Multispecific antibodies targeting IL-13 and IL-18

Abstract

Described herein are multispecific antibodies that target IL-13 and IL-18. The multispecific antibodies may be antagonistic and / or therapeutic antibodies that target IL-13 and IL-18. Also described herein are methods of making the multispecific antibodies, methods of simultaneously inhibiting IL-13 and IL-18 with the multispecific antibodies, and methods of treating IL-13 / IL-18 mediated disorders, such as atopic dermatitis, by administering the multispecific antibodies described herein.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202380035364.0 (PCT / IB2023 / 054248), titled "Multispecific Antibodies Targeting IL-13 and IL-18", with a filing date of April 25, 2023. Technical Field

[0002] The present invention relates to the field of immunology. In particular, the present invention relates to multispecific antibodies targeting interleukin 13 (IL-13) and interleukin 18 (IL-18), and methods for their preparation and use. Background Art

[0003] Atopic dermatitis (AD) is a chronic / recurrent inflammatory skin disease characterized by symptoms including intense pruritus (e.g., severe itching) and squamous and dry eczematous lesions. Severe cases can lead to extreme disability due to severe psychological problems, severe sleep deprivation, and impaired quality of life, resulting in high socioeconomic costs. The pathophysiology of AD is influenced by the complex interplay between immunoglobulin E (IgE)-mediated sensitization, the immune system, and environmental factors. Primary skin defects may be immune dysregulation leading to IgE-mediated sensitization, accompanied by epithelial barrier dysfunction, which is the result of both gene mutations and local inflammation. AD typically begins in childhood before the age of 5 and may persist into adulthood.

[0004] Typical treatments for AD include topical lotions and emollients, topical corticosteroid ointments, creams, or injections. However, most treatment options only provide temporary and incomplete symptom relief. In addition, many patients with moderate to severe AD are 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.

[0005] The pathogenesis of AD is multifactorial, and the immune-mediated mechanism is characterized by the inappropriate activation of type 2 T helper cells (Th2) and type 2 innate lymphoid cells (ILC2), and increased expression of inflammatory cytokines, particularly interleukin IL-4 and IL-13 (Moyle et al. (2019) Exp Dermatol. [Clinical and Experimental Dermatology] 28(7):756 - 768; Roediger et al. (2013) Nat Immunol. [Nature Immunology] 14(6):564 - 573). IL-13 is one of the major cytokines in the pathophysiology of AD due to its prominent role in the production and maintenance of the inflammatory process and epidermal barrier dysfunction (Tsoi et al. (2019) J Invest Dermatol. [Journal of Investigative Dermatology] 139(7):1480–1489).

[0006] Currently, dupilumab, an anti-IL4Ra antibody, has been 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 under development.

[0007] IL-18 is thought to be involved in the pathogenesis of AD because IL-18 induces the production and secretion of IFN-γ and IL-13 by super Th1 cells (Terada et al. (2006) Proc Natl Acad Sci U S A. 103:8816–8821). IL-18 is released by keratinocytes and inflammatory dendritic cells, and serum IL-18 levels in patients with AD have been shown to be significantly correlated with skin scores 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 in mice and increases IgE production in a CD4+ T cell-, IL-4- and STAT6-dependent manner (Yoshimoto et al. (2000) Nat Immunol 1:132-137; Hoshino et al. (2000) Eur J Immunol 30:1998-2006).

[0008] Since IL-13 and IL-18 are pro-inflammatory cytokines that affect many different cell types associated with AD, there is still a need for an effective treatment that achieves co-blockade of IL-13 and IL-18 signaling. Multispecific antibodies that target both IL-13 and IL-18 (e.g., bispecific antibodies) can address the unmet medical needs in this chronic inflammatory disease.

[0009] One of the most common problems in generating bispecific IgG (BsIgG) by co-expressing two different antibodies is unwanted component heavy chain homodimerization and unwanted pairing of the component light chain with the incorrect heavy chain. Figure 1Possible misassembled products are shown. To overcome the problem of heavy chain homodimerization, a combination of engineered disulfide bonds and previously identified "knobs-into-holes" mutations can be used to reshape the heavy chain for heterodimerization. One of the variants, S354C:T366W / Y349'C:T366'S:L368'A:Y407V, can provide near-quantitative (-95%) heterodimerization (Merchant et al., 1998). However, this near-quantitative heterodimerization does not solve the light chain pairing problem. Thus, assuming random light chain pairing, only 25% of the resulting antibodies are the desired bispecific antibodies. There is still a need to further improve both heavy chain heterodimerization and light chain pairing to increase the purity, yield, and quality of bispecific antibodies. SUMMARY OF THE INVENTION

[0010] The present disclosure provides a method for treating an autoimmune or inflammatory disorder, wherein the method comprises administering an IL13 inhibitor and an IL18 inhibitor simultaneously or sequentially. Without wishing to be bound by theory, the inventors hypothesize that co-blockade of IL-13 and IL-18 provides unexpectedly superior efficacy in treating autoimmune or inflammatory disorders compared to blocking IL-13 or IL-18 alone. In some cases, the co-blockade comprises administering an IL13 antagonist and an IL-18 antagonist. In some cases, the co-blockade comprises administering 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.

[0011] The present invention provides a multispecific antibody or fragment thereof that targets both IL-13 and IL-18 for the treatment of AD, ensuring sufficient overall yield, purity, and product quality at a reasonable cost for clinical development and commercial manufacturing.

[0012] In some aspects, the multispecific antibody is a bispecific antibody. An exemplary bispecific antibody is 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 to 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 to interleukin-13 (IL-13).

[0013] 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.

[0014] Adverse interactions of Fc with Fcγ receptors and complement receptor C1q can be decoupled from binding to the neonatal Fc receptor (FcRn), and the binding to the neonatal Fc receptor (FcRn) can increase serum persistence. The in vivo serum persistence conferred by FcRn has been shown to be an adjustable property, which can be modulated by mutations in the IgG Fc. Increasing the affinity of Fc for FcRn under endosomal conditions (acidic pH) by Fc engineering is an effective method for extending the pharmacokinetics of monoclonal antibodies (Maeda, 2017). The YTE mutation set (M252Y, S254T, T256E according to EU numbering) or LS mutation set (M428L, N434S according to EU numbering) are examples of such developed mutation sets in the Fc CH2 domain.

[0015] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises M252Y / S254T / T256E (YTE) according to EU numbering. In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises M428L, N434S (LS) according to EU numbering.

[0016] Chain pairing mutations have been shown to effectively drive heavy chain heterodimerization by introducing complementarity at the CH3-CH3 interface of bispecific or multispecific antibodies. Many chain pairing mutation sets are used to generate multispecific antibodies: increasing / decreasing side chain volume (T366W / S354C-T366S / L368A / Y407V / Y349C, the "staple" structure) (Ridgway, 1996), charge inversion (K409D / K392D-D399K / E356K, electrostatic steering) (Gunasekaran, 2010), or multiple IgA substitutions (SEEDbody) (Davis, 2010).

[0017] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises chain-pairing amino acid substitutions, e.g., in combination with silent and / or half-life extension mutations. In some cases, the chain-pairing amino acid substitutions are knobs-into-holes (KiH) mutations, e.g., wherein the engineered 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, and the amino acid residues are numbered according to EU numbering.

[0018] In another embodiment, the chain-pairing amino acid substitutions are knobs-into-holes (KiH) mutations, wherein the first constant heavy chain has the amino acid substitutions S354C and T366W and the second constant heavy chain has the amino acid substitutions Y349C, T366S, L368A, and Y407V, and the amino acid residues are numbered according to EU numbering.

[0019] In other embodiments, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises both T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and M252Y / S254T / T256E (YTE), and the amino acid residues are numbered according to EU numbering.

[0020] Without being bound by theory, in some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one or more mutations for silencing ADCC and / or CDC effector functions, such as within hFc. Various sets of mutations have been described in the art, such as LALA (L234A, L235A according to EU numbering) (Wines et al., 2000) or DAPA (D265A, P329A according to EU numbering) (Genentech, US 6,737,056). Several researchers have employed a cross-subclass approach to reduce effector function. In a further refinement of the cross-subclass approach, IgG2 variants were generated by point mutations in IgG4 (i.e., H268Q, V309L, A330S, P331S according to EU numbering) (An et al., 2009). Another silenced IgG1 antibody contains the N297A mutation, which results in an aglycosylated / non-glycosylated antibody (Strohl et al., 2009). Some of the mutation sets used combine previously described techniques to achieve higher levels of silencing up to and including complete elimination of some or all effector functions. DANAPA is an example (D265A, N297A, P329A) (Janssen's WO 2019068632). Other alternative methods of engineering or mutating key residues responsible for effector function in the Fc region have been reported. See, for example, PCT Publication WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), US2006 / 0134709 (Macrogenics), US 6,737,056 (Genentech), US2010 / 0166740 (Roche).

[0021] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises D265A / P329A (DAPA), and wherein the amino acid residues are numbered according to EU numbering.

[0022] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234A / L235A (LALA), and wherein the amino acid residues are numbered according to EU numbering.

[0023] 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, and wherein the amino acid residues are numbered according to EU numbering.

[0024] In other embodiments, 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.

[0025] In some embodiments, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one or more amino acid substitutions that reduce Fc effector function; and one or more amino acid substitutions that enhance the half-life of the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof via enhanced FcRn binding; and / or one or more amino acid substitutions that promote correct chain pairing.

[0026] Thus, for example, in some embodiments, according to EU numbering, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and a half-life extending mutation selected from the group consisting of YTE (M252Y, S254T, T256E) and LS (M428L, N434S). In some embodiments, according to EU numbering, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises T366W / S354C-T366S / L368A / Y407V / Y349C (KiH) and an Fc silencing mutation selected from the group consisting of LALA (L234A, L235A), DAPA (D265A, P329A), and N297. In some embodiments, according to EU numbering, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), a half-life extending mutation selected from the group consisting of YTE (M252Y, S254T, T256E) and LS (M428L, N434S), and an Fc silencing mutation selected from the group consisting of LALA (L234A, L235A), DAPA (D265A, P329A), and N297.

[0027] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234A / L235A (LALA) and M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering.

[0028] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234C and M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering. In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L235C and M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering. In another embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises G236C and M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering.

[0029] In one embodiment, the engineered 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), and wherein the amino acid residues are numbered according to EU numbering.

[0030] In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L234C, M252Y / S254T / T256E (YTE), and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), and wherein the amino acid residues are numbered according to EU numbering. In one embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises L235C, M252Y / S254T / T256E (YTE), and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), and wherein the amino acid residues are numbered according to EU numbering. In another embodiment, the engineered multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises G236C, M252Y / S254T / T256E (YTE), and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH), and wherein the amino acid residues are numbered according to EU numbering.

[0031] In some embodiments, the VH1 and VH2 domains of the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprise complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3; and the VL1 and the VL2 comprise LCDR1, LCDR2, LCDR3, wherein:

[0032] a. The VH1 domain comprises (e.g., in sequence):

[0033] 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

[0034] 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

[0035] 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

[0036] b. The VL1 domain comprises (e.g., in sequence):

[0037] 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

[0038] 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

[0039] 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

[0040] c. The VH2 domain comprises (e.g., in sequence):

[0041] 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

[0042] ii. said HCDR1 having the amino acid sequence SEQ ID NO:49, said HCDR2 having the amino acid sequence SEQ ID NO:50, and said HCDR3 having the amino acid sequence SEQ ID NO:51; or

[0043] iii. said HCDR1 having the amino acid sequence SEQ ID NO:52, said HCDR2 having the amino acid sequence SEQ ID NO:53, and said HCDR3 having the amino acid sequence SEQ ID NO:54; and

[0044] d. said VL2 domain comprises (e.g., in sequence):

[0045] i. said LCDR1 having the amino acid sequence SEQ ID NO:18, said LCDR2 having the amino acid sequence SEQ ID NO:19, and said LCDR3 having the amino acid sequence SEQ ID NO:20; or

[0046] ii. said LCDR1 having the amino acid sequence SEQ ID NO:21, said LCDR2 having the amino acid sequence SEQ ID NO:22, and said LCDR3 having the amino acid sequence SEQ ID NO:23; or

[0047] iii. said LCDR1 having the amino acid sequence SEQ ID NO:24, said LCDR2 having the amino acid sequence SEQ ID NO:25, and said LCDR3 having the amino acid sequence SEQ ID NO:26.

[0048] In some embodiments, said first light chain is of the λ type and said second light chain is of the κ type.

[0049] In some embodiments, said first light chain is of the λ1 type and said second light chain is of the κ4 type.

[0050] In some embodiments, said multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises: a VL1 domain having the amino acid sequence SEQ ID NO:13 and a VL2 domain having the amino acid sequence SEQ ID NO:27.

[0051] 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.

[0052] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises a first light chain comprising the amino acid sequence as set forth in SEQ ID NO:14 and a second light chain comprising the amino acid sequence as set forth in SEQ ID NO:28.

[0053] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises 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.

[0054] In some embodiments, the first constant heavy chain and the second constant heavy chain are human IgG1, which comprise one or more heterodimerization modifications and:

[0055] a) the heterodimerization modification of the first immunoglobulin heavy chain comprises serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises tryptophan at position 366; or

[0056] b) the heterodimerization modification of the second immunoglobulin heavy chain comprises serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises tryptophan at position 366,

[0057] and wherein the amino acid residues are numbered according to EU numbering.

[0058] In some embodiments, the multispecific antibody is a bispecific antibody, and the bispecific antibody comprises a mutation that enhances the half-life of the bispecific antibody via enhanced FcRn binding.

[0059] In some embodiments, the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering.

[0060] In some embodiments, 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.

[0061] In some embodiments, 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.

[0062] In some embodiments, 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.

[0063] In some embodiments, 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.

[0064] Also disclosed herein is a pharmaceutical composition comprising a multispecific antibody (e.g., bispecific antibody) or a fragment thereof of the present disclosure in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0065] In some embodiments, the pharmaceutical composition further comprises one or more additional active agents.

[0066] Also disclosed herein is an isolated nucleic acid molecule encoding a multispecific antibody of the present disclosure.

[0067] The present disclosure also discloses a cloning or expression vector comprising one or more nucleic acid sequences as outlined above, wherein the vector is suitable for recombinantly producing the multispecific antibodies of the present disclosure. In some embodiments, a set of two cloning or expression vectors is provided herein. 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, wherein the heavy chain and the light chain encoded by the first vector are capable of combining to form an anti-IL-18 arm of a bispecific IgG antibody; 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, wherein the heavy chain and the light chain encoded by the second vector are capable of combining to form an anti-IL-13 arm of the bispecific IgG antibody as described herein. In some embodiments, the first vector and the second vector are expression vectors, and co-expression of the first vector and the second vector in a common host cell provides an anti-IL-18 / IL-13 bispecific IgG-like antibody with high yield, purity, and activity.

[0068] The present disclosure also discloses a host cell comprising one or more cloning or expression vectors as outlined above.

[0069] The present disclosure also discloses a process for producing the multispecific antibodies of the present disclosure, the process comprising culturing a host cell as outlined above under conditions sufficient to express the multispecific antibody, and then purifying and recovering the multispecific antibody from the host cell culture.

[0070] The present disclosure also discloses a kit comprising one or more cloning and / or expression vectors of the present disclosure, wherein the kit further comprises instructions for producing the multispecific (e.g., bispecific) antibodies disclosed herein.

[0071] The present disclosure also discloses a kit comprising the multispecific antibody of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the kit further comprises instructions for use and a device for administering the multispecific antibody or the pharmaceutical composition to a subject in need thereof.

[0072] In some embodiments, the 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 a needle.

[0073] The present disclosure also discloses a method of simultaneously binding IL-13 and IL-18, the method comprising contacting IL-13 and IL-18 with an effective amount of the multispecific antibody of the present disclosure. In some cases, the contacting is carried out in vitro. In some cases, the contacting is carried out ex vivo. In some cases, the contacting is carried out in a subject (such as a human patient in need of IL-13 and IL-18 inhibition).

[0074] The present disclosure also discloses a method for simultaneously inhibiting the activities of IL-13 and IL-18, the method 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 carried out in vitro or in an ex vivo culture. In some cases, the contacting is carried out in a non-human animal (such as a non-human primate). In some cases, the contacting is carried out in a patient in need of IL-18 and IL-13 inhibition (such as a patient suffering from atopic dermatitis). In some cases, the activities of IL-13 and IL-18 are reduced by at least 10%, at least 25%, at least 50%, at least 75% or at least 90%. In some cases, the reduction in the activity of IL-13 is measured by a reduction in STAT-6 signaling. In some cases, the reduction in the activity of IL-18 is measured by a reduction in IFNγ production (such as a reduction in LPS / IL-12-induced IFNγ production). In some cases, the reduction in the activities of IL-18 and / or IL-13 is measured by an increase in the level of IL-18 or IL-13 that binds to the multispecific antibody described herein, respectively, or by a decrease in the level of free IL-18 or IL-13, respectively.

[0075] The present disclosure also discloses a method for simultaneously inhibiting the activities of IL-13 and IL-18 in a subject, the method 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 co-blockade of IL-13 and Il-18 may have a complementary (e.g., synergistic) effect compared to blockade of IL-18 or blockade of IL-13. In some embodiments, co-blockade of IL-13 and Il-18 may have a complementary (e.g., synergistic) effect compared to blockade of IL-18.

[0076] The present disclosure also discloses 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 (e.g., bispecific) antibody of the present disclosure. In some embodiments, the method is an improved treatment as compared to treatment with a monospecific anti-IL-13 antagonist. In some embodiments, the method is an improved treatment as compared to treatment with a monospecific anti-IL-18 antagonist. In some embodiments, the method is an improved treatment as compared to treatment with an anti-IL-13 antagonist or an anti-IL-18 antagonist. In some embodiments, the improvement is indicated by a better Eczema Area and Severity Index (EASI) score, a better Investigator's Global Assessment (IGA) score, a better Pruritus Numerical Rating Scale score, and / or a better Dermatology Life Quality Index score after 16, 24, 36, or 52 weeks of treatment. In some embodiments, the method is an improved treatment as compared to treatment with an anti-IL-13 antagonist or an anti-IL-18 antagonist, wherein the improvement is indicated by a lower Severity of Atopic Dermatitis (SCORAD) score after 16, 24, 36, or 52 weeks of treatment.

[0077] In some embodiments, the method of the present disclosure reduces the expression level of one or more AD-related biomarkers (especially one or more AD-related biomarkers selected from 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 expression level of the one or more AD-related biomarkers before co-blockade of IL13 and IL18 (e.g., treatment with the multispecific antibody (e.g., bispecific antibody) or a fragment thereof).

[0078] The present disclosure also discloses a method for inhibiting the production of IgE antibodies in a subject, the method comprising inhibiting IL13 and IL18 simultaneously or sequentially (e.g., by administering to the subject an effective amount of a multispecific antibody of the present disclosure).

[0079] The present disclosure also discloses a method for inhibiting the production of IFN-γ in a subject, the method comprising inhibiting IL13 and IL18 simultaneously or sequentially (e.g., by administering to the subject an effective amount of a multispecific antibody of the present disclosure).

[0080] Also disclosed herein is a method for treating and / or preventing an inflammatory or immune disorder, the method comprising inhibiting IL13 and IL18 simultaneously or sequentially (e.g., by administering a therapeutically effective amount of a multispecific antibody of the present disclosure to a subject in need thereof). In a preferred embodiment, the inflammatory or immune disorder is a skin disorder. In a preferred embodiment, the skin disorder 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 standard score, and wherein the Rajka / Lengeland standard score is determined to be between 4.5 and 9. In some embodiments, the method further comprises administering one or more topical corticosteroids. In some embodiments, the atopic dermatitis cannot be adequately controlled by administering the one or more topical corticosteroids.

[0081] In other aspects, disclosed herein are uses of the multispecific antibodies (eg, bispecific antibodies) or fragments thereof of the present disclosure in treating and / or preventing AD.

[0082] In other aspects, disclosed herein is the use of a multispecific antibody (eg, a bispecific antibody) or a fragment thereof of the present disclosure for the manufacture of a medicament for treating and / or preventing AD.

[0083] Additional details and examples are provided in the following paragraphs. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Possible products when expressing two KiH-modified mAbs in the same host cell line are shown. LHHL: Light chain-heavy chain-heavy chain-light chain, and this fraction includes the final bispecific antibody. HHL: Heavy chain-heavy chain-light chain. HL: Heavy chain-light chain = half mAb. H: Heavy chain. L: Light chain. LL: Light chain heterodimers and homodimers.

[0085] Figure 2 Schematic diagram of plasmids A to D for expression of IL-13 and IL-18. Plasmids A and C encode the expression of anti-IL13κ LC and anti-L13 knob HC; plasmids B and D encode the expression of anti-IL18λ LC and anti-IL18 knob HC.

