol, spray, mist, or drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure may be formulated in a suitable propellant (e.g., For example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro ethane, carbon dioxide, or other suitable gas) from a pressurized pack or nebulizer In the case of a pressurized aerosol, the dosage may be conveniently delivered in the form of an aerosol spray presentation. The dosage may be determined by providing a valve to deliver a metered amount. and a suitable powder base, such as lactose or starch, capsules and cartridges (e.g., composed of gelatin) for use in inhalers (composed of) may be blended.

[0276] The multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof may also be administered to patients. The drug may be administered periodically.

[0277] In certain embodiments, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or their fragments are The fragment may be formulated to ensure proper distribution in vivo. The barium duct barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure crossing of the BB (if necessary), these may be incorporated into e.g. liposomes. For methods of producing liposomes, see, for example, U.S. Patent No. 4,522 ,811; U.S. Pat. No. 5,374,548; and U.S. Pat. No. 5,399 See, e.g., 331. Liposomes are selectively delivered to specific cells or organs. and thus may contain one or more moieties that enhance targeted drug delivery (e.g., , Ranade VV (1989) J. Clin. Pharmacol. 29:685 Exemplary targeting moieties include folic acid or biotin (e.g., Low See U.S. Pat. No. 5,416,016 to Mannosi et al. (Umezawa et al., (1988)Biochem.Biophys.R es.Commun.153:1038);Antibodies(PGBloeman et al (1995)FEBS Lett.357:140;M.Owais et al.( 1995)Antimicrob.Agents Chemother.39:180) ; Surfactant protein A receptor (Briscoe et al. (1995) A mJPhysiol.1233:134);p120(Schreier et a l. (1994) J. Biol. Chem. 269:9090); K. Kei nanen;MLLaukkanen(1994)FEBS Lett.346:1 23;JJKillion;IJFidler(1994)Immunomet See also Hods 4:273.

[0278] The present application also provides multispecific antibodies (e.g., bispecific antibodies) of the present disclosure in combination with other treatments or therapeutic agents. Co-administration or treatment of patients using pharmaceutical compositions containing a specific antibody or fragment thereof Protocols for administering additional therapeutic agents, such as cytokines, steroids, and chemotherapeutics, are also provided. Methods of co-administration or co-treatment with therapeutic agents, antibiotics or radiation are known in the art. (e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis o f Therapeutics,10.sup.th ed.,McGraw-Hill ,New York,NY;Poole and Peterson(eds.)( 2001)Pharmacotherapeutics for Advanced P practice:A Practical Approach, Lippincott, Williams&Wilkins,Phila.,Pa.;Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, (See, e.g., Phila., Pa.) An effective amount of a therapeutic agent is one that reduces disease symptoms by at least 10%. at least 20%; at least about 30%; at least 40% or at least 50% reduction It can be done.

[0279] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise one or more additional therapeutic agents. nothing.

[0280] In addition to the above treatment regimens, patients may be subjected to surgery and other forms of physical therapy.

[0281] C. Therapeutic Applications for IL-13 and IL-18-Mediated Pathological Disorders The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used in a variety of Human diseases, such as pathological disorders mediated by IL-13 and IL-18, e.g., autoimmune diseases and inflammatory diseases or IL-13 and / or IL-18 dysregulation (e.g., inappropriate expression, These compounds have therapeutic uses for treating conditions involving alterations in the expression levels, signal transduction, etc. In embodiments, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof It is used in the treatment of atopic dermatitis.

[0282] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof The compounds are intended to treat, reduce the likelihood of, or inhibit the growth of IL-13 and IL-18 mediated pathological disorders. The phrase "IL-13 and IL-18 mediated pathological disorders" is useful for the alleviation of such disorders. IL-13 and IL-18 are not directly or indirectly involved in the causation, development, or progression of any disease or condition. All diseases and medical conditions, including the progression, development, persistence or pathology of a disease or medical condition Thus, these terms encompass conditions in which abnormal IL-13 and IL-18 levels IL-13 and IL-14 expression in conditions associated with or characterized by IL-13 and IL-14 expression in target cells or tissues Diseases or conditions that can be treated by reducing or inhibiting -18-induced activity Pathological disorders mediated by IL-13 and IL-18 include IL-13 and IL-1 Includes autoimmune and / or inflammatory conditions and disorders with eight components.

[0283] In some embodiments, the pathological disorder is associated with inappropriate IL-13 and IL-18 expression. In some embodiments, the pathological disorder is associated with inappropriate IL-13 and IL-1 8 associated with signal transduction.

[0284] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are not limited to: autoimmune and / or inflammatory conditions and disorders, particularly those with etiologies that include an autoimmune component, In one aspect, the present disclosure provides a method for treating, preventing, or ameliorating an inflammatory condition, such as an autoimmune disease. In one aspect, the present disclosure provides a method of treating an inflammatory disease or condition. In one embodiment, the subject of treatment is a human.

[0285] For use in the treatment and / or prevention of pathological disorders mediated by IL-13 and IL-18 a multispecific antibody (e.g., a bispecific antibody) of the present disclosure as described herein for Fragments and pharmaceutical compositions thereof are provided herein. In the manufacture of a medicament for use in the treatment of an mediated pathological disorder, Use of such multispecific antibodies or pharmaceutical compositions is provided herein. Provided herein are methods for treating and / or preventing pathological disorders mediated by β-18, The method comprises administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody as described herein. In some embodiments, the method comprises administering IL-13 and IL-14 to a subject or pharmaceutical composition. The pathological disorder mediated by -18 is an autoimmune disease or an inflammatory disorder or condition. In an embodiment, the autoimmune disease or inflammatory disorder or condition is atopic dermatitis.

[0286] The multispecific antibody of the present disclosure for use in the treatment and / or prevention of moderate to severe AD. An antibody (e.g., a bispecific antibody) or a fragment thereof and a medicament as described herein Compositions are provided herein.

[0287] The term "atopic dermatitis" (AD) or "eczema" as used herein means a strong Inflamed skin characterized by itching (e.g., severe itching) and scaly, dry, eczematous lesions The term "atopic dermatitis" or "eczema" refers to a skin disease characterized by epidermal barrier dysfunction, atopic dermatitis, Allergies (e.g., skin allergies, allergies to certain foods, pollen, mold, dust mites, animals, etc.) allergies caused by or related to exposure to radiation and / or asthma The present disclosure covers mild, moderate to severe, or severe AD (eczema). As used herein, "moderate to severe AD" includes methods of treating patients with AD. D) is often complicated by persistent bacterial, viral or fungal infection and is accompanied by severe itching. It is characterized by widespread skin lesions. Moderate to severe AD also includes chronic AD in patients. In most cases, chronic lesions include thickened plaques of the skin, lichenification, and fibrous papules. Patients with mild to severe AD commonly present with lesions of the eyes, hands, and folds of the body. In addition, more than 10% or more than 20% of the skin on the body is affected, or 10% of the skin area is affected. Patients with moderate to severe AD generally In general, (i) an Investigator's Global Assessment (IGA) score of 3 or 4, (ii) eczema area and an EASI score of at least 10, preferably at least 12; iii) Itching. Moderate to severe AD requires frequent treatment with topical corticosteroids. It is believed that topical corticosteroids or calcineurin may be present in patients who need them. either by steroid inhibitors or any other commonly used therapeutic agent known in the art. Patients may also have moderate to severe AD if they are tolerant or resistant to treatment. It can be said that.

[0288] Suitably, the uses and methods of the present disclosure comprise administering to the subject a therapeutically effective serum level sufficient to achieve the therapeutically effective serum level. and administering a multispecific antibody (e.g., bispecific antibody) of the present disclosure or a fragment thereof at a dose of Suitably, the multispecific antibody (e.g., bispecific antibody) or fragment thereof Therapeutically effective serum levels of the fragment are maintained throughout the course of treatment.

