Multispecific antibodies targeting IL-13 and IL-18

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of immunology. Specifically, the present invention relates to multispecific antibodies that target interleukin 13 (IL-13) and interleukin 18 (IL-18), and methods for their production and use.

Background Art

[0002] Atopic dermatitis (AD) is a chronic / recurrent inflammatory skin disease characterized by symptoms including severe pruritus (e.g., intense itching) and scaly and dry eczematous lesions. Severe disease can severely disrupt daily life due to significant psychological problems, marked sleep deprivation, and reduced quality of life, leading to high socioeconomic costs. The pathophysiology of AD is influenced by immunoglobulin E (IgE)-mediated sensitization and complex interactions between the immune system and environmental factors. The major cutaneous abnormalities can be immunological disturbances that cause IgE-mediated sensitization, and epithelial barrier dysfunction is a result of both genetic mutations and local inflammation. AD often begins in early childhood before the age of 5 and can persist into adulthood.

[0003] Typical treatments for AD include topical lotions and moisturizers, topical corticosteroid ointments, creams, or injections. However, most treatment options only provide temporary and incomplete symptom relief. Furthermore, many patients with moderate to severe AD become resistant to treatment with topical corticosteroids or calcineurin inhibitors. Therefore, there is a need in the art for new targeted therapies for the treatment and / or prevention of AD.

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

[0005] 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 being developed.

[0006] IL-18 is thought to be involved in the pathogenesis of AD because it induces super Th1 cells that produce and secrete IFN-γ and IL-13 (Terada et al. (2006) Proc Natl Acad Sci USA. 103:8816-8821). IL-18 is released by keratinocytes and inflammatory dendritic cells, and serum IL-18 levels in AD patients have been shown to be significantly correlated with the skin score of AD lesions (Ikezawa et al. (2010) Allergy, Asthma & Immunology Research 2(4):235-246). Th2 differentiation also occurs upon in vivo administration of IL-18, and in mice, IgE production increases 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).

[0007] Since IL-13 and IL-18 are pro-inflammatory cytokines that affect many different cell types associated with AD, an effective treatment to achieve simultaneous blockade of IL-13 and IL-18 signaling is still needed. Multispecific antibodies (e.g., bispecific antibodies) that target both IL-13 and IL-18 can address the unmet medical need in this chronic inflammatory disease.

[0008] One of the most common problems in the production of bispecific IgG (BsIgG) by co-expressing two different antibodies is the unwanted homodimerization of the component heavy chains and the unwanted pairing of the component light chains with the wrong heavy chains. Figure 1 shows the potentially misassembled products. To overcome this problem of heavy chain homodimerization, the heavy chains can be remodeled for heterodimerization using disulfide bonds modified in combination with the previously identified "knob-into-hole" mutations. One mutant, S354C:T366W / Y349'C:T366'S:L368'A:Y407V, can result in nearly quantitative (about 95%) heterodimerization (Merchant et al, 1998). However, this nearly quantitative heterodimerization does not solve the problem of light chain pairing. Thus, random light chain pairing is assumed, and only 25% of the antibodies produced are the desired bispecific ones. There is still a need to further improve both heavy chain heterodimerization and light chain pairing and thereby improve the purity, yield, and quality of bispecific antibodies. SUMMARY OF THE INVENTION

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

[0010] 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 to advance clinical development and commercial manufacture at reasonable cost.

[0011] In some embodiments, the multispecific antibody is a bispecific antibody. A representative 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).

[0012] In some embodiments, the modified 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 modified multispecific antibody (e.g., bispecific antibody) is a human or humanized (e.g., CDR-grafted) IgG1 antibody.

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

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

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

[0016] In one embodiment, a modified multispecific antibody (e.g., a bispecific antibody) or a fragment thereof includes, for example, chain pairing amino acid substitutions combined with silencing and / or half-life extension mutations. In some cases, the chain pairing amino acid substitutions are knob-into-hole (KiH) mutations. For example, a modified multispecific antibody (e.g., a bispecific antibody) or a fragment thereof includes a first constant heavy chain with an amino acid substitution of T366W and a second constant heavy chain with amino acid substitutions of T366S, L368A, and Y407V, and these amino acid residues are numbered according to EU numbering.

[0017] In another embodiment, the chain pairing amino acid substitutions are knob-into-hole (KiH) mutations and include a first constant heavy chain with amino acid substitutions of S354C and T366W and a second constant heavy chain with amino acid substitutions of Y349C, T366S, L368A, and Y407V, and these amino acid residues are numbered according to EU numbering.

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

[0019] Although not bound by theory, in some embodiments, a multispecific antibody (e.g., a bispecific antibody) or fragment thereof comprises one or more mutations within hFc to, for example, suppress ADCC and / or CDC effector functions. Various sets of mutations are described in the art, for example, as LALA (L234A, L235A according to EU numbering) (Wines et al, 2000) or DAPA (D265A, P329A according to EU numbering) (Genentech, US Patent No. 6,737,056). Some 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 from IgG4 (i.e., H268Q, V309L, A330S, P331S according to EU numbering) (An et al., 2009). Another silent IgG1 antibody comprises the N297A mutation that results in an aglycosylated / non-glycosylated antibody (Strohl et al, 2009). Some of the sets of mutations used combine previously described techniques to achieve a higher level of silencing up to the complete ablation of some or all effector functions. DANAPA is an example (D265A, N297A, P329A) (WO 2019068632 pamphlet Janssen). Other alternative approaches for engineering or mutating key residues of the Fc region responsible for effector function have been reported. See, for example, PCT WO 2009 / 100309 pamphlet (Medimmune), WO 2006 / 076594 pamphlet (Xencor), US Patent Application Publication No. 2006 / 0134709 (Macrogenics), US Patent No. 6,737,056 (Genentech), US Patent Application Publication No. 2010 / 0166740 (Roche).

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

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

[0022] In another embodiment, the modified 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 these amino acid residues are numbered according to EU numbering.

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

[0024] In some embodiments, the modified 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 and / or one or more amino acid substitutions that promote proper chain pairing that enhance the half-life of the modified multispecific antibody (e.g., bispecific antibody) or fragments thereof via enhanced FcRn binding.

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

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

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

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

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

[0030] 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 VL1 and VL2 comprise LCDR1, LCDR2, LCDR3, a. The VH1 domain comprises, for example, (in order), i. said HCDR1 having the amino acid sequence SEQ ID NO: 32, said HCDR2 having the amino acid sequence SEQ ID NO: 33, and said HCDR3 having the amino acid sequence SEQ ID NO: 34, or ii. said HCDR1 having the amino acid sequence SEQ ID NO: 35, said HCDR2 having the amino acid sequence SEQ ID NO: 36, and said HCDR3 having the amino acid sequence SEQ ID NO: 37, or iii. said HCDR1 having the amino acid sequence SEQ ID NO: 38, said HCDR2 having the amino acid sequence SEQ ID NO: 39, and said HCDR3 having the amino acid sequence SEQ ID NO: 40 and b. The VL1 domain comprises, for example, (in order) i. said LCDR1 having the amino acid sequence SEQ ID NO: 4, said LCDR2 having the amino acid sequence SEQ ID NO: 5, and said LCDR3 having the amino acid sequence SEQ ID NO: 6, or ii. said LCDR1 having the amino acid sequence SEQ ID NO: 7, said LCDR2 having the amino acid sequence SEQ ID NO: 8, and said LCDR3 having the amino acid sequence SEQ ID NO: 9, or iii. said LCDR1 having the amino acid sequence SEQ ID NO: 10, said LCDR2 having the amino acid sequence SEQ ID NO: 11, and said LCDR3 having the amino acid sequence SEQ ID NO: 12 and c. The VH2 domain is (for example, in order) i. said HCDR1 having the amino acid sequence SEQ ID NO: 46, said HCDR2 having the amino acid sequence SEQ ID NO: 47, and said HCDR3 having the amino acid sequence SEQ ID NO: 48, or 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 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 d. The VL2 domain is (for example, in order) 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 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 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 including.

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

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

[0033] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof comprises a VL1 domain comprising the amino acid sequence SEQ ID NO: 13 and a VL2 domain comprising the amino acid sequence SEQ ID NO: 27.

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

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

[0036] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) or a 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.

[0037] In some embodiments, the first and second constant heavy chains are human IgG1 comprising a heterodimerization modification, and 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 b) The heterodimerization modification of the second immunoglobulin heavy chain includes serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain includes tryptophan at position 366. This amino acid residue is numbered according to the EU numbering.

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

[0039] In some embodiments, the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E (YTE), and this amino acid residue is numbered according to the EU numbering.

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

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

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

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

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

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

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

[0047] Also disclosed herein are cloning or expression vectors comprising one or more nucleic acid sequences as outlined above, which vectors are suitable for the recombinant production of the multispecific antibodies of the present disclosure. In some embodiments, a set of two cloning or expression vectors is provided herein, wherein 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 and a variable domain, and the heavy and light chains encoded by the first vector can be combined to form the anti-IL-18 arm of a bispecific IgG antibody, and the second vector encodes a full-length heavy chain comprising a constant domain and a variable domain and a full-length light chain comprising a constant and a variable domain, and the heavy and light chains encoded by the second vector can be combined to form the anti-IL-13 arm of a bispecific IgG antibody as described herein. In some embodiments, the first and second vectors are expression vectors, and co-expression of the first and second vectors in a common host cell provides an anti-IL-18 / IL-13 bispecific IgG-like antibody with high yield, purity and activity.

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

[0049] A process for the production of the multispecific antibodies of the present disclosure is also disclosed herein, which includes 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.

[0050] A kit comprising one or more cloning and / or expression vectors of the present disclosure is also disclosed herein, and this kit further includes instructions for the preparation of the multispecific (e.g., bispecific) antibodies disclosed herein.

[0051] A kit comprising the multispecific antibody of the present disclosure or the pharmaceutical composition of the present disclosure is also disclosed herein, and this kit additionally includes instructions for use and means for administering the multispecific antibody or the pharmaceutical composition to a subject in need thereof.

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

[0053] A method of simultaneously binding IL-13 and IL-18, which includes contacting IL-13 and IL-18 with an effective amount of the multispecific antibody of the present disclosure, is also disclosed herein. In some cases, the contacting is performed in vitro. In some cases, the contacting is performed ex vivo. In some cases, the contacting is performed in a subject such as a human patient in need of IL-13 and IL-18 inhibition.

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

[0055] Methods for simultaneously inhibiting the activities of IL-13 and IL-18 in a subject, including administering to the subject a therapeutically effective amount of a multispecific (e.g., bispecific) antibody of the present disclosure, are also disclosed herein. Without wishing to be bound by theory, the inventors have hypothesized that the simultaneous blockade of IL-13 and Il-18 may have a complementary (e.g., synergistic) effect compared to the blockade of IL-18 or compared to the blockade of IL-13. In some embodiments, compared to the blockade of IL-18, the simultaneous blockade of IL-13 and Il-18 may have a complementary (e.g., synergistic) effect.

