Multispecific antibodies targeting IL-13 and IL-18

Abstract

Multispecific antibodies targeting IL-13 and IL-18 are described herein. The multispecific antibodies can be antagonists and / or therapeutic antibodies targeting IL-13 and IL-18. Methods for making the multispecific antibodies, methods for simultaneously inhibiting IL-13 and IL-18 with the multispecific antibodies, and methods for treating IL-13 / IL-18-mediated disorders such as atopic dermatitis by administering the multispecific antibodies described herein are also described herein.

Description

[Technical Field]

[0001] The present invention relates to the field of immunology. In particular, the present invention relates to multispecific antibodies that target interleukin-13 (IL-13) and interleukin-18 (IL-18), and methods for making and using the same. [Background technology]

[0002] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease characterized by symptoms including intense pruritus (e.g., severe itching) and scaly, dry eczema lesions. Severe disease can be extremely disabling due to significant psychological problems, significant sleep deprivation, and reduced quality of life, leading to high socioeconomic costs. The pathophysiology of AD is influenced by immunoglobulin E (IgE)-mediated sensitization and the complex interplay between the immune system and environmental factors. The primary skin abnormality may be an immunological derangement that causes IgE-mediated sensitization, and epithelial barrier dysfunction is the result of both genetic mutations and local inflammation. AD often begins in childhood before the age of 5 and can persist into adulthood.

[0003] Typical treatments for AD include topical lotions and moisturizers, topical corticosteroid ointments, creams, or injections. However, most treatment options provide only temporary and incomplete symptom relief. Furthermore, many patients with moderate to severe AD become 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.

[0004] The pathogenesis of AD is multifactorial, with immune-mediated mechanisms characterized by inappropriate activation of type 2 T helper cells (Th2) and type 2 innate lymphoid cells (ILC2), accompanied by increased expression of proinflammatory cytokines, particularly interleukins IL-4 and IL-13 (Moyle et al. (2019) Exp Dermatol. 28(7):756-768; Roediger et al. (2013) Nat Immunol. 14(6):564-573). IL-13 stands out as one of the key cytokines in AD pathophysiology through its prominent role in inflammatory processes and the development and maintenance of epidermal barrier dysfunction (Tsoi et al. (2019) J Invest Dermatol. 139(7):1480-1489).

[0005] Currently, the anti-IL4Ra antibody dupilumab is approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of moderate to severe forms of AD. Antibodies that specifically target IL-13, such as lebrikizumab and tralokinumab, are also in development.

[0006] IL-18 is thought to be involved in the pathogenesis of AD because it induces super Th1 cells that produce and secrete IFN-γ and IL-13 (Terada et al. (2006) Proc Natl Acad Sci USA. 103:8816-8821). IL-18 is released by keratinocytes and inflammatory dendritic cells, and serum IL-18 levels in AD patients have been shown to significantly correlate with the skin score of AD lesions (Ikezawa et al. (2010) Allergy, Asthma & Immunology Research 2(4):235-246). In vivo administration of IL-18 also induces Th2 differentiation and increases IgE production in a CD4+ T cell-, IL-4-, and STAT6-dependent manner in mice (Yoshimoto et al. (2000) Nat Immunol 1:132-137; Hoshino et al. (2000) Eur J Immunol 30:1998-2006).

[0007] Because IL-13 and IL-18 are pro-inflammatory cytokines that affect many different cell types associated with AD, there remains a need for effective treatments that achieve simultaneous blockade of IL-13 and IL-18 signaling. Multispecific antibodies (e.g., bispecific antibodies) that target both IL-13 and IL-18 may address an unmet medical need in this chronic inflammatory disease.

[0008] One of the most common problems in producing bispecific IgG (BsIgG) by coexpressing two different antibodies is unwanted homodimerization of component heavy chains and unwanted pairing of component light chains with incorrect heavy chains. Figure 1 shows possible misassembled products. To overcome this heavy chain homodimerization problem, heavy chains can be remodeled for heterodimerization using engineered disulfide bonds in combination with previously identified "knob-into-hole" mutations. One mutant, S354C:T366W / Y349'C:T366'S:L368'A:Y407V, can result in nearly quantitative (approximately 95%) heterodimerization (Merchant et al., 1998). However, this nearly quantitative heterodimerization does not solve the problem of light chain pairing. Therefore, random light chain pairing is assumed, and only 25% of the antibodies produced are of the desired bispecificity. There remains a need to further improve both heavy chain heterodimerization and light chain pairing and thereby increase the purity, yield and quality of bispecific antibodies. Summary of the Invention

[0009] The present disclosure provides methods of treating an autoimmune or inflammatory disorder, the methods comprising administering an inhibitor of IL13 and an inhibitor of IL18 simultaneously or sequentially. Without wishing to be bound by theory, the inventors hypothesize that simultaneous blockade of IL13 and IL18 unexpectedly results in superior efficacy in treating autoimmune or inflammatory disorders compared to blockade of IL-13 or IL-18 alone. In some cases, simultaneous blockade involves administration of an IL13 antagonist and an IL-18 antagonist. In some cases, simultaneous blockade involves administration of an antagonist that inhibits both IL-13 and IL-18, such as a multispecific (e.g., bispecific) antibody that binds both IL-18 and IL-13.

[0010] The present invention provides multispecific antibodies or fragments thereof that target both IL-13 and IL-18 for the treatment of AD, ensuring sufficient overall yield, purity, and product quality to proceed with clinical development and commercial manufacturing at a reasonable cost.

[0011] In some embodiments, the multispecific antibody is a bispecific antibody. Exemplary bispecific antibodies are characterized by a) a first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically bind interleukin-18 (IL-18), and b) a second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically bind interleukin-13 (IL-13).

[0012] In some embodiments, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof is a human or humanized (e.g., CDR-grafted) IgG (e.g., IgG1, IgG2, IgG3, or IgG4) antibody. In some cases, the engineered multispecific antibody (e.g., bispecific antibody) is a human or humanized (e.g., CDR-grafted) IgG1 antibody.

[0013] Undesirable Fc interactions with Fcγ receptors and the complement receptor C1q can be separated from binding to the neonatal Fc receptor (FcRn), which can enhance serum persistence. The in vivo serum persistence conferred by FcRn has been shown to be a tunable property that can be adjusted by mutations in the IgG Fc. Improving Fc affinity to FcRn in endosomal conditions (acidic pH) through Fc modifications is an effective approach to prolonging the pharmacokinetics of monoclonal antibodies (Maeda, 2017). The YTE mutation set (M252Y, S254T, T256E according to EU numbering) or the LS mutation set (M428L, N434S according to EU numbering) in the Fc CH2 domain are examples of such mutation sets developed.

[0014] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises M252Y / S254T / T256E (YTE) according to EU numbering. In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises M428L, N434S (LS) according to EU numbering.

[0015] Chain-pairing mutations have been shown to be efficient in promoting chain heterodimerization by introducing complementarity at the CH3-CH3 interface of bispecific or multispecific antibodies. Many sets of chain-pairing mutations are used in the generation of multispecific antibodies: side-chain volume increase / decrease (T366W / S354C-T366S / L368A / Y407V / Y349C, knob-into-hole) (Ridgway, 1996), charge reversal (K409D / K392D-D399K / E356K, electrostatic steering) (Gunasekaran, 2010), or multiple IgA substitutions (SEEDbody) (Davis, 2010).

[0016] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises chain-pairing amino acid substitutions, e.g., in combination with silencing and / or half-life extending mutations. In some cases, the chain-pairing amino acid substitutions are knobs-into-holes (KiH) mutations, for example, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a first constant heavy chain with the amino acid substitution T366W and a second constant heavy chain with the amino acid substitutions T366S, L368A, and Y407V, where the amino acid residues are numbered according to EU numbering.

[0017] In another embodiment, the chain-paired amino acid substitutions are knobs-into-holes (KiH) mutations, comprising a first constant heavy chain with amino acid substitutions S354C and T366W and a second constant heavy chain with amino acid substitutions Y349C, T366S, L368A and Y407V, wherein the amino acid residues are numbered according to EU numbering.

[0018] In a further embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises both T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering.

[0019] Without being bound by theory, in some embodiments, a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprises one or more mutations in hFc to, for example, suppress ADCC and / or CDC effector function. Various mutation sets have been described in the art, for example, as LALA (L234A, L235A according to EU numbering) (Wines et al., 2000) or DAPA (D265A, P329A according to EU numbering) (Genentech, U.S. Pat. No. 6,737,056). Some researchers have adopted a cross-subclass approach to reduce effector function. A further refinement of the cross-subclass approach involved generating IgG2 variants with point mutations from IgG4 (i.e., H268Q, V309L, A330S, P331S according to EU numbering) (An et al., 2009). Another silent IgG1 antibody contains the N297A mutation, resulting in an aglycosylated / aglycosylated antibody (Strohl et al., 2009). Some mutation sets used combine previously described techniques to achieve higher levels of silencing, up to complete loss of some or all effector functions. DANAPA is one example (D265A, N297A, P329A) (WO 2019068632 Janssen). Other alternative approaches have been reported for engineering or mutating key residues in the Fc region responsible for effector function. See, e.g., PCT Publication No. WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), U.S. Patent Application Publication No. 2006 / 0134709 (Macrogenics), U.S. Patent No. 6,737,056 (Genentech), U.S. Patent Application Publication No. 2010 / 0166740 (Roche).

[0020] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises D265A / P329A (DAPA), where the amino acid residues are numbered according to EU numbering.

[0021] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234A / L235A (LALA), where the amino acid residues are numbered according to EU numbering.

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

[0023] In further embodiments, the one or more cysteine ​​substitutions of the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof are selected from positions 234, 235, and 236. In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) comprises a cysteine ​​substitution at position 234. In another embodiment, the engineered multispecific antibody (e.g., bispecific antibody) comprises a cysteine ​​substitution at position 235. In another embodiment, the engineered multispecific antibody (e.g., bispecific antibody) comprises a cysteine ​​substitution at position 236.

[0024] In some embodiments, the engineered multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprise one or more amino acid substitutions that reduce Fc effector function and enhance the half-life of the engineered multispecific antibodies (e.g., bispecific antibodies) or fragments thereof through enhanced FcRn binding and / or one or more amino acid substitutions that promote proper chain pairing.

[0025] Thus, for example, in some embodiments, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises half-life extending mutations selected from the group consisting of T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and YTE (M252Y, S254T, T256E) and LS (M428L, N434S) according to EU numbering. In some embodiments, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises Fc silencing mutations selected from the group consisting of T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and LALA (L234A, L235A), DAPA (D265A, P329A), and N297 according to EU numbering. In some embodiments, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises half-life extending mutations selected from the group consisting of T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), YTE (M252Y, S254T, T256E) and LS (M428L, N434S) according to EU numbering, and Fc silencing mutations selected from the group consisting of LALA (L234A, L235A), DAPA (D265A, P329A) and N297.

[0026] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234A / L235A (LALA) and M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering.

[0027] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234C and M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering. In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L235C and M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering. In another embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises G236C and M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering.

[0028] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234A / L235A (LALA), M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), where the amino acid residues are numbered according to EU numbering.

[0029] In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), where the amino acid residues are numbered according to EU numbering. In one embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L235C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), where the amino acid residues are numbered according to EU numbering. In another embodiment, the modified multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises G236C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), where the amino acid residues are numbered according to EU numbering.

[0030] In some embodiments, the VH1 and VH2 domains of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprise complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3, and the VL1 and VL2 domains comprise LCDR1, LCDR2, LCDR3; a. The VH1 domain comprises (e.g., in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 32, the HCDR2 having 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 HCDR2 having 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 HCDR2 having the amino acid sequence SEQ ID NO: 39, 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 LCDR2 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 LCDR2 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 LCDR2 having the amino acid sequence SEQ ID NO: 11, 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 HCDR2 having 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 HCDR2 having 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 HCDR2 having the amino acid sequence SEQ ID NO: 53, 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 LCDR2 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 LCDR2 having 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 LCDR2 having the amino acid sequence SEQ ID NO:25, 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.

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

[0033] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof 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:27.

[0034] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a VH1 domain comprising the amino acid sequence SEQ ID NO:41, a VL1 domain comprising the amino acid sequence SEQ ID NO:13, a VH2 domain comprising the amino acid sequence SEQ ID NO:55, and a VL2 domain comprising the amino acid sequence SEQ ID NO:27.

[0035] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 28.

[0036] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprises a first heavy chain that comprises a heterodimerization modification and a second heavy chain that comprises a heterodimerization modification that is complementary to the heterodimerization modification of the first heavy chain.

[0037] In some embodiments, the first and second constant heavy chains are human IgG1s that include heterodimerization modifications, and a) the heterodimerization modification of the first immunoglobulin heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises a tryptophan at position 366; or b) the heterodimerization modification of the second immunoglobulin heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises a tryptophan at position 366; The amino acid residues are numbered according to the EU numbering system.

[0038] In some embodiments, the multispecific antibody is a bispecific antibody comprising a mutation that enhances the half-life of the bispecific antibody through enhanced FcRn binding.

[0039] In some embodiments, the mutations that enhance the half-life of the bispecific antibody are M252Y / S254T / T256E (YTE), where the amino acid residues are numbered according to EU numbering.

[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 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.

[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 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.

[0044] Also disclosed herein are pharmaceutical compositions comprising a multispecific antibody (e.g., a bispecific antibody) of the present disclosure, or a fragment thereof, in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[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] Also disclosed herein are cloning or expression vectors comprising one or more nucleic acid sequences as outlined above, which vectors 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 herein, where the first vector encodes a full-length heavy chain comprising a constant domain and a variable domain and a full-length light chain comprising a constant domain and a variable domain, and the heavy and light chains encoded by the first vector can be combined to form the anti-IL-18 arm of a bispecific IgG antibody, and the second vector encodes a full-length heavy chain comprising a constant domain and a variable domain and a full-length light chain comprising a constant domain and a variable domain, and the heavy and light chains encoded by the second vector can be combined to form the anti-IL-13 arm of a bispecific IgG antibody as described herein. In some embodiments, the first and second vectors are expression vectors, and co-expression of the first and second vectors in a common host cell provides an anti-IL-18 / IL-13 bispecific IgG-like antibody with high yield, purity, and activity.

[0048] Also disclosed herein are host cells containing one or more cloning or expression vectors as outlined above.

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

[0050] Also disclosed herein are kits that include one or more cloning and / or expression vectors of the present disclosure, which kits further include instructions for making the multispecific (e.g., bispecific) antibodies disclosed herein.

[0051] Also disclosed herein are kits comprising the multispecific antibodies of the present disclosure or the pharmaceutical compositions of the present disclosure, which kits additionally comprise instructions for use and a means for administering the multispecific antibodies or pharmaceutical compositions to a subject in need thereof.

[0052] In some embodiments, the means for administration comprises a syringe, an autoinjector, an injection pen, a vial and syringe, an infusion pump, a patch, or an infusion bag and needle.

[0053] Also disclosed herein are methods for simultaneously binding IL-13 and IL-18, comprising contacting IL-13 and IL-18 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 is performed in a subject, such as a human patient, in need of IL-13 and IL-18 inhibition.

[0054] Also disclosed herein are methods for simultaneously inhibiting the activity of IL-13 and IL-18, comprising contacting a plurality of mammalian cells with an effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. In some cases, the contacting is performed in in vitro or ex vivo culture. In some cases, the contacting is performed in a non-human animal, such as a non-human primate. In some cases, the contacting is performed in a patient in need of IL-18 and IL-13 inhibition, such as a patient with atopic dermatitis. In some cases, IL-13 and IL-18 activity is reduced by at least 10%, at least 25%, at least 50%, at least 75%, or at least 90%. In some cases, the reduction in IL-13 activity is measured by a reduction in STAT-6 signaling. In some cases, the reduction in IL-18 activity is measured by a reduction in IFNγ production, such as a reduction in IFNγ production induced by LPS / IL-12. In some cases, decreased IL-18 and / or IL-13 activity is measured by an increase in the level of IL-18 or IL-13, respectively, bound to a multispecific antibody described herein or a decrease in the level of free IL-18 or IL-13, respectively.

[0055] Also disclosed herein are methods of simultaneously inhibiting the activity of IL-13 and IL-18 in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. Without wishing to be bound by theory, the inventors hypothesize that simultaneous blockade of IL-13 and IL-18 may have complementary (e.g., synergistic) effects compared to blockade of IL-18 or compared to blockade of IL-13. In some embodiments, simultaneous blockade of IL-13 and IL-18 may have complementary (e.g., synergistic) effects compared to blockade of IL-18.

[0056] Also disclosed herein are methods of treating an IL-13- and / or IL-18-mediated disorder in a subject, the methods comprising administering to the subject a therapeutically effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure. In some embodiments, the method is improved treatment compared to treatment with a monospecific anti-IL-13 antagonist. In some embodiments, the method is improved treatment compared to treatment with a monospecific anti-IL-18 antagonist. In some embodiments, the method is improved treatment compared to treatment with an anti-IL-13 or anti-IL-18 antagonist. In some embodiments, the improvement is indicated by a better Eczema Area and Severity Index (EASI) score, a better Physician's Global Assessment (IGA) score, a better Pruritus Numerical Rating Scale score, and / or a better Dermatology-Related Quality of Life Index score after 16, 24, 36, or 52 weeks of treatment. In some embodiments, the method is an improved treatment compared to treatment with an anti-IL-13 or anti-IL-18 antagonist, where the improvement is indicated by a lower Scoring of Severity of Atopic Dermatitis (SCORAD) score after 16, 24, 36, or 52 weeks of treatment.

[0057] In some embodiments, the methods of the disclosure reduce the expression level of one or more AD-associated biomarkers, particularly one or more AD-associated biomarkers selected from the list consisting of CCL17 / TARC, IgE (e.g., serum IgE), CCL26 / Eotaxin-3, CCL22 / MDC, hsCRP, 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-18BP), compared to the level before simultaneous blockade of IL13 and IL18 (e.g., treatment with a multispecific antibody (e.g., bispecific antibody) or fragments thereof).

[0058] Also disclosed herein are methods for inhibiting IgE antibody production in a subject, the method comprising simultaneously or sequentially inhibiting IL13 and IL18 (e.g., by administering to the subject an effective amount of a multispecific antibody of the present disclosure).

[0059] Also disclosed herein are methods of inhibiting IFN-γ production in a subject, the method comprising simultaneously or sequentially inhibiting IL13 and IL18 (e.g., by administering to the subject an effective amount of a multispecific antibody of the present disclosure).