[0289] As used herein, the term "therapeutically effective serum level" refers to a therapeutically effective serum level for a condition, disorder, or is the severity of a disease and / or its associated symptoms and / or the severity of a condition, disorder or disease and / or or is sufficient to reduce the duration and / or ameliorate the symptoms associated therewith, versus refers to the serum levels of a therapeutic agent (e.g., a bispecific antibody) in an elephant. "Therapeutically effective serum levels," as used herein, are levels that achieve a specified result, e.g., improvement in AD-related parameters, e.g., reduction in physician global assessment (IGA) scores; a decline from baseline in the Dermatology-Related Daily Living Quality Index (DLQI); Decrease from baseline in Patient Global Impression of Severity (PGIS); Decreased improvement from baseline in Personal Impression of Change (PGIC); Reduction in skin surface area involvement (BSA) score; Eczema Area and Severity Index (EASI) score a decrease in SCORAD score; and / or a decrease in pruritus numerical rating scale (NRS) score A multispecific antibody (e.g., bispecific antibody) or its fragment in the serum of a subject that achieves a reduction It also refers to the amount of fragmentation.

[0290] In some embodiments, a "therapeutically effective serum level," as used herein, means: As a result of the designation, for example, the multispecific antibody (e.g., bispecific antibody) or a fragment thereof can be produced. one or more AD-related biomarkers, particularly CC, compared to pre-treatment levels in the control group L17 / TARC, IgE (e.g., serum IgE), CCL26 / eotaxin-3, C CL22 / MDC, hsCRP, CD40, IL-13, IL-24, IL-22, IL -18 (e.g., serum IL-18, serum-free IL-18 (bioactive)) and IL-18 One or more AD-related BPs selected from the list consisting of BP (e.g., serum IL-18BP) a multispecific antibody (e.g., The term also refers to the amount of a specific antibody (e.g., bispecific antibody) or fragment thereof.

[0291] Suitably, the uses and methods of the present disclosure involve the use of multispecific antibodies (e.g. bispecific antibodies) or The fragments are taken once a week, once every two weeks, once every three weeks, once every four weeks, and once every eight weeks. or once every 12 weeks. According to certain exemplary embodiments, the use of the present disclosure The use and method involves administering a multispecific antibody (e.g., a bispecific antibody) or fragment thereof once every four weeks. The method includes administering a therapeutic fragment.

[0292] Also provided herein is a method for inhibiting IgE antibody production in a subject, comprising administering an effective amount of Administering a multispecific antibody (e.g., bispecific antibody) of the present disclosure or a fragment thereof to a subject This includes:

[0293] Also provided herein is a method for inhibiting IFN-γ production in a subject, comprising administering an effective amount of Administering a multispecific antibody (e.g., bispecific antibody) of the present disclosure or a fragment thereof to a subject This includes:

[0294] In certain embodiments, provided herein are methods of treating an IgE-mediated disorder in a subject. The method comprises administering an effective amount of a multispecific antibody (e.g., a bispecific antibody) of the present disclosure or a method thereof. and administering to a subject a fragment of the antibody or fragment thereof, inhibits the binding of L13 to its receptor and is involved in the binding of interleukins to said receptor. inhibit one or more functions of the

[0295] D. Combination Therapy The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used in a variety of May be used in combination with other drugs and therapeutic agents used in the treatment of diseases, disorders and conditions (herein "additional therapeutic agent").

[0296] Administered "in combination" refers to a series of therapeutic agents associated with a disorder, with respect to the additional therapeutic agent. During a disease, two (or more) different treatments are delivered to a subject, e.g., two or more treatments are after a subject has been diagnosed with a disorder and after the disorder has been cured or eliminated or other It is meant to be delivered before treatment is stopped for any reason. In some cases, delivery of one treatment is still occurring when delivery of the second treatment begins, and There will be an overlap in the given time period. This is sometimes referred to herein as "simultaneity." This is called "multianeous" or "concurrent delivery." In some cases, delivery of one treatment ends before delivery of the other treatment begins. In some embodiments, the treatment is more effective due to the combined administration. For example, the second treatment may be more effective, e.g., a lower amount of the second treatment may be given to achieve the same effect. or the second treatment results in a greater improvement than that seen when administered without the first treatment. The second treatment alleviates the symptoms to a greater extent, or a similar condition is observed with the first treatment. In some embodiments, delivery is associated with a reduction in symptoms or other parameters associated with the disorder. reduction is greater than that observed when one treatment is delivered in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more than additive. The delivery can be as large as the amount of the first treatment delivered when the second treatment is delivered. The effect may be such that it is still detectable.

[0297] The term "concurrently" refers to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time. Rather, it is not limited to the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or their Pharmaceutical compositions containing the fragments, in turn, may provide a greater degree of benefit than if they were not administered. Within a time interval such that the molecules of the present disclosure may act together with an additional therapeutic agent to produce the above. administered to a subject in any order, e.g., at the same time or at different times. Each treatment may be administered to a subject sequentially, but if not administered simultaneously, they may be administered sequentially as desired. They should be administered sufficiently close in time to provide a therapeutic or prophylactic effect. The treatments may be administered separately to a subject in any suitable form and by any suitable route. It can be done.

[0298] Additional Therapeutic Agents (e.g., Additional Prophylactic or Therapeutic Agents) That May Be Administered in Combination with the Molecules of the Present Application are separated by less than 5 minutes, less than 30 minutes, and less than 1 hour from the molecules or fragments thereof of the present disclosure. Away, about 1 hour away, about 1 to about 2 hours away, about 2 to about 3 hours away, about 3 hours ~About 4 hours apart, about 4 hours~About 5 hours apart, about 5 hours~About 6 hours apart, about 6 hours~ Approximately 7 hours apart, approximately 7 to 8 hours apart, approximately 8 to 9 hours apart, approximately 9 to 10 hours apart 10 hours apart, about 10 to 11 hours apart, about 11 to 12 hours apart, about 1 2 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 Hours ~ 48 hours apart, 48 hours ~ 52 hours apart, 52 hours ~ 60 hours apart, 60 hours Between 72 hours, 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours In other embodiments, two or more additional therapeutic agents may be administered to the same patient from 1 to 120 hours apart. It is administered to patients during their visit.

[0299] Multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof and additional The therapeutic agents may be administered simultaneously, in the same or separate pharmaceutical compositions disclosed, or sequentially. In the case of sequential administration, the multispecific antibody (e.g., bispecific antibody) of the present disclosure or its The fragment may be administered first and the additional agent may be administered second, or the order of administration may be different. The order can be reversed. The additional therapeutic agent can be used in combination with the disclosed polyspecific binding molecules and fragments. The compounds may be administered to a subject by the same or different routes of administration.

[0300] a multispecific antibody (e.g., a bispecific antibody) of the present disclosure or a fragment thereof and / or Additional therapeutic agents, procedures, or modalities may be administered during the period of active disease or during remission or active disease. The multispecific antibodies of the present disclosure (e.g., bispecific The antibody) or fragment thereof may be administered before, concurrently with, or after other treatments or during remission of the disorder. can be given.

[0301] The additional therapeutic agents of the combination therapy of the disclosure may also be administered cyclically. Ring therapy involves administering a first treatment (a first preventative or therapeutic agent) for a period of time, followed by a second Treatment (second preventative or therapeutic agent) is administered for a period of time, and this sequential administration is repeated (all (i.e., cycle), reducing the development of resistance to one of the treatments (e.g., drugs) Avoiding or alleviating one of the side effects of (e.g., a drug) and / or improving the effectiveness of treatment This includes improving

[0302] When administered in combination, the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or The fragment and the additional therapeutic agent (e.g., a second or third agent), or all, may be administered individually. , e.g., greater than, less than, or the same amount or dosage of each drug used as monotherapy. In certain embodiments, the polyspecific binding molecule, e.g., a bispecific binding molecule, can be administered in an amount or dose that is A multispecific molecule, e.g., a bispecific antibody-like molecule as described herein, an additional agent (e.g., The amounts or dosages of any of the agents (e.g., second or third agents) administered individually, e.g., as monotherapy, may be used. As a rule, the amount or dose of each drug used should be less than the amount or dose of each drug used (e.g., at least 20%). , at least 30%, at least 40%, or at least 50%. So, we will discuss the use of multispecific antibodies (e.g., bispecific antibodies) of the present disclosure that produce the desired effect. the fragment, additional agent (e.g., second or third agent), or total amount or dosage (e.g., treatment of an autoimmune or inflammatory disease or condition) to achieve the same therapeutic effect. less than the amount or dosage of each drug used individually, e.g., as monotherapy, required for Not (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less).