[0056] 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, is also disclosed herein. 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 or anti-IL-18 antagonist. In some embodiments, this 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 at 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 or anti-IL-18 antagonist, and this improvement is indicated by a lower Severity Scoring of Atopic Dermatitis (SCORAD) score at 16, 24, 36 or 52 weeks of treatment.

[0057] In some embodiments, the method of the present disclosure reduces the expression level of one or more AD-related biomarkers, particularly selected from the list consisting of CCL17 / TARC, IgE (e.g., serum IgE), CCL26 / eotaxin-3, CCL22 / MDC, hsCRP, CD40, IL-13, IL-24, IL-22, IL-18 (e.g., serum IL-18, serum free IL-18 (bioactive)) and IL-18BP (e.g., serum IL-18BP), as compared to the levels before co-blockade of IL13 and IL18 (e.g., treatment with a multispecific antibody (e.g., bispecific antibody) or a fragment thereof).

[0058] A method of inhibiting IgE antibody production 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), is also disclosed herein.

[0059] A method of inhibiting IFN-γ production in a subject, 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) is also disclosed herein.

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

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

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

[0063] Further details and embodiments are provided in the following sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0064]

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Mode for Carrying Out the Invention

[0065] To make the present disclosure more readily understandable, certain terms are specifically defined throughout the detailed description. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present disclosure pertains.

[0066] Definitions Interleukin (IL)-18 (simply referred to herein 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). Under normal physiological conditions, IL-18 is associated with the induction of cell-mediated immunity after infection with microbial products such as lipopolysaccharide (LPS) in a synergistic effect with IL-12 (Sareneva T et al (2000) J Immunol;165(4):1933-8). After stimulation with IL-18, natural killer (NK) cells and T cells release interferon gamma (INF-γ), a cytokine that plays an important role in the activation of macrophages and other cells. In addition to its ability to induce interferon gamma, IL-18 also has various functions. These biological properties include activation of NF-κB, induction of both Fas ligand expression, CC and CXC chemokines, and enhancement of the production of competent human immunodeficiency virus.

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

[0068] (Interleukin (IL)-13, simply referred to herein as "IL-13") is a pleiotropic cytokine mainly produced by Th2 cells and ILC2, but also, to a lesser extent, by mast cells, basophils, eosinophils, natural killer cells, macrophages, dendritic cells and monocytes. Free IL-13 binds to the a1 subunit of the IL-13 receptor (IL-13Ra1) on all cells of the human body, particularly monocytes and B cells. In the cascade reaction, this binding supports the recruitment of IL-4Ra, which induces the formation of a signal transducer that activates Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) by dimerization, leading to the phosphorylation of signal transducer and activator of transcription 6 (STAT6), a transcription factor that promotes TH2 differentiation, and class switching to IgE (Silverberg et al (2017) Dermatol Clin. 35(3):327-334; Goenka et al (2011) Immunol Res. 50(1):87-96). The term "IL-13" is synonymous with the IL-13 polypeptide or interleukin-13 polypeptide.

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

[0070] As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can bind reversibly and specifically to an antigen by non-covalent bonding. For example, a natural "antibody" of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain. The heavy chain constant domain is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (abbreviated herein as CL). The VH and VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs), with more conserved regions called framework regions (FRs) interspersed. Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen, which may be referred to herein as the antigen-binding domain. The constant region of the antibody 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 (C1q).

[0071] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-Id) antibodies. The present antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG 1 IgG 2 IgG 3 IgG 4 IgA 1 IgA 2 )).

[0072] 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 is recognized that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distant 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 and CL domains include the carboxy termini of the heavy and light chains, respectively.

[0073] The phrase "antibody fragment" as used herein refers to one or more portions of an antibody. In some embodiments, these portions are part of the constant domain of the antibody, such as the crystallizable fragment (Fc), the constant (C) domain, etc. In some other embodiments, these portions are antigen-binding fragments that retain the ability to bind reversibly and specifically to an antigen non-covalently, and may be referred to herein as antigen-binding domains. The phrase "antigen-binding fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., bind, sterically hinder, stabilize / destabilize, by spatial distribution) an epitope of an antigen. Examples of binding fragments include single-chain Fv (scFv) (with or without internal cysteine cross-linking), disulfide-linked Fv (sdFv), F(ab) 2 fragment, Fab fragment, F(ab’) 2 , fragment F(ab’) fragment, 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; V HdAb fragments consisting of domains (Ward et al., (1989) Nature, 341:544-546); and isolated complementarity determining regions (CDRs) or other epitope binding fragments of antibodies are included, but not limited to.

[0074] 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 the Fc polypeptide chain. This half-antibody can pair with another half-antibody such that the two Fc polypeptide chains dimerize into the Fc region of the multispecific antibody of the present disclosure. Similar to all polypeptide chains, the Fc polypeptide chain contains an N-terminus and a C-terminus, each of which can be linked to an antigen-binding domain (e.g., an IL-18 binding domain or an IL-13 binding domain).

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

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

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

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

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

[0080] A bispecific antibody is "heterodimeric", which means that one part is derived from a first antibody specific for a first target and another part is derived from a second antibody specific for a second target. "Heterodimerization modification" is a modification to one or both parts of an antibody that aims to promote such formation to form a heterodimeric bispecific antibody. An example of a heterodimerization modification of the Fc domains of two IgG1 parts of an antibody aimed at forming bispecificity is a "knob" (S354C, T366W) with a protruding amino acid (aa) side chain in the first heavy chain, a "hole" (Y349C, T366S, L368A, Y407V) with a small aa side chain is introduced into the second heavy chain, and a further disulfide bridge is introduced in the CH3 region connecting the two heavy chains (Merchant et al., Nat. Biotechnol., 16:677-681 (1998), page 678, Table 1).

[0081] The terms "mismatch", or "misassembly", or "assembled incorrectly" mean that different parts of a protein complex such as a bispecific antibody do not complex and bind as intended, which means that the protein complex does not appear or behave as intended. An example of a mismatch in the context of a bispecific antibody is shown in Figure 1.

[0082] As used herein, the terms "recognize" or "bind" refer to a binding molecule, antibody, or antigen-binding fragment thereof that finds and interacts with (e.g., binds or recognizes) an epitope, whether the epitope is linear, discontinuous, or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. An epitope can be formed from both adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a particular spatial conformation. Methods for determining the spatial conformation of an epitope include techniques in the art such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)) or electron microscopy. A "paratope" is the part of an antibody that recognizes an epitope of an antigen.

[0083] The phrases "specifically binds" or "selectively binds", when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refer to a binding reaction that is a determining factor for the presence of the antigen in a heterogeneous population of proteins and other biological substances, such as in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under specified particular immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 2-fold above background and does not substantially bind in large amounts to other antigens present in the sample. In one aspect, under the specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least 10-fold above background and does not substantially bind in large amounts to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or agent has been selected for its specificity for a particular protein. If desired or appropriate, this selection can be achieved by subtracting antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some aspects, an antibody or antibody fragment that cross-reacts with a particular desired molecule is selected.

[0084] The term "antigen-binding site" refers to the portion of an antibody that contains determinants that form 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 antigen-binding moiety. With respect to a protein (or protein mimic), the antigen-binding site generally includes one or more loops (of at least 4 amino acids or amino acid mimics) that form an interface for binding to the antigen polypeptide. Generally, the antigen-binding site of an antibody molecule includes at least 1 or 2 CDRs and / or hypervariable loops or, more generally, at least 3, 4, 5, or 6 CDRs and / or hypervariable loops.

[0085] The term "complementary determining region" or "CDR", as used herein, refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3) and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The positions of the CDRs and framework regions can be determined using various well-known definitions in the art, such as Kabat, Chothia, IMGT, AbM, and combined definitions (e.g., see Kabat et al., (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD; Johnson et al., (2001) Nucleic Acids Res., 29:205-206; Chothia & Lesk, (1987) J. Mol. Biol., 196:901-917; Chothia et al., (1989) Nature, 342:877-883; Chothia et al., (1992) J. Mol. Biol., 227:799-817; Lefranc MP (2001) Nucleic Acids Res., 29:207-209; Al-Lazikani et al., (1997) J. Mol. Biol., 273:927-748). Definitions of the antigen-binding site are 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).Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined numbering scheme of Kabat and Chothia, in some embodiments, the CDRs correspond to amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, such as mammalian VH, such as human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL, such as mammalian VL, such as human VL. Under 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) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0086] The term "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. For the most part, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable regions are replaced by residues from the hypervariable regions of a non-human species such as mouse, rat, rabbit, or non-human primate that have the desired specificity, affinity, and capacity (donor antibody). In some examples, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Further, a humanized antibody can contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains substantially all of at least one and generally two variable domains, 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 λ sequence. A humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc), generally that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

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

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

[0089] As used herein, "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, where the change results from a sequence modification in which the amino acid residue / position is involved. For example, typical modifications include substitution of a residue (or at said position) with another amino acid (e.g., conservative substitution or non-conservative substitution), insertion of one or more amino acids adjacent to the residue / position, and deletion of the residue / position. "Amino acid substitution" or a mutation thereof refers to the replacement of an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Generally preferably, the modification results in an alteration of at least one physicochemical activity of the mutant polypeptide as compared to the polypeptide containing the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the physicochemical activity that is altered can be the binding affinity, binding capacity, and / or binding effect for a target molecule.

[0090] The term "conservatively modified variant" is used for both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence or, when the nucleic acid does not encode an amino acid sequence, to an essentially the same sequence. Because of 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 code for the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent mutations" and are one species of conservatively modified variation. All nucleic acid sequences herein that encode polypeptides also describe every possible silent mutation of the nucleic acids. One of skill in the art will recognize that each codon in a nucleic acid (except the ATG, which is ordinarily the only codon for methionine, and the TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent mutation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0091] For a polypeptide sequence, a "conservatively modified variant" includes an individual substitution, deletion or addition to the polypeptide sequence that substitutes an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are known in the art. Such conservatively modified variants are included in, and do not exclude, the variants, interspecies homologs and alleles of the polymorphisms of the present invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the phrase "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody or antibody-like molecule containing the amino acid sequence.

[0092] In reference to two or more nucleic acid or polypeptide sequences, the terms "identical percent" or "percent identity" refer to two or more sequences or subsequences that are the same. Two sequences are "substantially the same" if, when measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection on a comparison window or specified region, they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity over a specified region or, if not specified, over the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity). Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, more preferably over a region that is 100 - 500 nucleotides or 1000 nucleotides or more (or 20, 50, 200 amino acids or more) in length.