[0060] Also disclosed herein are methods of treating and / or preventing an inflammatory or immune condition, comprising simultaneously or sequentially inhibiting IL13 and IL18 (e.g., by administering a therapeutically effective amount of a multispecific antibody of the present disclosure to a subject in need of such treatment and / or prevention). In preferred embodiments, the inflammatory or immune condition is a skin condition. In preferred embodiments, the skin condition is atopic dermatitis. In some embodiments, the atopic dermatitis is moderate to severe atopic dermatitis. In some cases, the atopic dermatitis is moderate to severe atopic dermatitis. In some cases, the atopic dermatitis is moderate to severe as determined by the Rajka / Langeland scale score, where the Rajka / Langeland scale score is determined to be 4.5 to 9. In some embodiments, the method further comprises administering one or more topical corticosteroids. In some embodiments, the atopic dermatitis is inadequately controlled by administration of one or more topical corticosteroids.

[0061] In a further aspect, disclosed herein is the use of a multispecific antibody (e.g., a bispecific antibody) of the present disclosure, or a fragment thereof, in the treatment and / or prevention of AD.

[0062] In a further aspect, disclosed herein is the use of a multispecific antibody (e.g., a bispecific antibody) of the present disclosure, or a fragment thereof, for the manufacture of a medicament for the treatment and / or prevention of AD.

[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 this disclosure may be more readily understood, certain terms are specifically defined throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0066] definition Interleukin (IL)-18 (referred to herein simply as "IL-18") is primarily produced by macrophages and T cells as a precursor protein (pro-IL-18) and secreted as an active protein after cleavage by caspase-1 (Dinarello CA et al (1999) J Allergy Clin Immunol;103:11-24). Under normal physiological conditions, IL-18, in synergy with IL-12, is associated with the induction of cell-mediated immunity after infection with microbial products such as lipopolysaccharide (LPS) (Sareneva T et al (2000) J Immunol;165(4):1933-8). Following stimulation with IL-18, natural killer (NK) cells and T cells release interferon gamma (IFN-γ), a cytokine that plays an important role in the activation of macrophages and other cells. In addition to its ability to induce interferon gamma, IL-18 also has various functions. These biological properties include activation of NF-κB, Fas ligand expression, induction of both CC and CXC chemokines, and enhanced production of competent human immunodeficiency virus.

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

[0068] Interleukin (IL)-13 (referred to herein simply as "IL-13") is a pleiotropic cytokine produced primarily by Th2 cells and ILC2s, but also to a lesser extent by mast cells, basophils, eosinophils, natural killer cells, macrophages, dendritic cells, and monocytes. Free IL-13 binds to the a1 subunit of the IL-13 receptor (IL-13Ra1) on all cells of the human body, but particularly on monocytes and B cells. In a cascade reaction, this binding supports the recruitment of IL-4Ra, which by dimerization induces the formation of signal transducer and activator of transcription 6 (STAT6), which activates Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), leading to phosphorylation of signal transducer and activator of transcription 6 (STAT6), a transcription factor that promotes TH2 differentiation, and class switching to IgE (Silverberg et al (2017) Dermatol Clin. 35(3):327-334; Goenka et al (2011) Immunol Res. 50(1):87-96). The term "IL-13" is synonymous with IL-13 polypeptide or interleukin-13 polypeptide.

[0069] In all cases, the terms "comprise," "comprises," "comprising," and the like are used in reference to a sequence (e.g., an amino acid sequence), and it should be understood that the sequence may also be qualified by terms such as "consist," "consists," "consisting of," and the like. As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agent included in a method or composition, as well as any excipients that are inert for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the multispecific antibody of the present disclosure. In some embodiments, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the multispecific antibody of the present disclosure and a second co-administered agent.

[0070] The term "antibody," as used herein, refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that is capable of non-covalently, reversibly, and specifically binding to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. 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. Each light chain is composed of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (abbreviated herein as CL). The VH and VL regions can be further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, which is sometimes referred to herein as the antigen-binding domain. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

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

[0072] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is recognized that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy chains (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental transport, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distant from the antigen-binding site or amino terminus of the antibody. 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 portions of the constant domain of an antibody, such as the fragment crystallizable (Fc), constant (C) domain, etc. In some other embodiments, these portions are antigen-binding fragments that retain the ability to non-covalently, reversibly, and specifically bind to an antigen, and may be referred to herein as an antigen-binding domain. The phrase "antigen-binding fragment," as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) with an epitope of the antigen. 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 fragment, F(ab')2 fragment, monovalent fragment consisting of the VL, VH, CL, and CH1 domains; bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; V HThese include, but are not limited to, dAb fragments consisting of domains (Ward et al., (1989) Nature, 341:544-546); and isolated complementarity determining regions (CDRs) or other epitope-binding fragments of antibodies.

[0074] As used herein, "Fc" or "Fc region" includes CH2 and CH3, and optionally any portion of an antibody hinge region. The Fc region is composed of two polypeptide chains that dimerize to form the Fc region. Each half antibody of the present disclosure contains one Fc polypeptide chain; for example, a half antibody having an IL-18 scFv includes the IL-18 scFv linked to an Fc polypeptide chain. This half antibody can pair with another half antibody, resulting in the two Fc polypeptide chains dimerizing into the Fc region of the multispecific antibody of the present disclosure. Like all polypeptide chains, the Fc polypeptide chain contains an N-terminus and a C-terminus, each of which can be linked to an antigen-binding domain (e.g., an IL-18-binding domain or an IL-13-binding domain).

[0075] Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).

[0076] Antibody fragments can be assembled into single-chain molecules comprising a pair of tandem Fv segments (e.g., VH-CH1-VH-CH1) together with complementary light chain polypeptides (e.g., VL-CL-VL-CL) that form a pair of antigen-binding regions (Zapata et al., (1995) Protein Eng., 8:1057-1062; and U.S. Pat. No. 5,641,870).

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

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

[0079] The term "multispecific antibody" refers to an antibody capable of recognizing two or more epitopes of one antigen or two or more antigens. Recognition of each antigen is generally achieved via an "antigen-binding domain." In particular, bispecific antibodies recognize two different epitopes, either on the same antigen or on different antigens. All bispecific IgG molecules, i.e., bispecific antibodies indistinguishable in their composition from natural immunoglobulins, are bivalent and maintain an asymmetric structure due to the presence of at least different Fv regions. Depending on the method of preparation and the source of the heavy and light chains, they may also differ in the constant regions of the heavy or light chains (Brinkmann and Kontermann, 2017).

[0080] Bispecific antibodies are "heterodimeric," meaning that one portion is derived from a first antibody specific for a first target and another portion is derived from a second antibody specific for a second target. A "heterodimerization modification" is a modification to one or both portions of an antibody that allows the formation of heterodimeric bispecific antibodies, with the purpose of promoting such formation. An example of a heterodimerization modification of the Fc domains of two IgG1 portions of an antibody intended to form a bispecific antibody is the introduction of a "knob" (S354C, T366W) with a bulky amino acid (aa) side chain in the first heavy chain, a "hole" (Y349C, T366S, L368A, Y407V) with a small aa side chain in the second heavy chain, and an additional disulfide bridge in the CH3 region linking both heavy chains (Merchant et al., Nat. Biotechnol., 16:677-681 (1998), p. 678, Table 1).

[0081] The terms "mismatch," or "mismatch," or "misassembled" mean that different moieties of a protein complex of interest, such as a bispecific antibody, do not combine together as intended, meaning 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, refer to a binding molecule, antibody, or antigen-binding fragment thereof that finds and interacts with (e.g., binds to or recognizes) an epitope, whether the epitope is linear, punctuated, or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids arranged by tertiary folding of a protein. Epitopes formed from contiguous amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)) or electron microscopy. A "paratope" is the part of an antibody that recognizes an epitope of an antigen.

[0083] The phrases "specifically bind" or "selectively bind," when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refer to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biological materials, e.g., in a biological sample, such as a blood, serum, plasma, or tissue sample. Thus, under specified, particular immunoassay conditions, an antibody or binding agent with a particular binding specificity will bind to the particular antigen at least twice as much as background and will not significantly bind to other antigens present in the sample. In one aspect, under specified, particular immunoassay conditions, an antibody or binding agent with a particular binding specificity will bind to the particular antigen at least 10 times as much as background and will not significantly bind to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent be selected for its specificity for a particular protein. Where desired or appropriate, this selection can be achieved by subtracting out antibodies from other species (e.g., mouse or rat) or other subtypes that cross-react with the molecule. Alternatively, in some aspects, antibodies or antibody fragments are selected that cross-react with a particular desired molecule.

[0084] The term "antigen-binding site" refers to the portion of an antibody that contains the determinants that form the interface that binds to an antigen or its epitope. The term "antigen-binding site" can be used interchangeably with the term "antigen-binding domain" or antigen-binding portion. In the context of a protein (or protein mimetic), the antigen-binding site typically comprises one or more loops (of at least four amino acids or amino acid mimetics) that form an interface that binds to the antigen polypeptide. Typically, the antigen-binding site of an antibody molecule comprises at least one or two CDRs and / or hypervariable loops, or more typically, at least three, four, five, or six CDRs and / or hypervariable loops.

[0085] The term "complementarity determining region" or "CDR" as used herein refers to a sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are generally three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The locations of CDRs and framework regions can be determined using various definitions known in the art, such as Kabat, Chothia, IMGT, AbM, and combined definitions (e.g., Kabat et al., (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; Johnson et al., (2001) Nucleic Acids Res., 29:205-206; Chothia & 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; see Al-Lazikani et al., (1997) J. Mol. Biol., 273:927-748). The definition of antigen-binding sites is also described in the following references: Ruiz et al., (2000) Nucleic Acids Res., 28:219-221; MacCallum et al., (1996) 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.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the numbering scheme, in some embodiments, the CDR amino acid residues in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a human VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a human VL, e.g., a mammalian VL, e.g., a human VL. Under IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0086] The term "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin Io sequences. The humanized antibody optionally also comprises at least a portion of an 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., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0087] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from a human sequence, such as a human germline sequence or a mutated version of a human germline sequence, or an antibody-containing consensus framework sequence derived from an analysis of human framework sequences, e.g., as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

[0088] The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo, or conservative substitutions that promote stability or manufacturing). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0089] As used herein, a "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the original amino acid sequence, resulting from a sequence alteration involving the amino acid residue / position. For example, typical modifications include substitution of the residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more amino acids adjacent to the residue / position, and deletion of the residue / position. An "amino acid substitution" or mutation thereof refers to the replacement of an existing amino acid residue in a predetermined (original) amino acid sequence with a different amino acid residue. Generally, and preferably, the modification results in an alteration of at least one physico-biochemical activity of the variant polypeptide compared to a polypeptide comprising the original (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physico-biochemical activity can be binding affinity, binding ability, 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 a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes all possible silent variations of the nucleic acid. Those of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit within each described sequence.

[0091] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that substitute an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 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), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the phrase "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody-like molecule containing the amino acid sequence.

[0092] The term "percent identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if a specified percentage of amino acid residues or nucleotides are the same (i.e., 60% identity over a specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms below or by manual alignment and visual inspection, over a comparison window, or designated region. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100-500 nucleotides or 1000 nucleotides or more (or 20, 50, 200 amino acids or more) in length.

[0093] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared.Using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0094] As used herein, the term "comparison window" includes reference to any one segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, within which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0095] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters 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. Extension of word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses an alignment (B) of 50, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0096] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0097] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0098] Another indication that two nucleic acid sequences or polypeptides are substantially identical, other than the percentage of sequence identity noted above, is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0099] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0100] The nucleotides in a "polynucleotide" or "nucleic acid" may contain modifications such as base modifications such as bromouridine and inosine derivatives, ribose modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0101] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that is suitable for transformation or transfection of a host cell and contains a nucleic acid sequence that drives and / or regulates (in conjunction with the host cell) the expression of one or more heterologous coding regions to which it is operably linked.

[0102] As used herein, the term "operably linked" or operably linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Typically, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent to or positioned closely adjacent to the coding sequence whose transcription they enhance.

[0103] The term "co-expression" means that different polypeptides are expressed together in a single host cell that is common to all of the polypeptides. Co-expression of a bispecific antibody means that the different portions that form a functional bispecific antibody are expressed in a single common host cell. Co-expression can be achieved by incorporating several expression vectors into the expression host cell, such as one for each half of the bispecific antibody, or by incorporating one expression vector that encodes all portions of the bispecific antibody. As used herein, "C-terminus" refers to the carboxyl-terminal amino acid of a polypeptide chain having a free carboxyl group (-COOH). As used herein, "N-terminus" refers to the amino-terminal amino acid of a polypeptide chain having a free amine group (-NH).

[0104] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This phrase applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

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

[0106] Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize human variable region 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 now available in which the mouse immunoglobulin loci have been replaced with human immunoglobulin gene segments (Tomizuka K, (2000) Proc Natl Acad Sci, 97:722-727; Fishwild DM (1996) Nature Biotechnol 14:845-851; Mendez MJ, (1997) Nature Genetics 15:146-156). Upon antigen challenge, these mice can produce a repertoire of human antibodies from which antibodies of interest can be selected. Of particular note are the Trimera™ system (Eren R et al, (1988) Immunology 93:154-161), in which human lymphocytes are transplanted into irradiated mice; the Selective Lymphocyte Isolation Antibody System (SLAM, Babcook et al, Proc Natl Acad Sci (1996) 93:7843-7848), in which large numbers of pooled human (or other species) lymphocytes are effectively used in an in vitro isolation antibody generation procedure followed by deconvolution; limiting dilution and selection procedures; and the Xenomouse™ (Abgenix Inc). An alternative approach is available from Morphotek Inc using Morphodoma™ technology.

[0108] Phage display technology can be used to generate human antibodies and fragments thereof (McCafferty; (1990) Nature, 348:552-553 and Griffiths AD et al (1994) EMBO 13:3245-3260). According to this technology, isolated antibody variable domain genes are cloned in-frame into either the major or minor protein gene coat of a filamentous bacteriophage, such as M13 or fd, and displayed as functional isolated antibody fragments on the surface of the phage particle (usually with the assistance of a helper phage). Selection based on the functional properties of the isolated antibody results in the selection of genes encoding isolated antibodies exhibiting these properties. Phage display technology can also be used to select antigen-specific antibodies from libraries generated from human B cells harvested from individuals suffering from a disease or disorder or, alternatively, from unimmunized human donors (Marks; J Mol Bio (1991) 222:581-591). If an intact human isolated antibody containing the Fc domain is desired, it is necessary to re-clone the phage-displayed resulting fragment into a mammalian expression vector containing the desired constant region and establishing a stable expressing cell line.

[0109] The technique of affinity maturation (Marks; Biotechnol (1992) 10:779-783) can be used to improve the binding affinity of a primary human isolated antibody by successively replacing the heavy and light chain variable regions with natural variants and selecting on the basis of improved binding affinity. Variations of this technique, such as "epitope imprinting," are also now available (WO 93 / 06213; Waterhouse; Nucl Acids Res (1993) 21:2265-2266).

[0110] The term "pure," when used in the context of purified bispecific antibodies, relates to the purity and identity of different bispecific antibody combinations and constructs after co-expression in selected cells under conditions in which the cells express the bispecific antibodies and after Protein-A purification using an intact UPLC-MS mass screening approach. Purity or purity refers to the relative quantitation of hetero- and homodimeric bbmAbs formed. Using the methods of the present invention, correctly formed heterodimeric bispecific antibodies can be observed with greater than 85% relative purity based on intact mass signal intensity.

[0111] The terms "therapeutically acceptable amount," or "therapeutically effective amount," or "therapeutically effective dose" refer interchangeably to an amount sufficient to produce a desired result (i.e., a decrease in disease activity, a decrease in disease progression, a reduction in disease signs and / or symptoms, etc.). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount may be determined by administering an initial low dose and then gradually increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of a molecule of the present disclosure may prevent the onset of, or result in a reduction in the severity of, disease symptoms, respectively, including conditions associated with IL-13 activity and IL-18 activity.

[0112] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., 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 interchangeably herein.

[0113] As used herein, the phrases "patient in need of treatment" or "subject in need of treatment" and the like include subjects, such as mammalian subjects, who would benefit from the administration of a molecule or pharmaceutical composition of the present disclosure for use in detection, diagnostic procedures, and / or treatment, and the like.

[0114] The terms "treat," "treating," "treatment," "prevent," "preventing," or "prevention" include therapeutic treatments, prophylactic treatments, and applications that reduce a subject's risk of developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses the reduction of symptoms or underlying risk factors. As used herein, a human antibody or fragment thereof includes a heavy or light chain variable region or a full-length heavy or light chain that is the "product" of or "derived from" a particular germline sequence when the variable region or full-length chain of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening human immunoglobulin gene libraries displayed on phage with the antigen of interest. Human antibodies or fragments thereof that are the "product of" or "derived from" human germline immunoglobulin sequences can be identified, for example, by comparing the amino acid sequence of a human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., has the greatest percent identity) to the sequence of the human antibody. A human antibody that is the "product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, e.g., due to natural somatic mutations or the intentional introduction of site-specific mutations. However, the selected human antibody will generally be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contain amino acid residues that identify the human antibody as human when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody can be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.Generally, a human antibody derived from a particular human germline sequence will display up to 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can display up to 5, or even up to 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[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) or fragments thereof engineered to bind to human IL-18. Recognition of IL-18 by the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure occurs via an "IL-18 antigen-binding domain," which is interchangeably referred to as an "IL-18 binding domain."

[0117] In one preferred embodiment, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-18 binding domain, making the multispecific binding molecule monovalent with respect to IL-18 binding. In another preferred embodiment, the multispecific binding molecule comprises multiple IL-18 binding domains, e.g., two IL-18 binding domains, making the multispecific binding molecule multivalent with respect to binding IL-18, preferably bivalent with respect to binding IL-18.

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

[0119] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof has a 10 -4 M~10 -8 M, e.g. 10 -5M~10 -7 M, e.g., 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, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-18 Fab. In another preferred embodiment, the anti-IL-18 binding domain comprises two IL-18 Fabs. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprising three or more IL-18 Fabs are also contemplated herein.