[0086] Figure 3 is a schematic diagram of the furin-2A peptide (F2A) plasmids E and F. The F2A technology enables the expression of more than one protein chain from one promoter combination. On plasmids E and F, the first expression cassette encodes anti-IL18λ LC and anti-IL18 hole HC, and the second expression cassette encodes anti-IL13κ LC and anti-L13 knob HC.

[0087] Figure 4 Schematic diagrams of the modified plasmids G and H. Plasmid G encodes the expression of anti-IL13 κLC and anti-L13 pro HC; plasmid H encodes the expression of anti-IL18 λLC and anti-IL18 pro HC.

[0088] Figure 5 Schematic diagram of furin-2A peptide (F2A) plasmid I with different protein chain combinations in the expression cassette compared to plasmids E and F. On plasmid I, the first expression cassette encodes anti-IL18 λLC, anti-IL18 pro HC, and anti-IL13 pro HC, and the second expression cassette encodes anti-IL13 κLC.

[0089] Figure 6 shows the melting curves of the IL-13 / IL-18 bispecific antibody. Figure 6A Melting curve of bbmAb1. Figure 6B Melting curve of bbmAb2. Figure 6C Melting curve of bbmAb5. Figure 6D Melting curve of bbmAb4. Figure 6E Melting curve of bbmAb3.

[0090] Figure 7 Comparison of the pharmacokinetic characteristics of Tg276 mice after administration of bbmAb1 and bbmAb2 and the Fc-silent variants bbmAb6, bbmAb7, bbmAb8, and bbmAb9 (pooled serum samples were plotted according to the sampling time).

[0091] Figure 8 Gene set variation analysis (GSVA) results of genes differentially expressed between the following samples: control samples (skin biopsies) not treated with the cytokine mixture that induces an atopic dermatitis (AD)-like transcriptome (control), samples treated with the cytokine mixture that induces an atopic dermatitis (AD)-like transcriptome and co-incubated with an isotype control antibody (AD + isotype) (induced samples); induced samples co-incubated with an anti-IL-18 antibody (AD + anti-IL18); induced samples co-incubated with an anti-IL-13 antibody (AD + anti-IL13); and induced samples co-incubated with a bispecific antibody that simultaneously inhibits the activities of IL-13 and IL-18 (AD + bbmAb1) showed different degrees of inhibition. Data from cells of five different donor samples are shown.

[0092] Figure 9 Results of t-distributed stochastic neighbor embedding (t-SNE) analysis (perplexity of 5) of the in vitro disease transcriptome (AD + isotype compared to control) based on 507 upregulated genes as described in Example 8.

[0093] Figure 10 Shows the results of t-SNE analysis (perplexity = 5) of an in vitro disease transcriptome (AD + isoform compared to control) based on 1485 differentially expressed genes as described in Example 8. Detailed Description

[0094] To facilitate a better understanding of the present disclosure, 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.

[0095] Definitions

[0096] Interleukin (IL)-18 (referred to herein simply as "IL-18") is mainly produced by macrophages and T cells as a precursor protein (pro-IL-18) and is secreted as an active protein after cleavage by caspase-1 (Dinarello CA et al. (1999) J Allergy Clin Immunol; 103:11-24). In normal physiology, IL-18, which acts in concert with IL-12, is associated with the induction of cell-mediated immunity following infection with microbial products such as lipopolysaccharide (LPS) (Sareneva T et al. (2000) J Immunol; 165(4):1933-8). Upon stimulation with IL-18, natural killer (NK) cells and T cells release the cytokine interferon gamma (INF-γ), which plays an important role in activating macrophages and other cells. In addition to its ability to induce interferon gamma, IL-18 has multiple functions. These biological properties include activation of NF-κB, expression of Fas ligand, induction of both CC and CXC chemokines, and an increase in the production of competent human immunodeficiency virus.

[0097] The term "IL-18" refers to an IL-18 polypeptide, also known as interleukin-18 polypeptide, IFN-γ inducing factor, interferon-gamma inducing factor, or INF-γ inducing factor. Throughout the specification, the term IL-18 may interchangeably encompass pro-IL-18 (the precursor of mature IL-18 before protease cleavage) and mature IL-18 (after protease cleavage), unless specifically stated to mean the pro- or mature form.

[0098] Interleukin (IL)-13 (referred to herein simply as “IL-13”) is a pleiotropic cytokine that is 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 (IL-13Ra1) of the IL-13 receptor in all cells of the human body, but binds particularly to monocytes and B cells. In a cascade, this binding facilitates the recruitment of IL-4Ra, leading to the formation of signal transducers that can activate Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) through dimerization, resulting in the phosphorylation of signal transducer and activator of transcription 6 (STAT6), a transcription factor that promotes TH2 differentiation, and class switch to IgE (Silverberg et al. (2017) Dermatol Clin. [Dermatology Clinical] 35(3):327–334; Goenka et al. (2011) Immunol Res. [Immunology Research] 50(1):87–96). The term “IL-13” is synonymous with the IL-13 polypeptide or interleukin-13 polypeptide.

[0099] In all cases where the term “comprising” etc. is used in reference to a sequence (e.g., an amino acid sequence), it should be understood that the sequence may also be limited by terms such as “consisting of” etc. As used herein, the phrase “consisting essentially of” refers to the genus or species of active agents included in a method or composition, as well as any excipients that are inactive for the intended purpose of those methods or compositions. In some aspects, the phrase “consisting essentially of” specifically excludes one or more additional active agents other than the multispecific antibodies of the present disclosure. In some aspects, the phrase “consisting essentially of” specifically excludes one or more additional active agents other than the multispecific antibodies of the present disclosure and a second co-administered agent.

[0100] As used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that is capable of non-covalently, reversibly, and specifically binding an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain. The heavy chain constant domain comprises three domains, namely CH1, CH2, and CH3. Each light chain comprises a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, 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 in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen, which are sometimes referred to herein as antigen-binding domains. The constant regions of these antibodies can 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 of the classical complement system (Clq).

[0101] 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. Antibodies can 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).

[0102] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of the light chain (VL) and heavy chain (VH) portions both determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the constant domain is numbered higher the farther it is from the antigen-binding site or the 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 domain and the CL domain contain the carboxyl termini of the heavy and light chains, respectively.

[0103] As used herein, the phrase "antibody fragment" refers to one or more portions of an antibody. In some embodiments, these portions are part of one or more constant domains of an antibody, such as the crystallizable fragment (Fc) constant (C) domain, etc. In some other embodiments, the one or more portions are antigen-binding fragments that retain the ability to non-covalently, reversibly, and specifically bind an antigen, and such antigen-binding fragments are sometimes referred to herein as antigen-binding domains. As used herein, the phrase "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv) (with or without an internal cysteine bridge), disulfide-linked Fv (sdFv), F(ab)2 fragments, Fab fragments, F(ab')2, fragment F(ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of V H domains; and isolated complementarity-determining regions (CDRs), or other epitope-binding fragments of an antibody.

[0104] As used herein, "Fc" or "Fc region" includes CH2 and CH3, and optionally any part of the 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 contains an IL-18 scFv linked to an Fc polypeptide chain. The half-antibody can pair with another half-antibody such that the two Fc polypeptide chains dimerize to form 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 is capable of being linked to an antigen-binding domain (e.g., an IL-18 binding domain or an IL-13 binding domain).

[0105] Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intracellular antibodies, diabodies, triabodies, tetra-bodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).

[0106] Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1), which together with complementary light chain polypeptides (e.g., VL-CL-VL-CL) form a pair of antigen-binding regions (Zapata et al., (1995) Protein Eng. 8:1057-1062; and US 5,641,870).

[0107] The term "half-antibody" refers to an antibody molecule, antibody fragment, antibody-like molecule, or part of a multispecific binding molecule that contains a single antigen-binding domain. In one embodiment, a half-antibody refers to, for example, a heavy and light chain pair of an IgG antibody. In one embodiment, a half-antibody refers to a polypeptide containing a VL domain and a CL domain, and a second polypeptide (i.e., Fd and Fc) containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains contain antigen-binding domains. In another embodiment, a half-antibody refers to a polypeptide containing an scFv domain and an Fc polypeptide chain (containing a CH2 domain and a CH3 domain, and optionally a hinge region). In some multispecific binding molecules, the first half-antibody, the second half-antibody, or both the first and second half-antibodies can contain additional antigen-binding domains. In some embodiments of multispecific binding molecules, the first half-antibody associates with the second half-antibody, e.g., heterodimerizes. In some multispecific binding molecules, the first half-antibody is covalently linked to the second half-antibody.

[0108] As used herein, the term "monospecific molecule" refers to an Fc-containing molecule that binds to one epitope on a target antigen. In some embodiments, the monospecific molecules of the present disclosure are monospecific antibody-like molecules. In some embodiments, the monospecific molecules of the present disclosure are monospecific antibodies. The term "bispecific molecule" refers to an Fc-containing multispecific molecule that binds two different antigens. The term "trispecific molecule" refers to an Fc-containing multispecific binding molecule that binds three different antigens via three different binding moieties. In some embodiments, the bispecific molecules of the present disclosure are bispecific antibody-like molecules. In some embodiments, the multispecific molecules of the present disclosure are multispecific antibody-like molecules.

[0109] The term "bispecific antibody" refers to an antibody that is able to recognize two or more epitopes of an antigen or two or more antigens. Recognition of each antigen is typically accomplished via an "antigen-binding domain". In particular, a bispecific antibody recognizes two different epitopes on the same or different antigens. All bispecific IgG molecules (i.e., bispecific antibodies that are indistinguishable in their composition from natural immunoglobulins) are bivalent and have an asymmetric structure due to the presence of at least different Fv regions. They may further differ in the constant regions of the heavy or light chains depending on the method of preparation and the origin of the heavy and light chains (Brinkmann and Kontermann, 2017).

[0110] A bispecific antibody is a "heterodimer", which means that one part is from a first antibody that is specific for a first target and the other part is from a second antibody that is specific for a second target. "Heterodimerization modification" is a modification of one or both parts of the antibody that forms a heterodimeric bispecific antibody, aimed at facilitating such formation. An example of a heterodimerization modification of the Fc domains of two IgG1 parts intended to form a bispecific antibody is the "stalk" with large amino acid (aa) side chains (S354C, T366W) in the first heavy chain and the "socket" with small amino acid side chains (Y349C, T366S, L368A, Y407V) introduced in the second heavy chain and an additional disulfide bridge connecting the two heavy chains in the CH3 region (Merchant et al., Nat. Biotechnol. [Nature Biotechnology], 16:677 - 681 (1998), page 678, Table 1).

[0111] The terms "mismatch" or "mis-pairing" or "mis-assembly" refer to the situation where different parts of an expected protein complex (e.g., a bispecific antibody) do not complex as expected, which means that the appearance or behavior of the protein complex does not conform to expectations. Examples of mismatches in the case of bispecific antibodies are Figure 1 shown.

[0112] As used herein, the terms "recognize" or "bind" refer to the discovery and interaction (e.g., binding or recognition) of a binding molecule, antibody, or antigen-binding fragment thereof with an epitope, whether the epitope is linear, discontinuous, or conformational. The term "epitope" refers to the site on an antigen that specifically binds to an antibody or antigen-binding fragment of the present disclosure. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids typically are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically are lost upon treatment with denaturing solvents. Epitopes typically include 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, e.g., x-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology [Epitope Mapping Protocols in Methods in Molecular Biology], Vol. 66, edited by G.E. Morris (1996)) or electron microscopy. A "paratope" is the antibody portion that recognizes an antigenic epitope.

[0113] 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, the phrases "specifically binds" or "selectively binds" refer to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological products, e.g., in a biological sample (e.g., blood, serum, plasma, or tissue sample). Thus, under certain specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least two-fold above background and these antibodies or binding agents do not substantially bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least ten (10)-fold above background and these antibodies or binding agents do not substantially bind to other antigens present in the sample in significant amounts. Binding specifically to an antibody or binding agent under such conditions may require the specific selection of an antibody or reagent that has been directed against a particular protein. If desired or appropriate, this selection can be accomplished by subtracting antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some aspects, antibodies or antibody fragments that cross-react with certain desired molecules are selected.

[0114] The term "antigen-binding site" refers to the part of an antibody that contains determinants forming an interface for binding to an antigen or its epitope. The term "antigen-binding site" may be used interchangeably with the term "antigen-binding domain" or antibody-binding portion. With respect to a protein (or protein mimic), the antigen-binding site typically includes one or more loops (having at least four amino acids or amino acid mimics) that form an interface for binding to an antigen polypeptide. Typically, the antigen-binding site of an antibody molecule includes 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.

[0115] As used herein, the term "complementary determining region" or "CDR" refers to the sequence of amino acids within the variable region of an antibody that confers antigen specificity and binding affinity. For example, generally, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and there are three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The positions of the CDRs and framework regions can be determined using various known definitions in the art such as Kabat, Chothia, IMGT, AbM, and combined definitions (see, 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 and Lesk, (1987) J. Mol. Biol., 196:901-917; Chothia et al., (1989) Nature, 342:877-883; Chothia et al., (1992) J. Mol. Biol., 227:799-817; Lefranc MP (2001) Nucleic Acids Res., 29:207-209; Al-Lazikani et al., (1997) J. 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 M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). According to 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). According to the Chothia numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDR corresponds to amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH (e.g., mammalian VH, e.g., human VH); and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL (e.g., mammalian VL, e.g., human VL). According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (according to the "Kabat" numbering).According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0116] The term "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that contains the minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the recipient hypervariable regions are replaced with residues from hypervariable regions (donor antibody) of a non-human species such as mouse, rat, rabbit, or non-human primate that have the desired specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. In addition, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain substantially all of the following: at least one, typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are those of the human immunoglobulin sequence. A humanized antibody optionally also contains an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. 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).

[0117] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework region and the CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences or antibodies containing consensus framework sequences derived from analysis of human framework sequences, e.g., as described by Knappik et al. (2000. J Mol Biol 296, 57-86).

[0118] The human antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations are introduced by random mutagenesis or site-specific mutagenesis in vitro, or by somatic mutations in vivo, or by conservative substitutions to promote stability or production). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted into human framework sequences.

[0119] As used herein, "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the change is caused by a sequence change involving the amino acid residue / position. For example, typical modifications include substituting a residue with another amino acid (or substituting at the position) (e.g., conservative or non-conservative substitution), inserting one or more amino acids near the residue / position, and deleting the residue / position. "Amino acid substitution" or its variants refers to replacing an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Generally and preferably, the modification alters at least one physical and biochemical activity of the variant polypeptide compared to the polypeptide containing the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physical and biochemical activity can be the binding affinity, binding capacity, and / or binding effect for the target molecule.

[0120] The term "conservative modified variant" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservative modified variants are those nucleic acids that encode the same or substantially the same amino acid sequence, or, in cases where the nucleic acid does not encode an amino acid sequence, are substantially the same sequences. 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 a codon specifies alanine, the codon can be changed to any of the said corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservative modified variation. Each nucleic acid sequence encoding a polypeptide described herein also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for the AUG, which is typically the only codon for methionine, and the TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, every silent variation of the nucleic acid encoding a polypeptide is implicit in each such sequence.

[0121] For a polypeptide sequence, "conservative modified variants" include single substitutions, deletions or additions to the polypeptide sequence, which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. Such conservative modified variants are in addition to the polymorphic variants, interspecies homologs and alleles of the present invention, and do not exclude these polymorphic variants, interspecies homologs and alleles. 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);

[0122] 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 modification" 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.

[0123] In the case of two or more nucleic acid or polypeptide sequences, the terms "percent identical" or "percent identity" refer to two or more identical sequences or subsequences. When comparing and aligning in a comparison window or specified region to find the maximum correspondence measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity over the specified region or, when no region is specified, over the entire sequence). Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids), or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids).

[0124] For sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and the sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0125] As used herein, the term "comparison window" includes reference to a segment of any one of a plurality of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence can be compared to a reference sequence having 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 alignment of sequences for comparison can be conducted by, for example, the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman, 1988, Proc. Nat’l. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the 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).

[0126] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, 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 through the National Center for Biotechnology Information. The 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 satisfy some positive-valued threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch residue; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction terminates when: the cumulative alignment score drops from its maximum achieved value by the quantity X; the cumulative score goes to zero or below 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 a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and both strands comparison as defaults. For amino acid sequences, the BLASTP program uses a word length of 3 and an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) 50, expectation value (E) 10, M = 5, N = -4, and both strands comparison as defaults.

[0127] 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 minimum sum probability (P(N)), which gives an indication of the probability that a match occurs by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001 in a comparison of a test nucleic acid with a reference nucleic acid, the nucleic acid is considered similar to the reference sequence.

[0128] 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)), 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. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453 (1970)) algorithm, incorporated into the GAP program (available from www.gcg.com) in the GCG software package, using a Blossom 62 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.

[0129] In addition to the above percent sequence identity, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide typically is substantially identical to a second polypeptide, e.g., where 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 complementary sequences 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.

[0130] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases 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)).

[0131] The nucleotides in "polynucleotide" or "nucleic acid" may contain modifications, including base modifications such as bromouridine and inosine derivatives; ribose modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, and phosphoroamidate.

[0132] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) suitable for transformation or transfection of a host cell and containing a nucleic acid sequence that directs and / or controls (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto.

[0133] As used herein, the term "operably linked" or functionally linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to a functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter or enhancer sequence is operably linked to the coding sequence. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically contiguous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically contiguous or located in extremely close proximity to the coding sequence whose transcription they enhance.

[0134] The term "co-expression" refers to the expression of different polypeptides together in a single host cell common to all of the polypeptides. Co-expression of a bispecific antibody means that the different parts that form the 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 expression vector for each of the individual halves of the bispecific antibody), or by incorporating an expression vector encoding all parts of the bispecific antibody. As used herein, "C-terminus" refers to the carboxy-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 amino group (-NH2).

[0135] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These phrases also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses its conservatively modified variants.

[0136] As used herein, the term "in vivo half-life" refers to the half-life of a molecule of interest or a variant thereof circulating in the blood of a given mammal.

[0137] Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be prepared by hybridoma methods using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which use human variable region libraries (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23).

[0138] A number of transgenic mouse strains are now available in which their murine 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). After antigen stimulation, such mice are capable of generating a human antibody repertoire from which antibodies of interest can be selected. Of particular note is the TrimeraTM system (Eren R et al., (1988) Immunology 93:154-161), in which human lymphocytes were transplanted into irradiated mice; the Selective Lymphocyte Isolation Antibody System (SLAM, Babcock et al., Proc Natl Acad Sci (1996) 93:7843-7848), in which human (or other species) lymphocytes were effectively passed through a large pooled in vitro antibody generation procedure followed by deconvolution, limiting dilution and selection procedures and XenomouseTM (Abgenix Inc). Alternative methods are available from Morphotek Inc using MorphodomaTM technology.

[0139] Phage display technology can be used to produce human antibodies and their fragments (McCafferty; (1990) Nature, 348:552-553 and Griffiths AD et al. (1994) EMBO J. 13:3245-3260). According to this technology, the isolated antibody variable domain genes are cloned in-frame into the major or minor coat of the protein gene of a filamentous phage (such as M13 or fd) and displayed as functionally isolated antibody fragments (usually with the help of a helper phage) on the surface of the phage particles. Selection based on the functional properties of the isolated antibodies leads to the selection of genes encoding the isolated antibodies that exhibit these properties. Phage display technology can be used to select antigen-specific antibodies from libraries prepared from human B cells taken from an individual suffering from a disease or disorder or alternatively from non-immunized human donors (Marks; J MolBio (1991) 222:581-591). When full-length human isolated antibodies containing the Fc domain are required, the phage-displayed derived fragments must be recloned into a mammalian expression vector containing the desired constant regions and a stable expression cell line established.

[0140] Techniques for affinity maturation (Marks; Biotechnol (1992) 10:779-783) can be used to provide binding affinity, where the affinity of a primary human isolated antibody is improved by sequentially substituting the variable regions of the H and L chains with naturally occurring variants and selecting on the basis of improved binding affinity. Variants of this technology are also now available, such as "epitope imprinting" (WO 93 / 06213; Waterhouse; Nucl Acids Res (1993) 21:2265-2266).

[0141] When used in the context of purified bispecific antibodies, the term "pure" relates to the purity and identity of different bispecific antibody combinations and constructs after co-expression in a selected cell under conditions where the cell expresses the bispecific antibody and after protein A purification using a complete UPLC-MS mass screening method. Pure or purity refers to the relative quantification of the heterodimers and homodimers of the bbmAb formed. Using the methods of the present invention, correctly formed heterodimeric bispecific antibodies can be observed, with a relative purity of more than 85% based on the intact mass signal intensity.