[0093] For array comparison, typically, one array functions as a reference array to which a test array is compared. Using an array comparison algorithm, the test array and the reference array are input into a computer, and sub-array coordinates are specified as needed, and array algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. Next, the array comparison algorithm calculates the percent sequence identity of the test array relative to the reference array based on the program parameters.

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

[0095] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is generally available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits function as seeds to initiate a search for longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatching 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 stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-valued scoring residue alignments; or the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and comparison of both strands.For amino acid sequences, the BLASTP program, by default, uses a word length of 3, an expect (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), while the BLAST algorithm uses 50 alignments (B), an expect (E) of 10, M = 5, N = -4, and comparison of both strands.

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

[0097] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weighted 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, which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0098] As another indicator that two nucleic acid sequences or polypeptides are substantially the same, other than the percentage of sequence identity noted above, the polypeptide encoded by the first nucleic acid may be immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid, as follows. Thus, the polypeptide is typically substantially the same as the second polypeptide, for example, when the two polypeptides differ only by conservative substitutions. As another indicator that two nucleic acid sequences are substantially the same, the two molecules or their complements may hybridize to each other under stringent conditions, as follows. As yet another indicator that two nucleic acid sequences are substantially the same, the same primers may be used to amplify the sequences.

[0099] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single-stranded or double-stranded form. Unless specifically limited, 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 natural nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 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)).

[0100] The nucleotides in a "polynucleotide" or "nucleic acid" may include modifications such as base modifications such as bromouridine and inosine derivatives, phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoranoanilothioate, phosphoroanilidate and phosphoramidate, and ribose modifications.

[0101] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) containing a nucleic acid sequence that is suitable for transformation or transfection of a host cell and that (together with the host cell) promotes and / or regulates the expression of one or more heterologous coding regions operably linked thereto.

[0102] As used herein, the term "operably linked" or functionally linked refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. As a typical example, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system. Usually, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically adjacent to or positioned in close proximity to the coding sequence that enhances transcription.

[0103] The term "co-expression" means that different polypeptides are expressed together in a single host cell that is common to all of the polypeptides. Co-expression of a bispecific antibody means that the different moieties 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 for each half of the bispecific antibody, or by incorporating one expression vector encoding all parts of the bispecific antibody. As used herein, "C-terminus" refers to the carboxyl-terminal amino acid of a polypeptide chain having a free carboxyl group (-COOH). As used herein, "N-terminus" refers to the amino-terminal amino acid of a polypeptide chain having a free amine group (-NH2).

[0104] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This phrase is used for amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as for naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence implicitly encompasses its conservatively modified variants as well.

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

[0106] Human antibodies can be produced by many methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize the human variable region repertoire (Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:11-23).

[0107] Several strains of transgenic mice are currently available, in which their mouse immunoglobulin loci have been replaced with human immunoglobulin gene segments (Tomizuka K, (2000) Proc Natl Acad Sci, 97:722-727; Fishwild DM (1996) Nature Biotechnol 14:845-851; Mendez MJ, (1997) Nature Genetics 15:146-156). Upon antigen loading, such mice are capable of generating a repertoire of human antibodies from which the antibody of interest can be selected. Of particular note are the TrimeraTM system in which human lymphocytes are transplanted into irradiated mice (Eren R et al, (1988) Immunology 93:154-161), the Selective Lymphocyte Isolation Antibody System (SLAM), which effectively achieves and subsequently deconvolutes in vitro isolated antibody production procedures with large pools of human (or other species) lymphocytes (Babcook et al, Proc Natl Acad Sci (1996) 93:7843-7848), limiting dilution and selection procedures, and XenomouseTM (Abgenix Inc). Alternative approaches are available from Morphotek Inc using the MorphodomaTM technology.

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

[0109] Techniques for affinity maturation (Marks; Biotechnol (1992) 10:779-783) can be used to provide binding affinity by successively replacing the H and L chain variable regions with natural variants and selecting based on improved binding affinity, thereby improving the affinity of primary human isolated antibodies. Variant forms of this technique such as "epitope imprinting" are also currently available (WO 93 / 06213 pamphlet; Waterhouse; Nucl Acids Res (1993) 21:2265-2266).

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

[0111] The terms "therapeutically acceptable amount", or "therapeutically effective amount", or "therapeutically effective dose" are used interchangeably to refer to an amount sufficient to produce a desired result (i.e., reduction of disease activity, reduction of disease progression, alleviation of disease signs and / or symptoms, etc.). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved. The "prophylactically effective dosage" and "therapeutically effective dosage" of the molecules of the present disclosure can each prevent the onset of disease symptoms or, as a result, reduce the severity of disease symptoms, including symptoms associated with IL-13 activity and IL-18 activity.

[0112] The term "subject" includes humans 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. Except where indicated otherwise, the terms "patient" or "subject" are used interchangeably herein.

[0113] 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 in whom administration of the molecules or pharmaceutical compositions of the present disclosure used for detection, diagnostic procedures, and / or treatment, etc., is expected to be effective.

[0114] The terms "treating", "treatment", "treating", "preventing", "prevention" or "prevent" 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 includes reduction of symptoms or underlying risk factors. As used herein, a human antibody or fragment thereof is a "product" of or "derived from" a particular germline sequence if the variable region or full-length chain of the antibody is obtained from a system using human germline immunoglobulin genes, including heavy or light chain variable regions or full-length heavy or light chains. 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, for example, by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence with the closest sequence (i.e., greatest % identity) to the human antibody sequence. A human antibody that is a "product" of or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to natural somatic mutations or intentional introduction of site-specific mutations. However, the selected human antibody is generally at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human compared to immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, 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 in amino acid sequence to the amino acid sequence encoded by a germline immunoglobulin gene.Generally, a human antibody derived from a specific human germline sequence shows a maximum of 10 amino acid differences from the amino acid sequence encoded by the immunoglobulin gene of the human germline. In certain cases, the human antibody may show a maximum of 5 or even a maximum of 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0115] The various aspects of the present invention are described in further detail in the following sections and subsections.

[0116] Multispecific antibodies that bind to IL-13 and IL-18 I. IL-18 binding domain The present disclosure provides a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof that has been modified to bind to human IL-18. Recognition of IL-18 by the multispecific antibody (e.g., bispecific antibody) of the present disclosure occurs via an "IL-18 antigen-binding domain", which is interchangeably referred to as an "IL-18 binding domain".

[0117] In a preferred embodiment, the multispecific antibody (e.g., bispecific antibody) or a fragment thereof comprises one IL-18 binding domain such that the multispecific binding molecule is monovalent with respect to IL-18 binding. In another preferred embodiment, the multispecific binding molecule comprises a plurality of IL-18 binding domains, e.g., two IL-18 binding domains, such that the multispecific binding molecule is multivalent, preferably divalent, with respect to binding IL-18.

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

[0119] In some embodiments, the multispecific antibody (e.g., bispecific antibody) or a fragment thereof has a dissociation constant (Kd) for IL-18 of 10 -4 M to 10 -8 M, e.g., 10 -5M~10 -7 M, for example, 10 -6 M or 10 -7 The binding affinity K of M D comprises an IL-18 binding domain having

[0120] In preferred embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof comprises one IL-18 Fab. In another preferred embodiment, the anti-IL-18 binding domain comprises two IL-18 Fabs. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof comprising three or more IL-18 Fabs are also contemplated herein.

[0121] In some examples, the Fab can be prepared according to methods known in the art. Enzyme papain can be used to cleave immunoglobulin monomers into two Fab fragments and an Fc fragment. Enzyme pepsin cleaves under the hinge region to form an F(ab’)2 fragment and a pFc’ fragment. The F(ab’)2 fragment can be separated into two Fab’ fragments by gentle reduction. The Fab fragment is very stable due to non-covalent interactions occurring across the large interface between the heavy and light chain polypeptides and the presence of stabilizing disulfide bonds between the CH1 and CL domains (see Glover & Humphreys, Chapter 2, Antibodies, Vol 1: Production and Purification, Kluwer Academic / Plenum Publishers, New York 2004, edited by G Subramanian). For examples of linker orientation and size, see, for example, Hollinger et al. (1993) PNAS U.S.A. 90:6444-6448, US Patent Application Publication No. 2005 / 0100543, US Patent Application Publication No. 2005 / 0175606, US Patent Application Publication No. 2007 / 0014794, and International Publication No. 2006 / 020258 Pamphlet and International Publication No. 2007 / 024715 Pamphlet, which are incorporated herein by reference.

[0122] The terms "Fab that binds to human IL-18" and "IL-18 Fab" refer to a Fab that binds to human IL-18. In one aspect, the IL-18 Fab retains equivalent binding affinity and binds to IL-18, for example, with efficacy equivalent to that of a full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity, for example, it binds to IL-18 with a lower binding affinity than a full-length antibody, but it still provides the biological responses described herein.

[0123] Preferred IL-18 Fabs for use in the disclosed multispecific antibodies (e.g., bispecific antibodies) or fragments thereof include a VH comprising the amino acid sequence shown in SEQ ID NO: 41 and a VL comprising the amino acid sequence shown in SEQ ID NO: 13.

[0124] In one aspect, the disclosure provides a polynucleotide encoding a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof that comprises an IL-18 binding domain, such as an IL-18 Fab. The disclosure also provides isolated nucleic acid molecules encoding these Fabs.

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

[0126] In one aspect, the IL-18 binding domain (e.g., IL-18 Fab) of a multispecific antibody (e.g., bispecific antibody) or a fragment thereof is encoded by a transgene whose sequence is 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 is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the occurrence frequency of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA is biased in different species. Such codon degeneracy enables 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 U.S. Patent No. 5,786,464 and U.S. Patent No. 6,114,148.

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

[0128] In a preferred aspect, the multispecific antibody (e.g., bispecific antibody) or a fragment thereof contains one IL-13 binding domain such that the multispecific binding molecule is monovalent with respect to binding IL-13. In another preferred aspect, the multispecific binding molecule contains a plurality of IL-13 binding domains, e.g., two IL-18 binding domains, such that the multispecific binding molecule is multivalent with respect to binding IL-18, preferably divalent with respect to binding IL-13.

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

[0130] In some embodiments, the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof has a binding affinity K -4 M to 10 -8 M, e.g., 10 -5 M to 10 -7 M, e.g., 10 -6 M or 10 -7 for IL-13 and comprises an IL-13 binding domain. D

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

[0132] The terms "Fab that binds to human IL-13" and "IL-13 Fab" refer to a Fab that binds to human IL-13. In one embodiment, the IL-13 Fab retains an equivalent binding affinity and binds to IL-13 with, for example, equivalent potency as a full-length antibody. In other embodiments, the IL-18 Fab has a lower binding affinity and binds to IL-13 with, for example, a lower binding affinity than a full-length antibody, but nevertheless provides the biological responses described herein.