[0121] In some examples, Fab can be prepared according to methods known in the art. The enzyme papain can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment. The enzyme pepsin cleaves below the hinge region to form an F(ab')2 fragment and a pFc' fragment. The F(ab')2 fragment can be separated into two Fab' fragments by mild reduction. Fab fragments are very stable due to non-covalent interactions that occur across the large interface between the heavy and light chain polypeptides and the presence of a stabilizing disulfide bond between the CH1 and CL domains (see Glover & Humphreys, Chapter 2, Antibodies, Vol 1: Production and Purification, Kluwer Academic / Plenum Publishers, New York 2004, edited by G Subramanian). For examples of linker orientations and sizes, see, e.g., Hollinger et al. (1993) PNAS USA 90:6444-6448, U.S. Patent Application Publication No. 2005 / 0100543, U.S. Patent Application Publication No. 2005 / 0175606, U.S. Patent Application Publication No. 2007 / 0014794, and WO 2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0122] The phrases "Fab that binds to human IL-18" and "IL-18 Fab" refer to a Fab that binds to human IL-18. In one aspect, the IL-18 Fab retains comparable binding affinity, e.g., binds to IL-18 with comparable efficacy as the full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity, e.g., it binds to IL-18 with lower binding affinity than the full-length antibody, but it still provides the biological response described herein.

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

[0124] In one aspect, the present disclosure provides polynucleotides encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that include an IL-18 binding domain, e.g., an IL-18 Fab. The present disclosure also provides isolated nucleic acid molecules encoding these Fabs.

[0125] Disclosed herein is an isolated nucleic acid molecule encoding a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprising an IL-18 binding domain, wherein the multispecific antibody comprises a VH having the amino acid sequence set forth in SEQ ID NO:41, and the VL comprises the amino acid sequence set forth in SEQ ID NO:13.

[0126] In one embodiment, the IL-18 binding domain of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof (e.g., an IL-18 Fab) is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In one embodiment, the entire construct of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof of the present disclosure is encoded by a transgene whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows identical polypeptides to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

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

[0128] In one preferred embodiment, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-13 binding domain, such that the multispecific binding molecule is monovalent with respect to binding IL-13. In another preferred embodiment, the multispecific binding molecule comprises multiple IL-13 binding domains, e.g., two IL-18 binding domains, such that the multispecific binding molecule is multivalent with respect to binding IL-18, preferably bivalent with respect to binding IL-13.

[0129] In some embodiments, the IL-13 binding domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises an IL-13 scFv or an IL-13 Fab, preferably an IL-13 Fab.

[0130] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof has a 10 -4 M~10 -8 M, e.g. 10 -5 M~10 -7 M, e.g. 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, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-13 Fab. In another preferred embodiment, the anti-IL-13 binding domain comprises two IL-13 Fabs. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprising three or more IL-13 Fabs are also contemplated herein.

[0132] The phrases "Fab that binds to human IL-13" and "IL-13 Fab" refer to a Fab that binds to human IL-13. In one aspect, the IL-13 Fab retains comparable binding affinity, e.g., binds to IL-13 with comparable potency as the full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity, e.g., binds to IL-13 with a lower binding affinity than the full-length antibody, yet still provides the biological response described herein.

[0133] A preferred IL-13 Fab for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:55 and a VL comprising the amino acid sequence set forth in SEQ ID NO:27.

[0134] Another preferred IL-13 Fab for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 85 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 71.

[0135] In one aspect, the present disclosure provides polynucleotides encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that include an IL-13 binding domain, e.g., an IL-13Fab. The present disclosure also provides isolated nucleic acid molecules encoding these Fabs.

[0136] Disclosed herein is an isolated nucleic acid molecule encoding a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprising an IL-13 binding domain comprising a VH having the amino acid sequence set forth in SEQ ID NO:55, and a VL having the amino acid sequence set forth in SEQ ID NO:27.

[0137] Also disclosed herein is an isolated nucleic acid molecule encoding a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprising an IL-13 binding domain comprising a VH having the amino acid sequence set forth in SEQ ID NO: 85, and a VL having the amino acid sequence set forth in SEQ ID NO: 71.

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

[0139] In one aspect, the present disclosure encompasses a recombinant nucleic acid construct comprising a polynucleotide encoding a multispecific antibody (e.g., a bispecific antibody) disclosed herein, or a fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence encoding an IL-13 binding domain or a fragment thereof.

[0140] III. Linkage and Orientation of Domains and Regions of Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof As used herein, the term "linked" or "linking" refers to the direct or indirect linking of one portion of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof to another portion of the molecule. Direct linkage is a form of linkage and is referred to herein as "fused" or "fusion." Using molecules with the format ABC as an example: portion A is directly linked to portion B and indirectly linked to portion C (portion A may also be said to be fused to portion B). As another example, using an scFv in the format VH-internal linker-VL, the VH is indirectly linked to the VL and directly linked to the internal linker (the linker may also be described as being fused to both the VL and the VH).

[0141] In some embodiments, the IL-18 binding domain and the IL-13 binding domain of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof are linked. In some embodiments, the linkage is direct, such that the regions are fused to one another. In some embodiments, the IL-18 binding domain and / or the IL-13 binding domain are fused to an Fc polypeptide chain. In some embodiments, the IL-13 binding domain (e.g., an IL-13 Fab) is linked to the N-terminus of the IL-18 binding domain, e.g., via a polypeptide linker. In some embodiments, the IL-13 binding domain (e.g., an IL-13 Fab) is linked, e.g., fused, to the N-terminus of the IL-18 binding domain.

[0142] IV. Multispecific Antibody Formats In some aspects, a multispecific antibody is a bispecific antibody. In some aspects, a bispecific antibody can be multivalent, e.g., bivalent with respect to one antigen and monovalent with respect to another antigen. A typical bispecific antibody is characterized by a first antigen-binding domain (e.g., comprising a first VL and a first VH) that has binding specificity for a first antigen or epitope (e.g., IL-18) and a second antigen-binding domain that has binding specificity for a second antigen or epitope (e.g., IL-13). In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes are on different antigens, e.g., two proteins (or subunits of a multimeric protein). In embodiments of the disclosure, a bispecific antibody comprises heavy chain and light chain variable domain sequences that have binding specificity for a first epitope or antigen (e.g., IL-18) and an additional binding domain that has binding specificity for a second epitope or antigen (e.g., IL-13).

[0143] Protocols for making bispecific or heterodimeric antibodies are known in the art; for example, the "knobs-in-holes" approach, as described, for example, in U.S. Pat. No. 5,731,168; electrostatic steering Fc pairing, as described, for example, in WO 2009 / 089004, WO 2006 / 106905, and WO 2010 / 129304; strand exchange engineering domain (SEED) heterodimerization, as described, for example, in WO 2007 / 110205; Fab arm exchange, as described in International Publication Nos. 2008 / 119353, 2011 / 131746, and 2013 / 060867; bispecific antibody conjugates, e.g., by antibody cross-linking, using heterobifunctional reagents with amine-reactive groups and sulfhydryl-reactive groups to generate bispecific structures, e.g., as described in U.S. Pat. No. 4,433,059; and bispecific antibody conjugates, e.g., by antibody cross-linking, using heterobifunctional reagents with amine-reactive groups and sulfhydryl-reactive groups to generate bispecific structures, e.g., as described in U.S. Pat. No. 4,444,878; and Bispecific antibody or antibody-like molecule determinants produced by recombining half antibodies (heavy chain-light chain pairs or Fab) from corresponding antibodies or antibody-like molecules; e.g., trifunctional antibodies, e.g., three Fab' fragments cross-linked through sulfhydryl-reactive groups, such as those described in U.S. Pat. No. 5,273,743; biosynthetic binding proteins, e.g., pairs of scFvs cross-linked through their C-terminal tails, preferably through disulfide or amine-reactive chemical bridges, such as those described in U.S. Pat. No. 5,534,254; bifunctional antibodies, e.g., antibodies with constant domains Fab fragments with different binding specificities dimerized through a leucine zipper with a substituted nucleotide, such as those described in U.S. Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, such as the VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with associated light chains, such as those described in U.S. Pat. No. 5,591,828; bispecific DNA-antibody conjugates,No. 5,637,481; multivalent and multispecific binding proteins, such as dimers of polypeptides having a first domain with an Ig heavy chain variable region binding region and a second domain with an Ig light chain variable region binding region, commonly referred to as diabodies (including generating higher order structures for bispecific, trispecific, or tetraspecific molecules, e.g., as described in U.S. Pat. No. 5,837,242); minibody constructs in which linked VL and VH chains are further joined by a peptide spacer to antibody hinge and CH3 regions, which can be dimerized to form bispecific / multivalent molecules, e.g., as described in U.S. Pat. No. 5,837,821; short fragments of antibody fragments in either orientation that can dimerize to form bispecific diabodies. VL and VH domains linked by a peptide linker (e.g., 5 or 10 amino acids) or no linker at all; trimers and tetramers, such as those described in U.S. Pat. No. 5,844,094; strings of VH domains (or VL domains in family members) linked by peptide bonds with crosslinkable groups at the C-terminus, which are further associated with VL domains to form a series of Fvs (or scFvs), such as those described in U.S. Pat. No. 5,864,019; and single-chain binding polypeptides in which both VL and VH domains are linked via a peptide linker, as described in U.S. Pat. No. 5,869,620, using both scFv or diabody-type formats, which are combined into multivalent structures through non-covalent or chemical crosslinking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures, although other examples include VL and VH domains, scFv or Fab, which bind one antigen monovalently and one antigen bivalently, optionally comprising a heterodimeric Fc region, as described in U.S. Pat. No. 5,869,620; and single-chain binding polypeptides in which both VL and VH domains are linked via a peptide linker, as described in U.S. Pat. No. 5,869,620, using both scFv or diabody-type formats, which are combined into multivalent structures through non-covalent or chemical crosslinking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures.Non-limiting examples of additional representative multispecific and bispecific molecules and methods of making the same are described in, e.g., U.S. Patent Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, and 6,670,453. Nos. 6,743,896, 6,809,185, 6,833,441, 7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 2003211078A1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005 100543A1, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1,US Patent Application Publication No. 2007087381A1, US Patent Application Publication No. 2007128150A1, US Patent Application Publication No. 2007141049A1, US Patent Application Publication No. 2007154901A1, US Patent Application Publication No. 2007274985A1, US Patent Application Publication No. 2008050370A1, US Patent Application Publication No. 2008069820A1, US Patent Application Publication No. 2008152645A1, US Patent Application Publication No. 2008171855A1, US Patent Application Publication No. 20082 41884A1, U.S. Patent Application Publication No. 2008254512A1, U.S. Patent Application Publication No. 2008260738A1, U.S. Patent Application Publication No. 2009130106A1, U.S. Patent Application Publication No. 2009148905A1, U.S. Patent Application Publication No. 2009155275A1, U.S. Patent Application Publication No. 2009162359A1, U.S. Patent Application Publication No. 2009162360A1, U.S. Patent Application Publication No. 2009175851A1, U.S. Patent Application Publication No. 2009175867A1, U.S. Patent Publication No. 2009232811A1, U.S. Patent Application Publication No. 2009234105A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent No. 346087A2, International Publication No. 2000 / 06605A2, International Publication No. 2007 / 2635A2, International Publication No. 2004 / 081051A1, International Publication No. 2006 / 020258A2, International Publication No. 2007 / 044887A2, International Publication No. 2 International Publication No. 007 / 095338A2, International Publication No. 2007 / 137760A2, International Publication No. 2008 / 119353A1, International Publication No. 2009 / 021754A2, International Publication No. 2009 / 068630A1, International Publication No. 1991 / 03493A1, International Publication No. 1993 / 23537A1, International Publication No. 1994 / 09131A1, International Publication No. 1994 / 12625A2, International Publication No. 1995 / 09917A1,and WO 1996 / 37621 A2 and WO 1999 / 64460 A1. The contents of the aforementioned applications are incorporated herein by reference in their entireties. Thus, in some embodiments, the IL-13 / IL-18 multispecific antibodies (e.g., bispecific antibodies) of the present disclosure comprise an IL-13 binding domain and an IL-18 binding domain in any one of the multispecific or bispecific formats known in the art and described above. Preferred formats for the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are described in more detail below.

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

[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 are bispecific antibodies comprising amino acid sequences with one, two, or three substitutions, deletions, or insertions compared to the sequences in Table 1.

[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 are based on a human IgG1 format. All contain YTE half-life extending mutations, while two additionally contain LALA silencing mutations. More specifically, bbmAb2, bbmAb1, and bbmAb3 contain YTE half-life extending mutations in the Fc. bbmAb4 and bbmAb5 contain both LALA silencing and YTE half-life extending mutations. bbmAb2, bbmAb1, bbmAb4, and bbmAb5 combine the variable domains of mAb1 and mAb2, while bbmAb3 combines the variable domains of mAb1 and mAb3.

[0168] bbmAb2, bbmAb4, and bbmAb3 possess a KiH knob heterodimerization mutation in the anti-IL-18 heavy chain Fc, while the KiH hole mutation is in the anti-IL-13 heavy chain Fc. bbmAb2, bbmAb4, and bbmAb3 possess a KiH knob mutation in the anti-IL-18 heavy chain Fc 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 encompasses variants of the molecules and / or fragments thereof described herein, as well as fusions and conjugates of the disclosed molecules, with various modifications in the binding domains, variable domains, and / or constant regions. For example, the Fc region of the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be wild-type, or it can be modified to achieve various outcomes. Preferred modifications to the Fc region include "LS" mutations (M428L, N434S, (EU numbering)) and "YTE" mutations (M252Y, S254T, T256E (EU numbering)) for half-life extension, "DAPA" mutations (D265A, P329A (EU numbering)) for effector silencing, and knob-in-hole mutations (e.g., knob S354C, T366W; hole Y349C, T366S, L368A, Y407V (EU numbering)) to promote proper chain pairing.

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

[0172] Furthermore, in certain instances, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of an antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.). Molecules of the present disclosure (e.g., antibodies or antibody-like molecules) can be modified by introducing such mutations into their variable region frameworks to improve binding characteristics.

[0173] Another type of variable region modification is known as "affinity mutation," in which amino acid residues are mutated within the VH and / or VL CDR1, CDR2, and / or CDR3 domains to thereby improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect on antibody binding or other functional properties of interest can be assessed in in vitro or in vivo assays such as those described herein and provided in the Examples. Conservative modifications (as discussed above) can be introduced. Mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically, up to 1, 2, 3, 4, or 5 residues, preferably 1 or 2, within the CDR regions are altered.

[0174] Amino acid sequence variants of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be prepared by introducing appropriate nucleotide changes into the encoding DNA, or by synthesizing the desired variant. Such variants include, for example, deletions from, insertions into, or substitutions of residues within the amino acid sequence of the molecule. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct retains the desired antigen-binding characteristics. Amino acid changes can also alter post-translational processing of the molecule, such as changing the number or location of glycosylation sites.

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

[0176] B. Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof with Enhanced Heterodimerization Improper heterodimerization of two antibody heavy chain domains can be an obstacle to improving the yield of a desired multispecific antibody (e.g., bispecific antibody) or fragment thereof and represents a purification challenge. Various approaches are available for promoting the dimerization of the two heavy chain domains of a bispecific or multispecific antibody or antibody-like molecule, such as those disclosed in EP 1870459 A1; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006204493 A1; and WO 2009 / 089004 A1.

[0177] The present disclosure provides methods for promoting dimerization (heterodimerization) of two interacting heterologous polypeptides and / or reducing dimerization (homodimerization) of two identical polypeptides. Typically, each of the two interacting polypeptides comprises an Fc region having antibody CH2 and CH3 domains. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, and preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class, most preferably IgG1.

[0178] Typically, polypeptides of the present disclosure comprise other antibody fragments in addition to a CH3 domain, such as a CH1 domain, CH2 domain, hinge domain, VH domain, VL domain, CDRs, etc., and / or antigen-binding fragments described herein, e.g., scFv and / or Fab. These antibody fragments are derived from the various types of antibodies described herein, 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 conjugates. Heterodimerization of two different heavy chains at the CH3 domain generates the desired antibody or antibody-like molecule, while homodimerization of identical heavy chains reduces the yield of the desired antibody or molecule. In a representative embodiment, two or more heteropolypeptide chains comprise two chains that comprise a CH3 domain and form a molecule of any of the above-described multispecific antibody (e.g., bispecific antibody) or fragment formats of the present disclosure. In one embodiment, two heteropolypeptide chains comprising a CH3 domain comprise a modification that favors heterodimeric association of the polypeptides compared to the unmodified chains. Various examples of modification strategies are provided below.

[0179] Knob-in-Hole (KIH) (also known as "Key-in-Hole") A multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure can comprise one or more, e.g., multiple, mutations in one or more of the constant domains, e.g., to the CH3 domain. In one example, a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure comprises two polypeptides, each comprising an antibody heavy chain Fc or constant domain, e.g., a CH2 or CH3 domain. In one example, the two heavy chain constant domains, e.g., the CH2 or CH3 domains, of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprise one or more mutations that enable heterodimer association between the two chains. In one embodiment, one or more mutations are located in the CH2 domains of the two heavy chains of the multispecific antibody (e.g., bispecific antibody) or fragment thereof. In one embodiment, one or more mutations are located in the CH3 domains of at least two polypeptides of the multispecific antibody (e.g., bispecific antibody) or fragment thereof. In one aspect, one or more mutations to a first polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising heavy chain constant domains generate a "knob," one or more mutations to a second polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising heavy chain constant domains form a "hole," and heterodimerization of the polypeptides of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising heavy chain constant domains brings together the "hole" and the "knob" (e.g., interacting, e.g., the CH2 domain of the first polypeptide interacting with the CH2 domain of the second polypeptide or 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 at least one amino acid side chain that protrudes from the interface of a first polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising, for example, a heavy chain constant domain, and is therefore capable of positioning itself in a compensatory "hole" at the interface with a second polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising a heavy chain constant domain, so as to stabilize heteromultimers, thereby favoring heteromultimer formation over homomultimer formation.The knob can be present at the original interface or can be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). Preferred import residues for forming the knob are generally natural amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In preferred embodiments, the original residue for forming the overhang has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0180] A "hole" refers to at least one amino acid side chain that is recessed from the interface of a second polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising a heavy chain constant domain, and thus accommodates a corresponding knob on the adjacent interface of a first polypeptide of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising a heavy chain constant domain. The hole can be present in the original interface or can be synthetically introduced (e.g., by altering the nucleic acid encoding the interface). Preferred import residues for hole formation are typically natural amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Most preferred are serine, alanine, or threonine. In preferred embodiments, the original residue for hole formation has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0181] In one embodiment, the first CH3 domain has a mutation at residue 366, 405, or 407 according to the EU numbering scheme of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.) pp. 688-696) to generate either a "knob" or a "hole" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain has a mutation at residue 407 if the first CH3 domain has a mutation at residue 366, at residue 394 if the first CH3 domain has a mutation at residue 405, or at residue 366 if the first CH3 domain has a mutation at residue 407 (EU numbering), to generate a "hole" or "knob" complementary to the "knob" or "hole" of the first CH3 domain.