[0142] The terms "therapeutically acceptable amount" or "therapeutically effective amount" or "therapeutically effective dose" are used interchangeably to refer to an amount sufficient to achieve a desired result (i.e., reducing disease activity, reducing disease progression, reducing disease signs and / or symptoms, etc.). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. The therapeutically acceptable amount can be determined by first administering a low dose and then incrementally increasing the dose until the desired effect is achieved. The "prophylactically effective dose" and "therapeutically effective dose" of the molecules of the present invention can respectively prevent the onset of disease symptoms or reduce the severity of disease symptoms, said disease symptoms including those related to IL-13 activity and IL-18 activity.

[0143] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless indicated otherwise, the terms "patient" or "subject" are used interchangeably herein.

[0144] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of a molecule or pharmaceutical composition of the present disclosure, for example, for detection, diagnostic procedures, and / or treatment.

[0145] The terms "treat", "treating", "treatment", "prevent", "preventing", "prevention" include therapeutic treatment, prophylactic treatment and applications that reduce the risk of a subject developing a disorder or other risk factors. Treatment does not require complete cure of the disorder and encompasses alleviating symptoms or underlying risk factors. As used herein, a human antibody or fragment thereof comprises a heavy or light chain variable region or full-length heavy or light chain that is a "product of" or "derived from" a specific germline sequence if the variable region or full-length chain of the antibody is obtained from a system using human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening a human immunoglobulin gene library displayed on phage with an antigen of interest. A human antibody or fragment thereof that is a "product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of the germline immunoglobulin and selecting the germline immunoglobulin sequence that is most closely related (i.e., highest % identity) to the sequence of the human antibody. A human antibody that is a "product of" or "derived from" a specific human germline immunoglobulin sequence can contain amino acid differences compared to the germline sequence due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, the amino acid sequence of the selected human antibody typically is at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, the amino acid sequence of the human antibody can be at least 60%, 70%, 80%, 90% or at least 95% or even at least 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a specific germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene. In some cases, the human antibody can exhibit no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0146] Various aspects of the invention are described in further detail in the following paragraphs and subparagraphs.

[0147] Multispecific antibodies that bind IL-13 and IL-18

[0148] I. IL-18 binding domain

[0149] The present disclosure provides multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that are engineered to bind human IL-18. Recognition of IL-18 by the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure occurs through an "IL-18 antigen-binding domain", which may interchangeably be referred to as an "IL-18 binding domain".

[0150] In a preferred aspect, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-18 binding domain such that the multispecific binding molecule is monovalent for binding IL-18. In another preferred aspect, the multispecific binding molecule comprises more than one IL-18 binding domain, e.g., two IL-18 binding domains, such that the multispecific binding molecule is multivalent for binding IL-18, preferably divalent for binding IL-18.

[0151] In some aspects, the IL-18 binding domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises an IL-18 scFv or an IL-18 Fab, preferably an IL-18 Fab.

[0152] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises an IL-18 binding domain that has a K D of 10 -4 M to 10 -8 M, e.g., 10 -5 M to 10 -7 M, e.g., 10 -6 M or 10 -7 M binding affinity for IL-18.

[0153] 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 more than two IL-18 Fabs are also contemplated herein.

[0154] In some cases, Fabs can be prepared by methods known in the art. Papain can be used to cleave immunoglobulin monomers into two Fab fragments and one Fc fragment. Pepsin cleaves below the hinge region, thus forming F(ab')2 fragments and pFc’ fragments. The F(ab')2 fragments can be split into two Fab’ fragments by mild reduction. Fab fragments are highly stable due to non-covalent interactions occurring at the large interface between the heavy and light chain polypeptides, and the presence of a stable disulfide bond between the CH1 and CL domains (see Glover and Humphreys, Chapter 2, Antibodies, Volume 1: Production and Purification, Kluwer Academic / Plenum Publishers, New York 2004, edited by G Subramanian). For examples of linker orientation and size, see, e.g., Hollinger et al. (1993) PNAS U.S.A. 90:6444-6448, US2005 / 0100543, US2005 / 0175606, US2007 / 0014794, and WO 2006 / 020258, and WO 2007 / 024715, which are incorporated herein by reference.

[0155] The phrases “Fab that binds 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 equivalent binding affinity, e.g., binds IL-18 with comparable efficacy to a full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity compared to a full-length antibody, e.g., it binds IL-18 with a lower binding affinity, but still provides the biological responses described herein.

[0156] Preferred IL-18 Fabs for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprise a VH containing the amino acid sequence shown in SEQ ID NO:41 and a VL containing the amino acid sequence shown in SEQ ID NO:13.

[0157] In one aspect, the present disclosure provides polynucleotides encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that comprise an IL-18 binding domain (e.g., an IL-18 Fab). The present disclosure also provides isolated nucleic acid molecules encoding such Fabs.

[0158] The present disclosure provides an isolated nucleic acid molecule encoding a multispecific antibody (e.g., bispecific antibody) or a fragment thereof that comprises an IL-18 binding domain, wherein the IL-18 binding domain comprises a VH having the amino acid sequence shown in SEQ ID NO: 41 and comprises a VL having the amino acid sequence shown in SEQ ID NO: 13.

[0159] In one aspect, the IL-18 binding domain of the multispecific antibody (e.g., bispecific antibody) or a fragment thereof (e.g., IL-18 Fab) is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In one aspect, the entire construct of the multispecific antibody (e.g., bispecific antibody) or a 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 encoding the same amino acid) in coding DNA is biased among different species. This codon degeneracy allows the same polypeptide to be encoded by various nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least US 5,786,464 and US 6,114,148.

[0160] II. IL-13 Binding Domain

[0161] The present disclosure provides a multispecific antibody (e.g., bispecific antibody) or a fragment thereof that has been engineered to bind human IL-13. Recognition of IL-13 by the multispecific antibody (e.g., bispecific antibody) of the present disclosure occurs through an "IL-13 antigen-binding domain", which is interchangeably referred to as an "IL-13 binding domain".

[0162] In a preferred aspect, the multispecific antibody (e.g., bispecific antibody) or a fragment thereof comprises one IL-13 binding domain such that the multispecific binding molecule is monovalent for binding IL-13. In another preferred aspect, the multispecific binding molecule comprises more than one IL-13 binding domain, e.g., two IL-18 binding domains, such that the multispecific binding molecule is multivalent for binding IL-18 and preferably bivalent for binding IL-13.

[0163] In some aspects, the IL-13 binding domain of the multispecific antibody (e.g., bispecific antibody) or a fragment thereof comprises an IL-13 scFv or an IL-13 Fab, preferably an IL-13 Fab.

[0164] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises an IL-13 binding domain that has a K D of 10 -4 M to 10 -8 M, e.g., 10 -5 M to 10 -7 M, e.g., 10 -6 M or 10 -7 M binding affinity for IL-13.

[0165] 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 more than two IL-13 Fabs are also contemplated herein.

[0166] The phrases "Fab that binds 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 equivalent binding affinity, e.g., binds IL-13 with comparable efficacy to a full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity compared to a full-length antibody, e.g., it binds IL-13 with a lower binding affinity, but it still provides the biological responses described herein.

[0167] A preferred IL-13 Fab for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprises a VH containing the amino acid sequence shown in SEQ ID NO:55 and a VL containing the amino acid sequence shown in SEQ ID NO:27.

[0168] Another preferred IL-13 Fab for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprises a VH containing the amino acid sequence shown in SEQ ID NO:85 and a VL containing the amino acid sequence shown in SEQ ID NO:71.

[0169] In one aspect, the present disclosure provides polynucleotides encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that comprise an IL-13 binding domain (e.g., IL-13 Fab). The present disclosure also provides isolated nucleic acid molecules encoding these Fabs.

[0170] The present disclosure provides isolated nucleic acid molecules encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that comprise an IL-13 binding domain, wherein the IL-13 binding domain comprises a VH having the amino acid sequence shown in SEQ ID NO:55 and a VL having the amino acid sequence shown in SEQ ID NO:27.

[0171] The present disclosure also provides isolated nucleic acid molecules encoding multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that comprise an IL-13 binding domain, wherein the IL-13 binding domain comprises a VH having the amino acid sequence shown in SEQ ID NO:85 and a VL having the amino acid sequence shown in SEQ ID NO:71.

[0172] The present disclosure also includes constructs (e.g., cloning or expression vectors) comprising one or more of the above-described isolated nucleic acid molecules and polynucleotides. The present disclosure also includes host cells comprising one or more of the above-described constructs (e.g., cloning or expression vectors).

[0173] In one aspect, the present disclosure includes recombinant nucleic acid constructs comprising a polynucleotide encoding a multispecific antibody (e.g., bispecific antibody) or fragment thereof as disclosed herein, wherein the polynucleotide comprises a nucleic acid sequence encoding an IL-13 binding domain or fragment thereof.

[0174] III. Linkage and Orientation of Domains and Regions of Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof

[0175] As used herein, the terms "linked" or "linkage" refer to one part of a multispecific antibody (e.g., bispecific antibody) or fragment thereof being directly or indirectly attached to another part of the molecule. Direct attachment is one form of linkage and is referred to herein as "fusion". As an example of a molecule having the form A-B-C: part A is directly linked to part B and indirectly linked to part C (part A can also be described as being fused to part B). Using an scFv having the form VH-internal linker-VL as another example, VH is indirectly linked to VL and directly linked to the internal linker (the linker can also be described as being fused to both VL and VH).

[0176] In some embodiments, the IL-18 binding domain and the IL-13 binding domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof are linked. In some embodiments, the linkage is a direct linkage and thus the regions are fused to each other. 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., IL-13Fab) is linked, e.g., by a polypeptide linker, to the N-terminus of the IL-18 binding domain. In some embodiments, the IL-13 binding domain (e.g., IL-13Fab) is linked, e.g., fused, to the N-terminus of the IL-18 binding domain.

[0177] IV. Forms of Multispecific Antibodies

[0178] In some aspects, the multispecific antibody is a bispecific antibody. In some aspects, the bispecific antibody can be multivalent, e.g., bivalent, for one antigen and monovalent for another antigen. Exemplary bispecific antibodies are characterized by a first antigen-binding domain (e.g., comprising a first VL and a first VH) having binding specificity for a first antigen or epitope (e.g., IL-18) and a second antigen-binding domain having 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 epitope and the second epitope are on different antigens, e.g., two proteins (or subunits of a multimeric protein). In embodiments of the disclosure, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope or antigen (e.g., IL-18), and another binding domain having binding specificity for a second epitope or antibody (e.g., IL-13).

[0179] Protocols for generating bispecific or heterodimeric antibodies are known in the art; these protocols include, but are not limited to: for example, the "milling and mortaring" approach, as described, for example, in US 5,731,168; electrostatic steering Fc pairing, as described, for example, in WO2009 / 089004, WO 2006 / 106905, and WO 2010 / 129304; chain exchange engineered domain (SEED) heterodimer formation, as described, for example, in WO 2007 / 110205; Fab arm exchange, as described, for example, in WO2008 / 119353, WO 2011 / 131746, and WO 2013 / 060867; bispecific antibody conjugates, for example using heterobifunctional reagents having amine-reactive and thiol-reactive groups, cross-linking antibodies to generate bispecific structures, as described, for example, in US 4,433,059; bispecific antibodies or antibody-like molecule determinants generated by recombining half-antibodies (heavy chain-light chain pairs or Fabs) from different antibodies or antibody-like molecules, the recombination being carried out by cycles of reduction and oxidation of disulfide bonds between two heavy chains, as described, for example, in US 4,444,878; trifunctional antibodies, for example three Fab' fragments cross-linked by thiol-reactive groups, as described, for example, in US5,273,743; biosynthetic binding proteins, for example scFv pairs cross-linked by C-terminal tails preferably by disulfide bonds or amine-reactive chemical cross-linking, as described, for example, in US 5,534,254; bispecific antibodies, for example Fab fragments with different binding specificities, which dimerize via leucine zippers that have replaced constant domains, as described, for example, in US 5,582,996; bispecific and oligospecific monovalent receptors and oligovalent receptors, for example the VH-CH1 regions of two antibodies (two Fab fragments), which are linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody (typically with an associated light chain), as described, for example, in US 5,591,828; bispecific DNA-antibody conjugates, for example antibodies or Fab fragments cross-linked by double-stranded segments of DNA, as described, for example, in US 5,635,602; bispecific fusion proteins, for example expression constructs containing two scFvs (with a hydrophilic helical peptide linker between them) and a complete constant region, as described, for example, in US 5,637,481; multivalent and multispecific binding proteins, for example polypeptide dimers having a first domain containing an Ig heavy chain variable region binding domain and a second domain containing an Ig light chain variable region binding domain, commonly referred to as diabodies (also encompassing higher order structures, thus generating bispecific, trispecific, or tetraspecific molecules), as described, for example, in USas described in US 5,837,242; minibody constructs having linked VL and VH chains which are further linked via a peptide spacer to the antibody hinge region and CH3 region, which can dimerize to form bispecific / multivalent molecules, as for example described in US 5,837,821; VL and VH domains linked by a short peptide linker (e.g., 5 or 10 amino acids) or completely without a linker in either orientation, which VL and VH domains can form dimers to form bispecific diabodies; trimers and tetramers, as for example described in US 5,844,094; strings of VH domains (or VL domains in a family member) linked by peptide bonds to a crosslinkable group at the C-terminus, which crosslinkable groups further associate with VL domains to form a series of Fv (or scFv), as for example described in US 5,864,019; VL and VH domains, scFv or Fab, where one of the antigens binds monovalently and one of the antigens binds bivalently, optionally comprising a heterodimeric Fc region, as for example described in WO 2011 / 028952; single-chain binding polypeptides having both VL and VH domains linked by a peptide linker are combined into multivalent structures by non-covalent or chemical crosslinking to form, for example, homodivalent, heterodivalent, trivalent and tetravalent structures in scFv or diabody type forms, as for example described in US 5,869,620. Additional exemplary multispecific and bispecific molecules and methods for their preparation are found, for example, in the following documents: US5,910,573, US 5,932,448, US 5,959,083, US 5,989,830, US6,005,079, US 6,239,259, US 6,294,353, US 6,333,396, US 6,476,198, US 6,511,663, US6,670,453, US 6,743,896, US 6,809,185, US 6,833,441, US 7,129,330, US 7,183,076, US7,521,056, US 7,527,787, US 7,534,866, US 7,612,181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078 A1, US2004219643 A1, US2004220388A1, US2004242847 A1, US2005003403 A1, US2005004352 A1, US2005069552A1, US2005079170 A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782 A1, US2005266425 A1, US2006083747A1, US2006120960 A1, US2006204493 A1, US2006263367A1, US2007004909 A1, US2007087381 A1, US2007128150A1, US2007141049A1, US2007154901 A1, US2007274985 A1, US2008050370A1, US2008069820A1, US2008152645 A1, US2008171855 A1, US2008241884A1, US2008254512 A1, US2008260738 A1, US2009130106A1, US2009148905 A1, US2009155275 A1, US2009162359A1, US2009162360A1, US2009175851 A1, US2009175867 A1, US2009232811A1, US2009234105A1, US2009263392 A1, US2009274649 A1, EP346087A2, WO 2000 / 06605A2, WO 2007 / 2635A2, WO 2004 / 081051 A1, WO 2006 / 020258A2, WO 2007 / 044887A2, WO 2007 / 095338A2, WO2007 / 137760A2, WO 2008 / 119353 A1, WO 2009 / 021754 A2, WO2009 / 068630A1, WO 1991 / 03493A1, WO 1993 / 23537A1, WO 1994 / 09131A1, WO 1994 / 12625A2, WO 1995 / 09917A1, WO 1996 / 37621A2, WO1999 / 64460A1. The content of the applications cited above is incorporated herein by reference in its entirety. 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 of the multispecific or bispecific forms known in the art and described above. The preferred forms of the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are described in more detail below.

[0180] V. Exemplary Anti-IL-13 / IL-18 Bispecific Antibodies

[0181] The amino acid sequences in Table 1 are examples of the IL-13 / IL-18 bispecific antibody and its parts.

[0182] Table 1. Exemplary IL-13 / IL-18 bispecific antibody sequences.

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] In some embodiments, the multispecific antibodies described herein are bispecific antibodies that comprise an amino acid sequence having 1, 2, or 3 substitutions, deletions, or insertions relative to the sequences of Table 1.

[0213] Exemplary forms of the IL-13 / IL-18 bispecific antibodies are shown in Table 2. All bbmAbs combine an anti-IL13 binding domain and an anti-IL-18 binding domain and are based on the human IgG1 form, wherein all bbmAbs contain the YTE half-life extension mutation, and two of the bbmAbs also contain the LALA silent mutation. More specifically, bbmAb2, bbmAb1, and bbmAb3 contain the YTE half-life extension mutation in the Fc. bbmAb4 and bbmAb5 contain both the LALA silent mutation and the YTE half-life extension mutation. bbmAb2, bbmAb1, bbmAb4, and bbmAb5 combine the variable domains of mAb1 and MAb2, while bbmAb3 combines the variable domains of mAb1 and mAb3.

[0214] bbmAb2, bbmAb4, and bbmAb3 carry the KiH stud heterodimerization mutation in the anti-IL-18 heavy chain Fc, while the KiH socket mutation is in the anti-IL-13 heavy chain Fc. bbmAb2, bbmAb4, and bbmAb3 carry the KiH stud mutation in the anti-IL-18 heavy chain Fc and the KiH socket mutation in the anti-IL-13 heavy chain Fc.

[0215] Table 2. Forms of Exemplary IL-13 / IL-18 Bispecific Antibodies

[0216]

[0217] VI. Modifications of the Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof of the Present Disclosure

[0218] This application includes variants and / or fragments of the molecules described herein having various modifications in the binding domain, variable domain, and / or constant region, as well as fusions and conjugates of the disclosed molecules. For example, the Fc region of the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be wild-type or can be modified to obtain various outcomes. Preferred modifications of Fc include the "LS" mutations (M428L, N434S (EU numbering)) and "YTE" mutations (M252Y, S254T, T256E (EU numbering)) for half-life extension, the "DAPA" mutations (D265A, P329A (EU numbering)) for effector silencing, and the "stalk and socket" mutations (e.g., stalk S354C, T366W; socket Y349C, T366S, L368A, Y407V (EU numbering)) that facilitate correct chain pairing.

[0219] A. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof with variable region modifications

[0220] Each of the VH and VL domains of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure contains the hypervariable region CDR1, CDR2, and CDR3 sequences. In certain embodiments, one or more of these CDR sequences can have conservative modifications of the amino acid sequence, and wherein the modified molecule retains or has enhanced binding properties compared to the parental antibody.

[0221] In addition, it has been found that in certain cases it is beneficial to mutate residues in the framework region to maintain or enhance the antigen-binding ability of the antibody (see, e.g., US 5,530,101, US 5,585,089, US 5,693,762, and US 6,180,370 to Queen et al.). The 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 properties.

[0222] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 domains to improve one or more binding properties (e.g., affinity) of the antibody of interest, referred to as "affinity maturation". Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effects on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays as described herein and provided in the examples. Conservative modifications (discussed above) can be introduced. The mutations can be amino acid substitutions, additions, or deletions. Moreover, typically no more than one, two, three, four, or five residues, preferably one or two residues, within the CDR region are altered.

[0223] Amino acid sequence variants of the multispecific antibody (e.g., bispecific antibody) or fragment 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, insertions, or substitutions of residues within the amino acid sequence of the molecule of the invention. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired antigen-binding characteristics. Amino acid changes can also alter post-translational processes of the molecule, such as changing the number or location of glycosylation sites.

[0224] This application includes variants of the multispecific antibody (e.g., bispecific antibody) or fragment thereof having amino acid conservative modifications in the variable region and / or constant region.

[0225] B. Multispecific antibody (e.g., bispecific antibody) or fragment thereof with enhanced heterodimerization

[0226] Insufficient heterodimerization of the two heavy chain domains of an antibody can be an obstacle to increasing the yield of the desired multispecific antibody (e.g., bispecific antibody) or fragment thereof and represents a purification challenge. A variety of available methods can be used to enhance the dimerization of the two heavy chain domains of a bispecific or multispecific antibody or antibody-like molecule, as disclosed in the following documents: EP 1870459A1; US5,582,996; US 5,731,168; US 5,910,573; US 5,932,448; US 6,833,441; US 7,183,076; US2006204493 A1; and WO 2009 / 089004 A1.

[0227] This disclosure provides methods for enhancing the dimerization (heterodimerization) of two interacting heterologous polypeptides and / or reducing the dimerization (homodimerization) of two identical polypeptides. Typically, each of the two interacting polypeptides comprises an Fc region having the CH2 and CH3 domains of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, type, or subclass, and preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) type, most preferably IgG1.