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

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

[0135] In one aspect, the disclosure provides a polynucleotide encoding a multispecific antibody (e.g., bispecific antibody) or fragment thereof comprising an IL-13 binding domain, e.g., an IL-13 Fab. The disclosure also provides an isolated nucleic acid molecule encoding these Fabs.

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

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

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

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

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

[0141] In some embodiments, the IL-18 binding domain and the IL-13 binding domain of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof are linked. In some embodiments, the linkage is 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., an IL-13 Fab) is linked to the N-terminus of the IL-18 binding domain, for example via a polypeptide linker. In some embodiments, the IL-13 binding domain (e.g., an IL-13 Fab) is linked, for example fused, to the N-terminus of the IL-18 binding domain.

[0142] IV. Formats of Multispecific Antibodies In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody can be multivalent, e.g., bivalent with respect to one antigen and monovalent with respect to another antigen. A representative bispecific antibody is 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., on the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes are on different antigens, e.g., on two proteins (or subunits of a multimeric protein). In embodiments of the present 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 a further binding domain having binding specificity for a second epitope or antigen (e.g., IL-13).

[0143] Protocols for making bispecific or heterodimeric antibodies are known in the art; for example, the "knob-in-hole" approach described in, for example, U.S. Patent No. 5,731,168; electrostatic steering Fc pairing such as described in International Publication No. WO 2009 / 089004, International Publication No. WO 2006 / 106905, and International Publication No. WO 2010 / 129304; chain exchange domain (SEED) heterodimer formation such as described in International Publication No. WO 2007 / 110205; Fab arm exchange such as described in International Publication No. WO 2008 / 119353, International Publication No. WO 2011 / 131746, and International Publication No. WO 2013 / 060867; bispecific antibody conjugates for generating bispecific structures using, for example, heterobifunctional reagents having amine-reactive and sulfhydryl-reactive groups such as described in U.S. Patent No. 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 through reduction and oxidation cycles of disulfide bonds between two heavy chains, such as described in U.S. Patent No. 4,444,878; trifunctional antibodies, such as three Fab' fragments crosslinked through sulfhydryl-reactive groups, such as described in U.S. Patent No. 5,273,743; biosynthetic binding proteins, such as pairs of scFvs crosslinked through their C-terminal tails, preferably through disulfide or amine-reactive chemical crosslinks, such as described in U.S. Patent No. 5,534,254; bifunctional antibodies, such as Fab fragments having different binding specificities dimerized through leucine zippers with constant domains replaced, such as described in U.S. Patent No. 5,582,996; bispecific and oligospecific monovalent and oligo-valent receptors, such as the VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of another antibody, generally together with a bound light chain, such as described in U.S. Patent No. 5,591,828; bispecific DNA-antibody conjugates,For example, cross-linking of antibodies or Fab fragments through double-stranded DNA fragments, such as those described in U.S. Patent No. 5,635,602; bispecific fusion proteins, such as expression constructs containing two scFvs with a hydrophilic helical peptide linker between the full constant regions, such as those described in U.S. Patent No. 5,637,481; multivalent and multispecific binding proteins, such as dimers of polypeptides having a first domain with a binding region of an Ig heavy chain variable region and a second domain with a binding region of an Ig light chain variable region, generally called diabodies (higher-order structures are included for bispecific, trispecific or tetra-specific molecules, such as those described in U.S. Patent No. 5,837,242; minibody constructs in which linked VL and VH chains are further connected to the antibody hinge region and CH3 region by a peptide spacer and can be dimerized to form bispecific / multivalent molecules, such as those described in U.S. Patent No. 5,837,821; VL and VH domains linked by a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in any orientation and capable of forming a dimer to form a bispecific diabody; trimers and tetramers, such as those described in U.S. Patent No. 5,844,094; a string of VH domains (or VL domains in a family member) linked by a peptide bond to a cross-linkable group at the C-terminus and further associated with a VL domain to form a series of Fvs (or scFvs), such as those described in International Publication No. WO 2011 / 028952; VL and VH domains, scFvs or Fabs in which one antigen binds monovalently and one antigen binds divalently and optionally contains a heterodimeric Fc region, such as those described in International Publication No. WO 2011 / 028952; and single-chain binding polypeptides in which both VL and VH domains are linked through a peptide linker and combined into a multivalent structure through non-covalent or chemical cross-linking using either an scFv or diabody-type format to form, for example, homo-2-valent, hetero-2-valent, trivalent and tetravalent structures,is not limited. Further representative multi-specific and bispecific molecules and methods for making them are described, for example, in U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 5,959,083, U.S. Patent No. 5,989,830, U.S. Patent No. 6,005,079, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294,353, U.S. Patent No. 6,333,396, U.S. Patent No. 6,476,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453, U.S. Patent No. 6,743,896, U.S. Patent No. 6,809,185, U.S. Patent No. 6,833,441, U.S. Patent No. 7,129,330, U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A1, U.S. Patent Application Publication No. 2003211078A1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005100543A1, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1,U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1, U.S. Patent Application Publication No. 2007141049A1, U.S. Patent Application Publication No. 2007154901A1, U.S. Patent Application Publication No. 2007274985A1, U.S. Patent Application Publication No. 2008050370A1, U.S. Patent Application Publication No. 2008069820A1, U.S. Patent Application Publication No. 2008152645A1, U.S. Patent Application Publication No. 2008171855A1, U.S. Patent Application Publication No. 2008241884A1, U.S. Patent Application Publication No. 2008254512A1, U.S. Patent Application Publication No. 2008260738A1, U.S. Patent Application Publication No. 2009130106A1, U.S. Patent Application Publication No. 2009148905A1, U.S. Patent Application Publication No. 2009155275A1, U.S. Patent Application Publication No. 2009162359A1, U.S. Patent Application Publication No. 2009162360A1, U.S. Patent Application Publication No. 2009175851A1, U.S. Patent Application Publication No. 2009175867A1, U.S. Patent Application Publication No. 2009232811A1, U.S. Patent Application Publication No. 2009234105A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent No. 346087A2, International Publication No. 2000 / 06605A2 Pamphlet, International Publication No. 2007 / 2635A2 Pamphlet, International Publication No. 2004 / 081051A1 Pamphlet, International Publication No. 2006 / 020258A2 Pamphlet, International Publication No. 2007 / 044887A2 Pamphlet, International Publication No. 2007 / 095338A2 Pamphlet, International Publication No. 2007 / 137760A2 Pamphlet, International Publication No. 2008 / 119353A1 Pamphlet, International Publication No. 2009 / 021754A2 Pamphlet, International Publication No. 2009 / 068630A1 Pamphlet, International Publication No. 1991 / 03493A1 Pamphlet, International Publication No. 1993 / 23537A1 Pamphlet, International Publication No. 1994 / 09131A1 Pamphlet, International Publication No. 1994 / 12625A2 Pamphlet, International Publication No. 1995 / 09917A1 Pamphlet,International Publication No. WO 1996 / 37621 A2 and International Publication No. WO 1999 / 64460 A1. The content of the above applications is hereby incorporated by reference in its entirety. Accordingly, in some embodiments, the IL-13 / IL-18 multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are known in the art and comprise an IL-13 binding domain and an IL-18 binding domain in any one of the multispecific or bispecific formats described above. Preferred formats for the multispecific antibodies (e.g., bispecific antibodies) of the present disclosure are described in more detail below.,

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

[0145] [Table 1]

[0146] [Table 2]

[0147] [Table 3]

[0148] [Table 4]

[0149] [Table 5]

[0150] [Table 6]

[0151]

Table 7

[0152]

Table 8

[0153]

Table 9

[0154]

Table 10

[0155]

Table 11

[0156]

Table 12

[0157]

Table 13

[0158]

Table 14

[0159]

Table 15

[0160]

Table 16

[0161]

Table 17

[0162] [Table 18]

[0163] [Table 19]

[0164] [Table 20]

[0165] [Table 21]

[0166] In some embodiments, the multispecific antibodies described herein are bispecific antibodies comprising an amino acid sequence having 1, 2, or 3 substitutions, deletions, or insertions compared to the sequences in Table 1.

[0167] Representative formats of IL-13 / IL-18 bispecific antibodies are exemplified in Table 2. All bbmAbs combine anti-IL13 and anti-IL-18 binding domains, are based on the human IgG1 format, and all contain the YTE half-life extension mutation, while two further contain the LALA silencing mutation. More specifically, bbmAb2, bbmAb1, and bbmAb3 contain the YTE half-life extension mutation in the Fc. bbmAb4 and bbmAb5 contain both the LALA silencing and the YTE half-life extension mutations. bbmAb2, bbmAb1, bbmAb4, and bbmAb5 combine the variable domains of mAb1 and MAb2, while bbmAb3 combines the variable domains of mAb1 and mAb3.

[0168] bbmAb2, bbmAb4, and bbmAb3 possess the KiH knob heterodimerization mutation in the anti-IL-18 heavy chain Fc, while the KiH hole mutation is in the anti-IL-13 heavy chain Fc. bbmAb2, bbmAb4, and bbmAb3 possess the KiH knob mutation in the anti-IL-18 heavy chain Fc and the KiH hole mutation in the anti-IL-13 heavy chain Fc.

[0169]

Table 22

[0170] VI. Modifications of the Multispecific Antibodies (e.g., Bispecific Antibodies) or Fragments Thereof of the Present Disclosure This application includes molecules described herein and / or variants of fragments thereof having various modifications in the binding domains, variable domains, and / or constant regions, as well as fusions and complexes 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 it can be modified to achieve various outcomes. Preferred modifications to 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 knob-in-hole mutations (e.g., knob S354C, T366W; hole Y349C, T366S, L368A, Y407V (EU numbering)) to promote proper chain pairing.

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

[0172] Furthermore, in certain examples, it has been found beneficial to mutate residues within the framework region to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent No. 5,530,101 to Queen et al; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,762 and U.S. Patent No. 6,180,370). 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.

[0173] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 domains, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity mutagenesis." Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the 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 (as discussed above) can be introduced. The mutations can be amino acid substitutions, additions, or deletions. Furthermore, generally, within the CDR regions, up to 1, 2, 3, 4, or 5 residues, preferably 1 or 2 residues, are changed.

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

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

[0176] B. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof with enhanced heterodimerization Inappropriate heterodimerization of two antibody heavy chain domains can be an obstacle to improving the yield of the desired multispecific antibody (e.g., bispecific antibody) or fragment thereof and corresponds to a challenge for purification. Various approaches available for promoting dimerization of two heavy chain domains of bispecific or multispecific antibodies or antibody-like molecules, as disclosed in EP 1870459 A1; US 5,582,996; US 5,731,168; US 5,910,573; US 5,932,448; US 6,833,441; US 7,183,076; US Patent Application Publication No. 2006 / 204493 A1; and WO 2009 / 089004 A1 pamphlet, can be used.