[0303] Preferably, the additional therapeutic agent (e.g., a second or third agent) is an AD agent, e.g., a small molecule, Biological therapy or AD modalities, such as topical therapy, systemic therapy, phototherapy, and their "AD drugs" are drugs that use phototherapy, including a combination of creams, ointments, and lotions. ointments, gels or sprays (e.g. low- to medium-potency corticosteroids [WHO guidelines] Groups IV-VII according to the guidelines, Bolognia JL, Jorizzo JL, S chaffer JV.Glucocorticosteroids.Dermatol ogy.3rd ed.2012.Ch 125,2075-88;Ference J D,Last AR.Choosing topical cocorticoster oids.Am Fam Physician.2009 Jan 15;79(2): 135-40]); over-the-counter (OTC) emollients and medical devices or so-called barrier creams (such as atopiclair); and itching and / or pain lubricants for the treatment of ulcerative colitis, such as menthol, pramoxine, or antihistamines; anti-itch lotions; local anesthetics, systemic medications (e.g., biologic agents, e.g., I L-4R inhibitors, such as dupilumab; IL-13Ra1 inhibitors, such as ASLAN -004; IL-13Ra2 inhibitors, etc.; IL-31 inhibitors, such as nemolizumab; TNF-alpha inhibitors, such as adalimumab, infliximab, certolizumab, and entrectinib Tanercept, alefacept, etc.; IL-1α inhibitors, e.g., bermekimab (MABp 1) etc.; IL-23 inhibitors, such as briakinumab, ustekinumab, guselkumab, risankizumab, tildrakizumab, etc.; IL-17 inhibitors, e.g., brodalumab, iki Cefalizumab, etc.; CD11a inhibitors, e.g., efalizumab; IL-22 inhibitors, e.g., IL-22 binding proteins, such as featherimumab; IL-5 inhibitors, such as mepolizumab , benralizumab, etc.; synthetic forms of IL-2, such as aldesleukin; interleukin Recombinant IL-2 approaches targeting the IL-2 receptor complex, e.g., LY3471 851, etc.; OSMR inhibitors, such as KPL-716, etc.; VAP-1 inhibitors; OX-4 0 inhibitors or OX40L inhibitors, such as GBR830, KY1005, etc.; IgE inhibitors , such as omalizumab and ligelizumab;TSLP inhibitors, such as tezepelumab; IL-33 inhibitors, such as MEDI3506; IL-36 inhibitors, such as Spesolima B-cell modulating approaches, e.g., rituximab, ocrelizumab etc.; non-biologic immunomodulatory treatments, such as cyclosporine and other calcineurin inhibitors , JAK inhibitors, such as tofacitinib, upadacitinib, abrocitinib, and baricitinib TYK2 inhibitors, such as duravacitinib; methotrexate; PDE 4 inhibitors, such as apremilast; Siglec inhibitors, such as AK-002; S1 P agonists or antagonists, such as etrasimod or SCD-044; BTK inhibitors, such as TAS-5315, IRAK4 antagonists and CCR4-inhibitory approaches anticoagulants, such as RPT-193; systemic corticosteroids, cyclophosphamide, Rufasalazine, azathioprine, mycophenolate mofetil, dapsone, hydroxybenzoates chloroquine); retinoids (e.g., alitretinoin); leukotriene inhibitors or anti- Leukotriene agents such as montelukast, pranlukast or zafirlukast, and 5-LO inhibitors, such as zileuton, and LTA4H inhibitors, such as acevirstitium intralesional corticosteroid injections; phototherapy (e.g., high doses of UVB and UVA). Chemotherapy (e.g., psoralens and UVA (PUVA)); topical calcineurin inhibitors ( cyclosporine, tacrolimus, pimecrolimus) or topical PDE4 inhibitors, e.g. such as saborole, difamilast, or roflumilast; topical JAK inhibitors, such as luxo Litinib, delgocitinib or topical vitamin D analogues and topical aryl hydrocarbon receptor ( AhR) inhibitors, e.g. benvitimod / tapinarof; high to very high efficacy (WHO definition topical corticosteroids (classes I, II, and III); antifungal agents with known anti-inflammatory properties Antibacterial agents, such as griseofulvin, itraconazole, betamethasone, dexamethasone, NCB018424, triamcinolone, apremilast, turmeric paste, gluco Samin sulfate, triamcinolone acetonide, sesame oil, betamethasone dipropionate clobetasol propionate, probiotics (e.g., bifidobacterium Bifidobacterium animalis subspecies La Lactis HN019, Lactobacillus reuteri i reuteri), omega-3, prednisone, prednisolone, platelet-rich blood Jelly, Orabase paste, lycopene, topical chamomile, green tea, CO2 laser treatment, allergy Genome-specific immunotherapy, polybiotics, photobiomodulation, metronidazole, doxycycline Cycline, minocycline, cedar honey, purslane, curcuminoids, a Lefacept, hexaminolevulinate, hydroxychloroquine, Adcortil, F Falizumab, fluocinolone, coenzyme Q10 mucoadhesive tablets, chemomereum novi chamaemelum nobile, sirolimus, tacrolimus, king cucumber Yuan Decoction, NSAID Topical Rinse, NSAID, Quercetin, NAVS Naphthalane, Ba Appropriately, topical AD therapies include topical steroids, fluoxetine, bupivacaine, and oatmeal baths. steroids, e.g., corticosteroids, tacrolimus, cyclophosphamide, azathioprine Purine, methotrexate, mycophenolate mofetil, apremilast, calcinue calcineurin inhibitors, e.g., topical calcineurin inhibitors, phosphodiesterase 4 (PDE 4) inhibitors, e.g., topical PDE4 inhibitors, e.g., crisaborole, adrenocorticotropic hormone Analogues, dupilumab, etanercept, adalimumab, infliximab, omalizumab and atopic dermatitis prescription drug therapies, including but not limited to secukinumab and secukinumab.

[0304] E. Kit The present disclosure relates to IL-13 and IL-18 mediated pathological disorders, such as autoimmune diseases or The present invention also encompasses kits for treating a patient with an inflammatory disorder or condition. The kit comprises a therapeutically effective amount of the multispecific antibody of the present disclosure. A means for administering the indicated multispecific antibodies (e.g., autoinjectors, syringes, and vials) The product may include a pre-filled syringe, pre-filled pen, or other suitable container and instructions for use. These kits are useful for treating pathological disorders mediated by IL-13 and IL-18, such as autoimmune diseases. or may contain additional therapeutic agents (described above) for treating those with inflammatory disorders or conditions. Such kits may include instructions for administration of the multispecific antibodies of the present disclosure to treat a patient. Such instructions may include dosages, routes of administration, regimens, and instructions for use with the enclosed disclosure. The total treatment period for use with the multispecific antibody may be provided in the range of 100-1500 mg / kg.

[0305] The phrase "means for administration" includes pre-filled syringes, vials and syringes, injection pens, including syringes, auto-injectors, IV drips and bags, infusion pumps, patches, infusion bags and needles. This refers to any available device for systemic administration of drugs to a patient, including but not limited to: With such an article, patients can self-administer drugs (i.e., without the assistance of a physician). The patient may administer the drug unassisted (self-administered) or the drug may be administered by a physician. [Example]

[0306] The following examples are provided to further illustrate the present disclosure, but not to limit its scope. Other variations of the present disclosure will be readily apparent to those skilled in the art and are within the scope of the appended claims. Covered by a range.

[0307] Example 1: Generation of IL-13 / IL-18 bispecific antibodies in CHO cell lines 1. Expression Vector Construction The vector used in the examples contains the following elements: hCMV promoter / antibody construct Enhancers that drive the expression of individual genes required for assembly of the complex, Adenylation signal (polyA), folate receptor (FolR, FAR), DHFR, selection Puromycin and / or hygromycin genes as markers, E. coli (EC oli) origin of replication and base for ampicillin resistance to allow amplification in bacteria. The tarlactamase gene is included. Different plasmid arrangements were evaluated and further details are shown in Figure 1. Provided within.