[0228] Typically, in addition to the CH3 domain, the polypeptides of the present disclosure further comprise other antibody fragments, such as the CH1 domain, CH2 domain, hinge domain, one or more VH domains, one or more VL domains, one or more CDRs and / or antigen-binding fragments (such as scFv and / or Fab) as described herein. These antibody fragments are derived from various types of antibodies described herein, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific or multispecific antibodies, camelized antibodies, anti-idiotypic (anti-Id) antibodies and antibody conjugates. The heterodimerization of two different heavy chains at the CH3 domain gives rise to the desired antibody or antibody-like molecule, while the homodimerization of the same heavy chain will reduce the yield of the desired antibody or molecule. In an exemplary embodiment, two or more heteropolypeptide chains comprise two chains that comprise the CH3 domain and form a molecule in any of the multivalent antibody (e.g., bispecific antibody) or fragment form thereof as described above in the present disclosure. In an embodiment, the two heteropolypeptide chains comprising the CH3 domain comprise modifications (relative to the unmodified chains) that favor the heterodimeric association of the polypeptides. Various examples of modification strategies are provided below.

[0229] Knobs-into-Holes (KIH) (also known as "keyhole structure")

[0230] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure may comprise one or more (e.g., multiple) mutations to one or more constant domains (e.g., to the CH3 domain). In one instance, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure comprise two polypeptides, each polypeptide comprising the heavy chain Fc or constant domain of an antibody, e.g., the CH2 or CH3 domain. In instances, the two heavy chain constant domains, e.g., the CH2 or CH3 domains of the multispecific antibody (e.g., bispecific antibody) or fragment thereof, comprise one or more mutations that permit heterodimeric association between the two chains. In one aspect, the one or more mutations are disposed on the CH2 domains of the two heavy chains of the multispecific antibody (e.g., bispecific antibody) or fragment thereof. In one aspect, the one or more mutations are disposed on 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 the first polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that comprises a heavy chain constant domain generate a "stalk" and one or more mutations to the second polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that comprises a heavy chain constant domain generate a "socket" such that heterodimerization of the polypeptides of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that comprises a heavy chain constant domain results in the "stalk" engaging (e.g., interacting, e.g., the CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacts with the CH3 domain of the second polypeptide) the "socket" interface. As used herein, the term "stalk" refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that comprises a heavy chain constant domain and can thus be positioned in the complementary "socket" in the interface of the second polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that comprises a heavy chain constant domain to stabilize the heteropolymer and thus facilitate heteropolymer formation (e.g., relative to the homopolymer). The stalk may be present in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Preferred input residues for forming the stalk are typically naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In a preferred embodiment, the original residue for forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0231] "Mortar" refers to at least one amino acid side chain that indents into the interface of a second polypeptide of a multispecific antibody (e.g., bispecific antibody) or a fragment thereof that contains a heavy chain constant domain and thus accommodates a corresponding pestle on the adjacent junction surface of a first polypeptide of a multispecific antibody (e.g., bispecific antibody) or a fragment thereof that contains a heavy chain constant domain. The mortar can be present in the original interface or can be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Preferred input residues for forming the mortar are typically naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Most preferred are serine, alanine, or threonine. In a preferred embodiment, the original residue for forming the mortar has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0232] In one embodiment, the first CH3 domain is mutated at residue 366, 405, or 407 according to the EU numbering scheme of Kabat et al. (pp. 688 - 696, in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Maryland)) to generate a "pestle" or "mortar" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at the following positions: residue 407 (if residue 366 in the first CH3 domain is mutated), residue 394 (if residue 405 in the first CH3 domain is mutated), or residue 366 (if residue 407 in the first CH3 domain is mutated) (EU numbering) to generate a "pestle" or "mortar" that is complementary to the "mortar" or "pestle" of the first CH3 domain.

[0233] In another embodiment, the first CH3 domain is mutated at residue 366 (EU numbering) to create a "peg" or "socket" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at residues 366, 368, and / or 407 (EU numbering) to create a "peg" or "socket" that is complementary to the "socket" or "peg" of the first CH3 domain. In one embodiment, the mutation of the first CH3 domain introduces a tyrosine (Y) residue at position 366. In an embodiment, the mutation of the first CH3 is T366Y. In one embodiment, the mutation of the first CH3 domain introduces a tryptophan (W) residue at position 366. In an embodiment, the mutation of the first CH3 is T366W. In an embodiment, the mutation of the second CH3 domain that heterodimerizes with the first CH3 domain mutated at position 366 (e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., comprising the mutation T366Y or T366W) comprises a mutation at position 366, a mutation at position 368, and a mutation at position 407 (EU numbering). In an embodiment, 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 an embodiment, the mutations comprise T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of a 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, and vice versa. In one embodiment, the first CH3 domain of a 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, and vice versa.

[0234] Additional peg-socket structure mutations for any multispecific antibody (e.g., bispecific antibody) or fragment thereof suitable for use in the present invention are further described, for example, in WO 1996 / 027011 and Merchant et al., (1998) Nat. Biotechnol. [Nature Biotechnology], 16:677-681, the contents of which are hereby incorporated by reference in their entirety.

[0235] In any of the embodiments described herein, the CH3 domain may be additionally mutated to introduce pairs of cysteine residues. Without being bound by theory, it is believed that introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to the heterodimeric antibody. In an embodiment, the first CH3 domain comprises a cysteine at position 354 (EU numbering) and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine at position 349 (EU numbering). In an embodiment, the first CH3 domain of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof comprises a cysteine at position 354 (e.g., comprising the mutation S354C) and a tyrosine (Y) at position 366 (e.g., comprising the mutation T366Y), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine at position 349 (e.g., comprising the mutation Y349C), a serine at position 366 (e.g., comprising the mutation T366S), an alanine at position 368 (e.g., comprising the mutation L368A), and a valine at position 407 (e.g., comprising the mutation Y407V). In an embodiment, the first CH3 domain of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof comprises a cysteine at position 354 (e.g., comprising the mutation S354C) and a tryptophan (W) at position 366 (e.g., comprising the mutation T366W), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine at position 349 (e.g., comprising the mutation Y349C), a serine at position 366 (e.g., comprising the mutation T366S), an alanine at position 368 (e.g., comprising the mutation L368A), and a valine at position 407 (e.g., comprising the mutation Y407V).

[0236] IgG heterodimerization

[0237] In one aspect, heterodimerization of a polypeptide chain (e.g., a half-antibody) of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof is increased by introducing one or more mutations in a CH3 domain derived from the IgG1 antibody class. In an embodiment, the mutation comprises a K409R mutation of one CH3 domain paired with an F405L mutation in a second CH3 domain (EU numbering). Additional mutations may also, or alternatively, be at positions 366, 368, 370, 399, 405, 407, and 409 (EU numbering). Preferably, heterodimerization of a polypeptide comprising such a mutation is achieved under reducing conditions, e.g., at 25 °C - 37 °C, e.g., 25 °C or 37 °C, in 10 - 100 mM 2-MEA (e.g., 25, 50, or 100 mM 2-MEA) for 1 - 10 hours, e.g., 1.5 - 5 hours, e.g., 5 hours.

[0238] The amino acid substitutions described herein are introduced into the CH3 domain using techniques known in the art. Generally, the DNA encoding one or more heavy chains is genetically engineered using the techniques described in Mutagenesis: a Practical Approach. Oligonucleotide-mediated mutagenesis is the preferred method for preparing substitution variants of DNA encoding two heterologous heavy chains. This technique is known in the art as described by Adelman et al., (1983) DNA, 2:183.

[0239] Suitable IgG heterodimerization strategies are described, for example, in WO 2008 / 119353, WO 2011 / 131746, and WO 2013 / 060867, the contents of which are hereby incorporated by reference in their entirety.

[0240] In any of the embodiments described herein, the CH3 domain may be further mutated to introduce pairs of cysteine residues. Without being bound by theory, it is believed that introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to a multispecific antibody (e.g., a bispecific antibody) or fragment thereof that heterodimerizes. In an embodiment, the first CH3 domain comprises a cysteine at position 354 (EU numbering), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine at position 349 (EU numbering).

[0241] Polar bridge

[0242] In one aspect, heterodimerization of the polypeptide chains (e.g., half-antibodies) of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof is increased by introducing mutations based on the "polar bridging" principle, which is to create residues at the binding interface of two polypeptide chains that interact with residues having similar (or complementary) physical properties in the heterodimer configuration while interacting with residues having different physical properties in the homodimer configuration. In particular, these mutations are designed such that in heterodimer formation, polar residues interact with polar residues and hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, the residues are mutated such that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer configuration and the unfavorable interactions in the homodimer configuration together act to make it more likely for the CH3 domains to form heterodimers than homodimers.

[0243] In an exemplary embodiment, the above mutations are made at one or more positions among residues 364, 368, 399, 405, 409, and 411 (EU numbering) of the CH3 domain.

[0244] In one aspect, 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 residue of serine at position 364 is replaced by leucine; Thr366Val: the original residue of threonine at position 366 is replaced by valine; Leu368Gln: the original residue of leucine at position 368 is replaced by glutamine; Asp399Lys: the original residue aspartic acid at position 399 is replaced by lysine; Phe405Ser: the original residue phenylalanine at position 405 is replaced by serine; Lys409Phe: the original residue lysine at position 409 is replaced by phenylalanine; Thr411Lys: the original residue of threonine at position 411 is replaced by lysine.

[0245] In another aspect, one or more mutations selected from the group consisting of: Tyr407Phe, Lys409Gln, and Thr411Asp can be introduced into the other CH3 (e.g., Tyr407Phe: the original residue tyrosine at position 407 is replaced by phenylalanine; Lys409Glu: the original residue lysine at position 409 is replaced by glutamic acid; Thr411Asp: the original residue of threonine at position 411 is replaced by aspartic acid).

[0246] In another aspect, one CH3 domain has one or more mutations selected from the group consisting of: Ser364Leu, Thr366Val, Leu368Gln, Asp399Lys, Phe405Ser, Lys409Phe, and Thr411Lys, while the other CH3 domain has one or more mutations selected from the group consisting of: Tyr407Phe, Lys409Gln, and Thr411Asp.

[0247] In an exemplary embodiment, the original residue of threonine at position 366 in one CH3 domain is replaced by valine, while the original residue of tyrosine at position 407 in the other CH3 domain is replaced by phenylalanine.

[0248] In another exemplary embodiment, the original residue of serine at position 364 in one CH3 domain is replaced by leucine, and the original residue of leucine at position 368 in the same CH3 domain is replaced by glutamine.

[0249] In yet another exemplary embodiment, the initial residue of phenylalanine at position 405 of one CH3 domain is replaced with serine and the initial residue of lysine at position 409 of this CH3 domain is replaced with phenylalanine, while the initial residue of lysine at position 409 of another CH3 domain is replaced with glutamine.

[0250] In yet another exemplary embodiment, the initial residue of aspartic acid at position 399 of one CH3 domain is replaced with lysine, and the initial residue of threonine at position 411 of the same CH3 domain is replaced with lysine, while the initial residue of threonine at position 411 of another CH3 domain is replaced with aspartic acid.

[0251] The amino acid substitutions described herein can be introduced into the CH3 domain using techniques known in the art. Generally, the DNA encoding one or more heavy chains is genetically engineered using the techniques described in Mutagenesis: a Practical Approach. Oligonucleotide-mediated mutagenesis is the preferred method for preparing substitution variants of the DNA encoding two heterologous heavy chains. This technique is known in the art as described by Adelman et al., (1983) DNA, 2:183.

[0252] The polar bridge strategy is 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 hereby incorporated by reference in their entirety.

[0253] In any of the embodiments described herein, the CH3 domain can be further mutated to introduce a pair of cysteine residues. Without being bound by theory, it is believed that introducing a pair of cysteine residues capable of forming a disulfide bond provides stability to a heterodimerizing multispecific antibody (e.g., a bispecific antibody). In an embodiment, 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).

[0254] C. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof having an extended in vivo half-life.

[0255] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present invention can be further modified to have an extended in vivo half-life.

[0256] Multiple strategies can be used to extend the half-life of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure. For example, by chemical conjugation with polyethylene glycol (PEG), reCODEPEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; by genetic fusion and transfer with proteins that bind serum proteins such as albumin, IgG, FcRn; by coupling (genetically or chemically) with other binding moieties that bind serum proteins such as nanobodies, Fab, DARPin, avimer, affibody, and anticalin; by genetic fusion with rPEG, albumin, albumin domains, albumin-binding proteins, and Fc; or by incorporation into nanocarriers, sustained-release formulations, or medical devices.

[0257] In addition, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure having an increased in vivo half-life can be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or its FcRn-binding fragment (preferably the Fc region or a fragment thereof). See, e.g., WO 1998 / 23289, WO 1997 / 34631, and US6,277,375. Preferred modifications to the Fc of the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof for extending the half-life include the "LS" mutations (M428L, N434S (EU numbering)) and the "YTE" mutations (M252Y, S254T, T256E (EU numbering)).

[0258] Furthermore, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be conjugated or fused with one or more human serum albumin (HSA) polypeptides or a portion thereof. Additionally, albumin is used to make the molecule more stable in vivo or have a longer half-life in vivo. The techniques are known in the art, see, e.g., WO 1993 / 15199, WO 1993 / 15200, and WO 2001 / 77137; and EP 413622. It has also been proposed to use the N-terminal fragment of HSA for fusion with polypeptides (e.g., EP 399666). Thus, genetically or chemically fusing or conjugating the molecule to albumin can stabilize or extend the shelf life, and / or retain the activity of the molecule in solution, in vitro, and / or in vivo for an extended period of time. Other methods involving HSA fusion can be found, e.g., in WO 2001 / 077137 and WO 2003 / 06007, which are incorporated herein by reference. In a particular embodiment, the expression of the fusion protein is carried out in a mammalian cell line, e.g., a CHO cell line.

[0259] D. Fc silencing

[0260] In embodiments of the present disclosure incorporating one or more constant domains (such as heavy chain constant regions), it may be beneficial to include one or more mutations within the hFc to silence effector functions such as ADCC and / or CDC. Activation of immune cells preferentially occurs in the presence of cross-linking with target cells. However, human Fc can bind to both high-affinity and low-affinity FcRγ receptors. Thus, cross-linking of receptors (such as CD3) on immune cells and subsequent agonism may occur after binding in the absence of tumor targeting. Additionally, Fc cross-linking via γ receptors can induce antibody-dependent cell cytotoxicity (ADCC). When human Fc is complexed on the cell surface, it can also bind complement proteins and induce complement-dependent cytotoxicity (CDC). Mutations to residues in Fc that reduce or eliminate these interactions can thus limit these effects and focus the impact of the molecules described herein on tumor target cells.

[0261] In embodiments, one or more (such as all) of the heavy chain constant region domains of a multispecific antibody (such as a bispecific antibody) or a fragment thereof comprise DAPA mutations (such as D265A and P329A in EU numbering). See, for example, Shields RL et al., (2001) J Biol Chem., 276(9):6591-604; US2015 / 0320880 A1, the contents of each of which are incorporated herein by reference in their entirety.

[0262] In embodiments, one or more (such as all) of the heavy chain constant region domains of the multispecific antibody (such as a bispecific antibody) or a fragment thereof comprise LALA mutations (such as L234A and L235A in EU numbering). For example, Hezareh M et al., (2001) Journal of Virology, 75(24):12161-12168; Shields RL et al., (2001) ibid., the contents of each of which are incorporated herein by reference in their entirety.

[0263] In embodiments, one or more (such as all) of the heavy chain constant region domains of the multispecific antibody (such as a bispecific antibody) or a fragment thereof comprise an N279A mutation (according to EU numbering) (see, for example, Tao MH and Morrison SL (1989) J Immunol. 143(8):2595-601; Shields RL et al., (2001) ibid., the contents of each of which are incorporated herein by reference in their entirety).

[0264] Additional Fc mutations for providing silent effector function are described in WO2014 / 145806 (e.g., in Figure 7 ), which is incorporated herein by reference in its entirety. An example of a silent IgG1 antibody from WO 2014 / 145806 contains the E233P, L234V, L235A, and S267K mutations and a deletion of G236 (G236del). Another example of a silent IgG1 antibody from WO 2014 / 145806 contains the E233P, L234V, and L235A mutations, and a G236 deletion (G236del). Another example of a silent IgG1 antibody from WO2014 / 145806 contains the S267K mutation.

[0265] E. Conjugates

[0266] This disclosure includes multispecific antibodies (e.g., bispecific antibodies) or fragments thereof that are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or a 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 produce a fusion protein. Methods for fusing or conjugating a protein, polypeptide, or peptide to an antibody or antibody fragment are known in the art. See, for example, US 5,336,603, US5,622,929, US 5,359,046, US 5,349,053, US5,447,851, and US 5,112,946; EP 307434 and EP 367166; WO 1996 / 04388 and WO 1991 / 06570; Ashkenazi et al., (1991) PNAS.USA [Proceedings of the National Academy of Sciences of the United States of America] 88:10535-10539; Zheng et al., (1995), J. Immunol. [Journal of Immunology] 154:5590-5600; and Vil et al., (1992) PNAS.USA [Proceedings of the National Academy of Sciences of the United States of America] 89:11337-11341.

[0267] Additional fusion proteins can be generated by techniques such as 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. Generally, see US 5,605,793, US 5,811,238, US 5,830,721, US 5,834,252, and US 5,837,458; Patten et al., (1997), Curr. Opinion Biotechnol. [Current Opinion in Biotechnology] 8:724-33; Harayama, (1998), Trends Biotechnol. [Trends in Biotechnology] 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. [Journal of Molecular Biology] 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques [Biotechnology] 24(2):308-313 (each of these patents and publications is hereby incorporated by reference in its entirety). The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof described herein can be altered by random mutagenesis, such as by error-prone PCR, random nucleotide insertion, or other methods, prior to recombination. The polynucleotides encoding fragments of the molecules of the invention can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules.

[0268] In addition, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be fused to a tag sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the tag amino acid sequence is a hexahistidine peptide, particularly a tag such as that provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), of which a variety are commercially available. As described in Gentz et al., (1989) PNAS. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:821-824, for example, hexahistidine provides convenience for the purification of fusion proteins. Other peptide tags for purification include, but are not limited to, the hemagglutinin ("HA") tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37:767) and the "flag" tag.

[0269] In other embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof is conjugated to a diagnostic or detectable agent. Such molecules can be used to monitor or prognose the onset, development, progression, and / or severity of a disease or disorder as part of a clinical testing procedure, such as to determine the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by conjugating the molecule to a detectable substance, including but not limited to various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; cofactors such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive substances such as, but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin; and positron-emitting metals and non-radioactive paramagnetic metal ions for use in various positron emission tomography.

[0270] This application further encompasses the use of a multispecific antibody (e.g., bispecific antibody) or fragment thereof conjugated to a therapeutic moiety. The molecules or fragments of the present disclosure 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 such as an α-emitter. Cytotoxic or cytostatic agents include any agent that is detrimental to cells.

[0271] In addition, the multispecific antibody (e.g., bispecific antibody) or fragment thereof can be conjugated to a therapeutic or pharmaceutical moiety that modulates a given biological response. The therapeutic or pharmaceutical moiety should not be construed to be limited to classical chemotherapeutic agents. For example, the pharmaceutical moiety can be a protein, peptide, or polypeptide having the 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.

[0272] For further discussion of the types of cytotoxins, linkers, and methods for conjugating therapeutic agents to the molecule, see also Saito et al., (2003) Adv. Drug Deliv. Rev. [Advanced Drug Delivery Reviews] 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. [Cancer Immunology and Immunotherapy] 52:328-337; Payne (2003) Cancer Cell [Cancer Cell] 3:207-212; Allen (2002) Nat. Rev. Cancer [Nature Reviews - Cancer] 2:750-763; Pastan and Kreitman (2002) Curr. Opin. Investig. Drugs [Current Opinion in Investigational Drugs], 3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. [Advanced Drug Delivery Reviews] 53:247-264.

[0273] The multispecific antibody (e.g., bispecific antibody) or fragment thereof can also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceutical, also referred to as a radioimmunoconjugate. Examples of radioisotopes that can be conjugated to the molecule 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. [Clinical Cancer Research] 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. [Bioconjugate Chemistry] 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. [Nuclear Medicine and Biology] 26(8):943-50, each of which is incorporated by reference in its entirety.

[0274] Techniques for conjugating a therapeutic moiety to an antibody are known, see, for example, 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 Prospective Of The Therapeutic Use Of 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.

[0275] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof may also be attached 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.