[0177] The present disclosure provides methods for promoting the dimerization (heterodimerization) of two interacting heterologous polypeptides and / or reducing the dimerization (homodimerization) of two identical polypeptides. Generally, 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, class or subclass and preferably of the IgG (IgG1, IgG2, IgG3 and IgG4) class, most preferably IgG1.

[0178] Generally, the polypeptides of the present disclosure comprise, in addition to the CH3 domain, other antibody fragments such as the CH1 domain, CH2 domain, hinge domain, VH domain, VL domain, CDRs, etc., and / or antigen-binding fragments described herein, such as scFv and / or Fab. These antibody fragments are derived from various types of antibodies described herein, such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific or multispecific antibodies, camelized antibodies, anti-idiotype (anti-Id) antibodies and antibody conjugates. Heterodimerization of two different heavy chains at the CH3 domain results in the desired antibody or antibody-like molecule, while homodimerization of identical heavy chains reduces the yield of the desired antibody or molecule. In a representative embodiment, two or more heteropolypeptide chains comprise a CH3 domain and form a molecule in either the above-described multispecific antibody (e.g., bispecific antibody) or its fragment format of the present disclosure, comprising two chains. In one embodiment, the two heteropolypeptide chains comprising the CH3 domain comprise modifications that advantageously act on the heterodimeric association of the polypeptides as compared to unmodified chains. Various examples of modification strategies are provided below.

[0179] Knob-in-hole (KIH) (also known as "key-in-hole") The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure may include one or more, e.g., a plurality of mutations, in one or more of the constant domains, e.g., in the CH3 domain. In one example, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure include two polypeptides each including a heavy chain Fc or constant domain of the antibody, e.g., the CH2 or CH3 domain. In one example, the two heavy chain constant domains, e.g., the CH2 or CH3 domain of the multispecific antibody (e.g., bispecific antibody) or fragment thereof, include one or more mutations that enable heterodimeric association between the two chains. In one aspect, one or more mutations are located on the CH2 domain of the two heavy chains of the multispecific antibody (e.g., bispecific antibody) or fragment thereof. In one aspect, one or more mutations are located on the CH3 domain 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 includes a heavy chain constant domain generate a "knob", and one or more mutations to the second polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that includes a heavy chain constant domain form a "hole", and the heterodimerization of the polypeptides of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that includes a heavy chain constant domain associates (e.g., interacts, 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 "hole" and the "knob". As used herein, the term "knob" protrudes from the interface of the first polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof that includes a heavy chain constant domain, and thus can be positioned at the interface with the second polypeptide of the multispecific antibody (e.g., bispecific antibody) or fragment thereof in a compensatory "hole" so as to stabilize the heteromultimer, thereby referring to at least one amino acid side chain that makes heteromultimer formation more favorable than homomultimer formation.The knob can be present at the original interface or introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Preferred import residues for knob formation are generally natural amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In preferred embodiments, the original residue for protrusion formation has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

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

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

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

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

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

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

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

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

[0188] In any of the embodiments described herein, the CH3 domain can be further mutated to introduce pairs of cysteine residues. Without being bound by theory, the introduction of pairs of cysteine residues capable of forming disulfide bonds is thought to confer stability to heterodimerized multispecific antibodies (e.g., bispecific antibodies) or fragments thereof. 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).

[0189] Polar crosslink In one aspect, heterodimerization of the polypeptide chains (e.g., half-antibodies) of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof is increased by introducing mutations based on a rational "polar-crosslink", which involves residues with similar (or complementary) physical properties in the heterodimer conformation interacting with residues with different physical properties in the homodimer conformation and residues at the binding interface of the two polypeptide chains. In particular, these mutations are designed such that polar residues interact with polar residues and hydrophobic residues interact with hydrophobic residues in heterodimer formation. In contrast, in homodimer formation, residues are mutated such that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimer conformation and the unfavorable interactions in the homodimer conformation function together to increase the likelihood that the CH3 domain will form a heterodimer rather than a homodimer.

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

[0191] 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 with leucine; Thr366Val: the original residue of threonine at position 366 is replaced with valine; Leu368Gln: the original residue of leucine at position 368 is replaced with glutamine; Asp399Lys: the original residue aspartic acid at position 399 is replaced with lysine; Phe405Ser: the original residue phenylalanine at position 405 is replaced with serine; Lys409Phe: the original residue lysine at position 409 is replaced with phenylalanine; Thr411Lys: the original residue of threonine at position 411 is replaced with lysine.

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

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

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

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

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

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

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

[0199] The polar crosslinking strategy is described, for example, in WO 2006 / 106905, WO 2009 / 089004, the contents of which are incorporated herein by reference in their entirety, and Gunasekaran K et al., (2010) J Biol Chem., 285: 19637 - 19646.

[0200] 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, the introduction of a pair of cysteine residues capable of forming a disulfide bond is thought to confer stability to heterodimerized multispecific antibodies (e.g., bispecific antibodies). 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).

[0201] C. Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof with an extended in vivo half-life The present multispecific antibody (e.g., bispecific antibody) or fragment thereof can be further modified to extend its in vivo half-life.

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

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

[0204] Furthermore, the multispecific antibody (e.g., bispecific antibody) or fragment thereof can be complexed or fused with one or more human serum albumin (HSA) polypeptides or portions thereof. The use of albumin is for the molecule to be more stable in vivo or to have a longer half-life in vivo. This technique is known in the art; see, for example, WO 1993 / 15199, WO 1993 / 15200, and WO 2001 / 77137; and EP 413622. The use of the N-terminal fragment of HSA for fusion to polypeptides has also been proposed (e.g., EP 399666). Thus, the molecule can be stabilized or its shelf life extended by genetically or chemically fusing or complexing the molecule to albumin, and / or the activity of the molecule can be maintained in solution for an extended period in vitro and / or in vivo. Further methods regarding HSA fusion can be found, for example, in WO 2001 / 077137 and WO 2003 / 06007, which are incorporated herein by reference. In certain embodiments, expression of the fusion protein is performed in a mammalian cell line, such as a CHO cell line.

[0205] D. Fc silencing In embodiments of the present disclosure that incorporate one or more constant domains, such as heavy chain constant regions, it may be beneficial to include one or more mutations to silence, for example, ADCC and / or CDC effector functions within the hFc. Activation of immune cells preferentially occurs in the presence of cross-linking to target cells. However, human Fc can bind to both high and low affinity FcR gamma receptors. Thus, cross-linking of receptors (e.g., CD3) on immune cells and subsequent agonism can occur upon binding in the absence of tumor targeting. Additionally, cross-linking of Fc via the gamma receptor can induce antibody-dependent cell cytotoxicity (ADCC). Human Fc can also bind to complement proteins when complexed at the cell surface and induce complement-dependent cytotoxicity (CDC). Thus, mutations to residues in Fc that reduce or abrogate these interactions can limit these effects and focus the effects of the molecules described herein on tumor target cells.

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

[0207] In embodiments, one or more, for example all, of the heavy chain constant region domains of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof include LALA mutations (e.g., L234A and L235A in EU numbering). See, for example, Hezareh M et al., (2001) Journal of Virology, 75(24):12161-12168; Shields RL et al., (2001) supra.

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

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

[0210] E. Complex The present disclosure includes multispecific antibodies (e.g., bispecific antibodies) or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate a fusion protein. Methods of fusing or conjugating a protein, polypeptide or peptide to an antibody or antibody fragment are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851 and 5,112,946; European Patent Nos. 307434 and 367166; International Publication Nos. WO 96 / 4388 and WO 91 / 06570; Ashkenazi et al., (1991) PNAS.USA 88:10535-10539; Zheng et al., (1995) J. Immunol. 154:5590-5600; and Vil et al., (1992) PNAS.USA 89:11337-11341.

[0211] Additional fusion proteins can be made through techniques of gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to vary the activity of multispecific antibodies (e.g., bispecific antibodies) or fragments thereof. Generally, see U.S. Patent No. 5,605,793, U.S. Patent No. 5,811,238, U.S. Patent No. 5,830,721, U.S. Patent No. 5,834,252 and U.S. Patent No. 5,837,458; Patten et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo & Blasco (1998) Biotechniques, 24(2):308-313 (each of these patents and publications is hereby incorporated by reference in its entirety). Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can be varied by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Polynucleotides encoding fragments of the molecule can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

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

[0213] In other embodiments, the multispecific antibody (e.g., bispecific antibody) or fragment thereof is conjugated to a diagnostic or detectable agent. Such molecules can be useful for monitoring or prognosticating the onset, occurrence, progression and / or severity of a disease or disorder as part of clinical testing procedures, such as determining the effectiveness of a particular treatment. Such diagnosis and detection can be achieved by coupling the molecule to a detectable substance including, but not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin and aequorin; radioactive substances such as, but not limited to, iodine (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 various positron emitting metals and non-radioactive paramagnetic metal ions for use in various positron emission tomography, among others.

[0214] This application further encompasses the use of a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof conjugated to a therapeutic moiety. The molecules of the present disclosure or fragments thereof can be conjugated to a therapeutic agent such as a cytotoxin, e.g., a cell cycle arrest or cell disruptive agent, or a radioactive metal ion, e.g., an alpha emitter. Cytotoxins or cytotoxic agents include any agent that is harmful to cells.

[0215] Furthermore, a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof can be conjugated to a therapeutic moiety or a drug moiety that modifies a certain biological response. The therapeutic moiety or drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein, peptide or polypeptide that retains the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis agents, anti-angiogenesis agents; or biological response modifiers such as lymphokines and the like.

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

[0217] Multispecific antibodies (e.g., bispecific antibodies) or fragments thereof can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to molecules for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90, and lutetium 177. Methods for preparing radioimmunoconjugates are established in the art. See, for example, Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

[0218] Techniques for conjugating 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.

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

[0220] VII. Method for Producing the Antibodies of the Present Invention When the polypeptides of the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are crosslinked, these functional linkages can be achieved using methods known in the art. Various coupling or crosslinking agents can be used for covalent conjugation. 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 sulfo-succinimidyl 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). The conjugating agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0221] Alternatively, the multispecific antibody (e.g., bispecific antibody) or fragment thereof can be recombinantly produced by introducing a DNA construct encoding the desired molecule into an expression vector, expressing and assembling the desired molecule in the same host cell.

[0222] A. Preparation of Polypeptide Chains Polypeptides, antibodies, and fragments thereof (e.g., half antibodies) can be produced 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 producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can be used.