[0308] Figure 2 is a schematic diagram of NVS standard plasmids A to D. Plasmids A and C contain anti-I Plasmids B and D encode expression of L13 kappa LC and anti-L13 knob HC. encodes the expression of anti-IL18 lambda LC and anti-IL18 hole HC. Expression of the protein chains is driven by separate CMV promoters. In TD parent cells, linearized plasmids A and B or C and D were simultaneously co-transfected. In the first selection round, the selection markers DHFR and FAR were used. For the second selection, additional selection markers, hygromycin and pi were added. Plasmids are highly productive during FACS-assisted single cell sorting. Leaky Stop Transmembrane Technology (LS-TM) to enable staining and enrichment of clones Plasmids C and D contain the information of the LC and HC coding expression cassettes. Minimizing the risk of homologous recombination by deleting the repetitive partial phage f1 region Plasmid D was designed with lower sequence homology to allow for different codon optimization and I Plasmids A and B contain the signal peptide for the L18 hole HC. The DNA sequences of IL18 knob CH and IL13 hole CH are KiH Only the base that codes for the mutation differs.

[0309] FIG. 3 is a schematic diagram of NVS Furin-2A peptide (F2A) plasmids E and F. F2A technology allows for the combinatorial expression of multiple protein chains from a single promoter. On plasmids E and F, the first expression cassette contains the anti-IL18 lambda LC and and anti-IL18 hole HC, and the second expression cassette encodes anti-IL13 kappa L The linearized plasmids E and F encode the anti-L13 knob HC and the HC. O-C8TD parent cells were transfected with the selectable markers DHFR and FAR. Plasmid F was used to select cells. Using different codon optimization for obHC reduces the risk of homologous recombination. On the other hand, on plasmid E, IL18 is designed with lower sequence homology to minimize The DNA sequences of knob CH and IL13 hole CH encode the KiH mutation. Only the bases they contain differ.

[0310] Figure 4 is a schematic diagram of the adapted NVS standard plasmids G and H. Plasmids G and H / or H expressing plasmids E or F to increase the number of integrated plasmids The pool is used for supertransfection. Plasmid G contains the Plasmid H encodes expression of IL13 kappa LC and anti-IL13 knob HC; Each gene encodes the expression of anti-IL18 lambda LC and anti-IL18 hole HC. Expression of the protein chains is driven by separate CMV promoters. Different approaches of extraction and selection were performed: (I) Plasmids G and H were cloned into CH O-C8TD parent cells and the selection markers hygromycin and puromycin were used. (II) Plasmid G was co-transfected into selected cells. First round transfection, selection using hygromycin, and / or or plasmid H was transfected in the second round and puromycin was used. or (III) transfecting plasmid H in the first round, Select using puromycin and / or transfect plasmid G in the second round. Plasmids G and H were transfected into the GFP-positive cells and selected using hygromycin. S-assisted leaky sorting to allow staining and enrichment of highly productive clones during single-cell sorting It holds information on stop-transmembrane technology (LS-TM). To minimize the risk of recombination, the expression vectors encoding LC and HC were placed on plasmids G and H. The partial phage f1 region between the current cassettes was deleted, and the codon optimization of LC and HC was performed and differs from plasmids E and F.

[0311] Figure 5 shows the protein chain combinations in the expression cassettes compared to plasmids E and F. Schematic diagram of NVS Furin-2A peptide (F2A) plasmid I with different sequences. On plasmid I, the first expression cassette contains anti-IL18 lambda LC, anti-IL18 ho The second expression cassette encodes anti-IL13 knob HC and anti-IL14 knob HC. The linearized plasmid I encoding the 13kappa LC was transfected into CHO-C8TD parental cells. The cells were transfected with DHFR and FAR. The partial phage f1 region between the two expression cassettes was deleted, resulting in a different codon for CH. Optimization was used to reduce sequence homology to minimize the risk of homologous recombination.

[0312] 2. Cell Lines, Culture, Transfection and Selection The parental CHO cell line was used as the host cell line for the production of the antibody constructs. A single vial from the CHO line was used to CHO cell lines were prepared in shake flasks in a non-humidified shake cabinet. Culture in suspension in their own chemically defined culture medium at 150 rpm, 10% CO2, and 36.5°C. The cells were cultured using an automated system (ViCell, Beckman Coulter). Cell viability and proliferation rate were monitored. Cells were passaged into fresh medium 2-3 times a week to maintain logarithmic growth. Maintained during the breeding season.

[0313] Electroporation (Amaxa Nucleofection system, The antibody constructs were linearized with Swal by the manufacturer (Lonza, Germany). Expression plasmids were transfected into the chemically defined nucleoside analogs according to the manufacturer's instructions. The transfection reaction was carried out in the culture medium used for transfection. The parental CHO cells were in exponential growth phase with cell viability greater than 95%. 6 Transfection was performed at 1000 cells / transfection. Afterwards, the cells were transferred to shake flasks containing chemically defined medium. Incubate the cell pool for 48 h at 36.5 °C and 10% CO2 before starting did.

[0314] The selection procedure was carried out using the selectable markers encoded by the individual expression vectors. First transfection and selection round using folate receptor and DHFR Both proteins are involved in the same molecular pathway; FolR regulates folate and folate analogs. The analog MTX is transported into the cell, where DHFR transports it to the nucleus, which is important for purine and methionine synthesis. These are combined as a selection principle to produce both recombinant proteins. Certain strong selection regimes can be employed to enrich for recombinant cells expressing the .

[0315] After 48 hours of transfection and growth under low folate conditions, chemically defined Further selection pressure was applied by adding 10 nM MTX to the culture medium. Afterwards, the material was supplemented with 7.5% DMSO and prepared for further analysis as described below. The harvested cells were frozen in the medium they were fed.

[0316] Design of the plasmids used for the first transfection and selection round Depending on the pool, various possibilities for progression emerge. They were either used directly for single cell cloning or subjected to a second selection or Supertransfection was performed.

[0317] For the second selection, the collected pool was maintained under low folate conditions with 10 nM MTX and purified. A biologically defined culture medium containing 0.5ug / ml puromycin and 0.8mg / ml hyaluronan was used. Further selection pressure was applied by adding gromycin. After pool recovery, 7.5% Cells were cultured in medium supplemented with DMSO and materials prepared for further analysis as described below. The cells were frozen.

[0318] For supertransfection, the antibody constructs were linearized with SwaI. The expression plasmid was electroporated (Amaxa Nucleofection Transfection was performed using the NIH system (Lonza, Germany). Transfection reactions were performed in a chemically defined medium according to the manufacturer's instructions. The CHO pool recovered after transfection from the selection was used for the second or third transfection. The transfection was carried out as described above and incubated at 36.5°C. and 10% CO2 for 48 hours, followed by 0.5ug / ml puromycin or 0.8mg / m Selection was performed by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 After pool collection, the solution was diluted with 7.5% DMSO and prepared for further analysis as described below. The cells were frozen in medium supplemented with the indicated substances.

[0319] 3. Single-cell Cloning After selection, cells are analyzed using a Cytena cell printer device or by flow cytometry. or by single-cell cloning to obtain a cell line of monoclonal origin. Ta.

[0320] The Cytena cell printer is a printer that contains a microfluidic chip into which a cell suspension is placed. Contains a disposable dispense cartridge from which the 96-well plate is dispensed. During this process, the nozzle area is observed by a microscope system. The image is recorded. Automated image analysis algorithms detect cells on the image and identify the size and They are classified according to morphological criteria such as thickness and roundness. Based on image analysis of the droplet formation area, droplets containing single cells were separated into individual wells of a 96-well plate. Droplets that do not contain a single cell (empty droplets or droplets with multiple cells) are moved to the well. Transfer to waste.

[0321] Prior to flow cytometry, a surface-attached antibody was used to facilitate the selection of high-producing clones. FITC-labeled in-house produced BDC A directed against the Fc portion of ABC123 Cells were stained with b.

[0322] Use 100 µm disposable sorting tips to sort the wells with a 96-well plate holder. Single cell cloning was performed using the Cell Sorter instrument. To ensure that the cells were sorted, the settings were adjusted to single-cell mode and 3-droplet sorting. With these settings, only droplets containing cells will be detected if the previous and subsequent droplets are both empty. To improve the likelihood that each droplet contains no more than one single cell, the yield is The cell concentration and flow rate were optimized by sacrificing the cells. Selecting single, live cells with strong fluorescence. To achieve this, multiple gates were set up.