[0276] VII. Methods for Generating the Antibodies of the Invention

[0277] When the polypeptides or fragments thereof of the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are crosslinked, these functional linkages can be achieved using methods known in the art. Covalent conjugation can be carried out using a variety of coupling agents or crosslinking agents. Examples of crosslinking 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, for example, Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al. (1985) PNAS. USA 82:8648). Other methods include those described in 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). Conjugating agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0278] Alternatively, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present invention can be recombinantly produced by introducing a DNA construct encoding the desired molecule into an expression vector and expressing and assembling the desired molecule in the same host cell.

[0279] A. Preparation of Polypeptide Chains

[0280] Polypeptides, antibodies, and their fragments (e.g., half antibodies) can be generated by a variety of techniques, including conventional monoclonal antibody methods such as the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Many techniques for generating monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes.

[0281] The animal system used for preparing hybridomas is the murine system. The generation of hybridomas in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. The fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0282] Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequences of murine monoclonal antibodies prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered using standard molecular biology techniques to contain non-murine (e.g., human) immunoglobulin sequences. For example, to generate chimeric antibodies, murine variable regions can be joined to human constant regions using methods known in the art (see, e.g., US 4,816,567 to Cabilly et al.). To generate humanized antibodies, murine CDR regions can be inserted into a human framework using methods known in the art. See, e.g., US 5,225,539 and US 5,530,101 to Winter; US 5,585,089; US 5,693,762 and US 6,180,370 to Queen et al.

[0283] 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 a partial human immune system rather than a murine system. These transgenic and transchromosomic mice include mice that are herein referred to as HUmAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0284] HUmAb mice (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unrearranged human heavy chains (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al., 1994 Nature 368(6474):856 - 859). Thus, the mice exhibit reduced expression of murine IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic hypermutation to produce high-affinity human IgG. KMonoclonal (Lonberg et al., (1994) supra; in 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. N.Y. Acad. Sci. 764:536-546). The preparation and use of HUmAb mice and genomic modifications made in such mice are further described in the following references: 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 hereby incorporated by reference in their entirety. See also US 5,545,806; US 5,569,825; US 5,625,126; US 5,633,425; US 5,789,650; US 5,877,397; US 5,661,016; US 5,814,318; US 5,874,299; and US 5,770,429; all to Lonberg and Kay; US 5,545,807 to Surani et al.; WO 1992 / 103918, WO 1993 / 12227, WO 1994 / 25585, WO1997113852, WO 1998 / 24884 and WO 1999 / 45962, all to Lonberg and Kay; and WO 2001 / 14424 to Korman et al.

[0285] In another embodiment, the human antibodies used in the present disclosure can be produced using mice that are transgenic and transchromosomal and carry human immunoglobulin sequences, such as mice that carry 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 by Ishida et al.

[0286] Still further, alternative transgenic animal systems that express human immunoglobulin genes are available in the art and can be used to produce the human antibodies used in the present disclosure. For example, an alternative transgenic system known as Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in US 5,939,598; US 6,075,181; US 6,114,598; US 6,150,584; and US 6,162,963 (granted to Kucherlapati et al.).

[0287] In addition, alternative transchromosomal animal systems that express human immunoglobulin genes are available in the art and can be used to produce the human antibodies used in the present disclosure. For example, mice known as "TC mice" that carry a human heavy chain transchromosome and a human light chain transchromosome can be used; such mice are described in Tomizuka et al., (2000) PNAS USA 97:722-727. In addition, cattle that carry human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894), and these cattle can be used to produce the human antibodies used in the present application.

[0288] Phage display methods for screening human immunoglobulin gene libraries can also be used to prepare human monoclonal antibodies. Such phage display methods for isolating human antibodies are established in the art or described in the following examples. See, for example: US 5,223,409; US 5,403,484; and US 5,571,698 (granted to Ladner et al.); US 5,427,908 and US 5,580,717 (granted to Dower et al.); US 5,969,108 and US 6,172,197 (granted to McCafferty et al.); and US 5,885,793; US 6,521,404; US 6,544,731; US 6,555,313; US 6,582,915; and US 6,593,081 (granted to Griffiths et al.).

[0289] The human monoclonal antibodies used in this disclosure can also be prepared using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described, for example, in US 5,476,996 and US 5,698,767 (issued to Wilson et al.).

[0290] Methods for preparing bispecific antibodies are known in the art and are discussed in this application.

[0291] B. Methods for generating recombinant molecules

[0292] In one embodiment, this application provides a method for recombinantly generating one or more primary polypeptide chains of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof, the method comprising: 1) generating one or more DNA constructs comprising nucleic acid molecules encoding each of the polypeptide chains of a multispecific binding molecule; 2) introducing the one or more DNA constructs into one or more expression vectors; 3) co-transfecting the one or more expression vectors into one or more host cells; and 4) expressing and assembling the molecule in the host cell or solution.

[0293] In this regard, the present disclosure provides isolated nucleic acids, such as one or more polynucleotides, encoding the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof described herein, such as multispecific binding molecules comprising, for example, 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 continuous polynucleotide. In other embodiments, the isolated polynucleotide is arranged on two or more continuous nucleic acid sequences.

[0294] In some aspects, the isolated nucleic acid comprises a sequence encoding an IL-13 binding domain or a fragment thereof and a sequence encoding an IL-18 binding domain or a fragment thereof. In some aspects, the sequence encoding the IL-13 binding domain or a fragment thereof and the sequence encoding the IL-18 binding domain are arranged on separate polynucleotides, which are also referred to as "sets of nucleic acid molecules".

[0295] In some aspects, the sequence encoding the IL-13 binding domain or a fragment thereof and the sequence encoding the IL-18 binding domain are arranged on a single polynucleotide.

[0296] In an 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 host cells at a ratio that results in optimal assembly. The encoded heavy and light chains are expressed and assembled into a functional molecule in the host cell.

[0297] 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 a functional molecule.

[0298] Provided herein are cloning and expression vectors comprising one or more nucleic acid molecules or a set of nucleic acid molecules that encode a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof as described herein, wherein the vector is suitable for recombinantly producing a multispecific binding molecule. Also provided herein are processes for producing a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof as described herein, the process comprising culturing a host cell as disclosed herein under conditions sufficient to express the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof, and then purifying and recovering the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof from the host cell culture.

[0299] As described herein, desired mutations on the variable or constant regions of the molecules described herein can be introduced at this stage, such as mutations for enhancing heterodimerization.

[0300] DNA sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding the heavy or light chains of the molecules of the invention (e.g., the sequences as described in the following examples). Direct chemical synthesis of nucleic acids can be accomplished 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 diethylphosphoramidite method of Beaucage et al., (1981) Tetra. Lett. 22:1859; and the solid support method of U.S. 4,458,066. Introduction of mutations into polynucleotide sequences by PCR can be carried out as described in the following references, such as PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (ed.), Freeman Press, New York, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al., (ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., (1991) Nucleic Acids Res. 19:967; and Eckert et al., (1991) PCR Methods and Applications 1:17.

[0301] Also provided in the present disclosure are expression vectors and host cells for generating the above molecules. The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) suitable for transforming or transfecting a host cell and containing a nucleic acid sequence that directs and / or controls (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. A variety of expression vectors can be used to express the polynucleotide encoding the chain or binding domain of the molecule. Both virus-based expression vectors and non-virus-based expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically having an expression cassette for expressing a protein or RNA), and artificial human chromosomes (see, e.g., Harrington et al., (1997) Nat Genet 15:345). For example, non-viral vectors that can be used to express polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, pThioHis B, and pThioHis C, pcDNA3.1 / His, pEBVHis A, pEBVHis B, and pEBVHis C (Invitrogen, San Diego, CA), MPSV vectors, and a variety of other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vectors based on SV40, papillomaviruses, HBP Epstein Barr virus, 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.

[0302] The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of the inserted sequence under conditions other than the inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The transformed organism can be cultured under non-inducing conditions without biasing the population towards coding sequences for which the host cell better tolerates their expression products. In addition to the promoter, other regulatory elements may be required or desired for efficient expression of the heavy and light chains of a multispecific antibody (e.g., bispecific antibody) or its fragment. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Additionally, expression efficiency can be increased by including an enhancer suitable for the cell system in use (see, for example, Scharf et al., (1994) Results Probl. Cell Differ. [Results and Problems in Cell Differentiation] 20:125; and Bittner et al., (1987) Meth. Enzymol. [Methods in Enzymology], 153:516). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0303] The expression vector can also provide a position for a secretion signal sequence to form a fusion protein with the polypeptide encoded by inserting the above heavy chain and / or light chain sequences or fragments thereof. More commonly, the inserted antibody or antibody-like molecule sequence is linked to a signal sequence before being included in the vector. Vectors for receiving sequences encoding the variable domains of the light and heavy chains sometimes also encode the constant region or portions thereof. Such vectors allow the expression of the variable region as a fusion protein with the constant region, resulting in the production of a complete antibody or its fragment. Typically, such constant regions are human.

[0304] The host cells for accommodating and expressing the molecules of the present invention can be prokaryotic or eukaryotic. Escherichia coli (E. coli) is a prokaryotic host that can be used to clone and express 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), as well as various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. In addition, there are any number of known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system from bacteriophage λ. Promoters typically optionally control expression using operon sequences and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms such as yeast can also be used to express the antibodies of the present disclosure. Insect cells in combination with baculovirus vectors can also be used.

[0305] 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, they can be hybridoma cell lines that express endogenous immunoglobulin genes (e.g., the 1D6.C9 myeloma hybridoma clone) or mammalian cell lines containing exogenous expression vectors (e.g., SP2 / 0 myeloma cells). These include any normal non-immortalized or normal or abnormal immortalized animal or human cells. For example, many 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. Expression of polypeptides using mammalian tissue cell culture is generally discussed, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, New York, 1987. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen, et al., (1986) Immunol. Rev. 89:49-68) and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulatable or inducible. 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 polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0306] The methods for introducing an expression vector containing a polynucleotide sequence of interest vary according to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts. (See generally Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, biolistic methods, virosomes, immunoliposomes, polycation: nucleic acid conjugates, naked DNA, artificial virosomes, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, (1997) Cell 88:223), reagent-enhanced uptake of DNA, and ex vivo transduction. For long-term high-yield production of recombinant proteins, stable expression is generally desired. 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 selectable marker genes. After introducing the vector, the cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The purpose of the selectable marker is to confer selection resistance, and its presence allows the growth of cells that have successfully expressed the introduced sequence in selective medium. Resistant, stably transfected cells can be propagated using tissue culture techniques appropriate for the cell type.

[0307] The antibodies or fragments thereof of the present invention are typically recovered from the culture medium as secreted polypeptides, but can also be recovered from host cell lysates when produced directly without a secretion signal. If the molecule is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100).

[0308] When the molecule is produced in recombinant cells of non-human origin, it is completely free of human proteins or polypeptides. However, it is necessary to purify the molecule from recombinant cell proteins or polypeptides to obtain a substantially homogeneous preparation with respect to the heteromultimer. As a first step, the culture medium or lysate is typically centrifuged to remove particulate cell debris. The resulting molecule can be conveniently purified by hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, with affinity chromatography being the preferred purification technique. Other techniques for protein purification can also be used, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation. (mRNA).

[0309] VIII. Uses of the Antibodies of the Present Invention

[0310] A. Diagnostic and General Therapeutic Uses

[0311] The antibodies of the present disclosure have a variety of diagnostic and therapeutic applications. For example, these molecules can be used in enzyme immunoassays, where the arms bind to specific epitopes on an enzyme and other parts of the molecule bind to a solid matrix. Nolan et al. discussed enzyme immunoassays using antibody-like molecules (Nolan et al., (1990) Biochem. Biophys. Acta [Biochemistry and Biophysics Acta] 1040:1-11). The multispecific antibodies can also be used to diagnose various diseases, such as autoimmune diseases (Songsivilai et al., (1990) Clin. Exp. Immunol. [Clinical and Experimental Immunology] 79:315). In particular, one antigen-binding domain of the molecule can bind IL-13 or IL-18 in a tissue sample (in vitro, ex vivo, in vivo), while the other binding site can bind to a detectable label described herein, such as a chelator that tightly binds a radionuclide (LeDoussal et al., (1992) Int. J. Cancer Suppl. [International Journal of Cancer Supplement] 7:58-62; Le Doussal et al., (1993) J. Nucl. Med. [Journal of Nuclear Medicine] 34:1662-1671; Stickney et al., (1995) Cancer Res. [Cancer Research] 51:6650-6655).

[0312] The antibodies of the present disclosure have in vitro and in vivo diagnostic and therapeutic uses. For example, these antibodies can be administered to cells in culture (e.g., in vitro or in vivo) or to a subject (e.g., in vivo) to treat, prevent, or diagnose a variety of disorders.

[0313] In one aspect, the molecules of the present disclosure can be used to detect the presence of IL-13 and / or IL-18 in a biological sample. As used herein, the term "detect" encompasses quantitative or qualitative detection. In certain aspects, the biological sample comprises cells or tissue. In certain aspects, such tissue includes normal tissue and / or cancerous tissue that expresses higher levels of IL-13 and / or IL-18 relative to other tissue.

[0314] In one aspect, the present disclosure provides a method for detecting the presence of IL-13 and / or IL-18 in a biological sample. In certain aspects, the method comprises contacting the biological sample with a multispecific antibody of the present disclosure under conditions that permit binding of the antibody to the antigen, and detecting whether a complex forms between the antibody and the antigen. The biological sample can include, but is not limited to, urine or blood samples.

[0315] Also included is a method of diagnosing a disorder associated with IL-13 and / or IL-18 expression. In certain aspects, the method includes contacting a test cell with a multispecific antibody of the present disclosure; determining the expression level (quantitatively or qualitatively) of IL-13 and / or IL-18 in the test cell by detecting the binding of the multispecific molecule of the present disclosure; and comparing the expression level of IL-13 and / or IL-18 in the test cell with the expression level of IL-13 and / or IL-18 in a control cell (e.g., a normal cell or a non-virus-infected cell having the same tissue origin as the test cell), wherein a higher presence level of IL-13 and / or IL-18 in the test cell as compared to the control cell indicates the presence of a disorder associated with IL-13 and / or IL-18. In certain aspects, the test cell is obtained from an individual suspected of having a pathological disorder mediated by IL-13 and IL-18.

[0316] In certain aspects, diagnostic or detection methods such as the above methods include, for example, using a "FACS" assay to detect the binding of the multispecific molecule of the present disclosure.

[0317] Certain other methods can be used to detect the binding of the multispecific antibody of the present disclosure. Such methods include, but are not limited to, antigen-binding assays known in the art, such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, and immunohistochemistry (IHC).

[0318] In certain aspects, the multispecific antibody of the present disclosure is labeled. Labels include, but are not limited to, labels or moieties directly detectable (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties indirectly detectable (e.g., by enzymatic reaction or molecular interaction), such as enzymes or ligands.

[0319] B. Pharmaceutical Compositions and Administration Aspects

[0320] Provided herein are pharmaceutical compositions comprising a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure, which can be used in the uses and methods of the present disclosure in the treatment of atopic dermatitis or related conditions, and the compositions further comprise one or more pharmaceutically acceptable carriers and / or diluents.

[0321] The phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government of the United States or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals and more particularly in humans.

[0322] The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (e.g., an antibody or fragment thereof of the present disclosure) and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0323] Pharmaceutical compositions of therapeutic and diagnostic agents can be prepared by mixing them with a physiologically acceptable carrier, excipient, or stabilizer in the form of, for example, lyophilized powder, slurry, aqueous solution, lotion, or suspension (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: Oral Medications, Marcel Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, New York; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Massachusetts. The choice of administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In certain embodiments, the administration regimen maximizes the amount of therapeutic agent delivered to the patient that is consistent with an acceptable level of side effects. Thus, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated.Guidelines for selecting appropriate dosages of antibodies, cytokines, and small molecules are available (see, 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). Marcel Dekker, Inc., New York, NY).

[0324] The appropriate dosage is determined by the clinician, e.g., using parameters or factors known or suspected to affect or predicted to affect the treatment in the art. Generally, the dosage is started at an amount slightly less than the optimal dosage and is thereafter increased in small increments until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic magnitudes include those of symptoms (e.g., inflammation) or the levels of inflammatory cytokines produced.

[0325] The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure can be varied so as 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 upon a variety of pharmacokinetic factors including the activity of the particular composition of the present disclosure or its ester, salt or amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0326] The composition comprising the multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure can be provided by continuous infusion or at intervals of, for example, once a day, once a week, or 1 - 7 times a week. The dose can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranially, or by inhalation.

[0327] The required dose of the multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure, in moles per kilogram body weight, is substantially the same as the dose of the antibody or polypeptide. The dose administered to a subject can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times or more.

[0328] For the multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure, the dose administered to a patient can be from about 0.0001 mg to about 100 mg per kilogram of patient body weight, for example, about 1 mg - about 5 mg, about 5 mg to about 10 mg per kilogram of patient body weight. The unit dose of the multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure can be from about 0.1 mg to 100 mg, for example about 1 mg to 5 mg, about 5 mg - about 10 mg, about 10 mg - about 25 mg, about 25 mg - about 50 mg, about 50 mg - about 100 mg, about 100 mg - about 150 mg.

[0329] In the case of administering a series of doses, these doses can be administered, for example, about once a day, about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks (monthly), about once every 2 months, about once every 3 months (quarterly), about once every 6 months. These doses can be continued, for example, until disease progression, the occurrence of an adverse event, or other time determined by the physician. For example, about two, three, or four to about 17 or more fixed doses can be administered.

[0330] The effective amount for a particular patient can vary depending on factors such as the condition being treated, the patient's overall health, the method and route of administration and dose, 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).

[0331] When necessary, a therapeutic agent comprising a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure can be incorporated into a composition comprising a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site. Additionally, pulmonary administration can also be employed, e.g., by using an inhaler or a nebulizer and a formulation with a nebulizing agent. See, e.g., US 6,019,968, US 5,985,320, US 5,985,309, US 5,934,272, US 5,874,064, US 5,855,913, US 5,290,540, and US 4,880,078; and WO 1992 / 19244, WO1997 / 32572, WO1997 / 44013, WO 1998 / 31346, and WO 1999 / 66903, each of which is incorporated herein by reference in its entirety.

[0332] The multispecific antibodies of the present disclosure can also be administered by one or more of a variety of methods known in the art via one or more routes of administration. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration of the selected antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. Parenteral administration can represent a mode 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, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered via non-parenteral routes, such as local, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

[0333] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered by infusion. In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered subcutaneously. In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered intravenously.

[0334] The multispecific antibody (e.g., bispecific antibody) or fragment thereof can be administered by any of the above routes using, for example, an injection device, an injection pen, vials and syringes, prefilled syringes, autoinjectors, infusion pumps, patch pumps, infusion bags, and needles. If the multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure is administered in a controlled-release or sustained-release system, control or sustained release can be achieved using a pump (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). Polymer materials can be used to achieve controlled or sustained release of the therapeutic agent of the present disclosure (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, 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; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105); US 5,679,377; US 5,916,597; US 5,912,015; US 5,989,463; US 5,128,326; WO 1999 / 15154; and WO 1999 / 20253.Examples of polymers for use in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl acrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer for use in a sustained release formulation is inert, free of leachable impurities, stable upon storage, sterile, and biodegradable. A controlled or sustained release system can be placed near a prophylactic or therapeutic target, and thus only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0335] Controlled release systems are discussed in Langer (1990, Science 249:1527-1533). Any technique known to those of skill in the art can be used to generate sustained release formulations containing multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure. See, e.g., US 4,526,938, WO 1991 / 05548, 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,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

[0336] If a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure is administered locally, it can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or other forms 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, typically viscous to semi-solid or solid forms are used, which contain a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, which in some cases has a dynamic viscosity greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which, if desired, are sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers or salts) that affect various properties such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations, where in some cases the active ingredient is packaged in combination with a solid or liquid inert carrier in a mixture with a pressurized volatile substance (e.g., a gas propellant such as Freon) or a squeeze bottle. If desired, a humectant or wetting agent can also be added to the pharmaceutical composition and dosage form. Examples of such additional ingredients are known in the art.

[0337] If 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, nebulizer or in the form of drops. In particular, prophylactic and therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases). 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 to contain a powder mixture of the compound and a suitable bulking base (such as lactose or starch).

[0338] The multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure can also be administered to a patient periodically.