[0182] In another embodiment, the first CH3 domain is mutated at residue 366 (EU numbering) to generate either a "knob" or a "hole" (as described above) and a second CH3 domain that heterodimerizes with the first CH3 domain, and is mutated at residues 366, 368, and / or 407 (EU numbering) to generate a "hole" or "knob" that is complementary to the "knob" or "hole" of the first CH3 domain. In one embodiment, the mutation to the first CH3 domain introduces a tyrosine (Y) residue at position 366. In one embodiment, the mutation to the first CH3 is T366Y. In one embodiment, the mutation to the first CH3 domain introduces a tryptophan (W) residue at position 366. In one embodiment, the mutation to the first CH3 is T366W. In embodiments, mutations to a second CH3 domain that heterodimerizes with a first CH3 domain having a mutation at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., comprising a mutation T366Y or T366W) include a mutation at position 366, a mutation at position 368, and a mutation at position 407 (EU numbering). In embodiments, the mutation at position 366 introduces a serine (S) residue, the mutation at position 368 introduces an alanine (A), and the mutation at position 407 introduces a valine (V). In embodiments, the mutations include T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises the mutation T366Y and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the mutations T366S, L368A, and Y407V, or vice versa. In one embodiment, the first CH3 domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises the mutation T366W and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the mutations T366S, L368A, and Y407V, or vice versa.

[0183] Additional knobs-into-holes mutation pairs suitable for use in any of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are further described, for example, in WO 1996 / 027011 and Merchant et al., (1998) Nat. Biotechnol., 16:677-681, the contents of which are incorporated herein by reference in their entireties.

[0184] In any of the embodiments described herein, the CH3 domain may be further mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to the heterodimerized antibody. In embodiments, the first CH3 domain contains a cysteine ​​at position 354 (EU numbering), and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine ​​at position 349 (EU numbering). In embodiments, a first CH3 domain of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a cysteine ​​at position 354 (e.g., comprising a mutation S354C) and a tyrosine (Y) at position 366 (e.g., comprising a mutation T366Y), and a second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., comprising a mutation Y349C), a serine at position 366 (e.g., comprising a mutation T366S), an alanine at position 368 (e.g., comprising a mutation L368A), and a valine at position 407 (e.g., comprising a mutation Y407V). In embodiments, a first CH3 domain of a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a cysteine ​​at position 354 (e.g., comprising a mutation S354C) and a tryptophan (W) at position 366 (e.g., comprising a mutation T366W), and a second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., comprising a mutation Y349C), a serine at position 366 (e.g., comprising a mutation T366S), an alanine at position 368 (e.g., comprising a mutation L368A), and a valine at position 407 (e.g., comprising a mutation Y407V).

[0185] IgG heterodimerization In one aspect, heterodimerization of polypeptide chains of a multispecific antibody (e.g., a bispecific antibody) or fragments thereof (e.g., half antibodies) is increased by introducing one or more mutations into CH3 domains derived from the IgG1 antibody class. In one embodiment, the mutations include a K409R mutation in one CH3 domain paired with a F405L mutation (EU numbering scheme) in a second CH3 domain. Additional mutations may also or alternatively be at positions 366, 368, 370, 399, 405, 407, and 409 (EU numbering). Preferably, heterodimerization of polypeptides containing such mutations is achieved under reducing conditions, e.g., at 10 to 100 mM 2-MEA (e.g., 25, 50, or 100 mM 2-MEA) at 25 to 37°C, e.g., at 25°C or 37°C, for 1 to 10 hours, e.g., 1.5 to 5 hours, e.g., 5 hours.

[0186] The amino acid substitutions described herein are introduced into the CH3 domain using techniques known in the art. Typically, the DNA encoding the heavy chain is genetically modified using the techniques described in Mutagenesis: A Practical Approach. Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution mutants of the DNA encoding two hybrid heavy chains. This technique is known in the art, as described by Adelman et al., (1983) DNA, 2:183.

[0187] Suitable IgG heterodimerization strategies are described, for example, in WO 2008 / 119353, WO 2011 / 131746 and WO 2013 / 060867, the contents of which are incorporated herein by reference in their entireties.

[0188] In any of the embodiments described herein, the CH3 domain may be further mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to the heterodimerized multispecific antibody (e.g., bispecific antibody) or fragment thereof. In embodiments, the first CH3 domain contains a cysteine ​​at position 354 (EU numbering), and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine ​​at position 349 (EU numbering).

[0189] polar crosslinking In one aspect, heterodimerization of polypeptide chains of a multispecific antibody (e.g., a bispecific antibody) or fragments thereof (e.g., half antibodies) is increased by introducing mutations based on rational "polar-bridge" strategies, which allow residues at the binding interface of the two polypeptide chains to interact with residues of similar (or complementary) physical properties in the heterodimer configuration, and with residues of different physical properties in the homodimer configuration. In particular, these mutations are designed so that polar residues interact with polar residues, while hydrophobic residues interact with hydrophobic residues, in heterodimer formation. In contrast, for homodimer formation, residues are mutated so that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer configuration and the unfavorable interactions in the homodimer configuration work together to make the CH3 domain more likely to form heterodimers than homodimers.

[0190] In an exemplary embodiment, the above mutations are made at one or more of residues 364, 368, 399, 405, 409 and 411 (EU numbering) of the CH3 domain.

[0191] In one embodiment, one or more mutations selected from the group consisting of Ser364Leu, Thr366Val, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Phe and Thr411Lys are introduced into one of the two CH3 domains. For example, Ser364Leu: the original serine residue at position 364 is replaced with leucine; Thr366Val: the original threonine residue at position 366 is replaced with valine; Leu368Gln: the original leucine residue at position 368 is replaced with glutamine; Asp399Lys: the original aspartic acid residue at position 399 is replaced with lysine; Phe405Ser: the original phenylalanine residue at position 405 is replaced with serine; Lys409Phe: the original lysine residue at position 409 is replaced with phenylalanine; Thr411Lys: the original threonine residue at position 411 is replaced with lysine.

[0192] In another embodiment, another CH3 may be introduced along with one or more mutations selected from the group consisting of Tyr407Phe, Lys409Gln, and Thr411Asp (e.g., Tyr407Phe: the original tyrosine residue at position 407 is replaced with phenylalanine; Lys409Glu: the original lysine residue at position 409 is replaced with glutamic acid; Thr411Asp: the original threonine residue at position 411 is replaced with aspartic acid).

[0193] In a further aspect, one CH3 domain has one or more mutations selected from the group consisting of Ser364Leu, Thr366Val, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Phe and Thr411Ly, while the other CH3 domain has one or more mutations selected from the group consisting of Tyr407Phe, Lys409Gln and Thr411Asp.

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

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

[0196] In yet another exemplary embodiment, the original phenylalanine residue at position 405 of one CH3 domain is substituted with serine and the original lysine residue at position 409 of this CH3 domain is substituted with phenylalanine, while the original lysine residue at position 409 of the other CH3 domain is substituted with glutamine.

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

[0198] The amino acid substitutions described herein can be introduced into the CH3 domain using techniques known in the art. Typically, the DNA encoding the heavy chain is genetically modified using the techniques described in Mutagenesis: A Practical Approach. Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution mutants of the DNA encoding two hybrid heavy chains. This technique is known in the art, as described by Adelman et al., (1983) DNA, 2:183.

[0199] Polar cross-linking strategies are described, for example, in WO 2006 / 106905, WO 2009 / 089004, and Gunasekaran K et al., (2010) J Biol Chem., 285:19637-19646, the contents of which are incorporated herein by reference in their entireties.

[0200] In any of the embodiments described herein, the CH3 domain may be further mutated to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond provides stability to heterodimerized multispecific antibodies (e.g., bispecific antibodies). In embodiments, the first CH3 domain contains a cysteine ​​at position 354 (EU numbering), and the second CH3 domain that heterodimerizes with the first CH3 domain contains a cysteine ​​at position 349 (EU numbering).

[0201] C. Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof with Extended In Vivo Half-Life The present multispecific antibodies (eg, bispecific antibodies) or fragments thereof can be further modified to increase their half-life in vivo.

[0202] Various strategies can be used to extend the half-life of the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof, by, for example, chemical linkage to polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; genetic fusion to proteins that bind to serum proteins, such as albumin, IgG, FcRn, and transferrins; coupling (genetic or chemical) to other binding moieties that bind to serum proteins, such as nanobodies, Fabs, DARPins, avimers, affibodies, and anticalins; genetic fusion to rPEG, albumin, domains of albumin, albumin-binding proteins, and Fc; or incorporation into nanocarriers, slow-release formulations, or medical devices.

[0203] One or more amino acid modifications (i.e., substitutions, insertions, or deletions) can also be introduced into an IgG constant domain or an FcRn-binding fragment thereof (preferably the Fc region or fragment thereof) to generate multispecific antibodies (e.g., bispecific antibodies) of the present disclosure, or fragments thereof, that have extended half-lives in vivo. See, for example, WO 1998 / 23289, WO 1997 / 34631, and U.S. Pat. No. 6,277,375. Preferred modifications to the Fc of the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof include the "LS" mutation (M428L, N434S (EU numbering)) and the "YTE" mutation (M252Y, S254T, T256E (EU numbering)) for half-life extension.

[0204] Furthermore, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be conjugated or fused to one or more human serum albumin (HSA) polypeptides or portions thereof. The use of albumin renders the molecule more stable in vivo or has a longer half-life in vivo. This technology is known in the art; see, for example, WO 1993 / 15199, WO 1993 / 15200, and WO 2001 / 77137; and EP 413622. The use of N-terminal fragments of HSA for fusion to polypeptides has also been proposed (e.g., EP 399666). Thus, fusing or conjugating a molecule to albumin genetically or chemically can stabilize or extend its shelf life and / or retain its activity in solution in vitro and / or in vivo for extended periods of time. Further methods for HSA fusion can be found, for example, in WO 2001 / 077137 and WO 2003 / 06007, which are incorporated herein by reference. In certain embodiments, 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 present disclosure incorporating one or more constant domains, e.g., heavy chain constant regions, it may be beneficial to include one or more mutations within the hFc to, for example, silence ADCC and / or CDC effector functions. Immune cell activation occurs preferentially in the presence of cross-linking to target cells. However, human Fc can bind to high- and low-affinity FcR gamma receptors. Thus, cross-linking and subsequent agonism of receptors (e.g., CD3) on immune cells can occur upon binding in the absence of tumor targeting. Furthermore, cross-linking of Fc via gamma receptors can induce antibody-dependent cellular cytotoxicity (ADCC). When complexed on the cell surface, human Fc can also bind complement proteins and induce complement-dependent cytotoxicity (CDC). Therefore, mutations to residues in Fc that reduce or abrogate these interactions can limit these effects and focus the effects of the molecules described herein on tumor target cells.

[0206] In embodiments, one or more, e.g., all, of the heavy chain constant region domains of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprise a DAPA mutation (e.g., D265A and P329A according to EU numbering). See, e.g., Shields RL et al., (2001) J Biol Chem., 276(9):6591-604; U.S. Patent Application Publication No. 2015 / 0320880A1, the contents of each of which are incorporated by reference in their entirety.

[0207] In embodiments, one or more, e.g., all, of the heavy chain constant region domains of the multispecific antibody (e.g., bispecific antibody) or fragments thereof comprise the LALA mutation (e.g., L234A and L235A according to EU numbering). See, e.g., Hezareh M et al., (2001) Journal of Virology, 75(24):12161-12168; Shields RL et al., (2001) supra, the contents of each of which are incorporated by reference in their entirety.

[0208] In embodiments, one or more, e.g., all, of the heavy chain constant region domains of the multispecific antibody (e.g., bispecific antibody) or fragments thereof comprise the N279A mutation (according to EU numbering) (see, e.g., Tao MH & Morrison SL (1989) J Immunol. 143(8):2595-601; Shields RL et al., (2001) supra, the contents of each of which are incorporated by reference in their entirety).

[0209] Further Fc mutations for providing silenced effector functions are described in WO 2014 / 145806 (e.g., Figure 7), which is incorporated herein by reference in its entirety. One example of a silent IgG1 antibody from WO 2014 / 145806 includes E233P, L234V, L235A, and S267K mutations and a deletion of G236 (G236del). Another example of a silent IgG1 antibody from WO 2014 / 145806 includes E233P, L234V, and L235A mutations and a deletion of G236 (G236del). Another example of a silent IgG1 antibody from WO 2014 / 145806 includes a S267K mutation.

[0210] E. Compounds The present disclosure includes multispecific antibodies (e.g., bispecific antibodies) or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to generate a fusion protein. Methods for fusing or conjugating proteins, polypeptides, or peptides to antibodies or antibody fragments are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP 307434 and EP 367166; WO 1996 / 04388 and WO 1991 / 06570; 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] Additional fusion proteins can be generated through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof. See generally U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol. 16(2):76-82; Hansson 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 fragments thereof can be altered by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding fragments of the present molecules can be recombined with one or more components, motifs, sections, portions, domains, fragments, etc., of one or more heterologous molecules.

[0212] Furthermore, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be fused to a marker sequence, such as a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector, among many commercially available ones (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311). For example, hexa-histidine provides for convenient purification of the fusion protein, as described by Gentz ​​et al. (1989) PNAS.USA 86:821-824. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell 37:767) and the "flag" tag.

[0213] In other embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof is conjugated to a diagnostic or detectable agent. Such molecules may be useful for monitoring or prognosing the onset, development, progression, and / or severity of a disease or disorder, as part of a clinical testing procedure, such as determining the effectiveness of a particular treatment. Such diagnostics and detection may involve the use of enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances, such as, but not limited to, luminol; bioluminescent substances, such as, but not limited to, luciferase, luciferin, and aequorin; radioactive substances, such as, but not limited to, iodine (I, I, I, and I), carbon (C), sulfur (S), tritium (H), indium (In, In, In, and In), technetium (Tc), thallium (Ti), gallium (Ga, Ga), palladium (Pd), molybdenum (Mo), xenon (Xe), fluorine (F), Sm, Lu, Gd, Pm, La, Yb, Ho, Y, Sc, Re, Re, Pr, R and various positron-emitting metals and non-radioactive paramagnetic metal ions using positron emission tomography.

[0214] The present application further encompasses the use of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof conjugated to a therapeutic moiety. Molecules of the present disclosure or fragments thereof can be conjugated to a therapeutic moiety, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, e.g., an alpha emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

[0215] Furthermore, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be conjugated to therapeutic or drug moieties that modify certain biological responses. Therapeutic or drug moieties should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide that possesses a desired biological activity. Such proteins can include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents, anti-angiogenic agents; or biological response modifiers such as lymphokines.

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

[0217] Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can also be conjugated to radioisotopes to create cytotoxic radiopharmaceuticals, also called radioimmunoconjugates. Examples of radioisotopes that can be conjugated to molecules for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are established in the art. See, for example, Denardo et al., (1998) 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. 26(8):943-50, each of which is incorporated by reference in its entirety.

[0218] Techniques for conjugating therapeutic moieties to antibodies are known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use Of Antibodies," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); See "Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., (1982) Immunol. Rev. 62:119-58.

[0219] Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can also be linked to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0220] VII. Methods of Producing Antibodies of the Invention When the polypeptides of a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure are cross-linked, their functional linkage can be achieved using methods known in the art. Various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., (1984) J. Exp. Med. 160:1686; Liu et al. (1985) PNAS. USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78:118-132; Brennan et al. (1985) Science 229:81-83; and Glennie et al. (1987) J. Immunol. 139:2367-2375. Complexing agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0221] Alternatively, the present multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be produced recombinantly by introducing DNA constructs encoding the desired molecules into an expression vector and expressing and assembling the desired molecules in the same host cell.

[0222] A. Preparation of Polypeptide Chains Polypeptides and antibodies and fragments thereof (e.g., half antibodies) can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Many techniques for producing monoclonal antibodies can be used, e.g., viral or oncogenic transformation of B lymphocytes.

[0223] The animal system for preparing hybridomas is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0224] Chimeric or humanized antibodies for use in the present disclosure can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and modified to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create humanized antibodies, mouse CDR regions can be inserted into a human framework using methods known in the art. See, e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. No. 5,530,101 to Queen et al.; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and U.S. Pat. No. 6,180,370.

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

[0226] The HUmAb mouse (Medarex, Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al., (1994) Nature 368(6474):856-859). Thus, these mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonals (reviewed in Lonberg et al., (1994) supra; Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13:65-93 and Harding and Lonberg, (1995) Ann. NY Acad. Sci. 764:536-546). The preparation and use of HUmAb mice and the genomic modifications carried by such mice are described in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295; Chen et al., (1993) International Immunology 5:647-656; Tuaillon et al., (1993) PNAS USA 94:3720-3724; Choi et al., (1993) Nature Genetics 4:117-123; Chen et al., (1993) EMBO J. 12:821-830; Tuaillon et al., (1994) J. Immunol. 152:2912-2920; Taylor et al., (1994) International Immunology 579-591; and Fishwild et al., (1996) Nature Biotechnology 14:845-851, the contents of all of which are specifically incorporated herein by reference in their entireties.Further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429, all to Lonberg and Kay; Surani et al. See U.S. Pat. No. 5,545,807 to Lonberg et al.; WO 1992 / 103918, WO 1993 / 12227, WO 1994 / 25585, WO 1997113852, WO 1998 / 24884, and WO 1999 / 45962, all to Lonberg and Kay; and WO 2001 / 14424 to Korman et al.

[0227] In another embodiment, human antibodies used in the present disclosure can be produced using mice carrying human immunoglobulin sequences in a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in WO 2002 / 43478 to Ishida et al.

[0228] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate human antibodies for use in the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and 6,162,963 to Kucherlapati et al.

[0229] Additionally, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to generate human antibodies for use in the present disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used; such mice are described in Tomizuka et al., (2000) PNAS USA 97:722-727. Additionally, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894) and can be used to generate human antibodies for use in the present application.