[0323] Single cells using Cytena cell printer or Sony Cell Sorter After sorting and placing the cells into individual wells of a 96-well plate, high-resolution microscopic images of each well were obtained. Images were obtained to reveal a single clone and validate the single-cell cloning procedure. .

[0324] After single cell cloning, the clones are expanded to evaluate productivity, bioprocess suitability, and The transgene was characterized for integration and expression. By freezing cells from top performing clones in medium supplemented with MSO, P Primary seed lot (PSL) vials are prepared and finalized for MCB manufacturing. The selected clone PSL was used.

[0325] 4. Upstream processing After selection, material was produced in shake flask fed-batch cultures. The culture was inoculated and the addition of the original feed solution was started on the third day, and on the fifth day the culture temperature was increased to 33°C. In-process controls were performed to monitor the concentration of the antibody construct during incubation. Individual cultures were grown for 14 days. At the end of the cultivation process, the cultures were separated by centrifugation. Separation of cells from the supernatant followed by sterile filtration and subsequent further downstream processing and separation All product types and related impurities bearing the Fc moiety were determined. Protein A was measured in cell culture supernatants by HPLC or RP-LC to determine its activity. , the volumetric productivity of the selected pools was determined.

[0326] Example 2. LC-MS screening and purity of IL-13 / IL-18 bispecific antibodies evaluation 100 μg of 1 mg / ml in 20 mM Tris-HCl pH 7.5 Purified bispecific mAbs were diluted and treated with 2 μl PNGaseF enzyme (New England The PLRP-S RP was deglycosylated using a ELISA kit (Biolabs) at 37°C for 4 hours. Wate equipped with a column (3 μm, 2.1 × 150 mm, 300 Å, Agilent) ACQUITY UPLC Class and Dual Spray Ion Source (Scie x) Deglycosylated ribonucleotides were analyzed using a TripleTOF 6600 mass spectrometer. The sample was subjected to LC-MS system. The eluents were A: 0.1% TFA in water and B: 70% The solvent was 20% isopropanol, 20% acetonitrile, 10% water and 0.09% TFA. The temperature was set at 60°C. The flow rate was 0.2 ml / min. Proteins were added as follows: Elution was performed with a 40-minute gradient: 0–4 min 35% B, 4–28 min 35–50% B, 28– 29 minutes 50 to 80%B, 29 to 34 minutes 80%B, 34 to 35 minutes 80 to 35%, 35-40 min 35% B. Record the UV chromatogram at 214 nm. MS data were acquired using Analyst software TF1.7 (ABSciex). Data were acquired and analyzed using BioPharmaView (version 3.0, ABSciex) and and analyzed using PeakView (version 2.2, ABSciex) software. The identification and relative quantification of bbmAb species and mismatched variants was theoretically predicted. The relative mass signal intensities of the deconvoluted mass spectrum and the masses detected Based on the agreement, the results are shown in Table 3.

[0327] A wide range of valid heavy chain heterodimerization, heavy chain homodimerization, and half-molecules were detected. The desired degree of heterodimerization was high at >95%, and ideally close to 100%. To identify candidates for heterodimerization, bbmAb1, bbmAb2, and bbm Ab5, bbmAb4 and bbmAb3 were generated. Only bmAb5 exhibited >95% heterodimerization, whereas bbmAb4 and bbmAb3 exhibited <95% heterodimerization and were not suitable for therapeutic development.

[0328] [Table 23]

[0329] Example 3. Thermostability of CH2 and Fab domains of IL-13 / IL-18 bispecific antibodies Sexual evaluation The stability of an antibody greatly affects its performance (i.e., its specificity and affinity). Therefore, stability is crucial for the use of antibodies, especially in therapeutic, diagnostic and rapid analysis platforms. As its use increases, it becomes a major issue for researchers and manufacturers. -Thermal stability and melting temperature (T m ) Protein fusion Solution temperature (T m ) is defined as the temperature at which a protein denatures. m Value and Collapsed The percentage of soluble ions that are not present can predict the rate of aggregation (Robinson et al., 2018). .

[0330] Using the CFX96 Teal-Time PCR detection system (BioRad), The midpoint of the thermal transition was determined by differential scanning fluorimetry. Purified samples were diluted with 20 mM His Dilute to 0.3 mg / mL in a final volume of 43 μL in / His-HCl, pH 6.0, 7 1 μL SYPRO Orange dilution solution (1.4 μL SYPRO Orange stock solution) in 1 mL The thermocycler was set to a starting temperature of 20°C and a final temperature of 95°C and 0°C. The gradient rate was set at 0.5°C. BioRad CFX Manager Software Melting curves and thermal melting temperatures were obtained using re 3.1.

[0331] The melting temperatures of the CH2 and Fab domains of the modified antibodies are listed in Table 4. The melting curves of Ab1, bbmAb2, bbmAb5 and bbmAb4 are shown in FIG.

[0332] CH2 T m reflects the unfolding of the CH2 domain (Johns As shown in Table 4, all candidates have similar T m of showed.

[0333] Compared with the anti-13 Fab domain of bbmAb3 (64°C), bbmAb1, bbm The higher melting temperatures ( The CDR sequence in the anti-13 Fab domain of bbmAb3 was This result is for bbmAb1, bbmAb2, bbmAb5 and bbmAb6 because the columns are different from the other candidates. The specific CDR sequences in the anti-13 Fab domains of bbmAb4 and bbmAb5 improve the thermostability. This shows that this method can result in improved and more stable molecules.

[0334] [Table 24]

[0335] Example 4. Recombinant human and cynomolgus monkey IL-13 and IL-14 measured by SET Affinity for -18 The equilibrium dissociation constant (KD) was determined by solution equilibrium titration (SET) measurements, which are described as follows: The decision was reached.

[0336] Sample buffer (0.5% bovine serum albumin (BSA) and 0.02% Tween- 22 serial 2n dilutions of antigen (maximum concentration: hsIL-20) were made in PBS containing 20 18, 20 nM; cyIL-18, 40 nM; hsIL-13, 20 nM) were prepared and A fixed concentration of IL-13 was added (4 pM for hsIL-13 readout, 4 pM for hsIL-18 readout). (either 10 or 4 pM for the readout and 5 pM for the cyIL-18 readout) .

[0337] Add 60 μl / well of each antigen-antibody mixture to a 384-well polypropylene microplate. Duplicate aliquots were dispensed into a titer plate (MTP). Sample buffer was used as a negative control. A sample containing only the antibody served as a positive control (maximum electrochemiluminescence signal without antigen). Naru, B max Seal the plate and shake overnight (at least 16 h) at room temperature (RT). Incubated on a shaker.

[0338] The antigens and antibodies used are listed in Table 5.

[0339] [Table 25]

[0340] hsIL-18 and cyIL-18 reading: 50 μl / well of blocking buffer Blocked in PBS containing 5% BSA for at least 1 hour (h) at room temperature (RT). After the treatment or subsequent washing steps (TBST containing 0.05% Tween 20) TBS), followed by streptavidin Multi-Array® 384-well plate Platelet (MSD L21SA-5) in 30 μl / well of biotinylated human IL-18 (0 0.2 μg / ml in PBS) and incubated for at least 1 h at RT on a shaker. Incubated.

[0341] hsIL-13 reading: 50 μl / well of blocking buffer (containing 5% BSA) After blocking with PBS (containing PBS) for at least 1 hour (h) at room temperature (RT), After subsequent washing steps (TBST, TBS containing 0.05% Tween 20), Peptavidin Multi-Array® 384-well plate (MSD L 21SA-5) in 30 μl / well of biotinylated human IL-13 (0.2 μg / ml, P BS) and incubated on a shaker at RT for at least 1 h.

[0342] A volume of 30 μl / well of the equilibrated antigen-antibody mixture was dispensed from a polypropylene MTP. The plates were transferred to a coated MSD plate and incubated at RT for 20 min. After a washing step, 30 μl of sulfo-tagged anti-hsIgG diluted in sample buffer was added. Detection antibody (0.5 μg / ml) was added to each well and incubated for 30 min at RT on a shaker. Wash the MSD plate and add 35 μl / well of MSD read buffer. The solution was added and incubated at RT for 5 minutes. An electrochemiluminescence (ECL) signal was observed. , measured by MSD Sector Imager 6000.