[0339] In certain embodiments, multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be formulated to ensure appropriate in vivo distribution. 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 desired), they can be formulated in, for example, liposomes. For methods of making liposomes, see, e.g., US 4,522,811; US 5,374,548; and US 5,399,331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, e.g., Ranade VV (1989) J. Clin. Pharmacol., 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., US 5,416,016 of Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. 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); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

[0340] The present application also provides a protocol for co-administering or treating a patient with a pharmaceutical composition comprising a multispecific antibody (e.g., bispecific antibody) or a fragment thereof of the present disclosure and other therapies or one or more therapeutic agents in combination. Methods of co-administering or treating with other therapeutic agents, e.g., cytokines, steroids, chemotherapeutic agents, 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, N.Y.; 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 the therapeutic agent can reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[0341] In some embodiments, the pharmaceutical composition of the present disclosure further comprises one or more additional therapeutic agents.

[0342] In addition to the above treatment protocols, surgery and other forms of physical therapy can also be performed on the patient.

[0343] C. Therapeutic Applications for IL-13- and IL-18-Mediated Pathological Disorders

[0344] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be therapeutically used to treat various human diseases, such as pathological disorders mediated by IL-13 and IL-18, such as autoimmune diseases, and inflammatory diseases or conditions involving dysregulation of IL-13 and / or IL-18 (e.g., inappropriate expression, expression levels, signal transduction, etc.). In one embodiment, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are used to treat atopic dermatitis.

[0345] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used to treat, reduce the likelihood of, or ameliorate a pathological disorder mediated by IL-13 and IL-18. The phrase "pathological disorder mediated by IL-13 and IL-18" encompasses all diseases and medical conditions in which IL-13 and IL-18 directly or indirectly cause the disease or medical condition, including the cause, development, progression, persistence, or pathology of the disease or condition. Thus, these terms include conditions and / or diseases or disorders associated with or characterized by abnormal IL-13 and IL-18 levels and / or diseases or disorders that can be treated by reducing or inhibiting the activities induced by IL-13 and IL-18 in target cells or tissues. Pathological disorders mediated by IL-13 and IL-18 include autoimmune diseases and / or inflammatory conditions and disorders having IL-13 and IL-18 components.

[0346] 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.

[0347] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used for, but are not limited to, treating, preventing, or ameliorating autoimmune diseases and / or inflammatory conditions and disorders, particularly inflammatory conditions having an autoimmune component as a cause. In one aspect, the present disclosure provides methods for treating autoimmune diseases. In one aspect, the present disclosure provides methods for treating inflammatory diseases or conditions. In one aspect, the subject being treated is a human.

[0348] The present disclosure provides the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof and the pharmaceutical compositions as described herein, and the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof and the pharmaceutical compositions are for use in treating and / or preventing a pathological disorder mediated by IL-13 and IL-18. The present disclosure provides the use of the multispecific antibodies or pharmaceutical compositions as described herein in the preparation of a medicament for use in treating a pathological disorder mediated by IL-13 and IL-18. The present disclosure provides methods for treating and / or preventing a pathological disorder mediated by IL-13 and IL-18, the method comprising administering a therapeutically effective amount of the multispecific antibodies or pharmaceutical compositions as described herein to a subject in need thereof. In some embodiments, the pathological disorder mediated by IL-13 and IL-18 is an autoimmune disease or an inflammatory disorder or condition. In some embodiments, the autoimmune disease or inflammatory disorder or condition is atopic dermatitis.

[0349] The present disclosure provides multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure, and pharmaceutical compositions as described herein, for use in the treatment and / or prevention of moderate to severe AD.

[0350] As used herein, the term “atopic dermatitis” (AD) or “eczema” refers to an inflammatory skin disease characterized by intense pruritus (e.g., severe itching) and squamous and 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, dust mites, animals, etc.), radiation exposure, and / or asthma. The present disclosure includes methods for treating patients suffering from mild, moderate to severe, or severe AD. As used herein, “moderate to severe AD” is characterized by intense itching, extensive skin lesions, which are usually accompanied by persistent bacterial, viral, or fungal infections. Moderate to severe AD also includes chronic AD in patients. In many cases, chronic lesions include plaques with thickened skin, lichenification, and fibrotic papules. Generally, patients affected by moderate to severe AD also have more than 10% or more than 20% of the body skin affected, or 10% of the skin area affected in addition to involvement of the eyes, hands, and body folds. Generally, patients affected by moderate to severe AD also 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) itching. Moderate to severe AD is also considered to be present in patients who require frequent use of topical corticosteroid treatment. A patient can also be said to have moderate to severe AD when the patient is resistant or refractory to treatment with topical corticosteroids or calcineurin inhibitors or any other commonly used therapeutic agent known in the art.

[0351] Suitably, the uses and methods of the present disclosure include administering the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure at a dose sufficient to achieve a therapeutically effective serum level. Suitably, the therapeutically effective serum level of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof is maintained during treatment.

[0352] As used herein, the term "therapeutically effective serum level" refers to the serum level of a therapy (e.g., a bispecific antibody) in a subject that is sufficient to reduce and / or improve the severity and / or duration of a given disorder, condition, or disease and / or its associated symptoms. In some aspects, "therapeutically effective serum level" as used herein also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof in a subject's serum that achieves a particular outcome, e.g., an improvement in an AD-related parameter, e.g., a reduction in the Investigator's Global Assessment (IGA) score; a reduction in the Dermatology Life Quality Index (DLQI) from baseline; a reduction in the Patient's Global Impression of Severity (PGIS) from baseline; an improvement in the Patient's Global Impression of Change (PGIC), i.e., a reduction from baseline; a reduction in the Atopic Dermatitis Body Surface Area (BSA) involvement score; a reduction in the Eczema Area and Severity Index (EASI) score; a reduction in the SCORAD score; and / or a reduction in the Itch Numerical Rating Scale (NRS) score.

[0353] In some aspects, "therapeutically effective serum level" as used herein also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof in a subject's serum that achieves a particular outcome, e.g., a reduction in the expression level of one or more AD-related biomarkers, particularly one or more 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 prior to treatment with the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof.

[0354] Suitably, the uses and methods of the present disclosure include administering the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof once a week, once every two weeks, once every three weeks, once every four weeks, once every eight weeks, or once every 12 weeks. According to certain exemplary embodiments, the uses and methods of the present disclosure include administering the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof once every 4 weeks.

[0355] The present disclosure also provides a method of inhibiting IgE antibody production in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure.

[0356] The present disclosure also provides a method of inhibiting IFN-γ production in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure.

[0357] In certain embodiments provided herein are methods of treating IgE-mediated disorders in a subject, the methods comprising administering to the subject an effective amount of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure, 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 interleukin to the receptor.

[0358] D. Combination Therapies

[0359] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be used in combination with other agents and therapies (the "other therapeutic agents" herein) for treating a variety of diseases, disorders, and conditions.

[0360] "Combined" administration with other therapeutic agents means that during the course of a subject having a disorder, two (or more) different treatments are delivered to the subject, e.g., two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or treatment is terminated for other reasons. In some embodiments, the delivery of the first treatment is still ongoing at the start of the delivery of the second treatment, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous delivery" or "parallel delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of each case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed using less of the second treatment compared to the results observed when the second treatment is administered in the absence of the first treatment, or the second treatment results in a greater reduction in symptoms, or a similar situation is observed for the first treatment. In some embodiments, compared to the results observed when another treatment is delivered in the absence of one treatment, the delivery results in a greater reduction in symptoms or other parameters associated with the disorder. The effects of the two treatments can be partially additive, fully additive, or more than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0361] The term "in parallel" is not limited to administering therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that a pharmaceutical composition comprising a multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure is administered to a subject in a certain order and at certain time intervals such that the molecules of the present disclosure can act together with one or more other therapeutic agents to provide an increased benefit compared to administration in other ways. For example, each therapy can be administered to the subject at the same time or in any order sequentially at different time points; however, if not administered at the same time, the therapies should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to the subject separately in any suitable form and by any suitable route.

[0362] Other therapeutic agents (e.g., other prophylactic or therapeutic agents) that can be administered in combination with the molecules of the present application can be administered to the molecules or fragments thereof of the present disclosure less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. In other embodiments, two or more other therapeutic agents are administered to the patient during the same patient visit.

[0363] The multispecific antibody (e.g., bispecific antibody) or fragment thereof of the present disclosure and one or more additional therapeutic agents can be administered simultaneously or sequentially in the same or separate pharmaceutical compositions as disclosed. For sequential administration, the multispecific antibody (e.g., bispecific antibody) or fragment 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. One or more other therapeutic agents can be administered to the subject by the same or different routes of administration compared to the disclosed multispecific binding molecules and fragments.

[0364] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof and / or one or more additional therapeutic agents, procedures, or modalities of the present disclosure can be administered during the period of an active disorder or during a period of remission or lower disease activity. The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be administered before, simultaneously with, after, or during remission of the disorder, other therapies.

[0365] One or more additional therapeutic agents of the combination therapies of the present disclosure can also be administered in a cyclic manner. Cyclic combination therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating the sequential administration, i.e., the cycle, to reduce the development of resistance to one therapy (e.g., an agent), to avoid or reduce side effects of one therapy (e.g., an agent), and / or to improve the efficacy of the therapy.

[0366] When administered in combination, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof and additional therapeutic agents (e.g., second or third agents) of the present disclosure are administered either in higher, lower, or the same amounts or doses as each agent alone (e.g., as a monotherapy). In certain embodiments, the multispecific binding molecules as described herein, e.g., bispecific molecules, e.g., bispecific antibody-like molecules, other agents (e.g., second or third agents), or all are administered in amounts or doses lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amounts or doses of each agent alone (e.g., as a monotherapy). In other embodiments, the amounts or doses of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof, additional agents (e.g., second or third agents), or all of the present disclosure that result in a desired effect (e.g., treating an autoimmune disease or an inflammatory disease or disorder) are lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amounts or doses of each agent alone (e.g., as a monotherapy) required to achieve the same therapeutic effect.

[0367] Preferably, the additional therapeutic agent (e.g., a second agent or a third agent) is an AD agent, such as a small molecule, a biological therapy, or an agent that adopts an AD mode, such as phototherapy, including local therapy, systemic therapy, phototherapy, and combinations thereof. "AD agents" include topical therapies in the form of creams, ointments, lotions, gels or sprays (e.g., medium- and low-potency corticosteroids [according to WHO guidelines Group IV-VII, see Bolognia JL, Jorizzo JL, Schaffer JV. Glucocorticosteroids. [Glucocorticoids] Dermatology. [Dermatology] 3rd Edition 2012. Chapter 125, 2075-88; Ference JD, Last AR. Choosing topical corticosteroids. [Selection of local glucocorticoids] Am Fam Physician. [American Family Physician. 2009 Jan 15;79(2):135-40]); over-the-counter (OTC) moisturizers, as well as medical devices or so-called barrier creams (such as atopiclair); and lubricants for treating itching and / or pain, such as anti-itch lotions containing menthol, pramoxine, or antihistamines; local anesthetics, systemic agents (e.g., biologic agents, such as IL-4R inhibitors, such as doxorubicin, lumab; IL-13Ra1 inhibitors such as ASLAN-004; IL-13Ra2 inhibitors; IL-31 inhibitors such as nemolizumab; TNFα inhibitors such as adalimumab, infliximab, certolizumab and etanercept, alefacept; IL-1a inhibitors such as bermekimab (MABp1); IL-23 inhibitors such as briginumab, ustekinumab, gosekumab, risankizumab Antibodies, such as tetropezumab; IL-17 inhibitors, such as berdalumab and eselgizumab; CD11a inhibitors, such as efalizumab; IL-22 inhibitors, such as fezalimumab and IL-22 binding protein; IL-5 inhibitors, such as mepolizumab and benrelizumab; synthetic forms of IL-2, such as aldesleukin; recombinant IL-2 approaches targeting the interleukin-2 receptor complex, such as LY3471851; OS MR inhibitors, such as KPL-716; VAP-1 inhibitors; OX-40 inhibitors or OX40L inhibitors, such as GBR830 and KY1005; IgE inhibitors, such as omalizumab and ligalizumab; TSLP inhibitors, such as tezerumab; IL-33 inhibitors, such as MEDI3506; IL-36 inhibitors, such as sibsolizumab and ANB019; B cell regulation methods, such as rituximab and ocrelizumab;Non-biological immunomodulatory therapies such as cyclosporine and other calcineurin inhibitors, JAK inhibitors such as tofacitinib, upadacitinib, abrocitinib, baricitinib; TYK2 inhibitors such as deucravacitinib; 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 inhibition methods such as RPT-193; systemic corticosteroids, cyclophosphamide, sulfasalazine, azathioprine, mycophenolate mofetil, dapsone, hydroxychloroquine; retinoids (e.g., cis-retinoic acid); leukotriene inhibitors or anti-leukotrienes such as montelukast, pranlukast or zafirlukast, and 5-LO inhibitors such as zileuton, and LTA4H inhibitors such as abexinostat, intralesional corticosteroid injections; phototherapy (e.g., high-dose UVB and UVA). Photochemotherapy (e.g., psoralen 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, deucotinib, or topical vitamin D analogs and topical aryl hydrocarbon receptor (AhR) inhibitors such as bexarotene / tapinarof; high-potency - ultra-high-potency topical corticosteroids (Groups I, II, III according to WHO definition);Antifungal drugs with known anti-inflammatory properties, such as griseofulvin, itraconazole, betamethasone, dexamethasone, INCB018424, triamcinolone acetonide, apremilast, turmeric paste, glucosamine sulfate, triamcinolone acetonide acetate, sesame oil, betamethasone dipropionate, clobetasol propionate, probiotics (such as Bifidobacterium animalis subsp. lactis HN019, Lactobacillus reuteri), ω-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, aminolevulinic acid ester, hydroxychloroquine, adcortyl, efalizumab, fluocinonide, coenzyme Q10 mucoadhesive tablets, roman chamomile, sirolimus, tacrolimus, Qingxuan Decoction, NSAID topical irrigation, NSAID, quercetin, NAVS naphthalene, mechlorethamine, bupivacaine, oatmeal bath. Suitably, the topical AD therapy is a prescription therapy for atopic dermatitis, including but not limited to topical steroids, such as corticosteroids, tacrolimus, cyclophosphamide, azathioprine, methotrexate, mycophenolate mofetil, apremilast, calcineurin inhibitors, such as topical calcineurin inhibitors, phosphodiesterase 4 (PDE4) inhibitors, such as topical PDE4 inhibitors, such as crisaborole, adrenocorticotropic hormone analogs, dupilumab, etanercept, adalimumab, infliximab, omalizumab, secukinumab.;

[0368] E. Kit

[0369] The present disclosure also encompasses kits for treating patients suffering from a pathological disorder mediated by IL-13 and IL-18, such as an autoimmune disease or an inflammatory disorder or condition. Such kits contain a therapeutically effective amount of the multispecific antibodies of the present disclosure. Additionally, such kits may contain a device for administering the multispecific antibodies of the present disclosure (e.g., an autoinjector, a syringe and vial, a prefilled syringe, a prefilled pen) and instructions for use. These kits may contain other therapeutic agents (described below) for treating a pathological disorder mediated by IL-13 and IL-18, such as an autoimmune disease or an inflammatory disorder or condition. Such kits may also contain instructions for administering the multispecific antibodies of the present disclosure to treat a patient. Such instructions may provide the dosage, route of administration, regimen, and total treatment duration for the packaged multispecific antibodies of the present disclosure.

[0370] The phrase "device for administering" is used to indicate any available means for administering a drug systemically to a patient, including, but not limited to, prefilled syringes, vials and syringes, injection pens, autoinjectors, intravenous drip sets and bags, infusion pumps, patches, infusion bags and needles, etc. Using such items, a patient can self-administer the drug (i.e., administer the drug without the assistance of a physician) or a physician can administer the drug.

[0371] Examples

[0372] The following examples are provided to further illustrate the present disclosure, but do not limit the scope of the present disclosure. Other variations of the present disclosure will be clear to those of ordinary skill in the art and are also covered by the appended claims.

[0373] Example 1: Production of IL-13 / IL-18 bispecific antibodies in CHO cell lines

[0374] 1. Expression vector construction

[0375] The vector used in the examples consists of the following elements: hCMV promoter / enhancer driving the expression of the various genes required for assembly of the antibody construct, a polyadenylation signal (polyA), a folate receptor (FolR, FAR), DHFR, puromycin and / or hygromycin gene as a selection marker, an E. coli origin of replication, and a β-lactamase gene that provides ampicillin resistance to initiate amplification in bacteria. Different plasmid settings were evaluated and more details are provided in the figure.

[0376] Figure 2 Schematic diagram of NVS standard plasmids AD. Plasmids A and C encode the expression of anti-IL13κ LC and anti-L13 knob HC; plasmids B and D encode the expression of anti-IL18λ LC and anti-IL18 hole HC. The expression of each individual protein chain is driven by a separate CMV promoter. Linearized plasmids A and B or C and D are co-transfected simultaneously in CHO-C8TD parental cells. Cells are selected in the first round of selection using the selection markers DHFR and FAR. For the second selection, additional selection markers hygromycin and puromycin can be used. The plasmids carry the information of leaky transmembrane technology (LS-TM) to stain and enrich high-yielding clones during FACS-assisted single-cell sorting. Plasmids C and D are designed to have low sequence homology to minimize the risk of homologous recombination by deleting the repeated part of the phage f1 region between the expression cassettes encoding LC and HC. Plasmid D carries different codon optimization and signal peptide for IL18 hole HC, while on plasmids A and B, the DNA sequences of IL18 knob CH and IL13 hole CH differ only in the bases encoding the KiH mutation.

[0377] Figure 3Schematic diagrams of NVS furin-2A peptide (F2A) plasmids E and F. The F2A technology enables the co-expression of more than one protein chain from a single promoter cassette. On plasmids E and F, the first expression cassette encodes anti-IL18 λLC and anti-IL18 θHC, and the second expression cassette encodes anti-IL13 κLC and anti-IL13 ϕHC. The linearized plasmid E or F was transfected into CHO-C8TD parental cells. Selection markers DHFR and FAR were used to select cells. Plasmid F was designed to have lower sequence homology to minimize the risk of homologous recombination by using different codon optimizations for IL18 θHC and IL13 ϕHC, while on plasmid E, the DNA sequences of IL18 ϕCH and IL13 θCH only differ in the bases encoding the KiH mutation.

[0378] Figure 4 Schematic diagrams of the adapted NVS standard plasmids G and H. Plasmids G and / or H were used for supertransfection of the pool of plasmid E or F to increase the number of plasmid integrations. Plasmid G encodes the expression of anti-IL13 κLC and anti-IL13 ϕHC; plasmid H encodes the expression of anti-IL18 λLC and anti-IL18 θHC. The expression of each individual protein chain is driven by a separate CMV promoter. Different transfection and screening methods were performed, namely (I) co-transfecting plasmids G and H simultaneously in CHO-C8TD parental cells and selecting cells using the selection markers hygromycin and puromycin, or (II) transfecting plasmid G in the second round and selecting cells using hygromycin and / or transfecting plasmid H in the second round and selecting cells using puromycin, or (III) transfecting plasmid H in the first round and selecting cells using puromycin and / or transfecting plasmid G in the second round and selecting cells using hygromycin. Plasmids G and H carry the information of the leaky transmembrane technology (LS-TM) to stain and enrich high-producing clones during the FACS-assisted single-cell sorting process. The partial phage f1 region between the expression cassettes encoding LC and HC was deleted on plasmids G and H, and the codon optimizations of LC and HC were different from each other and from plasmids E and F to reduce sequence homology, thus minimizing the risk of homologous recombination.

[0379] Figure 5 Schematic diagram of the NVS furin-2A peptide (F2A) plasmid I with different protein chain combinations in the expression cassette compared to plasmids E and F. On plasmid I, the first expression cassette encodes anti-IL18 λLC, anti-IL18 θHC, and anti-IL13 ϕHC, and the second expression cassette encodes anti-IL13 κLC. The linearized plasmid I was transfected into CHO-C8TD parental cells. Selection markers DHFR and FAR were used to select cells. The partial phage f1 region between the two expression cassettes was deleted, and different codon optimizations of CH were used to reduce sequence homology to minimize the risk of homologous recombination.

[0380] 2. Cell Lines, Cultivation, Transfection, and Selection

[0381] The parental 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 line was used to prepare the recombinant cell line. The CHO cell line was cultivated in suspension in a proprietary chemically defined medium in shake flasks in a shaking cabinet at 150 rpm, 10% CO2, and 36.5 °C. Cell viability and growth rate were monitored by an automated system (ViCell, Beckman-Colter). The cells were passaged 2 - 3 times per week into fresh medium and maintained in the logarithmic growth phase.

[0382] The SwaI-linearized expression plasmid encoding the antibody construct was transfected by electroporation (Amaxa Nucleofector System, Lonza). The transfection reaction was carried out in a chemically defined medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in the exponential growth phase with a cell viability higher than 95%. 5x10 6 cells were used for each transfection. Immediately after transfection, the cells were transferred to a shake flask containing chemically defined medium. The cell bank was incubated at 36.5 °C and 10% CO2 for 48 hours before the start of the selection process.