[0230] Human monoclonal antibodies can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art or are described in the Examples below. See, for example, U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

[0231] Human monoclonal antibodies for use in the present disclosure can also be prepared using SCID mice that have been reconstituted with human immune cells so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[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 method for recombinantly producing one or more major polypeptide chains of a multispecific antibody (e.g., a bispecific antibody) or fragments thereof, comprising: 1) generating one or more DNA constructs comprising nucleic acid molecules encoding each of the polypeptide chains of the multispecific binding molecule; 2) introducing the DNA constructs into one or more expression vectors; 3) co-transfecting the expression vectors in one or more host cells; and 4) expressing and assembling the molecules in the host cells or in solution.

[0234] In this regard, the present disclosure provides isolated nucleic acids, e.g., one or more polynucleotides, encoding a multispecific antibody (e.g., a bispecific antibody) described herein or a fragment thereof, e.g., a multispecific binding molecule comprising an IL-13 binding domain and an IL-18 binding domain, as described herein. In embodiments, the isolated nucleic acid is arranged on a single contiguous polynucleotide. In other embodiments, the isolated polynucleotide is arranged on two or more contiguous nucleic acid sequences.

[0235] In embodiments, the isolated nucleic acid comprises a sequence encoding an IL-13-binding domain or fragment thereof and a sequence encoding an IL-18-binding domain or fragment thereof. In embodiments, the sequence encoding the IL-13-binding domain or fragment thereof and the sequence encoding the IL-18-binding domain are located on separate polynucleotides, also referred to as a "set of nucleic acid molecules."

[0236] In embodiments, the sequence encoding the IL-13 binding domain or fragment thereof and the sequence encoding the IL-18 binding domain are located on a single polynucleotide.

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

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

[0239] Provided herein are cloning and expression vectors comprising one or more nucleic acid molecules or sets of nucleic acid molecules encoding a multispecific antibody (e.g., bispecific antibody) or fragment thereof as described herein, which vectors are suitable for the recombinant production of the multispecific binding molecules. Provided herein are processes for the production of a multispecific antibody (e.g., bispecific antibody) or fragment thereof as described herein, which process comprises culturing a host cell disclosed herein under conditions sufficient to express the multispecific antibody (e.g., bispecific antibody) or fragment thereof, and then purifying and recovering the multispecific antibody (e.g., bispecific antibody) or fragment thereof from the host cell culture.

[0240] Desired mutations in the variable or constant regions of the molecules described herein, such as to promote heterodimerization, can be introduced at this stage as described herein.

[0241] The DNA sequence can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding the heavy or light chain of the molecule (e.g., a sequence as described in the Examples below). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109; the diethyl phosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22:1859; and the solid-support method of U.S. Pat. No. 4,458,066. For example, introduction of mutations into a polynucleotide sequence by PCR can be carried out as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., (1991) Nucleic Acids Res. 19:967; and Eckert et al., (1991) PCR Methods and Applications 1:17.

[0242] Expression vectors and host cells for producing the above-described molecules are also provided in the present disclosure. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that is suitable for transformation or transfection of a host cell and contains a nucleic acid sequence that directs and / or controls the expression of one or more heterologous coding regions operably linked thereto (in association with the host cell). Various expression vectors can be used to express polynucleotides encoding the chains or binding domains of molecules. Both viral-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids or episomal vectors (typically carrying expression cassettes for protein or RNA expression) and human artificial chromosomes (see, e.g., Harrington et al., (1997) Nat Genet 15:345). For example, non-viral vectors useful for expressing polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A,B&C, pcDNA3.1 / His, pEBVHis A,B&C, (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40, papilloma viruses, HBP Epstein-Barr virus-based vectors, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., (1995) supra; Smith, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., (1992) Cell 68:143.

[0243] The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the heavy and light chains of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by the inclusion of enhancers appropriate for the cell system used (see, e.g., Scharf et al., (1994) Results 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 increase expression in mammalian host cells.

[0244] The expression vector may also provide a secretory signal sequence site to form a fusion protein with the polypeptide encoded by the insertion of the above-mentioned sequences of the heavy and / or light chain or fragment thereof. More often, the inserted antibody or antibody-like molecule sequence is linked to a signal sequence before being included in the vector. The vector used to receive the sequences encoding the light and heavy chain variable domains sometimes also encodes constant regions or portions thereof. Such vectors can produce intact antibodies or fragments thereof by expressing the variable regions as fusion proteins with the constant regions. Typically, such constant regions are human.

[0245] Host cells for harboring and expressing the present molecules can be either prokaryotic or eukaryotic. Escherichia coli (E. coli) is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one skilled in the art can also construct expression vectors, which typically contain expression control sequences (e.g., an origin of replication) compatible with the host cell. In addition, any number of different known promoters will exist, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from phage lambda. The promoter typically controls expression, optionally with an operator sequence, and contains ribosome binding site sequences and the like, to initiate and complete transcription and translation. Other microorganisms, such as yeast, can also be used to express the antibodies of the present disclosure. Insect cells in conjunction with baculovirus vectors can also be used.

[0246] In some preferred embodiments, mammalian host cells are used to express and produce the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure. For example, mammalian host cells can be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., 1D6.C9 myeloma hybridoma clones) or mammalian cell lines harboring exogenous expression vectors (e.g., SP2 / 0 myeloma cells). These include any mortal or immortal, normal or abnormal animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, FROM GENES TO CLONES, VCH Publishers, NY 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., (1986) Immunol. Rev. 89:49-68), as well as essential processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell-type specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0247] Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts. (See generally, Sambrook, et al., supra.) Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic transfer, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to herpesvirus structural protein VP22 (Elliot and O'Hare, (1997) Cell 88:223), drug-enhanced DNA uptake, and ex vivo transduction. In many cases, stable expression will be desired for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing antibody chains or binding fragments can be prepared using the expression vectors of the present disclosure containing viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells can be grown for 1-2 days in an enriched media and then switched to a selective media. The purpose of the selectable marker is to confer resistance to selection; its presence allows growth of cells that successfully express the introduced sequences in a selective media. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type.

[0248] The antibody or fragment thereof is generally recovered from the culture medium as a secreted polypeptide, but can also be recovered from host cell lysates if directly produced without a secretory signal. If the molecule is membrane-bound, it can be released from the membrane using an appropriate detergent solution (e.g., Triton-X100).

[0249] When molecules are produced in recombinant cells other than those of human origin, they are completely free of proteins or polypeptides of human origin. However, to obtain preparations that are substantially homogeneous with respect to the heteromultimer, it is necessary to purify the molecules from recombinant cell proteins or polypeptides. As a first step, the culture medium or lysate is usually centrifuged to remove certain cellular debris. The produced molecules can be conveniently purified by hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, with affinity chromatography being the preferred purification technique. Other techniques for protein purification are also available, such as fractionation by ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate 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 for enzyme immunoassays, in which one arm binds to a specific epitope on an enzyme and the other portion of the molecule binds to an immobilization matrix. Enzyme immunoassays using antibody-like molecules are discussed by Nolan et al. (Nolan et al., (1990) Biochem. Biophys. Acta. 1040:1-11). Multispecific antibodies can also be used for the diagnosis of various diseases, such as autoimmune diseases (Songsivilai et al., (1990) Clin. Exp. Immunol. 79:315). In particular, one antigen-binding domain of the molecule may bind to IL-13 or IL-18 in a tissue sample (in vitro, ex vivo, in vivo), and the other binding site may bind to a detectable marker as described herein, e.g., a chelator that tightly binds a radionuclide (Le Doussal 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 disclosure have utility in in vitro and in vivo diagnostics and therapeutics. For example, the antibodies can be administered to cells in culture, e.g., in vitro or in vivo, or in a subject, e.g., in vivo, to treat, prevent, or diagnose various disorders.

[0252] In one embodiment, the molecules of the present disclosure are useful for detecting the presence of IL-13 and / or IL-18 in a biological sample. The term "detecting," as used herein, encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In certain embodiments, such tissues include normal and / or cancerous tissues that express IL-13 and / or IL-18 at higher levels compared to other tissues.

[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 a particular aspect, the method comprises contacting the biological sample with a multispecific antibody of the present disclosure under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine or blood sample.

[0254] Also included are methods for diagnosing disorders associated with IL-13 and / or IL-18 expression. In certain embodiments, the methods comprise contacting test cells with a multispecific antibody of the present disclosure; determining the expression level (either quantitatively or qualitatively) of IL-13 and / or IL-18 in the test cells by detecting binding of the multispecific molecule of the present disclosure; and comparing the expression level of IL-13 and / or IL-18 in the test cells with the expression level of IL-13 and / or IL-18 in control cells (e.g., normal cells of the same tissue origin as the test cells or non-virally infected cells), wherein higher levels of IL-13 and / or IL-18 in the test cells compared to the control cells indicate the presence of a disorder associated with IL-13 and / or IL-18. In certain embodiments, the test cells are obtained from an individual suspected of having a pathological disorder mediated by IL-13 and IL-18.

[0255] In certain aspects, methods of diagnosis or detection, such as those described above, involve detecting binding of a multispecific molecule of the present disclosure using, for example, a "FACS" assay.

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

[0257] In certain aspects, the multispecific antibodies of the present disclosure are labeled, including, but not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels) and moieties such as enzymes or ligands that are indirectly detected, for example, through an enzymatic reaction or molecular interaction.

[0258] B. Pharmaceutical Compositions and Dosage Forms Provided herein are pharmaceutical compositions comprising the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof useful in the uses and methods of the present disclosure in treating atopic dermatitis or related conditions, which compositions further comprise one or more pharmaceutically acceptable carriers and / or diluents.

[0259] The phrase "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

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

[0261] Pharmaceutical compositions of therapeutic and diagnostic agents can be prepared, for example, by mixing them with physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Medical Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY). (See, for example, "Forms: Disperse Systems," Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety." The choice of dosing regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of immunogenic symptoms of the entity, and the accessibility of target cells within the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient with an acceptable level of side effects. Thus, the amount of biologic delivered 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 (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; 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.(2000)New Engl.J.Med.342:613-619;Ghosh,et al.(2003)New Engl.J.Med.348:24-32;Lipsky,et al.(2000)New Engl.J.Med.343:1594-1602).rcel Dekker, Inc., New York, NY).

[0262] The determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art that affect or are expected to affect treatment. Typically, the dose is started somewhat less than optimal and then increased by small increments for any negative side effects until the desired or optimal effect is achieved. Important diagnostic measures include, for example, measures of inflammatory symptoms or levels of inflammatory cytokines produced.

[0263] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure or its esters, salts, or amides being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition being used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors known in the medical arts.

[0264] Compositions comprising the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be provided by continuous infusion or administration at intervals of, for example, daily, weekly, or 1 to 7 times per week. Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation.

[0265] The desired dose of a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure is approximately the same as that for an antibody or polypeptide on a moles / kg body weight basis. The dose administered to a subject can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more doses.

[0266] The dosage of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof of the present disclosure administered to a patient can be about 0.0001 mg / kg to about 100 mg / kg of patient body weight, e.g., about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg of patient body weight. The unit dose of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof of the present disclosure can be about 0.1 mg to 100 mg, e.g., about 1 mg to 5 mg, about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, or about 100 mg to about 150 mg.

[0267] When a series of doses is administered, they can be administered, for example, about daily, about weekly, about every two weeks, about every three weeks, about every four weeks (monthly), about every two months, about every three months (quarterly), or about every six months. The doses can be continued, for example, until disease progression, an adverse event occurs, or another time as determined by a physician. For example, fixed doses can be administered from about 2, 3, or 4 times, up to about 17 or more times.

[0268] The amount effective for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, the method, route and dose of administration, and the severity of side effects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0269] If necessary, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be incorporated into a composition containing a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the injection site. Additionally, pulmonary administration can also be employed, for example, by use of an inhalant or nebulizer and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and WO 1992 / 19244, WO 1997 / 32572, WO 1997 / 44013, WO 1998 / 31346, and WO 1999 / 66903, each of which is incorporated herein by reference in its entirety.

[0270] Multispecific antibodies of the present disclosure may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be recognized by those of skill in the art, the route and / or mode of administration will vary depending on the desired results. Routes of administration for a selected antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. Parenteral administration can refer to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, compositions of the present disclosure may be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

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

[0272] The multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof can be administered via any of the above routes using, for example, an injection device, an injection pen, a vial and syringe, a prefilled syringe, an autoinjector, an infusion pump, a patch pump, an infusion bag and needle, etc. If the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof are administered in a controlled- or sustained-release system, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). Polymeric materials can be used to achieve controlled or sustained release of the therapeutic agents of the present disclosure (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7:190). 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; WO 1999 / 15154; and WO 1999 / 20253.Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in sustained-release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Controlled- or sustained-release systems can be placed in proximity to the prophylactic or therapeutic target, thus requiring only a small systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0273] Controlled-release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to make sustained-release formulations comprising one or more multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure. See, for example, U.S. Pat. No. 4,526,938, International Publication No. WO 1991 / 05548, International Publication No. WO 1996 / 20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, all of which are incorporated herein by reference in their entireties. See, al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760.

[0274] When the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered topically, they can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous semi-solid or solid form containing a carrier or one or more excipients compatible with topical application, and in some cases having a dynamic viscosity greater than that of water, is typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure, as needed. Other suitable topical dosage forms include sprayable aerosol preparations, in some cases, where the active ingredient is combined with a solid or liquid inert carrier and is packaged in a mixture with a pressurized volatile substance (for example, gaseous propellant, such as Freon) or in a squeeze bottle.If necessary, moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms.Examples of such additional ingredients are known in the art.

[0275] When a composition comprising a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure is administered intranasally, it can be formulated in the form of an aerosol, spray, mist, or drops. In particular, prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0276] Multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof can also be cyclically administered to a patient.

[0277] In certain embodiments, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport to specific cells or organs, thus enhancing targeted drug delivery (see, e.g., Ranade VV (1989) J. Clin. Pharmacol. 29:685). Representative targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman 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) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); K. Keinanen; M. L. Laukkanen (1994) FEBS See also Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0278] The present application also provides protocols for co-administration or treatment of patients using pharmaceutical compositions comprising the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure in combination with other therapies or therapeutic agents. Methods for co-administration or co-treatment with additional therapeutic agents, such as cytokines, steroids, chemotherapeutics, antibiotics, or radiation, are known in the art (see, e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Philadelphia, Pa.). An effective amount of a therapeutic agent may reduce disease symptoms by at least 10%; at least 20%; at least about 30%; at least 40% or at least 50%.

[0279] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise one or more additional therapeutic agents.

[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 find therapeutic use in treating a variety of human diseases, e.g., pathological disorders mediated by IL-13 and IL-18, such as autoimmune and inflammatory diseases or conditions involving IL-13 and / or IL-18 dysregulation (e.g., inappropriate expression, expression levels, signaling, etc.). In one embodiment, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are used in the treatment of atopic dermatitis.

[0282] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are useful for treating, reducing the likelihood of, or ameliorating pathological disorders mediated by IL-13 and IL-18. The phrase "pathological disorders mediated by IL-13 and IL-18" encompasses all diseases and medical conditions in which IL-13 and IL-18 are involved, directly or indirectly, in the causation, development, progression, persistence, or pathology of the disease or condition. Thus, these terms include conditions associated with or characterized by aberrant IL-13 and IL-18 levels and / or diseases or conditions that may be treated by reducing or inhibiting IL-13- and IL-18-induced activity in target cells or tissues. Pathological disorders mediated by IL-13 and IL-18 include autoimmune diseases and / or inflammatory conditions and disorders having an IL-13 and IL-18 component.

[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-18 signaling.

[0284] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are useful for, but not limited to, the treatment, prevention, or amelioration of autoimmune diseases and / or inflammatory conditions and disorders, particularly inflammatory conditions with an etiology that includes an autoimmune component. In one aspect, the present disclosure provides a method of treating an autoimmune disease. In one aspect, the present disclosure provides a method of treating an inflammatory disease or condition. In one aspect, the subject of treatment is a human.

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

[0286] Provided herein are multispecific antibodies (e.g., bispecific antibodies) of the present disclosure or fragments thereof and pharmaceutical compositions as described herein for use in the treatment and / or prevention of moderate to severe AD.

[0287] The term "atopic dermatitis" (AD) or "eczema," as used herein, refers to an inflammatory skin disease characterized by intense pruritus (e.g., severe itching) and scaly, dry, eczematous lesions. The term "atopic dermatitis" or "eczema" includes, but is not limited to, AD (eczema) caused by or associated with epidermal barrier dysfunction, allergies (e.g., skin allergies, allergies to certain foods, pollen, mold, house dust mites, animals, etc.), exposure to radiation, and / or asthma. The present disclosure encompasses methods of treating patients with mild, moderate-severe, or severe AD. As used herein, "moderate-severe AD" is characterized by extensive, intensely pruritic skin lesions that are often complicated by persistent bacterial, viral, or fungal infections. Moderate-severe AD also includes chronic AD in patients. Often, chronic lesions include thickened plaques, lichenification, and fibrous papules of the skin. Patients with moderate to severe AD generally have more than 10% or more than 20% of their body's skin affected, or even 10% of their skin area affected, in addition to lesions in the eyes, hands, and body folds. Patients with moderate to severe AD generally have (i) an Investigator's Global Assessment (IGA) score of 3 or 4, (ii) an Eczema Area and Severity Index (EASI) score of at least 10, preferably at least 12, and (iii) pruritus. Moderate to severe AD is also considered to be present in patients who require frequent treatment with topical corticosteroids. Patients may also be said to have moderate to severe AD if they are resistant or refractory to treatment with either topical corticosteroids or calcineurin inhibitors or any other commonly used therapeutic agent known in the art.

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

[0289] As used herein, the term "therapeutically effective serum level" refers to a serum level of a therapeutic agent (e.g., a bispecific antibody) in a subject that is sufficient to reduce the severity of a condition, disorder, or disease and / or its associated symptoms and / or the duration and / or ameliorate the condition, disorder, or disease and / or its associated symptoms. In some embodiments, "therapeutically effective serum level" as used herein also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof in the serum of a subject that achieves a specified result, such as an improvement in an AD-related parameter, such as a decrease in the Physician's Global Assessment (IGA) score; a decrease from baseline in the Dermatology-Related Quality of Life Index (DLQI); a decrease from baseline in the Patient's Global Impression of Severity (PGIS); a decrease from baseline in the Patient's Global Impression of Change (PGIC); a decrease in the Atopic Dermatitis Skin Surface Area Involvement (BSA) score; a decrease in the Eczema Area and Severity Index (EASI) score; a decrease in the SCORAD score; and / or a decrease in the Itch Numerical Rating Scale (NRS) score.