[0343] Export SET data to Xlfit, an MS Excel add-in software. The mean ECL signal was calculated from duplicate measurements within each assay. The lowest value was subtracted from all data points and plotted against the corresponding antigen concentration. The baseline was adjusted by creating a titration curve. Fit the plot using a 1:1 binding model for the 1:1 bispecific Ab. By doing so, K D value was determined.

number

[0344] The obtained K D The values ​​are shown in Table 6.

[0345] [Table 26]

[0346] Example 5. Recombinant human and cynomolgus monkey IL-13 and IL-14 measured by SPR Affinity for -18 Optical biosensor Biacore™ T200 (http: / / www.cyt Surface plasmon resonance (SPR) measurements using ivalifesciences.com By this determination, the determination of kinetic binding parameters was achieved.

[0347] This technique allows the binding of ligands to receptors (k a , association rate constant) and dissociation ( k d This allows for label-free determination of the kinetic rate constants for the equilibrium dissociation constant (dissociation rate constant). number K D is calculated from the kinetic rate constants.

[0348] Immobilization buffer (10 mM sodium acetate) on the chip surface via amine coupling NeutrAvidin™ (Thermo Scientific) in PBS (pH 5.0) For indirect binding of antibodies by immobilization of 80 μg / mL of FITC #31000 1. Prepare the surface of a sensor chip (Cytiva #BR100535) followed by HB Biotinylated protein G (Sigma #P8045) in S-EP buffer, 5 μg / m Saturation of NeutrAvidin™ with L was performed.

[0349] Add blank buffer HBS-EP (0.01 M HEPES) to a final concentration of 10 µg / mL pH7.4, 0.15M NaCl, 3mM EDTA, 0.05%v / v surfactant Antibodies were diluted in P20 (Cytiva #BR100669). 13 (two-fold increasing concentrations from 0.25 to 8.0 nM) and recombinant huIL-18 (0.125 Determination of kinetic constants for bbmAb2 or bbmAb1 for two-fold increasing concentrations of ~32 nM Between cycles, affinity measurements were performed in 10 mM glycine, pH 1.5, 0.5% Tw The surface was regenerated using een 20.

[0350] The antigens and antibodies used are listed in Table 7.

[0351] [Table 27]

[0352] Biacore(TM) T200 Control Software v2.0.1 The full set of these traces at increasing concentrations is called a run. A set of traces was analyzed using the Biacore T200 Evaluation Software The 1:1 binding model provided by R max set global) and Include a zero concentration sample (blank) in each run to allow for double referencing. Including.

[0353] The obtained K D The values ​​are shown in Table 8.

[0354] [Table 28]

[0355] Example 6. Responses to human Fc receptors measured by surface plasmon resonance (SPR) spectroscopy Affinity Binding characteristics of engineered IL-13 / IL-18 bispecific antibodies to human Fc receptors To investigate the binding signature, we performed a direct binding assay using surface plasmon resonance (SPR) spectroscopy. SPR provides a continuous readout of complex formation and dissociation, allowing for the detection of complexes on the sensor chip surface. Proteins are coupled to the surface to allow for analysis of interactions between analytes in solution and ligands. Protein-protein, protein-peptide, protein-DNA and protein-small molecules It is a commonly applied technique for analyzing affinity and kinetics of interactions.

[0356] 1. In vitro Fcγ receptor binding assay The Fc fragment of bbmAb2 (IL1 8 knob YTE IL13 hole YTE1+1bsAb) The affinity of bbmAb to the cγ receptor was determined on a Biacore 8K instrument. 5 (bispecific KiH LALA YTE) and bbmAb1 (IL18 hole Y Fc flag of TE (mAb1) IL13 (mAb2) knob YTE1 + 1bsAb) The affinity of human Fcγ receptors binding to the marker was determined using 10 mM sodium acetate pH 7.0. Samples were diluted to 5 μg / ml in 4.5 and run on a Biacore T200 instrument using standard Applying a simple amine coupling procedure, a peak of approximately 750 resonance units was observed on a CM5 sensor chip. A similar procedure was applied on a Biacore 8K instrument, but the immobilization was The density was approximately 1310 resonance units. Group 1 was the immobilized blank and served as the reference. There are eight channels in total, and flow cell 1 of each channel is left blank and serves as a reference surface. The flow rate was 30 μl / min in the Biacore T200 or Human F was measured on all flow cells at 50 μl / min at 8 K and 25°C. cγ receptors (CD64 / FcγRI, CD32a / FcγRIIA) R131, CD32b / FcγRIIB, CD16a / FcγRIIIA V176 and FcγRIIIA F176 , CD1 Subsequent injections of a 1:2 dilution series of FcγRIIIB / FcγRIIIB (FcγRIIIB / FcγRIIIB) were used to obtain kinetic binding data. Depending on the strength of the interaction, different concentration ranges of running buffer (PBS Fcγ receptors were diluted in a 5% PBS solution (pH 7.4, containing 0.005% Tween-20) (Bi On acore T200, the present inventors measured FcγRI: 0.20 to 100 nM, FcγRIIA R131 , FcγRIIB and FcγRIIIB: 7.81 to 4000 nM, FcγRIIIA V176: 1.95-1000nM and FcγRIIIA F176 :3.91~ 2000 nM was used; tested from 0.05 to 20 nM on a Biacore 8K. The same conditions were applied except for FcγRI, which was used for the Biacore T200. In FcγRI and FcγRIIIA V176 After each measurement cycle, 30 μ The chip surface was regenerated using a 10 mM glycine pH 2.0 solution at 1 / min. On the acore 8K, hFcγRs were analyzed at a flow rate of 50 μl / min at pH 2. The surface was regenerated with a single injection of 10 mM glycine at 0 for 30 s. Double referencing was performed during data evaluation. A zero concentration sample (blank run) was measured to allow for accurate analysis of each sample and buffer. Duplicate injections of buffer blank were run across all sides. Biacore T20 0 evaluation software version 3.0 and Biacore 8K evaluation software (v. Data were evaluated using the software (3.0.12.15655).

[0357] Double-reference raw data, i.e., measure flow cell for response of reference flow cell The response was corrected and the response of a blank injection was subtracted in a second step. The resulting sensorgram was Fit using either a steady-state model or a 1:1 Langmuir model. , equilibrium dissociation constant (K D ) was calculated.

[0358] 2. In Vitro FcRn Receptor Binding Measurements bbmAb2 (IL18 (mAb1) knob Y TE IL13 (mAb2)hole YTE1+1bsAb) to Fc fragment Human FcRn receptor binding affinity determination was performed. bbmAb5 (bispecific KiH LA LA YTE) and bbmAb1 (IL18 hole YTE (mAb1) IL13 ( Human FcR binding to the Fc fragment of mAb2)knob YTE1+1bsAb n receptor affinity determination was performed on a Biacore 8K instrument. 10 mM sodium acetate Samples were diluted to 5 μg / ml in pH 4.5 and run on a Biacore T200 instrument. A standard amine coupling procedure was applied to obtain approximately 75% ATP on a CM5 sensor chip. The same procedure was applied on a Biacore 8K instrument, but the immobilization density was 0.0175 MHz. Immobilization was at a density of approximately 1310 resonance units on a Biacore T200. In this case, flow cell 1 was the immobilized blank and served as the reference. On the 8K, there are a total of 8 channels, and the flow cell of each channel is left blank. Then, a 1:2 dilution series of human FcRn receptor was injected to measure the chromatin density. Kinetic binding data were obtained on all flow cells at a flow rate of 50 μl / min and a temperature of 25°C. The data was collected. The FcRn receptor was analyzed to cover the concentration range of 4.88 to 2500 nM. was diluted in two different running buffers to check pH-dependent binding: 0. PBS pH 5.8 with 0.005% Tween-20 and 0.005% Tween-20 PBS pH 7.4 containing 0.005% Tween-20 The chip surface was regenerated using a flow rate of 50 μl / min for 120 s. A sample (blank run) can be measured to allow double referencing during data evaluation. Duplicate injections of each sample and buffer blank were run on all sides. Biacore T200 evaluation software version 3.0 and Biacore 8K Data were evaluated using evaluation software (v.3.0.12.15655).