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

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

[0225] In certain embodiments, the antibodies or antibody-like molecules of the present disclosure are human monoclonal antibodies. Such human monoclonal antibodies can be produced using transgenic or translchromosomal mice that possess a part of the human immune system rather than a mouse system. These transgenic and translchromosomal 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".

[0226] The HUmAb mouse (Medarex, Inc.) contains human immunoglobulin gene miniloci encoding non-rearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with mutations targeted to inactivate the endogenous μ and κ locus genes (see, e.g., Lonberg, et al., (1994) Nature 368(6474):856-859). Thus, this mouse exhibits reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic hypermutation to generate high affinity human IgGκ monoclonal antibodies (Lonberg et al., (1994) supra; Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13:65-93 and Harding and Lonberg, (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HUmAb mice and the genomic modifications carried by such mice are further described in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295; Chen et al., (1993) International Immunology 5:647-656; Tuaillon et al., (1993) PNAS USA 94:3720-3724; Choi et al., (1993) Nature Genetics 4:117-123; Chen et al., (1993) EMBO J. 12:821-830; Tuaillon et al., (1994) J. Immunol. 152:2912-2920; Taylor et al., (1994) International Immunology 579-591; and Fishwild et al., (1996) Nature Biotechnology 14:845-851, the entire contents of all of which are specifically incorporated herein by reference.Furthermore, reference is made to U.S. Patent No. 5,545,806; U.S. Patent No. 5,569,825; U.S. Patent No. 5,625,126; U.S. Patent No. 5,633,425; U.S. Patent No. 5,789,650; U.S. Patent No. 5,877,397; U.S. Patent No. 5,661,016; U.S. Patent No. 5,814,318; U.S. Patent No. 5,874,299; and U.S. Patent No. 5,770,429 to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al.; International Publication No. WO 92 / 103918; International Publication No. WO 93 / 12227; International Publication No. WO 94 / 25585; International Publication No. WO 97 / 13852; International Publication No. WO 98 / 24884; and International Publication No. WO 99 / 45962 to Lonberg and Kay; and International Publication No. WO 01 / 14424 to Korman et al.

[0227] In another embodiment, the human antibodies used in the present disclosure can be produced using mice that carry human immunoglobulin sequences in transgenes and transloci, such as mice that carry a human heavy chain transgene and a human light chain translocus. Such mice, herein referred to as "KM mice," are described in detail in International Publication No. WO 02 / 43478 to Ishida et al.

[0228] Still further, alternative transgenic animal systems that express human immunoglobulin genes are available in the art and can be used to generate the human antibodies used in the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent No. 5,939,598; U.S. Patent No. 6,075,181; U.S. Patent No. 6,114,598; U.S. Patent No. 6,150,584; and U.S. Patent No. 6,162,963 to Kucherlapati et al.

[0229] Furthermore, alternative translchromosomal 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, a mouse that carries both a human heavy chain translchromosome and a human light chain translchromosome, called a "TC mouse", can be used; such mice are described in Tomizuka et al., (2000) PNAS USA 97:722-727. Furthermore, cows that carry human heavy and light chain translchromosomes have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894) and can be used to generate the human antibodies used in the present application.

[0230] Human monoclonal antibodies can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art or described in the examples below. See, for example, U.S. Patent No. 5,223,409 to Ladner et al.; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698; U.S. Patent No. 5,427,908 and U.S. Patent No. 5,580,717 to Dower et al.; U.S. Patent No. 5,969,108 and U.S. Patent No. 6,172,197 to McCafferty et al.; and U.S. Patent No. 5,885,793 to Griffiths et al.; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081.

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

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

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

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

[0235] In 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 aspects, the sequence encoding the IL-13 binding domain or a fragment thereof and the sequence encoding the IL-18 binding domain are disposed on separate polynucleotides, which is also referred to as a "set of nucleic acid molecules".

[0236] In one aspect, the sequences encoding the IL-13 binding domain or fragments thereof and the sequence encoding the IL-18 binding domain are arranged on a single polynucleotide.

[0237] In a representative 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. Next, the expression vectors are co-transfected into a host cell at a ratio that results in optimal assembly. The encoded heavy and light chains are expressed in the host cell and assembled into a functional molecule.

[0238] In another representative 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 one expression vector. Next, the expression vector can be transfected into a host cell. The encoded heavy and light chains are expressed in the host cell and assembled into a functional molecule.

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

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

[0241] 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 molecule (e.g., sequences such as those described in the examples below). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., (1979) Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., (1979) Meth. Enzymol. 68:109; the diethylphosphoramidite method of Beaucage et al., (1981) Tetra. Lett., 22:1859; and the solid support method of U.S. Patent No. 4,458,066. For example, introduction of mutations into polynucleotide sequences by PCR can be performed as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., (1991) Nucleic Acids Res. 19:967; and Eckert et al., (1991) PCR Methods and Applications 1:17.

[0242] Expression vectors and host cells for producing the above-described molecules are also provided by the present disclosure. The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) containing a nucleic acid sequence that is suitable for transformation or transfection of a host cell and that directs and / or controls the expression of one or more heterologous coding regions operably linked thereto (in conjunction with the host cell). Various expression vectors can be used to express polynucleotides encoding the chains or binding domains of the molecules. Both virus-based expression vectors and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Non-viral vectors and non-viral systems include plasmids or episomal vectors (typically having an expression cassette for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., (1997) Nat Genet 15:345). For example, non-viral vectors useful for the expression of polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1 / His, pEBVHis A, B & C, (Invitrogen, San Diego, CA), MPSV vectors and numerous other vectors known in the art for expressing other proteins. Useful virus-based vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, 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.

[0243] The selection of an expression vector depends on the intended host cell in which the vector will be expressed. As a typical example, 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 used to prevent expression of the inserted sequence except under inducing conditions. Examples of inducible promoters include, for example, the arabinose, lacZ, metallothionein promoter or heat shock promoter. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population towards a coding sequence whose expression product is better tolerated by the host cell. 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 fragment thereof. These elements typically include, for example, an ATG start codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be enhanced by inclusion of an enhancer suitable for the cell line used (see, for example, Scharf et al., (1994) Results Probl. Cell Differ. 20:125; and Bittner et al., (1987) Meth. Enzymol., 153:516). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0244] The expression vector may also provide a secretion signal sequence position for forming a fusion protein with the polypeptide encoded by insertion of the above sequences of the heavy chain and / or light chain or fragment thereof. More often, the inserted antibody or antibody-like molecule sequence is ligated to the signal sequence prior to being included in the vector. Vectors used to accept sequences encoding the light and heavy chain variable domains sometimes also encode a constant region or portion thereof. Such vectors can generate intact antibodies or fragments thereof by expressing the variable region as a fusion protein with the constant region. Typically, such a constant region is human.

[0245] The host cell for carrying and expressing this molecule can be either a prokaryote or a eukaryote. Escherichia coli (E. coli) is a useful prokaryotic host for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, as well as other enterobacteriaceae such as the genus Salmonella, the genus Serratia, and various species of the genus Pseudomonas. In these prokaryotic hosts, those skilled in the art can also construct expression vectors, which typically contain expression control sequences (such as an origin of replication) compatible with the host cell. In addition, any number of various known promoters will be present, for example, the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from phage lambda. The promoter typically controls expression, optionally together with an operator sequence, and has a ribosome binding site sequence, etc., to initiate and complete transcription and translation. It is also possible to use other microorganisms, such as yeast, to express the antibodies of the present disclosure. Insect cells combined with baculovirus vectors can also be used.

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

[0247] The method of introducing an expression vector containing the target polynucleotide sequence varies depending on 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. (Generally, see 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 virions, fusion to the herpes virus structural protein VP22 (Elliot and O’Hare, (1997) Cell 88:223), enhancement of DNA uptake by drugs, and ex vivo transduction. In many cases, stable expression is desired for the long-term high-yield production of recombinant proteins. For example, cell lines stably expressing antibody chains or binding fragments can be prepared using the expression vectors and selection marker genes of the present disclosure containing viral origins of replication or endogenous expression elements. After vector introduction, the cells can be grown in enriched medium for 1-2 days and then switched to selection medium. The purpose of the selection marker is to confer resistance to selection, and its presence allows the growth of cells that express the introduced sequence without problems in the selection medium. Stably transfected cells showing resistance can be grown using tissue culture techniques suitable for the cell type.

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

[0249] When a molecule is produced in a recombinant cell other than one of human origin, it is completely free of proteins or polypeptides of human origin. However, in order to obtain a preparation that is substantially homogeneous with respect to the heteromultimer, it is necessary to purify the molecule from the recombinant cell protein or polypeptide. As a first step, the culture or lysis solution is usually centrifuged to remove certain cell debris. The molecule produced can be conveniently purified by hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, and the preferred purification technique is affinity chromatography. Other techniques for protein purification are also available, such as ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, fractionation by SDS-PAGE and ammonium sulfate precipitation (mRNA).

[0250] VIII. Use of the Antibodies of the Invention A. Diagnostic and General Therapeutic Uses The antibodies of the present disclosure have many diagnostic and therapeutic applications. For example, these can be used for enzyme immunoassays, where the arm binds to a specific epitope on the enzyme and the other part of the molecule binds to an immobilization matrix. Enzyme immunoassays using antibody-like molecules have been discussed by Nolan et al. (Nolan et al., (1990) Biochem. Biophys. Acta. 1040:1-11). Bispecific antibodies can also be used for the diagnosis of various diseases, such as autoimmune diseases (Songsivilai et al., (1990) Clin. Exp. Immunol. 79:315). In particular, one antigen-binding domain of the molecule can bind to IL-13 or IL-18 in a tissue sample (in vitro, ex vivo, in vivo), and the other binding site can bind to a chelating agent that binds tightly to a detectable marker described herein, such as a radionuclide (Le Doussal et al., (1992) Int. J. Cancer Suppl. 7:58-62; Le Doussal et al., (1993) J. Nucl. Med. 34:1662-1671; Stickney et al., (1995) Cancer Res. 51:6650-6655).

[0251] The antibodies of the present disclosure are useful in in vitro and in vivo diagnosis and therapy. 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 various disorders.

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

[0253] In one aspect, the present disclosure provides a method for detecting the presence of IL-13 and / or IL-18 in a biological sample. In certain aspects, the method includes contacting the biological sample with the multiplexed specific antibody of the present disclosure under conditions that allow binding of the antibody to the antigen and detecting whether a complex is formed between the antibody and the antigen. The biological sample can include, but is not limited to, a urine or blood sample.