[0290] In some embodiments, a "therapeutically effective serum level," as used herein, also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof in the serum of a subject that achieves a specified result, e.g., a decrease in the expression level of one or more AD-associated biomarkers, particularly one or more AD-associated biomarkers selected from the list consisting of CCL17 / TARC, IgE (e.g., serum IgE), CCL26 / Eotaxin-3, CCL22 / MDC, hsCRP, 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-18BP), as compared to the level before treatment with the multispecific antibody (e.g., bispecific antibody) or fragment thereof.

[0291] Suitably, the uses and methods of the present disclosure comprise administering the multispecific antibody (e.g., bispecific antibody) or fragment thereof once per week, once per two weeks, once per three weeks, once per four weeks, once per eight weeks or once per twelve weeks. According to certain exemplary embodiments, the uses and methods of the present disclosure comprise administering the multispecific antibody (e.g., bispecific antibody) or fragment thereof once per four weeks.

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

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

[0294] In certain embodiments, provided herein is a method of treating an IgE-mediated disorder in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody (e.g., a bispecific antibody) of the present disclosure, or a fragment thereof, wherein the antibody or fragment thereof inhibits the binding of IL13 to its receptor and inhibits one or more functions associated with the binding of an interleukin to the receptor.

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

[0296] Administered "in combination," with respect to additional therapeutic agents, means that two (or more) different treatments are delivered to a subject during the course of the subject's illness associated with a disorder, e.g., two or more treatments are delivered after the subject is diagnosed with a disorder and before the treatment is stopped when the disorder is cured or eliminated or for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, such that there is an overlap in the period of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, in either case, the treatments are more effective because they are administered in combination. For example, the second treatment is more effective, e.g., a comparable effect is seen with a lower amount of the second treatment, or the second treatment alleviates symptoms to a greater extent than would be seen if the second treatment were administered without the first treatment, or a similar condition is seen with the first treatment. In some embodiments, delivery results in a reduction in symptoms or other parameters associated with the disorder that is greater than that observed when one treatment is delivered in the absence of the other. The effect of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0297] The term "concurrently" is not limited to administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that pharmaceutical compositions comprising a multispecific antibody (e.g., bispecific antibody) of the present disclosure or fragments thereof are administered to a subject within a time interval that allows the molecules of the present disclosure to act together with the additional therapeutic agent to provide an improved benefit over if they were not administered. For example, each treatment can be administered to a subject at the same time or sequentially in any order at different time points, but if not administered simultaneously, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each treatment can be administered separately to a subject in any suitable form and by any suitable route.

[0298] Additional therapeutic agents (e.g., additional prophylactic or therapeutic agents) that may be administered in combination with a molecule of the present application may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, about 12 to 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 48 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart from the molecule or fragment thereof of the present disclosure. In other embodiments, two or more additional therapeutic agents are administered to the patient within the same patient visit.

[0299] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure and the additional therapeutic agent can be administered simultaneously, in the same or separate disclosed pharmaceutical compositions, or sequentially. In the case of sequential administration, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be administered first and the additional agent can be administered second, or the order of administration can be reversed. The additional therapeutic agent can be administered to the subject by the same or a different route of administration compared to the disclosed multispecific binding molecules and fragments.

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

[0301] The additional therapeutic agents of the combination therapies disclosed herein may also be administered cyclically. Combination cycling therapy involves administering a first therapy (a first prophylactic or therapeutic agent) for a period of time, followed by a second therapy (a second prophylactic or therapeutic agent) for a period of time, repeating this sequential administration (i.e., cycling) to reduce the development of resistance to one of the therapies (e.g., agents), avoid or alleviate side effects of one of the therapies (e.g., agents), and / or improve the efficacy of the therapies.

[0302] When administered in combination, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure and the additional therapeutic agent (e.g., second or third agent), or all, can be administered in amounts or dosages that are greater than, less than, or the same as the amount or dosage of each agent used individually, e.g., as monotherapy. In certain embodiments, the administered amount or dosage of the multispecific binding molecule, e.g., bispecific molecule, e.g., bispecific antibody-like molecule as described herein, additional agent (e.g., second or third agent), or all, is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as monotherapy. In other embodiments, the amount or dosage of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof, additional agent (e.g., a second or third agent), or all of the disclosed multispecific antibodies (e.g., bispecific antibodies) that produces a desired effect (e.g., treating an autoimmune or inflammatory disease or condition) is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less) than the amount or dosage of each agent used individually, e.g., as monotherapy, required to achieve the same therapeutic effect.

[0303] Preferably, the additional therapeutic agent (e.g., a second or third agent) is an AD agent, such as a small molecule, a biological therapy, or an agent that uses an AD modality, such as topical therapy, systemic therapy, phototherapy, including phototherapy and combinations thereof. An "AD agent" can be a cream, ointment, lotion, gel, or spray (e.g., a low- to medium-potency corticosteroid [Groups IV to VII according to WHO guidelines, Bolognia JL, Jorizzo JL, Schaffer JV. Glucocorticosteroids. Dermatology. 3rd ed. 2012. Ch 125, 2075-88; Ference JD, Last AR. Choosing topical cocorticosteroids. Am Fam Physician. 2009 Jan 12, 2010]). 15;79(2):135-40]); over-the-counter (OTC) emollients and medical devices or so-called barrier creams (such as atopiclair); and lubricants for the treatment of itch and / or pain, for example, anti-itch lotions containing menthol, pramoxine, or antihistamines; local anesthetics, systemic agents (for example, biological agents, such as IL-4R inhibitors, for example, dupilumab; IL-13Ra1 inhibitors, for example, ASLAN-004; IL-13Ra2 inhibitors; IL-31 inhibitors, for example, nemolizumab; TNF-alpha inhibitors, for example, adalimumab, infliximab, certolizumab, and etanercept, alefacept; IL-1a inhibitors, for example, verumumab, kimab (MABp1), etc.; IL-23 inhibitors, such as briakinumab, ustekinumab, guselkumab, risankizumab, tildrakizumab, etc.; IL-17 inhibitors, such as brodalumab and ixekizumab; CD11a inhibitors, such as efalizumab; IL-22 inhibitors, such as featherimumab, IL-22 binding proteins; IL-5 inhibitors, such as mepolizumab and benralizumab; synthetic forms of IL-2, such as aldesleukin; recombinant IL-2 approaches targeting the interleukin-2 receptor complex, such as LY3471851; OSMR inhibitors, such as KPL-716; VAP-1 inhibitors; OX-40 or OX40L inhibitors, such as GBR830 and KY1005;IgE inhibitors, such as omalizumab and ligelizumab; TSLP inhibitors, such as tezepelumab; IL-33 inhibitors, such as MEDI3506; IL-36 inhibitors, such as spesolimab and ANB019; B-cell modulating approaches, such as rituximab and ocrelizumab; non-biological immunomodulatory treatments, such as cyclosporine and other calcineurin inhibitors, JAK inhibitors, such as tofacitinib, upadacitinib, abrocitinib, baricitinib, etc.; TYK2 inhibitors, such as deuclavacitinib, etc.; methotrexate; PDE4 inhibitors, such as apremilast; Siglec inhibitors, such as AK-002; S1P agonists or antagonists, such as etrasimod or SCD-044; BTK inhibitors, such as TAS-5315, IRAK4 antagonists and CCR4-inhibitory approaches, such as RPT-193; systemic corticosteroids, cyclophosphamide, sulfasalazine, azathioprine, mycophenolate mofetil, dapsone, hydroxychloroquine); retinoids (e.g., alitretinoin); leukotriene inhibitors or anti-leukotrienes, such as montelukast, pranlukast, or zafirlukast, and 5-LO inhibitors, such as zileuton, and LTA4H inhibitors, such as acebilstat, intralesional corticosteroid injections; phototherapy (e.g., high doses of UVB and UVA). Photochemotherapy (e.g., psoralens and UVA (PUVA)); topical calcineurin inhibitors (cyclosporine, tacrolimus, pimecrolimus) or topical PDE4 inhibitors such as crisaborole, difamilast, or roflumilast; topical JAK inhibitors such as ruxolitinib, delgocitinib, or topical vitamin D analogues and topical aryl hydrocarbon receptor (AhR) inhibitors such as benvitimod / tapinarof; topical corticosteroids of high to very high potency (WHO class I, II, III);Antifungal agents with known anti-inflammatory properties, such as griseofulvin, itraconazole, betamethasone, dexamethasone, INCB018424, triamcinolone, apremilast, turmeric paste, glucosamine sulfate, triamcinolone acetonide, sesame oil, betamethasone dipropionate, clobetasol propionate, probiotics (e.g., Bifidobacterium animalis subsp. lactis HN019, Lactobacillus reuteri), reuteri), omega-3, prednisone, prednisolone, platelet-rich plasma, Orabase paste, lycopene, topical chamomile, green tea, CO2 laser treatment, allergen-specific immunotherapy, polybiotics, photobiomodulation, metronidazole, doxycycline, minocycline, cedar honey, purslane, curcuminoids, alefacept, hexaminolevulinate, hydroxychloroquine, Adcortil, efalizumab, fluocinolone, coenzyme Q10 mucoadhesive tablets, chamaemelum nobile, sirolimus, tacrolimus, King Kushuang decoction, NSAID topical rinse, NSAIDs, quercetin, NAVS naphthalane, bulcrolfol, bupivacaine, and oatmeal baths. Suitably, the topical AD therapy is an atopic dermatitis prescription drug therapy including, but not limited to, topical steroids, e.g., corticosteroids, tacrolimus, cyclophosphamide, azathioprine, methotrexate, mycophenolate mofetil, apremilast, calcineurin inhibitors, e.g., topical calcineurin inhibitors, phosphodiesterase 4 (PDE4) inhibitors, e.g., topical PDE4 inhibitors, e.g., crisaborole, adrenocorticotropic hormone analogs, dupilumab, etanercept, adalimumab, infliximab, omalizumab, secukinumab;

[0304] E. Kit The present disclosure also encompasses kits for treating patients with pathological disorders mediated by IL-13 and IL-18, such as autoimmune or inflammatory disorders or conditions. Such kits comprise a therapeutically effective amount of a multispecific antibody of the present disclosure. Furthermore, such kits may include a means for administering the multispecific antibody of the present disclosure (e.g., an autoinjector, syringes and vials, prefilled syringes, prefilled pens) and instructions for use. These kits may contain additional therapeutic agents (described above) for treating those with pathological disorders mediated by IL-13 and IL-18, such as autoimmune or inflammatory disorders or conditions. Such kits may also include instructions for administering the multispecific antibody of the present disclosure to treat patients. Such instructions may provide the dose, route of administration, regimen, and total treatment duration for use with the enclosed multispecific antibody of the present disclosure.

[0305] The phrase "means for administration" is used to refer to any available device for systemically administering a drug to a patient, including, but not limited to, pre-filled syringes, vials and syringes, injection pens, autoinjectors, IV drips and bags, infusion pumps, patches, infusion bags and needles, etc. Using such items, a patient can self-administer a drug (i.e., administer the drug without the assistance of a physician) or a physician can administer the drug. [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 of ordinary skill in the art and are encompassed by the appended claims.

[0307] Example 1: Generation of IL-13 / IL-18 bispecific antibodies in CHO cell lines 1. Expression Vector Construction The vectors used in the examples consist of the following elements: hCMV promoter / enhancers driving expression of the individual genes required for assembly of the antibody construct, a polyadenylation signal (polyA), a folate receptor (FolR, FAR), DHFR, puromycin and / or hygromycin genes as selectable markers, an E. coli origin of replication and a beta-lactamase gene for ampicillin resistance to allow amplification in bacteria. Different plasmid devices were evaluated and further details are provided in the figures.

[0308] Figure 2 is a schematic diagram of the NVS standard plasmids A–D. Plasmids A and C encode the expression of anti-IL13 kappa LC and anti-IL13 knob HC; plasmids B and D encode the expression of anti-IL18 lambda LC and anti-IL18 hole HC. Expression of each individual protein chain is driven by a separate CMV promoter. Linearized plasmids A and B or C and D were simultaneously cotransfected into CHO-C8TD parental cells. Cells were selected in the first selection round using the selectable markers DHFR and FAR. For the second selection, additional selectable markers, hygromycin and puromycin, may be used. The plasmids carry leaky-stop transmembrane technology (LS-TM) information to enable staining and enrichment of highly productive clones during FACS-assisted single-cell sorting. Plasmids C and D were designed with lower sequence homology to minimize the risk of homologous recombination by deleting the repetitive partial phage f1 region between the expression cassettes encoding the LC and HC. Plasmid D carries a different codon optimization and signal peptide for IL18 hole HC, while in plasmids A and B, the DNA sequences of IL18 knob CH and IL13 hole CH differ only in the base that encodes the KiH mutation.

[0309] Figure 3 shows a schematic diagram of the NVS Furin-2A peptide (F2A) plasmids E and F. The F2A technology allows for the combinatorial expression of multiple protein chains from a single promoter. On plasmids E and F, the first expression cassette encodes the anti-IL18 lambda LC and anti-IL18 hole HC, and the second expression cassette encodes the anti-IL13 kappa LC and anti-IL13 knob HC. Linearized plasmids E or F were transfected into CHO-C8TD parental cells. Cells were selected using the selectable markers DHFR and FAR. Plasmid F was designed with lower sequence homology to minimize the risk of homologous recombination by using different codon optimization for the IL18 hole HC and IL13 knob HC, while on plasmid E, the DNA sequences of the IL18 knob CH and IL13 hole CH differ only at the base encoding the KiH mutation.

[0310] Figure 4 is a schematic diagram of the adapted NVS standard plasmids G and H. Plasmids G and / or H are used for supertransfection of pools expressing plasmids E or F to increase the number of integrated plasmids. Plasmid G encodes the expression of anti-IL13 kappa LC and anti-IL13 knob HC; plasmid H encodes the expression of anti-IL18 lambda LC and anti-IL18 hole HC. Expression of each individual protein chain is driven by a separate CMV promoter. Different transfection and selection approaches were used: (I) Plasmids G and H were simultaneously cotransfected into CHO-C8TD parental cells and selected using the selectable markers hygromycin and puromycin; (II) Plasmid G was transfected in the first round and selected using hygromycin, and / or Plasmid H was transfected in the second round and selected using puromycin; or (III) Plasmid H was transfected in the first round and selected using puromycin, and / or Plasmid G was transfected in the second round and selected using hygromycin. Plasmids G and H carry leaky-stop transmembrane technology (LS-TM) to enable staining and enrichment of highly productive clones during FACS-assisted single-cell sorting. To reduce sequence homology and minimize the risk of homologous recombination, a partial phage f1 region was deleted between the expression cassettes encoding LC and HC on plasmids G and H, and the codon optimization of LC and HC differed from each other and from plasmids E and F.

[0311] Figure 5 is a schematic diagram of the NVS Furin-2A peptide (F2A) plasmid I, which has a different protein chain combination in the expression cassette 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. Linearized plasmid I was transfected into CHO-C8TD parent cells. Cells were selected using the selectable markers DHFR and FAR. A partial phage f1 region between the two expression cassettes was deleted, and differential codon optimization of CH was used to reduce sequence homology and minimize the risk of homologous recombination.

[0312] 2. Cell Lines, Culture, Transfection and Selection The parent CHO cell line was used as the host cell line for the production of antibody constructs. The host cell line was derived from the CHO-K1 cell line. A single vial from the CHO cell line was used to prepare the recombinant cell line. The CHO cell line was cultured in suspension in a proprietary, chemically defined medium in shake flasks in a non-humidified shake cabinet at 150 rpm, 10% CO2, and 36.5°C. Cell viability and growth rate were monitored using an automated system (ViCell, Beckman Coulter). Cells were passaged into fresh medium two to three times a week and maintained in logarithmic growth phase.

[0313] The SwaI-linearized expression plasmids encoding the antibody constructs were transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). Transfection reactions were carried out in chemically defined medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in exponential growth phase with cell viability greater than 95%. 5 × 10 cells 6Transfections were performed at 1000 cells / transfection. Immediately after transfection, cells were transferred to shake flasks containing chemically defined medium. The cell pool was incubated at 36.5°C and 10% CO for 48 hours before starting the selection process.

[0314] Selection procedures were performed using selectable markers encoded by the individual expression vectors. The first transfection and selection round was performed using the folate receptor and DHFR. Both proteins are involved in the same molecular pathway; FolR transports folate and the folate analog MTX into cells, and DHFR converts them into important precursors for purine and methionine synthesis. Combining their selection principles, a specific, strong selection regime can be employed to enrich for recombinant cells expressing both recombinant proteins.

[0315] Forty-eight hours after transfection and growth under low folate conditions, further selection pressure was applied by adding 10 nM MTX to the chemically defined medium. After pool harvest, the harvested cells were frozen in medium supplemented with 7.5% DMSO and other materials prepared for further analysis as described below.

[0316] Depending on the design of the plasmids used for the first transfection and selection round, different possibilities for progression emerged: either pools were used directly for single-cell cloning as described below, or a second selection or supertransfection was performed before single-cell cloning.

[0317] For the second selection, the recovered pool was maintained under low folate conditions with 10 nM MTX, and further selection pressure was applied by adding 0.5 μg / ml puromycin and 0.8 mg / ml hygromycin to the chemically defined medium. After pool recovery, the cells were frozen in medium supplemented with 7.5% DMSO and the materials prepared for further analysis as described below.

[0318] For supertransfection, SwaI-linearized expression plasmids encoding antibody constructs were transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). Transfection reactions were performed in chemically defined medium according to the manufacturer's instructions. CHO pools recovered from selection after transfection were used for the second or third transfection. Transfections were performed as described above, and after 48 hours at 36.5°C and 10% CO2, selection was initiated by adding 0.5 μg / ml puromycin, 0.8 mg / ml hygromycin, or both to the chemically defined medium. After pool recovery, cells were frozen in medium supplemented with 7.5% DMSO and the materials prepared for further analysis as described below.

[0319] 3. Single-cell Cloning After selection, single cell cloning was performed to obtain cell lines of monoclonal origin either using a Cytena cell printer instrument or by flow cytometry.