[0359] The raw data is double-referenced, i.e., the response of the measurement flow cell is compared to the response of the reference flow cell. The response was corrected using a steady-state model or 1:1La. Fit the resulting sensorgram using one of the Ngmuir models and calculate the equilibrium Dissociation constant (K D ) was calculated.

[0360] 3. Measurement of C1q Binding in Vitro Affinity determination of human C1q binding to bbmAb2 on a Biacore T200 instrument Human C for bbmAb5 and bbmAb1 was performed on a Biacore 8K instrument. The affinity determination of 1q binding was performed. Samples were collected in 10 mM sodium acetate buffer, pH 4.5. The IgG was diluted to 50 μg / ml and analyzed using standard amine coupling assays on a Biacore T200 instrument. Applying a gelation procedure, immobilization was performed on a CM5 sensor chip at a density of approximately 8900 resonance units. A similar procedure was applied on a Biacore 8K instrument, but immobilization was approximately 940 On the Biacore T200, flow cell 1 had a density of 0.0 resonance units. A standardized blank was used as a reference. There are a total of 8 channels on the Biacore 8K. The flow cell of each channel was left blank to serve as a reference surface. A 1:2 dilution series of human C1q was run on all flow cells at a flow rate of 100 s and a temperature of 25 °C. Kinetic binding data were collected by post-injection of 0.49 nM to 250 nM. Human C1q was diluted in a range of concentrations in running buffer (HBS-EP + pH 7.4). After the measurement cycle, a 5 μL / min flow rate was performed for 30 s, including a 60 s stabilization period. The chip surface was regenerated using 0 mM NaOH. Zero concentration sample (blank run) was measured for each sample and buffer blank to allow for double referencing during data evaluation. Duplicate injections were run on all sides. Biacore T200 evaluation software bar Version 3.0 and Biacore 8K Evaluation Software (v.3.0.12.1565 5) was used to evaluate the data.

[0361] The raw data is double-referenced, i.e., the response of the measurement flow cell is compared to the response of the reference flow cell. The resulting sensorgrams were corrected by subtracting the response of a blank injection in a second step. The equilibrium dissociation constant (K D ) was calculated.

[0362] Results showing the binding affinity of Fcγ and FcRn receptors in vitro Binding affinities of bbmAb1, bbmAb2, and bbmAb5 to different Fc receptors are summarized in Table 9 below.

[0363] [Table 29]

[0364] Example 7. PK study of IL-13 / IL-18 bispecific antibodies PK studies conducted in hFcRn transgenic mice Derived from C57BL / 6 mice and purchased from Jackson Laboratory (USA). Tg276 B6.Cg-Fcgrttm1DcrTg(CAG-FCGRT Mouse experiments were performed using 276Dcr / DcrJ hemizygous mice. Rn(lineage 276) Tg mice express the mouse gene under the control of the ubiquitous CAG promoter. It carries a null mutation for the gene and a transgene expressing the hFcRn α-chain. All mice were naive males aged 8-12 weeks at the start of the study. Prepare the antibody in phosphate-buffered saline (1x PBS pH 7.3) at a dose volume of 5 mL / kg. The study was conducted in the left lateral tail vein at 10 mg / kg as a single intravenous dose. A serial sampling approach was used over a period of time (pre-dose, 1h, 1d, 2d, 3d, 6d, A total of three animal replicates were used for each antibody (9d, 17d, and 29d). A blood sample (30-50 μL) was collected in a serum separator tube and incubated at room temperature for 20 minutes. The samples were then processed and centrifuged (2000 g, room temperature, 1 Serum was obtained by centrifugation (0 min). The obtained serum was stored at -80°C until analysis.

[0365] Total antibodies in unknown mouse serum samples, calibration standards (Cs) and quality control samples (QC) For the measurement of the body mass index, a quantitative continuous electrochemiluminescence immunoassay (ECLIA) was used. The use of both targets for antibody capture and detection allows for the detection of these targets using their binding sites. The assay now allows detection of the total amount of bispecific therapeutic antibody. Biotinylated human IgG immobilized on a coated and blocked MSD plate The antibody was captured by IL-18. MSD Sulfo-TAG™ labeled cytochrome P450 (C14) sulfonate NHS ester Monkey IL-13 was added and allowed to bind for detection, which was achieved by electrochemiluminescence (ECL) analysis. ) and read on an ECL Sector Imager from MSD The obtained ECL values ​​are weighted by 1 / Y2 (Y denotes the ECL value of the corresponding calibration standard). Drugs present in the initial sample as determined through 5PL regression analysis using weighted coefficients is proportional to the amount of

[0366] Using the above experimental approach in a humanized mouse model, bbmAb1 and bbmAb 2 and Fc-silenced variants bbmAb6, bbmAb7, bbmAb8 and bbmAb7 and bbmAb8 were tested. bbmAb6 is an Fc-silenced variant of bbmAb1 containing the Fc-reactive nucleotides (numbering). , bbmA containing the L235C mutation and not the YTE mutation (EU numbering) bbmAb9 is an Fc-silenced mutant of b1. Fc-silenced variants of bbmAb1 containing the mutations (EU numbering).

[0367] All the above mentioned antibodies showed C max High, typical of antibodies with extended half-lives Good exposure and typical PK curve in the systemic circulation with a short distribution phase and a long elimination phase The half-lives of the antibodies examined herein were very similar, with almost This can be estimated from the terminal phase of the PK curve, which ranges from approximately 10 to 16 days (as shown in Figure 7).

[0368] Example 7 Simultaneous Inhibition of IL-13 / IL-18 Human peripheral blood mononuclear cells (pBMC) or human keratinocytes were treated with IL-18 or I, respectively. The cells were treated with IL-13 and analyzed for differences in gene expression. The overlap between the gene signature and the IL-18-induced gene signature was Both of these are observed only slightly in AD patients (data not shown). From the current dataset, there are elevated levels in published lesional skin compared to non-lesional skin. It has been shown that the 5 patients with AD (lesioned and non-lesioned) and 7 patients with AD (He et al. 2020) Skin biopsies from 1 HV subject; scRNA-seq data).

[0369] Antagonizes IL-4 receptor alpha (anti-IL-4Rα), thus inhibiting IL-13 and I Lesioned and non-lesioned skin tissue from AD patients treated with an antibody that blocks both L-4 signaling By mining the dataset in which the test samples were collected, genes induced by IL-13 were identified. The signature was significantly downregulated by anti-IL-4Rα treatment; however, it was downregulated by IL-18. The induced gene signature was not significantly downregulated by anti-IL-4Rα treatment This was revealed (data not shown).

[0370] These results suggest that the inhibition of IL-13 and IL-18 is more potent than that of IL-13 or IL-18 alone. It is suggested that dual inhibition of both IL-18 may improve treatment outcomes.

[0371] Four-mm skin biopsies were collected from 10 patients with atopic dermatitis and 8 healthy mullets. Four-mm skin biopsies from volunteers were collected as controls. Each biopsy was cut into four pieces. / -α-IL-18, α-IL-13 or both in 100 μl of medium The cells were cultured ex vivo for 24 hours at 150 μg / mL each. The cell culture supernatant was centrifuged at low speed. The Olink assay was used to measure the protein Changes in expression of proteins in the supernatants of lesion biopsy samples compared to control samples were assessed. There was a measurable decrease in protein expression in samples treated with anti-IL-13 compared to controls. and showed a decrease in anti-IL-18 treated samples compared to the control, and in samples treated with anti-IL-18 / anti-IL-13 compared to single-treated samples showed a further decrease in

[0372] Example 8 Simultaneous inhibition of IL-13 / IL-18 with bispecific antibodies inhibits AD-like transcription. Revealing synergistic inhibition of the rhiptone method Obtain 8 mm full thickness skin biopsies from surgical waste from five individual donors. 1% Pen / Strep and 10% NaCl in tissue culture inserts for 2-well plates On day 0, the biopsies were cultured in IMDM medium containing a control (30 μL) serum replacement. or in 30 μL of PBS at a final concentration of 500 mg each. Activation with a mixture of anti-CD28 and anti-CD3 antibodies. To induce proliferation, biopsies (except for the control) were incubated in IMDM medium with the following cytokines: Itokine 50ng / mL: IL-4, IL-13, IL-33, TSLP, IL18 and The cells were incubated with a mixture of IL-31 and IL-31 for 6 days, with medium changes on days 2, 4, and 5. During the 6-day induction period, 1 μM Ig containing the LALA silencing mutation was administered. G1 isotype control antibody (AD+isotype); 1 μM Anti-IL13 antibody; 1 μM Biopsy induced with anti-IL18 antibody or 1 μM anti-IL13 / IL18 bispecific antibody bbmAb1 Samples were processed. On day 6, supernatants were collected and subjected to histological and transcriptomic analysis. Biopsies were aliquoted for Ampliseq whole transcriptome protocol. .