[0383] The selection procedure was carried out using selectable markers encoded by separate expression vectors. Folate receptor and DHFR were used for the first round of transfection and selection. Both proteins are involved in the same molecular pathway; FolR transports folic acid as well as the folic acid analogue MTX into the cell, and DHFR converts it into an important precursor for purine and methionine synthesis. Combining them as a selection principle, a specific strong selection scheme can be employed to enrich recombinant cells expressing both recombinant proteins.

[0384] Forty-eight hours after transfection and growth under low folate conditions, an additional selection pressure was applied by adding 10 nM MTX to the chemically defined medium. After pool recovery, the cells were frozen in a medium supplemented with 7.5% DMSO and the materials generated for further analysis as described below.

[0385] Based on the plasmid design used for the first round of transfection and selection, different progression possibilities emerged. The pool was used directly for single cell cloning as described below, or a second selection or supertransfection was carried out before single cell cloning.

[0386] For the second selection, the recovered pool was maintained under low folate conditions with 10 nM MTX, and additional 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 materials generated for further analysis as described below.

[0387] For supertransfection, the SwaI-linearized expression plasmid encoding the antibody construct was transfected by electroporation (Amaxa Nucleofector System, Lonza, Germany). The transfection reaction was carried out in chemically defined medium according to the manufacturer's instructions. The CHO pool that had recovered from selection after transfection was used for a second or third transfection. Transfection was performed as described above, and selection was initiated 48 hours later at 36.5 °C and 10% CO2 by adding 0.5 μg / ml puromycin or 0.8 mg / ml hygromycin or both to the chemically defined medium. After pool recovery, the cells were frozen in medium supplemented with 7.5% DMSO and materials generated for further analysis as described below.

[0388] 3. Single cell cloning

[0389] After selection, single cell cloning was performed using a Cytena cell printer device or by flow cytometry to obtain cell lines of monoclonal origin.

[0390] The Cytena cell printer contains a disposable dispensing cartridge that includes a microfluidic chip loaded with a cell suspension. From this cartridge, droplets are ejected through a nozzle into a 96-well plate. During this process, an image of the nozzle area is recorded by a microscope system. An automated image analysis algorithm detects the cells on the image and classifies them according to morphological criteria such as size and roundness. Based on image analysis of the droplet formation area at the nozzle exit, droplets containing single cells are directed to separate wells of the 96-well plate, while droplets that do not contain single cells (blank droplets or droplets with multiple cells) are directed to waste.

[0391] Prior to flow cytometry, cells were stained with an internally generated BD Ab labeled with FITC against the Fc portion of ABC123 attached to the cell surface to facilitate the selection of high-producing clones.

[0392] Single cell cloning was performed using a Sony CellSorter instrument equipped with a 96-well plate support and a 100 μm disposable sorting chip. To ensure that only single cells were sorted, the settings were adjusted to single cell mode, i.e., 3-droplet sorting. With these settings, droplets containing cells were sorted only if the previous and the subsequent droplets were both empty. The cell concentration and flow rate were optimized at the expense of yield to increase the probability that each droplet contained no more than one single cell. Multiple gates were set to select single live cells with high fluorescence.

[0393] After sorting single cells into individual wells of a 96-well plate using a Cytena cell printer or Sony Cell Sorter, high-resolution microscope images of each well were obtained to record monoclonality and verify the single cell cloning procedure.

[0394] After single cell cloning, the clones were expanded and characterized with respect to productivity and bioprocess suitability as well as transgene integration and expression. Primary seed lot (PSL) vials were prepared by freezing cells from the best performing clones in medium supplemented with 7.5% dimethyl sulfoxide (DMSO), and the PSL of the ultimately selected clones was used for MCB manufacture.

[0395] 4. Upstream processing

[0396] After selection, material was generated in shake flask fed-batch cultures. The fed-batch cultures were inoculated at a defined cell seeding density, a proprietary feed solution was added starting from day 3, and the cultivation temperature was shifted to 33 °C on day 5. In-process controls were performed during cultivation to monitor the concentration of the antibody construct. The individual cultures were cultivated for 14 days. At the end of the cultivation process, the cells were separated from the culture supernatant, followed by sterile filtration and then further downstream processing and analytical characterization. The volumetric productivity of the selected pool was determined by Protein A HPLC in cell culture supernatant for all species of products and related impurities carrying the Fc moiety or by RP-LC assay.

[0397] Example 2. LC-MS screening and purity assessment of an IL-13 / IL-18 bispecific antibody

[0398] 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 of PNGase F enzyme (New England Biolabs) at 37 °C for 4 h. Using a PLRP-S RP column (3 μm, 2.1 x 150 mm, The Waters ACQUITY UPLC Class from Agilent Technologies and the TripleTOF 6600 mass spectrometer with a dual spray ion source (Sciex) subjected the deglycosylated samples to the LC-MS system. 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 at 60 °C. The flow rate was 0.2 ml / min. The protein was eluted with the following gradient over 40 min: 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%, 35 - 40 min 35% B. The UV chromatogram was recorded at 214 nm and MS data acquisition was performed in positive ES(+). Data were acquired using Analyst software TF 1.7 (ABSciex) and analyzed using BioPharmaView (version 3.0, ABSciex) and PeakView (version 2.2, ABSciex) software. The identification and relative quantification of bbmAb species and mispaired variants were based on the matching to the theoretical expected mass and the relative mass signal intensity of the deconvoluted mass spectra, and the results are shown in Table 3.

[0399] A wide range of correct heavy chain heterodimerization, heavy chain homodimerization, and half - molecules were detected. The required degree of heterodimerization was up to >95%, and ideally close to 100%. To identify candidates with >95% heterodimerization, bbmAb1, bbmAb2, bbmAb5, bbmAb4, and bbmAb3 were generated. Only bbmAb1, bbmAb2, and bbmAb5 showed >95% heterodimerization, while bbmAb4 and bbmAb3 showed <95% heterodimerization and did not meet the criteria for therapeutic development.

[0400] Table 3. Mispaired detected by LC - MS analysis

[0401]

[0402] Example 3. Evaluation of the thermal stability of the CH2 and Fab domains of an IL - 13 / IL - 18 bispecific antibody

[0403] The stability of antibodies greatly affects their performance (i.e., their specificity and affinity). Therefore, stability is a major issue for researchers and manufacturers, especially as antibodies are used more and more in therapeutic, diagnostic, and rapid analysis platforms. The key parameters are the thermal stability and melting temperature (T m ) of the CH2 and Fab domains. The protein melting temperature (T m) is defined as the temperature at which the protein denatures. T m values and the unfolding fractions can predict the aggregation rate (Robinson et al., 2018).

[0404] The thermal transition midpoint was determined by differential scanning fluorimetry using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The purified samples were diluted to 0.3 mg / mL in 20 mM His / His HCl (pH 6.0) with a final volume of 43 μL and mixed with 7 μL of SYPRO Orange dilution (1.4 μL of SYPRO Orange stock solution diluted in 1 mL of water). The starting temperature of the thermal cycler was set at 20 °C, the ending temperature was set at 95 °C, and the heating rate was set at 0.5 °C. The melting curve and the melting temperature were obtained using Bio-Rad CFX Manager Software 3.1.

[0405] The melting temperatures of the CH2 and Fab domains of the engineered antibodies are shown in Table 4, and the melting curves of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 are shown in Figure 6.

[0406] The T m of CH2 reflects the unfolding of the CH2 domain (Johnson, 2012). As shown in Table 4, all candidates showed similar T m .

[0407] Higher melting temperatures (67 °C - 68 °C) were observed in the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 compared to the anti-13 Fab domain of bbmAb3 (64 °C). Since the CDR sequences in the anti-13 Fab domain of bbmAb3 are different from those of the other candidates, the results suggest that the specific CDR sequences in the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 can generate more stable molecules with improved thermal stability.

[0408] Table 4. Melting temperatures (T m )

[0409]

[0410]

[0411] Example 4. Affinity for recombinant human and cynomolgus monkey IL-13 and IL-18 measured by SET

[0412] The determination of the equilibrium dissociation constant (KD) was achieved by solution equilibrium titration (SET) measurements as described below.

[0413] Twenty-two consecutive 2n dilutions of the antigen (highest concentration: 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 a constant concentration of antibody was added (for hsIL-13, the reading was 4 pM; for hsIL-18, the reading was 10 or 4 pM; and for cyIL-18, the reading was 5 pM).

[0414] Each antigen-antibody mixture with a volume of 60 μl / well was dispensed in duplicate into a 384-well polypropylene microtiter plate (MTP). The sample buffer served as a negative control, and the sample containing only the antibody served as a positive control (maximum electrochemiluminescence signal without antigen, B max ). The plate was sealed and incubated overnight (at least 16 hours) on a shaker at room temperature (RT).

[0415] The antigens and antibodies used are listed in Table 5.

[0416] Table 5: Antigens and antibodies used in SET measurements

[0417]

[0418]

[0419] hsIL-18 and cyIL-18 readings: After blocking with 50 μl / well blocking buffer (PBS containing 5% BSA) for at least 1 hour (h) at room temperature (RT) or following a washing step (TBST, TBS containing 0.05% Tween 20), the streptavidin Multi- 384-well plate (MSD L21SA-5) was coated with 30 μl / well biotinylated human IL-18 (0.2 μg / ml, PBS) and incubated on a shaker at RT for at least 1 h.

[0420] hsIL-13 readings: After blocking with 50 μl / well blocking buffer (PBS containing 5% BSA) for at least 1 hour (h) at room temperature (RT) and following a washing step (TBST, TBS containing 0.05% Tween 20), the streptavidin Multi- 384-well plate (MSD L21SA-5) was coated with 30 μl / well biotinylated human IL-13 (0.2 μg / ml, PBS) and incubated on a shaker at RT for at least 1 h.

[0421] Transfer 30 μl / well of the equilibration antigen-antibody mixture from the polypropylene MTP to the coated MSD plate and incubate at RT for 20 min. After an additional washing step, add 30 μl of the sulfo-tagged anti-hsIgG detection antibody (0.5 μg / ml) diluted in sample buffer to each well and incubate on a shaker at RT for 30 min. Wash the MSD plate and add 35 μl / well of MSD read buffer and incubate at RT for 5 min. The electrochemiluminescence (ECL) signal is generated and measured by the MSD Sector Imager 6000.

[0422] SET data was exported to the MS Excel add-in software Xlfit. The mean ECL signal was calculated from the replicate measurements in each assay. The data was baseline-adjusted by subtracting the lowest value from all data points and plotted against the corresponding antigen concentration to generate a titration curve. K D values were determined by fitting the plot with:

[0423] 1:1 binding model of the pestle and mortar structure bispecific Ab

[0424]

[0425] where

[0426] y: ECL signal minus blank

[0427] B max : maximum ECL signal at zero antigen concentration

[0428] [Fab]: concentration of the applied bispecific antibody

[0429] K D : dissociation equilibrium constant

[0430] x: applied antigen concentration

[0431] The obtained K D values are shown in Table 6.

[0432] Table 6: Affinities for recombinant human and cynomolgus monkey IL-13 and IL-18 measured by SET (single target binding assay)

[0433]

[0434] n.d., not determined; *, measured by SPR (see below); K D , calculated from 3 or 4 replicate experiments

[0435] Example 5. Affinities for recombinant human and cynomolgus monkey IL-13 and IL-18 measured by SPR

[0436] The determination of the kinetic binding parameters was carried out using the optical biosensor Biacore TM surface plasmon resonance (SPR) of the T200 (http: / / www.cytivalifesciences.com).

[0437] This technique allows the label-free determination of the kinetic rate constants of ligand-receptor binding (k a , association rate constant) and dissociation (k d , dissociation rate constant). The equilibrium dissociation constant K D is calculated from the kinetic rate constants.

[0438] The surface of a C1 sensor chip (Cytiva #BR100535) was prepared for indirect binding antibodies by immobilizing 80 μg / mL of NeutrAvidin TM (Thermo Scientific #31000) in immobilization buffer (10 mM sodium acetate (pH 5.0)) on the chip surface via amine coupling, followed by saturating the NeutrAvidin TM with 5 μg / mL of biotinylated protein G (Sigma #P8045) in HBS-EP buffer.

[0439] The antibodies were diluted to a final concentration of 10 μg / mL 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). Affinity measurements were performed on recombinant hulL-13 (concentrations ranging from 0.25 to 8.0 nM, 2-fold increments) and recombinant hulL-18 (concentrations ranging from 0.125 to 32 nM, 2-fold increments) to determine the kinetic constants of bbmAb2 or bbmAb1. The surface was regenerated with 10 mM glycine (pH 1.5), 0.5% Tween 20 between cycles.

[0440] The antigens and antibodies used are listed in Table 7.

[0441] Table 7: Antigens and antibodies used in SET measurements

[0442]

[0443]

[0444] Using Biacore TMT200 Control Software v2.0.1 evaluates kinetic traces. The complete set of these increasing concentration traces is called a run. The set of traces of the run is fitted with a 1:1 binding model (R max set population) provided by the Biacore T200 Evaluation Software v3.0. A zero concentration sample (blank) is included in each run to allow for dual referencing.

[0445] The obtained K D values are shown in Table 8.

[0446] Table 8: Kinetic rate constants and affinities for recombinant human and cynomolgus monkey IL-13 measured by SPR

[0447]

[0448] Example 6. Affinity for human Fc receptors measured by surface plasmon resonance (SPR) spectroscopy

[0449] To characterize the binding of the engineered IL-13 / IL-18 bispecific antibody to human Fc receptors, a direct binding assay was performed using surface plasmon resonance (SPR) spectroscopy. SPR is a technique commonly applied to the affinity and kinetic analysis of protein-protein, protein-peptide, protein-DNA, and protein-small molecule interactions because it allows the analysis of the interaction between an analyte in solution and a ligand attached to the surface of a sensor chip, providing continuous readings of complex formation and dissociation.

[0450] 1. Measurement of in vitro binding to Fcγ receptors

[0451] Affinity determination of human Fcγ receptor binding to the Fc fragment of bbmAb2 (IL18 knob YTEIL13 hole YTE 1+1bsAb) 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 YTE 1+1bsAb) was performed on a Biacore 8K instrument. The samples were diluted at 5 μg / ml with 10 mM sodium acetate (pH 4.5) and immobilized on a CM5 sensor chip at a density of about 750 resonance units by applying a standard amine coupling procedure on a Biacore T200 instrument. On a Biacore 8K instrument, a similar procedure was applied, but the fixed density was about 1310 resonance units. On the Biacore T200, flow cell 1 was blank fixed to serve as a reference. On the Biacore 8K, there are a total of 8 channels, and flow cell 1 of each channel is left blank to serve as a reference surface. A 1:2 dilution series of human Fcγ receptors (CD64 / FcγRI, CD32a / FcγRIIA) was then injected over all flow cells at a flow rate of 30 μl / min in Biacore T200 or 50 μl / min in Biacore 8K and a temperature of 25°C. R131 , CD32b / FcγRIIB, CD16a / FcγRIIIA V176 and FcγRIIIA F176 Kinetic binding data were collected using the ELISA kit (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 the Biacore T200, we used FcγRI: 0.20 to 100 nM; FcγRIIA: 0.20 to 100 nM). R131 , FcγRIIB and FcγRIIIB: 7.81 to 4000 nM; FcγRIIIA V176 : 1.95 to 1000 nM; and FcγRIIIA F176 : 3.91 to 2000 nM; on Biacore 8K, the same conditions were applied but FcγRI was tested from 0.05 to 20 nM). On Biacore T200, the expression of FcγRI and FcγRIIIA V176After each measurement cycle, the chip surface was regenerated with 10 mM glycine solution (pH 2.0) at 30 μl / min for 30 s. On the Biacore 8K, the surface was regenerated for hFcγR by injecting 10 mM glycine (pH 2.0) once at a flow rate of 50 μl / min for 30 s. Zero concentration samples (blank operating samples) were measured to allow for dual referencing during data evaluation. Replicate injections of each sample and buffer blank were flowed over all surfaces. The data was evaluated using BiacoreT200 evaluation software version 3.0 and Biacore 8K evaluation software (v. 3.0.12.15655).

[0452] The raw data was dual referenced, i.e., the response of the measurement flow cell was corrected with the response of the reference flow cell, and in a second step the response of the blank injection was subtracted. The resulting sensorgrams were fitted using a steady state model or a 1:1 Langmuir model to calculate the equilibrium dissociation constant (K D ).

[0453] 2. Measurement of in vitro binding to the FcRn receptor

[0454] The affinity assay of the human FcRn receptor binding to the Fc fragment of bbmAb2 (IL18(mAb1) pestle YTE IL13(mAb2) mortar YTE 1+1 bsAb) was performed on a Biacore T200 instrument. The affinity assay of the human FcRn receptor binding to the Fc fragments of bbmAb5 (bispecific KiH-LALA YTE) and bbmAb1 (IL18 mortar YTE (mAb1) IL13(mAb2) pestle YTE 1+1 bsAb) was performed on a Biacore 8K instrument. The samples were diluted to 5 μg / ml with 10 mM sodium acetate (pH 4.5) and immobilized on a CM5 sensor chip at a density of approximately 750 resonance units by applying a standard amine coupling procedure on the Biacore T200 instrument. On the Biacore 8K instrument, a similar procedure was applied, but the immobilization density was approximately 1310 resonance units. On the Biacore T200, flow cell 1 was blank immobilized to serve as a reference. On the Biacore 8K, there were a total of 8 channels, and flow cell 1 of each channel was left blank to serve as a reference surface. Kinetic binding data were collected by subsequently injecting a 1:2 dilution series of the human FcRn receptor at a flow rate of 50 μl / min over all flow cells at a temperature of 25 °C. The FcRn receptor was diluted in two different running buffers to examine pH-dependent binding: PBS (pH 5.8) with 0.005% Tween-20 and PBS (pH 7.4) with 0.005% Tween-20, thus covering a concentration range from 4.88 to 2500 nM. The chip surface was regenerated with PBS (pH 7.4) with 0.005% Tween-20 at a flow rate of 50 μl / min for 120 s. Zero-concentration samples (blank runs) were measured to allow for dual referencing during data evaluation. Replicate injections of each sample and buffer blank were flowed over all surfaces. The data were evaluated using Biacore T200 evaluation software version 3.0 and Biacore 8K evaluation software (v.3.0.12.15655).

[0455] The raw data were dual referenced, i.e., the response of the measurement flow cell was corrected with the response of the reference flow cell, and in a second step, the response of the blank injection was subtracted. The resulting sensorgrams were fitted using a steady-state model or a 1:1 Langmuir model to calculate the equilibrium dissociation constant (K D ).

[0456] 3. Measurement of in vitro binding to C1q

[0457] The affinity determination of human C1q binding to bbmAb2 was performed on a Biacore T200 instrument. The affinity determinations of human C1q binding to bbmAb5 and bbmAb1 were performed on a Biacore 8K instrument. The samples were diluted to 50 μg / ml with 10 mM sodium acetate buffer (pH 4.5) and immobilized on a CM5 sensor chip at a density of approximately 8900 resonance units by applying a standard amine coupling procedure on the Biacore T200 instrument. On the Biacore 8K instrument, a similar procedure was applied, but the immobilization density was approximately 9400 resonance units. On the Biacore T200, flow cell 1 was blank immobilized to serve as a reference. On the Biacore 8K, there were a total of 8 channels, and flow cell 1 of each channel was left blank to serve as a reference surface. Kinetic binding data were collected by subsequently injecting a 1:2 dilution series of human C1q at a flow rate of 30 μl / min at a temperature of 25 °C on all flow cells. Human C1q was diluted in running buffer (HBS-EP+ pH 7.4) at a concentration range of 0.49 nM - 250 nM. The chip surface was regenerated with 50 mM NaOH at a flow rate of 30 μl / min for 30 s, including a stabilization period of 60 s after each measurement cycle. Zero concentration samples (blank operation samples) were measured to allow for dual referencing during data evaluation. Replicate injections of each sample and buffer blank were flowed over all surfaces. The data were evaluated using Biacore T200 evaluation software version 3.0 and Biacore 8K evaluation software (v.3.0.12.15655).

[0458] The raw data were dual referenced, i.e., the reaction of the measurement flow cell was corrected with the reaction of the reference flow cell, and in a second step, the reaction of the blank injection was subtracted. The resulting sensorgrams were fitted using a steady-state model to calculate the equilibrium dissociation constant (K D ).

[0459] Results showing the in vitro binding affinities of Fcγ and FcRn receptors

[0460] The binding affinities of bbmAb1, bbmAb2, and bbmAb5 to different Fc receptors are summarized in Table 9 below.