[0320] The Cytena cell printer contains a disposable dispensing cartridge containing a microfluidic chip into which a cell suspension is placed. From this cartridge, droplets are ejected through a nozzle onto a 96-well plate. During this process, an image of the nozzle area is recorded by a microscope system. Automated image analysis algorithms detect cells on the image and classify them according to morphological criteria such as size and roundness. Based on image analysis of the droplet formation area at the nozzle outlet, droplets containing a single cell are transferred to individual wells of the 96-well plate, while droplets without a single cell (empty droplets or droplets with multiple cells) are sent to waste.

[0321] Prior to flow cytometry, cells were stained with FITC-labeled in-house produced BD Ab directed against the Fc portion of cell surface-attached ABC123 to facilitate selection of high-producing clones.

[0322] Single-cell cloning was performed using a Sony Cell Sorter instrument equipped with a 96-well plate holder using 100 μm disposable sorting tips. To ensure that only single cells were sorted, the settings were adjusted for single-cell mode, 3-droplet sorting. Using these settings, only droplets containing cells were sorted if both the previous and subsequent droplets were empty. To improve the likelihood that each droplet contained no more than one single cell, the cell concentration and flow rate were optimized, at the expense of yield. Multiple gates were set to select for strongly fluorescent, live single cells.

[0323] Single cells were sorted into individual wells of a 96-well plate using a Cytena cell printer or a Sony Cell Sorter, and high-resolution microscopic images of each well were then taken to reveal single clones and validate the single-cell cloning procedure.

[0324] After single-cell cloning, clones were expanded and characterized for productivity and bioprocess suitability, as well as transgene integration and expression. Primary seed lot (PSL) vials were prepared by freezing cells from top-performing clones in medium supplemented with 7.5% dimethyl sulfoxide (DMSO), and the PSL of the final selected clone was used for MCB production.

[0325] 4. Upstream processing After selection, material was produced in shake flask fed-batch cultures. The fed-batch cultures were inoculated at a defined cell seeding density, and the addition of a unique feed solution began on day 3, with the culture temperature shifted to 33°C on day 5. In-process controls were performed to monitor the concentration of the antibody construct during cultivation. Individual cultures were cultivated for 14 days. At the end of the cultivation process, cells were separated from the culture supernatant by centrifugation, followed by sterile filtration, before further downstream processing and analytical characterization. The volumetric productivity of selected pools was determined in the cell culture supernatant by Protein A HPLC or RP-LC to determine all product types and associated impurities bearing the Fc portion.

[0326] Example 2. LC-MS screening and purity assessment of IL-13 / IL-18 bispecific antibodies 100 μg of purified bispecific mAb was diluted to 1 mg / ml in 20 mM Tris-HCl pH 7.5 and deglycosylated using 2 μl PNGaseF enzyme (New England Biolabs) at 37°C for 4 h. The deglycosylated sample was subjected to LC-MS using a Waters ACQUITY UPLC Class 1000 with a PLRP-S RP column (3 μm, 2.1 × 150 mm, 300 Å, Agilent) and a TripleTOF 6600 with a dual-spray ion source (Sciex) mass analyzer. The eluents were A: 0.1% TFA in water and B: 70% isopropanol, 20% acetonitrile, 10% water, and 0.09% TFA. The column was set to 60°C. The flow rate was 0.2 ml / min. The protein was eluted with a 40-minute gradient: 0–4 min, 35% B; 4–28 min, 35–50% B; 28–29 min, 50–80% B; 29–34 min, 80% B; 34–35 min, 80–35% B; and 35–40 min, 35% B. UV chromatograms were recorded at 214 nm, and MS data acquisition was performed on positive ES (+). Data were acquired using Analyst software TF1.7 (ABSciex) and analyzed using BioPharmaView (version 3.0, ABSciex) and PeakView (version 2.2, ABSciex) software. Identification and relative quantification of bbmAb species and mismatched variants were based on agreement between theoretically predicted masses and relative mass signal intensities in deconvoluted mass spectra. The results are shown in Table 3.

[0327] A wide range of reasonable heavy chain heterodimerization, heavy chain homodimerization, and half-molecules were detected. The desired degree of heterodimerization was high at >95%, ideally approaching 100%. To identify candidates with >95% heterodimerization, bbmAb1, bbmAb2, bbmAb5, bbmAb4, and bbmAb3 were generated. Only bbmAb1, bbmAb2, and bbmAb5 exhibited >95% heterodimerization, while bbmAb4 and bbmAb3 exhibited <95% heterodimerization and were unsuitable for therapeutic development.

[0328] [Table 23]

[0329] Example 3. Evaluation of the thermal stability of the CH2 and Fab domains of IL-13 / IL-18 bispecific antibodies The stability of an antibody greatly influences its performance (i.e., its specificity and affinity). Therefore, stability is a major concern for researchers and manufacturers, especially with the increasing use of antibodies in therapeutic, diagnostic, and rapid analysis platforms. Important parameters are the thermal stability and melting temperature (T) of the CH2 and Fab domains. m ) Protein melting temperature (T m ) is defined as the temperature at which a protein denatures. m The value and unfolded fraction can predict the aggregation rate (Robinson et al., 2018).

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

[0331] The melting temperatures of the CH2 and Fab domains of the modified antibodies are listed in Table 4, and the melting curves of bbmAb1, bbmAb2, bbmAb5 and bbmAb4 are shown in FIG.

[0332] CH2 T m reflects the unfolding of the CH2 domain (Johnson, 2012). As shown in Table 4, all candidates showed similar T m showed.

[0333] Higher melting temperatures (67-68°C) were observed for the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 compared to the anti-13 Fab domain of bbmAb3 (64°C). Because the CDR sequences in the anti-13 Fab domain of bbmAb3 are different from the other candidates, this result indicates that the specific CDR sequences in the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 may result in more stable molecules with improved thermal stability.

[0334] [Table 24]

[0335] Example 4. Affinity for recombinant human and cynomolgus monkey IL-13 and IL-18 measured by SET Determination of the equilibrium dissociation constant (KD) was achieved by solution equilibrium titration (SET) measurements described as follows.

[0336] Twenty-two serial 2n dilutions of antigen (maximum concentrations: hsIL-18, 20 nM; cyIL-18, 40 nM; hsIL-13, 20 nM) were prepared in sample buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.02% Tween-20) and constant concentrations of antibody were added (4 pM for hsIL-13 readings, either 10 or 4 pM for hsIL-18 readings, and 5 pM for cyIL-18 readings).

[0337] A volume of 60 μl / well of each antigen-antibody mixture was dispensed in duplicate into a 384-well polypropylene microtiter plate (MTP). Sample buffer served as a negative control, and a sample containing only antibody served as a positive control (maximum electrochemiluminescence signal without antigen, B max The plates were sealed and incubated overnight (at least 16 h) at room temperature (RT) on a shaker.

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

[0339] [Table 25]

[0340] hsIL-18 and cyIL-18 readings: After blocking with 50 μl / well of blocking buffer (PBS containing 5% BSA) for at least 1 hour (h) at room temperature (RT) or a subsequent washing step (TBST, TBS containing 0.05% Tween 20), streptavidin Multi-Array® 384-well plates (MSD L21SA-5) were coated with 30 μl / well of biotinylated human IL-18 (0.2 μg / ml, PBS) and incubated on a shaker at RT for at least 1 h.

[0341] hsIL-13 readings: After blocking with 50 μl / well of blocking buffer (PBS containing 5% BSA) for at least 1 hour (h) at room temperature (RT) or a subsequent washing step (TBST, TBS containing 0.05% Tween 20), streptavidin Multi-Array® 384-well plates (MSD L21SA-5) were coated with 30 μl / well of biotinylated human IL-13 (0.2 μg / ml, PBS) 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 transferred from the polypropylene MTP to the coated MSD plate and incubated for 20 minutes at room temperature. After a further washing step, 30 μl of sulfo-tagged anti-hsIgG detection antibody (0.5 μg / ml) diluted in sample buffer was added to each well and incubated for 30 minutes at room temperature on a shaker. The MSD plate was washed, and 35 μl / well MSD read buffer was added and incubated for 5 minutes at room temperature. The electrochemiluminescence (ECL) signal was generated and measured by an MSD Sector Imager 6000.

[0343] SET data were exported to Xlfit, an MS Excel add-in software. The average ECL signal was calculated from duplicate measurements within each assay. Data were baseline adjusted by subtracting the lowest value from all data points and plotting them against the corresponding antigen concentration to generate a titration curve. K was calculated by fitting the plot using the following 1:1 binding model for knobs-in-holes bispecific Abs: D value was determined.

number

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

[0345] [Table 26]

[0346] Example 5. Affinity for recombinant human and cynomolgus IL-13 and IL-18 measured by SPR Determination of kinetic binding parameters was achieved by surface plasmon resonance (SPR) measurements using the optical biosensor Biacore™ T200 (http: / / www.cytivalifesciences.com).

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

[0348] The surface of a C1 sensor chip (Cytiva #BR100535) was prepared for indirect binding of antibodies by immobilization of 80 μg / mL of NeutrAvidin™ (Thermo Scientific #31000) in immobilization buffer (10 mM sodium acetate pH 5.0) on the chip surface via amine coupling, followed by saturation of NeutrAvidin™ with biotinylated Protein G (Sigma #P8045) at 5 μg / mL in HBS-EP buffer.

[0349] Antibodies were diluted in blank buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20; Cytiva #BR100669) to a final concentration of 10 μg / mL. Affinity measurements were performed to determine the kinetic constants of bbmAb2 or bbmAb1 against recombinant huIL-13 (2-fold increasing concentrations from 0.25 to 8.0 nM) and recombinant huIL-18 (2-fold increasing concentrations from 0.125 to 32 nM). The surface was regenerated between cycles using 10 mM glycine, pH 1.5, 0.5% Tween 20.

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

[0351] [Table 27]

[0352] Kinetic traces were evaluated with Biacore™ T200 Control Software v2.0.1. A full set of these traces at increasing concentrations is called a run. The set of traces in a run was analyzed using a 1:1 binding model (R) provided by Biacore T200 Evaluation Software v3.0. maxA zero concentration sample (blank) was included in each run to allow for double referencing.

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

[0354] [Table 28]

[0355] Example 6. Affinity for human Fc receptors measured by surface plasmon resonance (SPR) spectroscopy To characterize the binding of the engineered IL-13 / IL-18 bispecific antibody to human Fc receptors, direct binding assays were performed using surface plasmon resonance (SPR) spectroscopy. SPR is a commonly applied technique for affinity and kinetic analysis of protein-protein, protein-peptide, protein-DNA, and protein-small molecule interactions, as it provides a continuous readout of complex formation and dissociation, allowing for the analysis of interactions between analytes in solution and ligands linked to a sensor chip surface.

[0356] 1. In vitro Fcγ receptor binding assay Affinity determination of human Fcγ receptor binding to the Fc fragment of bbmAb2 (IL18 knob YTE IL13 hole YTE1 + 1 bsAb) was performed on a Biacore T200 instrument. Affinity determination of human Fcγ receptor binding to the Fc fragment of bbmAb5 (bispecific KiH LALA YTE) and bbmAb1 (IL18 hole YTE (mAb1) IL13 (mAb2) knob YTE1 + 1 bsAb) was performed on a Biacore 8K instrument. Samples were diluted to 5 μg / ml in 10 mM sodium acetate, pH 4.5, and immobilized to a density of approximately 750 resonance units on a CM5 sensor chip using the standard amine coupling procedure on the Biacore T200 instrument. A similar procedure was applied on the Biacore 8K instrument, but immobilization was performed at a density of approximately 1310 resonance units. On the Biacore T200, flow cell 1 served as the immobilized blank and reference. On the Biacore 8K, there were a total of eight channels, and flow cell 1 of each channel was left blank to serve as a reference surface. Human Fcγ receptors (CD64 / FcγRI, CD32a / FcγRIIA) were detected on all flow cells at a flow rate of 30 μl / min on the Biacore T200 or 50 μl / min on the Biacore 8K and a temperature of 25°C. R131 , CD32b / FcγRIIB, CD16a / FcγRIIIA V176 and FcγRIIIA F176 Kinetic binding data were collected by subsequent injection of a 1:2 dilution series of Fcγ receptors (CD16b / FcγRIIIB). Depending on the strength of the interaction, the Fcγ receptors were diluted in running buffer (PBS pH 7.4 with 0.005% Tween-20) at different concentration ranges (on a Biacore T200, we used FcγRI: 0.20-100 nM, FcγRIIA: 0.20-100 nM). R131 , FcγRIIB and FcγRIIIB: 7.81 to 4000 nM, FcγRIIIA V176: 1.95-1000nM and FcγRIIIA F176On the Biacore T200, FcγRI and FcγRIIIA were tested at 3.91-2000 nM; on the Biacore 8K, the same conditions were applied except for FcγRI, which was tested at 0.05-20 nM. V176 After each measurement cycle, the chip surface was regenerated with 10 mM glycine pH 2.0 solution for 30 seconds at 30 μl / min. On the Biacore 8K, the surface was regenerated with a single injection of 10 mM glycine at pH 2.0 for 30 seconds at a flow rate of 50 μl / min for hFcγRs. A zero concentration sample (blank run) was measured to allow double referencing during data evaluation. Duplicate injections of each sample and buffer blank were run across the entire surface. Data were evaluated using Biacore T200 evaluation software version 3.0 and Biacore 8K evaluation software (v.3.0.12.15655).

[0357] The raw data were double-referenced, i.e., the response of the measuring flow cell was corrected for the response of the reference flow cell, and in a second step the response of a blank injection was subtracted. The resulting sensorgrams were fitted using either a steady-state model or a 1:1 Langmuir model to determine the equilibrium dissociation constant (K D ) was calculated.

[0358] 2. In Vitro FcRn Receptor Binding Measurements Affinity determination of human FcRn receptor binding to the Fc fragment of bbmAb2 (IL18 (mAb1) knob YTE IL13 (mAb2) hole YTE1 + 1 bsAb) was performed on a Biacore T200 instrument. Affinity determination of human FcRn receptor binding to the Fc fragment of bbmAb5 (bispecific KiH LALA YTE) and bbmAb1 (IL18 hole YTE (mAb1) IL13 (mAb2) knob YTE1 + 1 bsAb) was performed on a Biacore 8K instrument. Samples were diluted to 5 μg / ml in 10 mM sodium acetate, pH 4.5, and immobilized on a CM5 sensor chip at a density of approximately 750 resonance units using the standard amine coupling procedure on the Biacore T200 instrument. A similar procedure was applied on the Biacore 8K instrument, but immobilization was at a density of approximately 1310 resonance units. On the Biacore T200, flow cell 1 was an immobilized blank and served as a reference. On the Biacore 8K, there were a total of eight channels, and the flow cell of each channel was left blank and served as a reference surface. Kinetic binding data were then collected on all flow cells at a flow rate of 50 μl / min and a temperature of 25°C by injecting a 1:2 dilution series of human FcRn receptor. pH-dependent binding was checked by diluting the FcRn receptor in two different running buffers to cover a concentration range of 4.88 to 2500 nM: PBS pH 5.8 with 0.005% Tween-20 and PBS pH 7.4 with 0.005% Tween-20. The chip surface was regenerated with PBS pH 7.4 with 0.005% Tween-20 for 120 s at a flow rate of 50 μl / min. A zero-concentration sample (blank run) was measured to allow for double referencing during data evaluation. Duplicate injections of each sample and buffer blank were run on all sides. Data were evaluated using Biacore T200 Evaluation Software version 3.0 and Biacore 8K Evaluation Software (v.3.0.12.15655).

[0359] The raw data were double-referenced, i.e., the response of the measurement flow cell was corrected to that of the reference flow cell, and in a second step the response of a blank injection was subtracted. The resulting sensorgrams were fitted using either a steady-state model or a 1:1 Langmuir model to determine the equilibrium dissociation constant (K D ) was calculated.

[0360] 3. Measurement of C1q Binding in Vitro Affinity determination of human C1q binding to bbmAb2 was performed on a Biacore T200 instrument. Affinity determination of human C1q binding to bbmAb5 and bbmAb1 was performed on a Biacore 8K instrument. Samples were diluted to 50 μg / ml in 10 mM sodium acetate buffer, pH 4.5, and immobilized on a CM5 sensor chip at a density of approximately 8,900 resonance units using the standard amine coupling procedure on the Biacore T200 instrument. A similar procedure was applied on the Biacore 8K instrument, but immobilization was at a density of approximately 9,400 resonance units. On the Biacore T200, flow cell 1 was the immobilized blank and served as the reference. There were a total of eight channels on the Biacore 8K, and the flow cell of each channel was left blank and served as the reference surface. Kinetic binding data were collected by subsequently injecting a 1:2 dilution series of human C1q over all flow cells at a flow rate of 30 μl / min and a temperature of 25°C. Human C1q was diluted in running buffer (HBS-EP + pH 7.4) at concentrations ranging from 0.49 nM to 250 nM. After each measurement cycle, the chip surface was regenerated with 50 mM NaOH for 30 s at a flow rate of 30 μl / min, including a 60 s stabilization period. A zero-concentration sample (blank run) was measured to allow double referencing during data evaluation. Duplicate injections of each sample and buffer blank were run on all surfaces. Data were evaluated using Biacore T200 Evaluation Software version 3.0 and Biacore 8K Evaluation Software (v. 3.0.12.15655).

[0361] The raw data were double-referenced, i.e., the response of the measurement flow cell was corrected to that of the reference flow cell, and in a second step the response of a blank injection was subtracted. The resulting sensorgrams were fitted using either a steady-state model to determine the equilibrium dissociation constant (K D ) was calculated.

[0362] Results showing the binding affinity of Fcγ and FcRn receptors in vitro The binding affinities of bbmAb1, bbmAb2 and bbmAb5 for 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 Mouse experiments were performed on Tg276 B6.Cg-Fcgrttm1DcrTg(CAG-FCGRT)276Dcr / DcrJ hemizygous mice derived from C57BL / 6 mice and purchased from Jackson Laboratory (USA). FcRn- / -hFcRn (lineage 276) Tg mice carry a null mutation for a transgene expressing the mouse gene and hFcRn α-chain under the control of the ubiquitous CAG promoter. All mice were naive males aged 8–12 weeks at the start of the study. For dosing, antibodies were prepared in phosphate-buffered saline (1x PBS, pH 7.3) and administered as a single intravenous dose of 10 mg / kg into the left lateral tail vein at a dose volume of 5 mL / kg. A total of three animal replicates were evaluated for each antibody using a serial sampling approach (pre-dose, 1 h, 1 d, 2 d, 3 d, 6 d, 9 d, 17 d, and 29 d) over the 29-day study period. Blood samples (30-50 μL) were collected in serum separator tubes and allowed to clot at room temperature for 20-30 minutes. Samples were then processed to obtain serum by centrifugation (2000 g, room temperature, 10 minutes). The resulting serum was stored at -80°C until analysis.