[0254] Also included is a method for diagnosing a disorder associated with the expression of IL-13 and / or IL-18. In certain aspects, the method includes contacting test cells with the multiplexed specific antibody of the present disclosure; determining the expression level (either quantitative or qualitative) of IL-13 and / or IL-18 in the test cells by detecting the binding of the multiplexed specific molecule of the present disclosure; and comparing the expression level of IL-13 and / or IL-18 in the test cells with the expression level of IL-13 and / or IL-18 in control cells (e.g., normal cells of the same tissue origin as the test cells or non-virus-infected cells), wherein if IL-13 and / or IL-18 are present at a higher level in the test cells when compared to the control cells, it indicates the presence of a disorder associated with IL-13 and / or IL-18. In certain aspects, the test cells are obtained from an individual suspected of having a pathological disorder mediated by IL-13 and IL-18.

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

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

[0257] In certain embodiments, the multispecific antibodies of the disclosure are labeled. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescence, chromophores, high electron density, chemiluminescence, and radiolabels) and moieties such as enzymes or ligands that are indirectly detected through, for example, enzymatic reactions or molecular interactions.

[0258] B. Pharmaceutical Compositions and Modes of Administration The present disclosure provides pharmaceutical compositions comprising a multispecific antibody (e.g., a bispecific antibody) or fragment thereof of the present disclosure useful in the methods and uses of the present disclosure for the treatment of atopic dermatitis or related conditions, the composition further comprising one or more pharmaceutically acceptable carriers and / or diluents.

[0259] The phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans.

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

[0261] Pharmaceutical compositions of therapeutic and diagnostic agents can be prepared, for example, by mixing them with a physiologically acceptable carrier, excipient or stabilizer in the form of a 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, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety). The selection of the dosing regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of the immunogenic disease signs of the entity and the accessibility of the target cells within the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient while keeping the acceptable level of side effects. Thus, the amount of biologic agent delivered depends in part on the particular entity and the severity of the symptoms to be treated.Guidance for selecting appropriate dosages of antibodies, cytokines, and small molecules is 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, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; 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). See Marcel Dekker, Inc., New York, N.Y.).

[0262] Determination of an appropriate dosage is made by a clinician, using, for example, parameters or factors that are known or suspected in the art to affect or be predictive of affecting the treatment. Usually, the dosage is started at a somewhat less than the appropriate amount and then increased in small increments against any negative side effects until the desired or optimal effect is achieved. Important diagnostic measures include, for example, measures of signs of inflammation or levels of inflammatory cytokines produced.

[0263] The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure may vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected depends on various pharmacokinetic factors including the activity of the particular composition of the present disclosure or its ester, salt, or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition being used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors known in the medical arts.

[0264] Compositions containing the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be provided by continuous infusion or by administration at intervals, for example, once a day, once a week, or 1 to 7 times a week. The dosage can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranially, or by inhalation.

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

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

[0267] When a series of dosages are administered, these can be administered, for example, approximately daily, approximately weekly, approximately every two weeks, approximately every three weeks, approximately every four weeks (monthly), approximately every two months, approximately every three months (four times a year), approximately every six months. This dosage can be continued until, for example, disease progression, adverse events or other times as determined by the physician. For example, a fixed dosage can be administered from about two, three or four times up to a maximum of about 17 times or more.

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

[0269] If necessary, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be incorporated into a composition containing a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Further, for example, pulmonary administration can also be used by the use of formulations with inhalers or nebulizers and aerosolizing agents. See, for example, U.S. Patent No. 6,019,968, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. 5,874,064, U.S. Patent No. 5,855,913, U.S. Patent No. 5,290,540 and U.S. Patent No. 4,880,078, each of which is incorporated herein by reference in its entirety; and International Publication No. 1992 / 19244 Pamphlet, International Publication No. 1997 / 32572 Pamphlet, International Publication No. 1997 / 44013 Pamphlet, International Publication No. 1998 / 31346 Pamphlet and International Publication No. 1999 / 66903 Pamphlet.

[0270] The multispecific antibodies of the present disclosure can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be recognized by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration for the selected antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal or other parenteral routes of administration, such as by injection or infusion. Parenteral administration can generally represent a route of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intramedullary, epidural and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered via parenteral routes, such as topical, epidermal or mucosal routes of administration, for example intranasally, orally, vaginally, rectally, sublingually or topically.

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

[0272] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be administered via any of the above routes using, for example, infusion devices, injection pens, vials and syringes, prefilled syringes, automatic infusion devices, infusion pumps, patch pumps, infusion bags, and needles. When the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered in a controlled release or sustained release system, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). Controlled release or sustained release of the therapeutic agent of the present disclosure can be achieved using polymeric materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; International Publication No. WO 1999 / 15154; and International Publication No. WO 1999 / 20253).Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, contains no leachable impurities, is stable upon storage, is sterile, and is biodegradable. The controlled release or sustained release system can be placed in proximity to the prophylactic or therapeutic target and thus requires only a small systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0273] The controlled release system has been discussed in a review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to prepare a sustained release formulation comprising one or more multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure. For example, see U.S. Patent No. 4,526,938, International Publication No. WO 1991 / 05548, International Publication No. WO 1996 / 20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” 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, which are hereby incorporated by reference in their entirety.

[0274] When the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are administered locally, they can be formulated in the form of ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions 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, carriers or one or more excipients compatible with topical application are included, and in some examples, viscous semi-solid or solid forms having a dynamic viscosity greater than water are typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, plasters, etc., which are sterilized if necessary or mixed with auxiliaries (e.g., preservatives, stabilizers, wetting agents, buffers or salts) to affect various properties, such as osmotic pressure, etc. As other suitable topical dosage forms, in some examples, an aerosol preparation that can be sprayed, in which the active ingredient combined with a solid or liquid inert carrier is packaged in a mixture with a pressurized volatile substance (e.g., a gaseous propellant, e.g., Freon) or in a squeeze bottle, is included. If necessary, humectants or humectants can also be added to the pharmaceutical composition and dosage form. Examples of such additional components are known in the art.

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

[0276] The multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure can also be administered to the patient periodically.

[0277] In certain embodiments, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB (if necessary), these can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patent No. 4,522,811; U.S. Patent No. 5,374,548; and U.S. Patent No. 5,399,331. Liposomes can be selectively transported to specific cells or organs and thus can contain one or more moieties that enhance targeted drug delivery (see, for example, Ranade VV (1989) J. Clin. Pharmacol. 29:685). Representative targeting moieties include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannose (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.

[0278] This application also provides a protocol for co - administration or treatment of a patient using a pharmaceutical composition comprising a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure in combination with another treatment or therapeutic agent. Methods of co - administration or co - treatment with additional therapeutic agents, such as 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, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of a therapeutic agent can reduce the symptoms by at least 10%; at least 20%; at least about 30%; at least 40% or at least 50%.

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

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

[0281] C. Therapeutic applications for pathologies mediated by IL - 13 and IL - 18 The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure have therapeutic use for treating various human diseases, such as pathological disorders mediated by IL-13 and IL-18, such as autoimmune diseases and inflammatory diseases or conditions involving IL-13 and / or IL-18 dysregulation (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 in the treatment of atopic dermatitis.

[0282] In one aspect, the multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are useful for the treatment, reduction of the likelihood, or remission of pathological disorders mediated by IL-13 and IL-18. The phrase "pathological disorders mediated by IL-13 and IL-18" encompasses all diseases and medical conditions in a disease or medical state, including the causality, development, progression, persistence, or pathology of the disease or condition, whether directly or indirectly, involving IL-13 and IL-18. Thus, these terms include diseases or conditions that can be treated by reducing or suppressing IL-13 and IL-18-induced activities in target cells or tissues in a state and / or associated with abnormal IL-13 and IL-18 levels. 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.

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

[0284] The multispecific antibodies (e.g., bispecific antibodies) or fragments thereof of the present disclosure are useful for the treatment, prevention, or remission of autoimmune diseases and / or inflammatory conditions and disorders, particularly inflammatory conditions with etiologies involving autoimmune components, without limitation. In one aspect, the present disclosure provides a method for treating an autoimmune disease. In one aspect, the present disclosure provides a method for treating an inflammatory disease or condition. In one aspect, the subject to be treated is human.

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

[0286] Provided herein are the 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.

[0287] The term "atopic dermatitis" (AD) or "eczema", as used herein, means an inflammatory skin disease characterized by intense pruritus (e.g., severe itching) and scaly 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.), exposure to radiation and / or asthma. The present disclosure encompasses methods of treating patients with mild, moderate-to-severe or severe AD. As used herein, "moderate-to-severe AD" is characterized by extensive skin lesions with intense pruritus, often complicated by persistent bacterial, viral or fungal infections. Moderate-to-severe AD also includes chronic AD in patients. In many cases, chronic lesions include hypertrophic plaques, lichenification and fibrotic papules of the skin. Patients suffering from moderate-to-severe AD generally have more than 10% or more than 20% of the body skin affected, in addition to lesions on the eyes, hands and body fold areas, or 10% of the skin area is also affected. Patients suffering from moderate-to-severe AD generally have (i) a Physician's Global Assessment (IGA) score of 3 or 4, (ii) an Eczema Area and Severity Index (EASI) score of at least 10, preferably at least 12, and (iii) pruritus. Moderate-to-severe AD is also considered to exist in patients who require frequent treatment with topical corticosteroids. A patient may also be said to have moderate-to-severe AD if the patient is resistant or refractory to treatment with any of topical corticosteroids or calcineurin inhibitors or any other commonly used therapeutic agent known in the art.

[0288] Suitably, the use and methods of the present disclosure involve administering a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure at a dosage sufficient to achieve a therapeutically effective serum level. Suitably, the therapeutically effective serum level of the multispecific antibody (e.g., a bispecific antibody) or a fragment thereof is maintained during the course of treatment.

[0289] As used herein, the term "therapeutically effective serum level" refers to the serum level of a therapeutic agent (e.g., a bispecific antibody) in a subject that is sufficient to reduce the severity and / or duration of a condition, disorder or disease and / or symptoms associated therewith, and / or to effect remission. In some embodiments, "therapeutically effective serum level", as used herein, also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof in the serum of a subject that achieves a specified result, such as improvement in AD-related parameters, such as a decrease in the overall assessment (IGA) score by a physician; a decrease from baseline in the Dermatology Life Quality Index (DLQI), an indicator related to the quality of daily life in dermatology; a decrease from baseline in the Patient Global Impression of Severity (PGIS); an improvement decrease from baseline in the Patient Global Impression of Change (PGIC); a decrease in the Body Surface Area (BSA) score of the skin surface area involved in atopic dermatitis; a decrease in the Eczema Area and Severity Index (EASI) score; a decrease in the SCORAD score; and / or a decrease in the Itch Numerical Rating Scale (NRS) score.