[0461] Table 9: K D values for bbmAb1, bbmAb2, and bbmAb5

[0462]

[0463]

[0464] Note: KD values marked with * are unreliable because the determined KD is close to or greater than the highest concentration applied.

[0465] Example 7. PK Study of an IL-13 / IL-18 Bispecific Antibody

[0466] PK study conducted in hFcRn transgenic mice

[0467] Mouse experiments were conducted in Tg276 B6.Cg-Fcgrttm1DcrTg(CAG-FCGRT)276Dcr / DcrJ hemizygous mice, which were derived from C57BL / 6 mice and purchased from The Jackson Laboratory (USA). The FcRn- / - hFcRn (line 276) Tg mice carry a null mutation of the murine gene and a transgene expressing the hFcRn α-chain under the control of the ubiquitous CAG promoter. At the start of the study, all mice were naive male mice between 8 and 12 weeks of age. For dosing, the antibody was prepared in phosphate-buffered saline (1x PBS (pH 7.3)) and administered as a single intravenous dose of 10 mg / kg at a dose volume of 5 mL / kg into the left lateral tail vein. During the 29-day study duration, the experiment was repeated for a total of 3 animals per antibody using a serial sampling method (before dosing, 1 h, 1 d, 2 d, 3 d, 6 d, 9 d, 17 d, and 29 d). Blood samples (30 - 50 μL) were collected into serum separator tubes and allowed to clot at room temperature for 20 - 30 minutes. The samples were then processed by centrifugation (2000 g, room temperature, 10 minutes) to obtain serum. The resulting serum was stored at -80 °C until analysis.

[0468] Total antibody in unknown mouse serum samples, calibration standards (Cs), and quality control samples (QC) was measured using a quantitative sequential electrochemiluminescence immunoassay (ECLIA). Two targets were used to capture and detect the antibody such that the assay was able to detect the total amount of the bispecific therapeutic antibody using its binding sites. Thus, the biotinylated human IL-18 capture antibody was immobilized on an SA-coated and blocked MSD plate. Ru(II) tris(bipyridine)-(4-methylsulfonate) NHS ester (MSD-Sulfo-TAG TM )-labeled cynomolgus monkey IL-13 was added and bound for electrochemiluminescence (ECL)-based detection and read on an ECL imager from MSD. The resulting ECL values were proportional to the amount of drug present in the initial sample, as determined by 5PL regression analysis with a weighting factor of 1 / Y2, where Y represents the ECL numerical value of the corresponding calibration standard.

[0469] The above experimental methods were used to study bbmAb1 and bbmAb2, as well as the Fc-silent variants bbmAb6, bbmAb7, bbmAb8 and bbmAb9 in a humanized mouse model. bbmAb7 and bbmAb8 are Fc-silent variants of bbmAb1 that contain the L235C mutation (EU numbering). bbmAb6 is an Fc-silent variant of bbmAb1 that contains the L235C mutation and does not contain the YTE mutation (EU numbering). bbmAb9 is an Fc-silent variant of bbmAb1 that contains the L235C / G236C mutation (EU numbering).

[0470] All of the above antibodies showed good exposure after intravenous administration, which is typical for antibodies with an extended half-life of high C max and showed a typical PK curve shape with a short distribution phase and an extended elimination phase in the systemic circulation. The half-lives of the antibodies studied here were very similar and could be estimated from the terminal phase of the PK curve, ranging from approximately 10 to 16 days (as Figure 7 shown).

[0471] Example 7 Simultaneous inhibition of IL-13 / IL-18

[0472] Human peripheral blood mononuclear cells (pBMC) or human keratinocytes were treated with IL-18 or IL-13 separately, and the differences in gene expression were analyzed. Little overlap was observed between the gene signatures induced by IL-13 and IL-18 (data not shown), while both were shown to be increased in published lesional skin compared to non-lesional skin from a gene expression dataset of AD patients (He et al. 2020: skin biopsies from 5 AD (lesional and non-lesional) and 7 HV subjects; scRNA-seq data).

[0473] Mining a dataset in which lesional and non-lesional skin biopsy samples had been collected from AD patients treated with an antibody that antagonizes the IL-4 receptor α (anti-IL-4Rα) to block both IL-13 and IL-4 signaling revealed that anti-IL-4Rα treatment significantly downregulated the gene signature induced by IL-13; however, anti-IL-4Rα treatment did not significantly downregulate the gene signature induced by IL-18 (data not shown).

[0474] These results suggest that dual inhibition of both IL-13 and IL-18 may improve treatment outcomes compared to inhibiting IL-13 or IL-18 alone.

[0475] Harvest 4 mm skin biopsies from 10 patients with atopic dermatitis. Harvest 4 mm skin biopsies from 8 healthy volunteers as control samples. Cut each biopsy into 4 pieces and culture ex vivo in 100 μl medium which is + / - α-IL-18, α-IL-13 or both (each at 150 μg / mL). Centrifuge the cell culture supernatants at low speed to remove cells without lysing them. Evaluate changes in protein expression using the Olink assay. Reductions in protein expression in the supernatants of lesional biopsy samples compared to control samples showed a measurable reduction in anti-IL-13 treated samples compared to control, a reduction in anti-IL-18 treated samples compared to control, and a further reduction in anti-IL-18 / anti-IL-13 treated samples compared to control and compared to samples treated alone (data not shown).

[0476] Example 8 Simultaneous inhibition of IL-13 / IL-18 with a bispecific antibody shows synergistic inhibition of the AD-like transcriptome

[0477] Methods

[0478] Obtain 8 mm full-thickness skin biopsies from surgical discard from 5 individual donors and culture them in IMDM medium with 1% Pen / Strep and 10% KnockOut Serum Replacement in tissue culture inserts of 12-well plates. On day 0, inject the biopsies with control (30 μL PBS), or activate the biopsies with a mixture of 30 μL of anti-CD28 and anti-CD3 antibodies in PBS at final concentrations of 500 ng each. To induce differentiation into an AD-like transcriptome, incubate the biopsies (except for controls) with a mixture of the following cytokines in IMDM medium at a concentration of 50 ng / mL each for 6 days: IL-4, IL-13, IL-33, TSLP, IL18 and IL-31, where the medium is changed on days 2, 4 and 5. During the 6-day induction period, treat the induced biopsy samples with the following antibodies: 1 μM IgG1 isotype control antibody with a LALA silent mutation (AD+ isotype); 1 μM anti-IL13 antibody; 1 μM anti-IL18 antibody or 1 μM anti-IL13 / 18 bispecific antibody bbmAb1. On day 6, collect the supernatants and halve the biopsies for histological and transcriptome analysis (Ampliseq whole transcriptome protocol).

[0479] The Ampliseq normalized values were imported into Qlucore Omics Explorer 3.8, and variables with values less than 0.5 in 90% of the samples were removed (if true), resulting in 16,719 variables. The threshold was set to 0.25, and the values were log2-transformed. On donor-corrected samples, "AD isotype" samples were compared to controls at q < 0.1 and FC > 2, generating a "disease transcriptome", 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.

[0480] Results

[0481] As Figure 8 shown, for all anti-IL18-treated samples, GSVA showed ineffective inhibition. Anti-IL13 inhibition was partially effective in 2 out of 5 samples. In contrast, the anti-IL18 / IL13 bispecific antibody bbmAb1 showed substantial inhibition of the AD-like disease transcriptome in 4 out of 5 samples, indicating synergistic effects of combined cytokine blockade.

[0482] As Figure 9 shown, t-SNE analysis (perplexity = 5) of the 507 upregulated genes showed a distinct disease effect, with controls and AD+ isotype samples being furthest apart on the x-axis. Four out of five samples treated with the anti-IL13 / 18 bispecific antibody bbmAb1 clustered near the control samples, indicating that cells from these samples had the least AD-like transcriptome among the induced cells. In contrast, only two anti-IL13-treated samples showed a similar effect, and anti-IL18-treated samples were indistinguishable from AD+ isotype samples, indicating no therapeutic effect. These results were further supported by Figure 10 t-SNE analysis (perplexity = 5) of the 1,485 differentially expressed genes (AD+ isotype compared to control) shown in Figure 10 . However, Figures 8 - 10 does suggest that blocking IL-18 alone has a modest therapeutic effect. Without wishing to be bound by theory, the inventors hypothesize that Figures 8 - 10 the data suggest that IL-18 blockade (e.g., using an anti-IL18 antibody) can exhibit a therapeutic effect on AD in patients, but IL13 / 18 co-blockade (e.g., by simultaneous or sequential administration of IL-13 and IL-18 antagonists) can be unexpectedly superior therapeutically compared to blocking IL-13 or blocking IL-18.

[0483] Example 9. Simultaneous inhibition of IL-13 / IL-18 with a bispecific antibody

[0484] In a test assay, multiple cells are contacted with an agent comprising the bispecific antibody disclosed herein. IL-13 and IL-18 activities are assayed and compared to a control assay in which multiple cells are contacted with only the agent. Both IL-13 activity and IL-18 activity are significantly reduced in the test assay compared to the control.

[0485] Example 10. Treatment of atopic dermatitis with an anti-IL-13 / IL-18 bispecific antibody

[0486] The bispecific antibody described herein is administered to a subject having atopic dermatitis. At week 16, the subject achieved a greater reduction in one or more signs and / or symptoms of atopic dermatitis compared to placebo-treated subjects.

[0487] Some embodiments of the present application:

[0488] 1. A multispecific antibody, wherein the antibody comprises

[0489] a. a first portion that comprises a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically bind interleukin-18 (IL-18), and

[0490] b. a second portion that comprises a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically bind interleukin-13 (IL-13).

[0491] 2. The multispecific antibody according to embodiment 1, wherein the antibody is a bispecific antibody.

[0492] 3. The multispecific antibody according to embodiment 1 or 2, wherein the VH1 and the VH2 comprise complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3; and the VL1 and the VL2 comprise LCDR1, LCDR2, LCDR3, wherein:

[0493] e. the VH1 domain comprises (e.g., in order):

[0494] 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

[0495] 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

[0496] iii. The said HCDR1 having the amino acid sequence SEQ ID NO:38, the said HCDR2 having the amino acid sequence SEQ ID NO:39, and the said HCDR3 having the amino acid sequence SEQ ID NO:40; and

[0497] f. The said VL1 domain comprises (for example, in sequence):

[0498] i. The said LCDR1 having the amino acid sequence SEQ ID NO:4, the said LCDR2 having the amino acid sequence SEQ ID NO:5, and the said LCDR3 having the amino acid sequence SEQ ID NO:6; or

[0499] ii. The said LCDR1 having the amino acid sequence SEQ ID NO:7, the said LCDR2 having the amino acid sequence SEQ ID NO:8, and the said LCDR3 having the amino acid sequence SEQ ID NO:9; or

[0500] iii. The said LCDR1 having the amino acid sequence SEQ ID NO:10, the said LCDR2 having the amino acid sequence SEQ ID NO:11, and the said LCDR3 having the amino acid sequence SEQ ID NO:12; and

[0501] g. The said VH2 domain comprises (for example, in sequence):

[0502] i. The said HCDR1 having the amino acid sequence SEQ ID NO:46, the said HCDR2 having the amino acid sequence SEQ ID NO:47, and the said HCDR3 having the amino acid sequence SEQ ID NO:48; or

[0503] ii. The said HCDR1 having the amino acid sequence SEQ ID NO:49, the said HCDR2 having the amino acid sequence SEQ ID NO:50, and the said HCDR3 having the amino acid sequence SEQ ID NO:51; or

[0504] iii. The said HCDR1 having the amino acid sequence SEQ ID NO:52, the said HCDR2 having the amino acid sequence SEQ ID NO:53, and the said HCDR3 having the amino acid sequence SEQ ID NO:54; and

[0505] h. The said VL2 domain comprises (for example, in sequence):

[0506] 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

[0507] 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

[0508] 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.

[0509] 4. The multispecific antibody according to embodiment 1, 2 or 3, wherein the multispecific antibody comprises a first light chain of the lambda type and a second light chain of the kappa type.

[0510] 5. The multispecific antibody according to embodiment 4, wherein the first light chain is of the lambda1 type and the second light chain is of the kappa4 type.

[0511] 6. The multispecific antibody according to any one of embodiments 1 - 5, wherein:

[0512] (a). The VL1 domain comprises the amino acid sequence SEQ ID NO:13, and

[0513] (b). The VL2 domain comprises the amino acid sequence SEQ ID NO:27.

[0514] 7. The multispecific antibody according to any one of embodiments 1 - 6, wherein:

[0515] a. The VH1 domain comprises the amino acid sequence SEQ ID NO:41, and

[0516] b. The VL1 domain comprises the amino acid sequence SEQ ID NO:13, and

[0517] c. The VH2 domain comprises the amino acid sequence SEQ ID NO:55, and

[0518] d. The VL2 domain comprises the amino acid sequence SEQ ID NO:27.

[0519] 8. The multispecific antibody according to any one of embodiments 1-7, wherein the multispecific antibody comprises a first light chain having the amino acid sequence shown in SEQ ID NO: 14 and a second light chain having the amino acid sequence shown in SEQ ID NO: 28.

[0520] 9. The multispecific antibody according to any one of embodiments 1-8, wherein the multispecific antibody comprises a first heavy chain having a heterodimerization modification and a second heavy chain having a heterodimerization modification complementary to the heterodimerization modification of the first heavy chain.

[0521] 10. The multispecific antibody according to embodiment 9, wherein the first heavy chain and the second heavy chain are human IgG1, and:

[0522] a) the heterodimerization modification of the first heavy chain comprises serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain comprises tryptophan at position 366; or

[0523] b) the heterodimerization modification of the second heavy chain comprises serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain comprises tryptophan at position 366,

[0524] and wherein the amino acid residues are numbered according to EU numbering.

[0525] 11. The multispecific antibody according to any one of embodiments 1-10, wherein the multispecific antibody is a bispecific antibody, and the bispecific antibody comprises a mutation that enhances the half-life of the bispecific antibody via enhanced FcRn binding.

[0526] 12. The bispecific antibody according to embodiment 11, wherein the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E (YTE), and wherein the amino acid residues are numbered according to EU numbering.

[0527] 13. The multispecific antibody according to any one of embodiments 1-12, 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.

[0528] 14. The multispecific antibody according to any one of embodiments 1-12, 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.

[0529] 15. The multispecific antibody according to any one of embodiments 1-12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 42, the first light chain comprises the amino acid sequence shown in SEQ ID NO: 14, the second heavy chain comprises the amino sequence shown in SEQ ID NO: 56, and the second light chain comprises the amino acid sequence shown in SEQ ID NO: 28.

[0530] 16. The multispecific antibody according to any one of embodiments 1-12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, the first light chain comprises the amino acid sequence shown in SEQ ID NO: 14, the second heavy chain comprises the amino sequence shown in SEQ ID NO: 58, and the second light chain comprises the amino acid sequence shown in SEQ ID NO: 28.

[0531] 17. A pharmaceutical composition comprising the multispecific antibody according to any one of the foregoing embodiments in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0532] 18. The pharmaceutical composition according to embodiment 17, wherein the pharmaceutical composition further comprises one or more additional active agents.

[0533] 19. An isolated nucleic acid molecule encoding the multispecific antibody according to any one of embodiments 1-16.

[0534] 20. A cloning or expression vector comprising one or more of the nucleic acid sequences according to embodiment 19, wherein the vector is suitable for recombinantly producing the multispecific antibody according to any one of embodiments 1-16.

[0535] 21. A host cell comprising one or more of the cloning or expression vectors according to embodiment 20.

[0536] 22. A process for producing the multispecific antibody according to any one of embodiments 1 to 16, the process comprising culturing the host cell according to embodiment 21 under conditions sufficient to express the multispecific antibody, and then purifying and recovering the multispecific antibody from the host cell culture.

[0537] 23. A kit, said kit comprising a multispecific antibody according to any one of embodiments 1 to 16 or a pharmaceutical composition according to embodiment 17 or 18, wherein said kit further comprises instructions for use and a drug delivery device for administering said multispecific antibody or said pharmaceutical composition to a subject in need thereof.

[0538] 24. The kit according to embodiment 23, wherein the drug 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 a needle.

[0539] 25. A method for simultaneously inhibiting the activities of IL-13 and IL-18, said method comprising contacting a plurality of mammalian cells with an effective amount of a multispecific antibody according to any one of embodiments 1 to 16.

[0540] 26. A method for simultaneously inhibiting the activities of IL-13 and IL-18 in a subject, said method comprising administering to said subject a therapeutically effective amount of a multispecific antibody according to any one of embodiments 1 to 16.

[0541] 27. A method for treating an IL-13- and / or IL-18-mediated disorder in a subject, said method comprising administering to said subject a therapeutically effective amount of a multispecific antibody according to any one of embodiments 1 to 16.

[0542] 28. A method for preventing or treating an inflammatory or immune disorder, said method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody according to any one of embodiments 1 to 16.

[0543] 29. A multispecific antibody according to any one of embodiments 1 to 16, said multispecific antibody for use in preventing or treating an inflammatory or immune disorder in a subject in need thereof.

[0544] 30. The method or use according to embodiment 28 or 29, wherein the inflammatory or immune disorder is a skin disorder.

[0545] 31. The method or use according to embodiment 29, wherein the skin disorder is atopic dermatitis.

[0546] 32. The method or use according to embodiment 31, wherein the method is an improved treatment as compared to treatment with a monospecific anti-IL-13 antagonist and / or a monospecific anti-IL-18 antagonist.

[0547] 33. The method or use according to embodiment 31 or 32, wherein after 16 weeks of treatment, the improvement is indicated by a better Eczema Area and Severity Index (EASI) score.

[0548] 34. The method or use according to any one of embodiments 31 to 33, wherein the atopic dermatitis is moderate to severe atopic dermatitis.

[0549] 35. The method or use according to any one of embodiments 31 to 34, wherein the atopic dermatitis cannot be adequately controlled with topical corticosteroids.

[0550] 36. A method of treating an inflammatory or immune disorder in a subject in need thereof, the method comprising administering an IL-13 antagonist and an I-18 antagonist to the subject simultaneously or sequentially.

[0551] 37. The method according to embodiment 36, wherein the method comprises administering a first antagonist selected from an IL-13 antagonist and an IL-18 antagonist and a second antagonist selected from an IL-13 antagonist and an IL-18 antagonist, wherein the first antagonist and the second antagonist are structurally different molecules.

[0552] 38. The method according to embodiment 36, wherein the method comprises administering an anti-IL18 antagonist antibody and an anti-IL13 antagonist antibody.

[0553] 39. The method according to embodiment 36, wherein the method comprises administering an anti-IL13 / 18 bispecific antibody.

[0554] 40. The method according to embodiment 36, wherein the inflammatory or immune disorder is a skin disorder.

[0555] 41. The method according to embodiment 36, wherein the inflammatory or immune disorder is atopic dermatitis, such as moderate to severe atopic dermatitis, or atopic dermatitis that cannot be adequately controlled with topical corticosteroids.

[0556] 42. The method according to embodiment 41, wherein the method is an improved treatment compared to treatment with a monospecific anti-IL-13 antagonist and / or a monospecific anti-IL-18 antagonist.

[0557] 43. The method according to embodiment 42, wherein after 16 weeks of treatment, the improvement is indicated by a better Eczema Area and Severity Index (EASI) score.

Claims

1. A multispecific antibody, wherein the antibody comprises a. a first part, the first part comprising a first heavy chain and a first light chain that specifically bind interleukin-18 (IL-18), 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 b. a second part, the second part comprising a second heavy chain and a second light chain that specifically bind interleukin-13 (IL-13), wherein 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.

2. A multispecific antibody, wherein the antibody comprises a. a first part, the first part comprising a first heavy chain and a first light chain that specifically bind interleukin-18 (IL-18), 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 b. a second part, the second part comprising a second heavy chain and a second light chain that specifically bind interleukin-13 (IL-13), wherein 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.

3. The multispecific antibody according to claim 1 or 2, wherein the antibody is a bispecific antibody.

4. A pharmaceutical composition, the pharmaceutical composition comprising the multispecific antibody according to any one of claims 1-3 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

5. The pharmaceutical composition according to claim 4, the pharmaceutical composition further comprising one or more additional active agents.

6. A kit, the kit comprising the multispecific antibody according to any one of claims 1 to 3, wherein the kit further comprises instructions for use and a drug delivery device for administering the multispecific antibody or the pharmaceutical composition to a subject in need thereof.

7. The kit according to claim 6, wherein the drug 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 a needle.

8. A kit, the kit comprising the pharmaceutical composition according to any one of claims 4 or 5, wherein the kit further comprises instructions for use and a drug delivery device for administering the multispecific antibody or the pharmaceutical composition to a subject in need thereof.

9. The kit according to claim 8, wherein the drug 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 a needle.

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