[0365] A quantitative sequential electrochemiluminescence immunoassay (ECLIA) was used to measure total antibodies in unknown mouse serum specimens, calibration standards (Cs), and quality control samples (QCs). The use of both targets for antibody capture and detection allowed this assay to detect the total amount of bispecific therapeutic antibodies using their binding sites. Therefore, antibodies were captured by biotinylated human IL-18 immobilized on SA-coated, blocked MSD plates. Ruthenium(II) tris-bipyridine-(4-methylsulfonate) NHS ester (MSD Sulfo-TAG™)-labeled cynomolgus monkey IL-13 was added and allowed to bind for detection, which is based on electrochemiluminescence (ECL) and was read on an ECL Sector Imager from MSD. The resulting ECL values ​​are proportional to the amount of drug present in the initial sample, as determined through 5PL regression analysis using a weighting factor of 1 / Y², where Y represents the ECL value of the corresponding calibration standard.

[0366] Using the above experimental approach in a humanized mouse model, we examined bbmAb1 and bbmAb2 and the Fc-silenced variants bbmAb6, bbmAb7, bbmAb8, and bbmAb9. bbmAb7 and bbmAb8 are Fc-silenced variants of bbmAb1 containing the L235C mutation (EU numbering). bbmAb6 is an Fc-silenced variant of bbmAb1 containing the L235C mutation but without the YTE mutation (EU numbering). bbmAb9 is an Fc-silenced variant of bbmAb1 containing the L235C / G236C mutation (EU numbering).

[0367] All the above mentioned antibodies showed C max The antibodies exhibited good exposure and typical PK curve shapes in the systemic circulation, with a short distribution phase and a long elimination phase, typical of antibodies with extended half-lives. The half-lives of the antibodies investigated herein were very similar and could be estimated from the terminal phase of the PK curves, ranging 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 (pBMCs) or human keratinocytes were treated with IL-18 or IL-13, respectively, and analyzed for differences in gene expression. While only minimal overlap between IL-13-induced and IL-18-induced gene signatures was observed (data not shown), both were shown to be elevated in lesional skin compared to non-lesional skin from published AD patient gene expression datasets (He et al. 2020: skin biopsies from five AD (lesional and non-lesional) and seven HV subjects; scRNA-seq data).

[0369] Mining a dataset in which lesional and non-lesional skin biopsy samples were collected from AD patients treated with an antibody that antagonizes IL-4 receptor alpha (anti-IL-4Rα), thus blocking both IL-13 and IL-4 signaling, revealed that the IL-13-induced gene signature was significantly downregulated by anti-IL-4Rα treatment; however, the IL-18-induced gene signature was not significantly downregulated by anti-IL-4Rα treatment (data not shown).

[0370] These results suggest that dual inhibition of both IL-13 and IL-18 may improve treatment outcomes compared to inhibition of IL-13 or IL-18 alone.

[0371] Four-mm skin biopsies were collected from 10 patients with atopic dermatitis. Four-mm skin biopsies from eight healthy volunteers were collected as controls. Each biopsy was cut into four small pieces and cultured ex vivo for 24 hours in 100 μl of medium containing either α-IL-18, α-IL-13, or both (150 μg / mL each). Cell culture supernatants were centrifuged at low speed to remove cells without lysis. Changes in protein expression were assessed using the Olink assay. Decreased protein expression in the supernatants of lesion biopsy samples compared to control samples showed a measurable decrease in anti-IL-13-treated samples compared to control, a decrease in anti-IL-18-treated samples compared to control, and a further decrease in anti-IL-18 / anti-IL-13-treated samples compared to control and compared to single-treatment samples (data not shown).

[0372] Example 8 Simultaneous Inhibition of IL-13 / IL-18 with Bispecific Antibodies Reveals Synergistic Suppression of the AD-Like Transcriptome method Eight-mm full-thickness skin biopsies were obtained from surgical waste from five individual donors and cultured in IMDM medium with 1% Pen / Strep and 10% knockout serum replacement in tissue culture inserts for 12-well plates. On day 0, biopsies were either injected with control (30 μL PBS) or activated with a mixture of anti-CD28 and anti-CD3 antibodies in 30 μL PBS at a final concentration of 500 μg each. To induce differentiation toward an AD-like transcriptome, biopsies (except for the control) were incubated in IMDM medium with a mixture of the following cytokines, each at 50 ng / mL: IL-4, IL-13, IL-33, TSLP, IL-18, and IL-31, for 6 days with medium changes on days 2, 4, and 5. During the 6-day induction period, induced biopsy samples were treated with 1 μM IgG1 isotype control antibody (AD+ isotype) carrying the LALA silencing mutation; 1 μM anti-IL13 antibody; 1 μM anti-IL18 antibody; or 1 μM anti-IL13 / IL18 bispecific antibody bbmAb1. On day 6, supernatants were collected and biopsies were aliquoted for histological and transcriptome analysis (Ampliseq whole transcriptome protocol).

[0373] Ampliseq normalized values ​​were imported into Qlucore Omics Explorer 3.8, and variables with values ​​below 0.5 were removed if applicable to 90% of samples, resulting in 16,719 variables. The threshold was set at 0.25, and values ​​were log2-transformed. A "disease transcriptome" was generated by comparing "AD isotype" samples with controls at q < 0.1 and FC > 2 in donor-corrected samples, resulting in 1,485 differentially expressed "disease genes." Of these, 507 were upregulated in the AD isotype samples. Gene set variation analysis (GSVA) ​​was performed on this gene set in unimodal mode.

[0374] result As shown in Figure 8, GSVA showed ineffective inhibition of all anti-IL18-treated samples. Anti-IL13 inhibition was partially effective in two out of five samples. Meanwhile, the anti-IL18 / IL13 bispecific antibody bbmAb1 showed robust suppression of the AD-like disease transcriptome in four out of five samples, indicating a synergistic effect for the combined cytokine blockade.

[0375] As shown in Figure 9, t-SNE analysis (perplexity 5) of 507 upregulated genes showed a clear disease effect, with the control and AD+ isotype samples furthest apart on the x-axis. Four of the five samples treated with the anti-IL13 / 18 bispecific antibody bbmAb1 clustered near the control sample, suggesting that cells from these samples had the least AD-like transcriptome of induced cells. In contrast, only two anti-IL13-treated samples showed a similar effect, and the anti-IL18-treated sample was indistinguishable from the AD+ isotype sample, indicating no therapeutic effect. These results were further supported by t-SNE analysis (perplexity 5) of 1,485 differentially expressed genes (AD+ isotype vs. control), illustrated in Figure 10. However, Figure 10 suggests a modest therapeutic effect for blockade of IL-18 alone. Without wishing to be bound by theory, the inventors hypothesize that the data in Figures 8-10 suggest that IL-18 blockade (e.g., using anti-IL18 antibodies) may have a therapeutic effect on AD in patients, but that IL13 / 18 co-blockade (e.g., by simultaneous or sequential administration of IL-13 and IL-18 antagonists) may unexpectedly be a superior treatment compared to blockade of either IL-13 or IL-18.

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

[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, and at week 16, the subject achieves a greater reduction in one or more signs and / or symptoms of atopic dermatitis compared to a placebo-treated subject. The inventions described in the original claims of this application are listed below. [Invention 1] A multispecific antibody, a. a first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1), which specifically binds interleukin-18 (IL-18); b. a second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically binds interleukin-13 (IL-13); The multispecific antibody comprising: [Invention 2] 2. The multispecific antibody according to claim 1, which is a bispecific antibody. [Invention 3] the VH1 and VH2 comprise complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL1 and VL2 comprise LCDR1, LCDR2, and LCDR3; e. the VH1 domain comprises (e.g., in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 32, the HCDR2 having 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 HCDR2 having 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 HCDR2 having the amino acid sequence SEQ ID NO: 39, and the HCDR3 having the amino acid sequence SEQ ID NO: 40 and f. The VL1 domain comprises (e.g., in order): i. the LCDR1 having the amino acid sequence SEQ ID NO:4, the LCDR2 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 LCDR2 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 LCDR2 having the amino acid sequence SEQ ID NO: 11, and the LCDR3 having the amino acid sequence SEQ ID NO: 12 and g. The VH2 domain comprises (e.g., in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 46, the HCDR2 having 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 HCDR2 having 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 HCDR2 having the amino acid sequence SEQ ID NO: 53, and the HCDR3 having the amino acid sequence SEQ ID NO: 54 and h. The VL2 domain comprises (e.g., in order): i. the LCDR1 having the amino acid sequence SEQ ID NO: 18, the LCDR2 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 LCDR2 having 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 LCDR2 having the amino acid sequence SEQ ID NO:25, and the LCDR3 having the amino acid sequence SEQ ID NO:26 3. The multispecific antibody according to claim 1 or 2, comprising: [Invention 4] 4. The multispecific antibody according to any one of Inventions 1 to 3, comprising a first light chain of lambda type and a second light chain of kappa type. [Invention 5] 5. The multispecific antibody according to claim 4, wherein the first light chain is of the lambda 1 type and the second light chain is of the kappa 4 type. [Invention 6] (a) the VL1 domain comprises the amino acid sequence SEQ ID NO: 13, and (b) The multispecific antibody according to any one of Inventions 1 to 5, wherein the VL2 domain comprises the amino acid sequence SEQ ID NO: 27. [Invention 7] a. the VH1 domain comprises the amino acid sequence SEQ ID NO:41, and b. the VL1 domain comprises the amino acid sequence SEQ ID NO: 13, and c. the VH2 domain comprises the amino acid sequence SEQ ID NO:55; and d. The multispecific antibody according to any one of Inventions 1 to 6, wherein the VL2 domain comprises the amino acid sequence SEQ ID NO: 27. [Invention 8] 8. The multispecific antibody according to any one of Inventions 1 to 7, comprising 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. [Invention 9] 9. The multispecific antibody according to any one of Inventions 1 to 8, comprising a first heavy chain comprising a heterodimerization modification and a second heavy chain comprising a heterodimerization modification complementary to the heterodimerization modification of the first heavy chain. [Invention 10] the first and second heavy chains are human IgG1; and a) the heterodimerization modification of the first heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises a tryptophan at position 366; or b) the heterodimerization modification of the second heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises a tryptophan at position 366; 10. The multispecific antibody according to invention 9, wherein the amino acid residues are numbered according to EU numbering. [Invention 11] 11. The multispecific antibody according to any one of Inventions 1 to 10, which is a bispecific antibody comprising a mutation that enhances the half-life of the bispecific antibody through enhanced FcRn binding. [Invention 12] 12. The bispecific antibody according to claim 11, wherein the mutations that enhance the half-life of the bispecific antibody are M252Y / S254T / T256E (YTE), and the amino acid residues are numbered according to EU numbering. [Invention 13] 13. The multispecific antibody according to any one of Inventions 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 42, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56. [Invention 14] 13. The multispecific antibody according to any one of Inventions 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 58. [Invention 15] 13. The multispecific antibody according to any one of Inventions 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 42 and the first light chain comprises the amino acid sequence shown in SEQ ID NO: 14, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56 and the second light chain comprises the amino acid sequence shown in SEQ ID NO: 28. [Invention 16] 13. The multispecific antibody according to any one of Inventions 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57 and the first light chain comprises the amino acid sequence shown in SEQ ID NO: 14, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 58 and the second light chain comprises the amino acid sequence shown in SEQ ID NO: 28. [Invention 17] 17. A pharmaceutical composition comprising a multispecific antibody according to any one of inventions 1 to 16 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. [Invention 18] 18. The pharmaceutical composition according to claim 17, further comprising one or more additional active agents. [Invention 19] 17. An isolated nucleic acid molecule encoding the multispecific antibody according to any one of inventions 1 to 16. [Invention 20] A cloning or expression vector comprising one or more nucleic acid sequences according to invention 19, which is suitable for the recombinant production of a multispecific antibody according to any one of inventions 1 to 16. [Invention 21] 21. A host cell comprising one or more cloning or expression vectors according to invention 20. [Invention 22] 17. A process for producing a multispecific antibody according to any one of claims 1 to 16, comprising culturing a host cell according to claim 21 under conditions sufficient to express said multispecific antibody, and subsequently purifying and recovering said multispecific antibody from the host cell culture. [Invention 23] 19. A kit comprising a multispecific antibody according to any one of Inventions 1 to 16 or a pharmaceutical composition according to Invention 17 or 18, the kit additionally comprising instructions for use and a pharmaceutical delivery device for administering said multispecific antibody or said pharmaceutical composition to a subject in need thereof. [Invention 24] 24. The kit of claim 23, wherein said pharmaceutical delivery device for administration comprises a syringe, an autoinjector, an injection pen, a vial and syringe, an infusion pump, a patch, or an infusion bag and needle. [Invention 25] A method for simultaneously inhibiting the activity of IL-13 and IL-18, the method comprising contacting a plurality of mammalian cells with an effective amount of the multispecific antibody according to any one of inventions 1 to 16. [Invention 26] 17. A method for simultaneously inhibiting the activity of IL-13 and IL-18 in a subject, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody according to any one of inventions 1 to 16. [Invention 27] 17. A method of treating an IL-13 and / or IL-18 mediated disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 16. [Invention 28] 17. A method for preventing or treating an inflammatory or immune condition, comprising administering a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 16 to a subject in need thereof. [Invention 29] 17. The multispecific antibody according to any one of Inventions 1 to 16, for use in preventing or treating an inflammatory or immune condition in a subject in need thereof. [Invention 30] 30. The method or use of invention 28 or 29, wherein the inflammatory or immune condition is a skin condition. [Invention 31] 31. The method or use according to claim 30, wherein the skin condition is atopic dermatitis. [Invention 32] 32. The method or use according to invention 31, wherein said method is an improved treatment compared to treatment with a monospecific anti-IL-13 antagonist and / or a monospecific anti-IL-18 antagonist. [Invention 33] 33. The method or use according to invention 31 or 32, wherein said improvement is indicated by a better Eczema Area and Severity Index (EASI) score after 16 weeks of treatment. [Invention 34] 34. The method or use according to any one of Inventions 31 to 33, wherein the atopic dermatitis is moderate to severe atopic dermatitis. [Invention 35] 35. The method or use according to any one of Inventions 31 to 34, wherein the atopic dermatitis is inadequately controlled with topical corticosteroids. [Invention 36] A method of treating an inflammatory or immune condition in a subject in need thereof, comprising administering to said subject IL-13 and IL-18 antagonists simultaneously or sequentially. [Invention 37] 37. The method of claim 36, comprising administering a first antagonist selected from an IL-13 and IL-18 antagonist and a second antagonist selected from an IL-13 and IL-18 antagonist, wherein the first and second antagonists are structurally distinct molecules. [Invention 38] 37. The method according to claim 36, comprising administering an anti-IL18 antagonist antibody and an anti-IL13 antagonist antibody. [Invention 39] 37. The method of claim 36, comprising administering an anti-IL13 / 18 bispecific antibody. [Invention 40] 37. The method of claim 36, wherein the inflammatory or immune condition is a skin condition. [Invention 41] 37. The method of claim 36, wherein said inflammatory or immune condition is atopic dermatitis, such as moderate to severe atopic dermatitis or atopic dermatitis that is inadequately controlled with, for example, topical corticosteroids. [Invention 42] 42. The method according to invention 41, which is an improved treatment compared to treatment with a monospecific anti-IL-13 antagonist and / or a monospecific anti-IL-18 antagonist. [Invention 43] 43. The method of claim 42, wherein said improvement is indicated by a better Eczema Area and Severity Index (EASI) score after 16 weeks of treatment.

Claims

1. 1. A bispecific antibody comprising: a. a first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically binds interleukin-18 (IL-18), the VL1 domain comprises the amino acid sequence of SEQ ID NO: 13; the VH1 domain comprises the amino acid sequence of SEQ ID NO: 41; the first portion; and b. a second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically binds interleukin-13 (IL-13); the VL2 domain comprises the amino acid sequence of SEQ ID NO: 27; the VH2 domain comprises the amino acid sequence of SEQ ID NO: 55; the second portion; The bispecific antibody comprising:

2. 2. The bispecific antibody of claim 1, comprising a first light chain of the lambda type and a second light chain of the kappa type.

3. 3. The bispecific antibody of claim 2, wherein the first light chain is of the lambda 1 type and the second light chain is of the kappa 4 type.

4. 2. The bispecific antibody of claim 1, comprising a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 14 and a second light chain comprising the amino acid sequence set forth in SEQ ID NO:

28.

5. 2. The bispecific antibody of claim 1, comprising a first heavy chain comprising a heterodimerization modification and a second heavy chain comprising a heterodimerization modification that is complementary to the heterodimerization modification of the first heavy chain.

6. the first and second heavy chains are human IgG1; and a) the heterodimerization modification of the first heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises a tryptophan at position 366; or b) the heterodimerization modification of the second heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises a tryptophan at position 366; The bispecific antibody of claim 5, wherein the amino acid residues are numbered according to EU numbering.

7. 2. The bispecific antibody of claim 1, wherein the bispecific antibody comprises a mutation that enhances the half-life of the bispecific antibody through enhanced FcRn binding.

8. 8. The bispecific antibody of claim 7, wherein the mutations that enhance the half-life of the bispecific antibody are M252Y / S254T / T256E (YTE), and the amino acid residues are numbered according to EU numbering.

9. 2. The bispecific antibody of claim 1 , wherein 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.

10. 2. The bispecific antibody of claim 1 , wherein 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.

11. 2. The bispecific antibody of claim 1 , wherein 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 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.

12. 2. The bispecific antibody of claim 1 , wherein 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 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.

13. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 12 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

14. 14. The pharmaceutical composition of claim 13, further comprising one or more additional active agents.

15. 13. A kit comprising the bispecific antibody of any one of claims 1 to 12, additionally comprising instructions for use and a pharmaceutical delivery device for administering the bispecific antibody to a subject in need thereof.

16. 16. The kit of claim 15, wherein the pharmaceutical delivery device for administration comprises a syringe, an autoinjector, an injection pen, a vial and syringe, an infusion pump, a patch, or an infusion bag and needle.

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