[0373] Ampliseq normalized values ​​in Qlucore Omics Explorer 3.8 and remove variables with values ​​less than 0.5 if they apply to 90% of the samples. This resulted in 16719 variables. The threshold was set to 0.25 and the values ​​were log2 Transformed. "AD isotype" with q<0.1 and FC>2 in donor-corrected samples Samples were compared with controls to generate a "disease transcriptome," resulting in 14 85 differentially expressed "disease genes" were obtained, 507 of which were associated with AD. In this gene set, the gene set variants were upregulated in the isotype sample. A variance analysis (GSVA) ​​was performed in unimodal mode.

[0374] result As shown in Figure 8, GSVA produced ineffective inhibition of all anti-IL18 treated samples. Anti-IL13 blockade was partially effective in two of five samples. On the other hand, the anti-IL18 / IL13 bispecific antibody bbmAb1 inhibited the expression of 5 samples. Four of the four showed robust suppression of the AD-like disease transcriptome, suggesting that the A synergistic effect for combined cytokine blockade is shown.

[0375] As shown in Figure 9, t-SNE analysis of 507 up-regulated genes (per Complexity 5) is the ratio of the control and AD+ isotype samples that are furthest apart on the x-axis. The anti-IL13 / IL18 bispecific antibody bbmAb1 showed a clear disease response. Four of the five samples treated with α-glucan clustered near the control sample, and this Therefore, cells from these samples showed minimal AD-like transcriptomes in the induced cells. In contrast, only two anti-IL13 treated samples showed the same pattern. showed a similar effect, with anti-IL18 treated samples indistinguishable from AD+ isotype samples. These results show that there is no therapeutic effect. t-SNE analysis of differentially expressed genes (AD+ isotype vs. control) However, Figure 10 shows that blocking IL-18 alone Without wishing to be bound by theory, the inventors 8 to 10, the data in Figures 8 to 10 show that IL-18 blockade (e.g., using anti-IL18 antibodies) Although it has been suggested that simultaneous IL13 / 18 blockade (e.g. , by simultaneous or sequential administration of IL-13 and IL-18 antagonists) We hypothesized that this treatment may be unexpectedly superior compared to blockade of IL-3 or IL-18. There are.

[0376] Example 9. Simultaneous inhibition of IL-13 / IL-18 with bispecific antibodies In a test assay, a carrier comprising the bispecific antibody disclosed herein and a plurality of cells IL-13 and IL-18 activity was assayed and multiple cells were contacted with the carrier alone. The test assays are compared to control assays in which the test cells are exposed to IL-13 and IL-14. Both -18 activity and ATP are significantly reduced.

[0377] Example 10. Treatment of atopic dermatitis with anti-IL-13 / IL-18 bispecific antibodies A subject with atopic dermatitis is administered a bispecific antibody described herein. At week 1, subjects had a significantly lower incidence of one or more symptoms of atopic dermatitis and / or eczema compared to placebo-treated subjects. or achieve greater relief of symptoms.

Claims

1. A pharmaceutical composition for use in the prevention or treatment of inflammatory or immune diseases or disorders in subjects requiring the same, The pharmaceutical composition comprises a multispecific antibody in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers. The aforementioned antibody a. A first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically bind to interleukin-18 (IL-18), wherein the VL1 domain includes sequence number 13 and the VH1 domain includes sequence number 41, b. A second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically bind to interleukin-13 (IL-13), wherein the VL2 domain includes SEQ ID NO: 27 and the VH2 domain includes SEQ ID NO:

55. The pharmaceutical composition comprising the above.

2. The pharmaceutical composition according to claim 1, wherein the antibody is a bispecific antibody.

3. The pharmaceutical composition according to claim 1, wherein the inflammatory or immune disease or disorder is related to dysregulation of IL-13 and / or IL-18.

4. The pharmaceutical composition according to claim 1, wherein the inflammatory or immune disease or disorder is a skin disease or disorder.

5. The pharmaceutical composition according to claim 1, wherein the inflammatory or immune disease or disorder is atopic dermatitis.

6. The pharmaceutical composition according to claim 5, wherein the atopic dermatitis is moderate to severe atopic dermatitis.

7. The pharmaceutical composition according to claim 6, wherein the atopic dermatitis is not adequately controlled with topical corticosteroids.

8. The pharmaceutical composition according to claim 1, wherein a therapeutically effective amount of the multispecific antibody is administered to the subject.

9. The pharmaceutical composition according to claim 1, wherein the treatment is an improved treatment compared to treatment with a single-specific anti-IL-18 antagonist or a single-specific anti-IL-13 antagonist.

10. The pharmaceutical composition according to claim 1, wherein the multispecific antibody comprises a first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, and a second light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

28.

11. The pharmaceutical composition according to claim 10, wherein the multispecific antibody comprises a first heavy chain containing a heterodimerization modification and a second heavy chain containing a heterodimerization modification complementary to the heterodimerization modification of the first heavy chain.

12. The pharmaceutical composition according to claim 11, wherein the first and second heavy chains are human IgG1, and: a) the heterodimerization modification of the first heavy chain comprises cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises tryptophan at position 366 and cysteine ​​at position 354; or b) the heterodimerization modification of the second heavy chain comprises cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises tryptophan at position 366 and cysteine ​​at position 354, and the amino acid residues are numbered according to EU numbering.

13. The pharmaceutical composition according to claim 12, wherein the multispecific antibody is a bispecific antibody containing a mutation that enhances the half-life of the bispecific antibody via enhanced FcRn binding.

14. The pharmaceutical composition according to claim 13, wherein the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E (YTE), and the amino acid residues are numbered according to EU numbering.

15. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises one or more additional activators.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the multispecific antibody comprises a first heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 42, a first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, a second heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, and a second light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

28.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the multispecific antibody comprises a first heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 57, a first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, a second heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 58, and a second light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

28.

18. Use of a pharmaceutical composition in the manufacture of an agent for the prevention or treatment of an inflammatory or immune disease or disorder in a subject requiring the use thereof, wherein the pharmaceutical composition comprises a multispecific antibody in combination with one or more pharmaceutically acceptable excipients, diluents or carriers, the antibody comprising: a. a first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically bind to interleukin-18 (IL-18), wherein the VL1 domain comprises SEQ ID NO: 13 and the VH1 domain comprises SEQ ID NO: 41; b. a second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically bind to interleukin-13 (IL-13), wherein the VL2 domain comprises SEQ ID NO: 27 and the VH2 domain comprises SEQ ID NO: 55; the use thereof.

19. The use according to claim 18, wherein the multispecific antibody is a bispecific antibody.

20. The use according to claim 18, wherein the inflammatory or immune disease or disorder is related to dysregulation of IL-13 and / or IL-18.

21. The use according to claim 18, wherein the inflammatory or immune disease or disorder is a skin disease or disorder.

22. The use according to claim 18, wherein the inflammatory or immune disease or disorder is atopic dermatitis.

23. The use according to claim 22, wherein the atopic dermatitis is moderate to severe atopic dermatitis.

24. The use according to claim 23, wherein the atopic dermatitis is not adequately controlled with topical corticosteroids.

25. The use according to claim 18, wherein the treatment is an improved treatment compared to treatment with a single-specific anti-IL-18 antagonist or a single-specific anti-IL-13 antagonist.

26. The use according to any one of claims 18 to 25, wherein the multispecific antibody comprises a first heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 42, a first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, a second heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, and a second light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

28.

27. ​​The use according to any one of claims 18 to 25, wherein the multispecific antibody comprises a first heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 57, a first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, a second heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 58, and a second light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 28.

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