[0290] In some embodiments, "therapeutically effective serum level", as used herein, also refers to the amount of a multispecific antibody (e.g., a bispecific antibody) or fragment thereof in the serum of a subject that achieves a specified result, such as a decrease 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), when compared to the level prior to treatment with the multispecific antibody (e.g., a bispecific antibody) or fragment thereof.

[0291] Suitably, the uses and methods of the present disclosure include administering a 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 twelve weeks. According to certain representative embodiments, the uses and methods of the present disclosure include administering a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof once every four weeks.

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

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

[0294] In certain embodiments, provided herein is a method of treating an IgE-mediated disorder in a subject, the method comprising administering to the subject an effective amount of a multispecific antibody (e.g., a bispecific antibody) 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.

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

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

[0297] The term "simultaneously" is not limited to the administration of therapeutic agents (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that a pharmaceutical composition comprising a multispecific antibody (e.g., a bispecific antibody) or a fragment thereof of the present disclosure is administered to a subject within a time interval such that, together with an additional therapeutic agent, the molecule of the present disclosure can act to provide an improved benefit compared to the case where they were not administered in sequence. For example, each treatment can be administered to the subject sequentially at any order, simultaneously or at different time points. If not administered simultaneously, these should be administered at times that are close enough in time to provide the desired therapeutic or prophylactic effect. Each treatment can be administered to the subject separately in any suitable form and by any suitable route.

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

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

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

[0301] The additional therapeutic agents of the combination therapies of the present disclosure can also be administered periodically. Cyclic therapy of the combination involves administering a first treatment (first prophylactic or therapeutic agent) for a period of time, then administering a second treatment (second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration (i.e., cycling) to reduce the development of resistance to one of the treatments (e.g., agents), avoid or mitigate a side effect of one of the treatments (e.g., agents), and / or improve the effectiveness of the treatment.

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

[0303] Preferably, the additional therapeutic agent (e.g., a second or third agent) is an AD agent, such as a small molecule, biological therapy, or AD modality, e.g., an agent that uses phototherapy, including topical therapy, systemic therapy, phototherapy, and combinations thereof. An “AD agent” is in the form of a cream, ointment, lotion, gel, or spray (e.g., corticosteroids of low to moderate potency [Groups IV - VII according to WHO guidelines, Bolognia JL, Jorizzo JL, Schaffer JV. Glucocorticosteroids. Dermatology. 3rd ed. 2012. Ch 125, 2075 - 88; Ference JD, Last AR. Choosing topical cocorticosteroids. Am Fam Physician. 2009 Jan 15;79(2):135 - 40]); over - the - counter (OTC) emollients and medical devices or so - called barrier creams (such as atopiclair); and an anti - itch lotion containing a lubricant for the treatment of itching and / or pain, such as menthol, pramoxine, or an anti - histamine; a local anesthetic, a systemic agent (e.g., a biological agent, such as an IL - 4R inhibitor, such as dupilumab, etc.; an IL - 13Ra1 inhibitor, such as ASLAN - 004; an IL - 13Ra2 inhibitor, etc.; an IL - 31 inhibitor, such as nemolizumab, etc.; a TNF alpha inhibitor, such as adalimumab, infliximab, certolizumab, and etanercept, alefacept, etc.; an IL - 1a inhibitor, such as belmekizumab (MABp1), etc.; an IL - 23 inhibitor, such as briakinumab, ustekinumab, guselkumab, risankizumab, tildrakizumab, etc.; an IL - 17 inhibitor, such as brodalumab, ixekizumab, etc.; a CD11a inhibitor, such as efalizumab; an IL - 22 inhibitor, such as fezakinumab, etc., an IL - 22 binding protein; an IL - 5 inhibitor, such as mepolizumab, benralizumab, etc.; a synthetic form of IL - 2, such as aldesleukin, etc.; a recombinant IL - 2 approach targeting the interleukin - 2 receptor complex, such as LY3471851, etc.; an OSMR inhibitor, such as KPL - 716, etc.; a VAP - 1 inhibitor; an OX - 40 inhibitor or OX40L inhibitor, such as GBR830, KY1005, etc.;IgE inhibitors, such as omalizumab, rigezumab, etc.; TSLP inhibitors, such as tezepelumab, etc.; IL-33 inhibitors, such as MEDI3506, etc.; IL-36 inhibitors, such as spesolimab, ANB019, etc.; B-cell modulation approaches, such as rituximab, ocrelizumab, etc.; non-biological immunomodulatory treatments, such as cyclosporine and other calcineurin inhibitors, JAK inhibitors, such as tofacitinib, upadacitinib, abrocitinib, baricitinib, etc.; TYK2 inhibitors, such as deucravacitinib, etc.; methotrexate; PDE4 inhibitors, such as apremilast; Siglec inhibitors, such as AK-002, etc.; S1P agonists or antagonists, such as etrasimod or SCD-044, etc.; BTK inhibitors, such as TAS-5315, IRAK4 antagonists and CCR4-inhibitory approaches, such as RPT-193, etc.; systemic corticosteroids, cyclophosphamide, sulfasalazine, azathioprine, mycophenolate mofetil, dapsone, hydroxychloroquine); retinoids (e.g., alitretinoin); leukotriene inhibitors or anti-leukotriene agents, such as montelukast, pranlukast or zafirlukast and 5-LO inhibitors, such as zileuton, etc., and LTA4H inhibitors, such as asvilstat, intralesional corticosteroid injection; 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, etc.; topical JAK inhibitors, such as ruxolitinib, delgocitinib or topical vitamin D analogs and topical aryl hydrocarbon receptor (AhR) inhibitors, such as benvitimod / tapinarof; high to very high potency (Groups I, II, III according to WHO definition) topical corticosteroids;Antifungal drugs with known anti-inflammatory properties, such as griseofulvin, itraconazole, betamethasone, dexamethasone, INCB018424, triamcinolone, apremilast, turmeric paste, glucosamine sulfate, triamcinolone acetonide, sesame oil, betamethasone dipropionate, clobetasol propionate, probiotics (e.g., Bifidobacterium animalis subsp. lactis HN019, Lactobacilli reuteri), omega-3, prednisone, prednisolone, platelet-rich plasma, Orabase paste, lycopene, topical chamomile, green tea, CO2 laser treatment, allergen-specific immunotherapy, polybiotics, photobiomodulation, metronidazole, doxycycline, minocycline, Himalayan cedar honey, Japanese butterbur, curcuminoid, alefacept, hexaminolevulinate, hydroxychloroquine, adcortyl, efalizumab, fluocinolone, coenzyme Q10 mucoadhesive tablets, Chamaemelum nobile, sirolimus, tacrolimus, King Kuxuan decoction, NSAID topical rinse, NSAID, quercetin, NAVS naphthalane, bulkroll, bupivacaine, oatmeal bath. Appropriately, topical AD therapies include 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, including but not limited to atopic dermatitis prescription drug therapies.;

[0304] E. Kit The present disclosure also encompasses kits for treating patients having a pathological disorder mediated by IL-13 and IL-18, such as an autoimmune disease or an inflammatory disorder or condition. Such kits include a therapeutically effective amount of the multispecific antibodies of the present disclosure. Further, such kits may include means for administering the multispecific antibodies of the present disclosure (e.g., an autoinjector device, syringe and vial, prefilled syringe, prefilled pen) and instructions for use. These kits may contain additional therapeutic agents (as described above) for treating individuals having 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 include instructions for administration of the multispecific antibodies of the present disclosure for treating a patient. Such instructions may provide dosage, route of administration, regimen, and total treatment period for use with the enclosed multispecific antibodies of the present disclosure.

[0305] The phrase "means for administration" is used to denote any available tool for systemic administration of a drug to a patient, including but not limited to prefilled syringes, vials and syringes, injection pens, autoinjectors, IV drips and bags, infusion pumps, patches, infusion bags and needles. Using such articles, a patient may self-administer (i.e., administer the drug without the aid of a physician) or a physician may administer the drug.

Examples

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

[0307] Example 1: Preparation of an IL-13 / IL-18 Bispecific Antibody in a CHO Cell Line 1. Expression Vector Construction The vectors used in the examples consist of the following elements: an enhancer that drives the expression of the individual genes required for the assembly of the hCMV promoter / antibody construct, a polyadenylation signal (polyA), folate receptor (FolR, FAR), DHFR, puromycin and / or hygromycin genes as selection markers, an origin of replication for E. coli, and a beta-lactamase gene for ampicillin resistance to enable amplification in bacteria. Different plasmid devices were evaluated and further details are provided in the drawings.

[0308] Figure 2 is a schematic diagram of NVS standard plasmids A - D. Plasmids A and C encode the expression of anti-IL13 kappa LC and anti-L13 knob HC; plasmids B and D encode the expression of anti-IL18 lambda LC and anti-IL18 hole HC. The expression of each individual protein chain is driven by a separate CMV promoter. In CHO-C8TD parental cells, linearized plasmids A and B or C and D were co-transfected simultaneously. The selection markers DHFR and FAR were used to select cells in the first selection round. For the second selection, additional selection markers, hygromycin and puromycin, may be used. The plasmids carry information on leaky stop transmembrane technology (LS-TM) to enable staining and enrichment of highly productive clones during FACS-assisted single cell sorting. Plasmids C and D were designed with lower sequence homology to minimize the risk of homologous recombination by deleting the repetitive partial phage f1 region between the expression cassettes encoding LC and HC. Plasmid D carries different codon optimization and a signal peptide for IL18 hole HC, while in plasmids A and B, the DNA sequences of IL18 knob CH and IL13 hole CH differ only in the bases encoding the KiH mutation.

[0309] Figure 3 is a schematic diagram of NVS Furin-2A peptide (F2A) plasmids E and F. The F2A technology enables the combinatorial expression of multiple protein chains from a single promoter. On plasmids E and F, the first expression cassette encodes anti-IL18 lambda LC and anti-IL18 hole HC, and the second expression cassette encodes anti-IL13 kappa LC and anti-L13 knob HC. Linearized plasmid E or F was transfected into CHO-C8TD parental cells. Cells were selected using the selection markers DHFR and FAR. Plasmid F is designed with lower sequence homology to minimize the risk of homologous recombination by using different codon optimizations for IL18 hole HC and IL13 knob HC, while on plasmid E, the DNA sequences of IL18 knob CH and IL13 hole CH differ only in the bases encoding the KiH mutation.

[0310] Figure 4 is a schematic diagram of the adapted NVS standard plasmids G and H. Plasmids G and / or H are used for supertransfection of a pool that expresses plasmid E or F to increase the number of plasmids incorporated. Plasmid G encodes the expression of anti-IL13 kappa LC and anti-L13 knob HC; plasmid H encodes the expression of anti-IL18 lambda LC and anti-IL18 hole HC. The expression of each individual protein chain is driven by a separate CMV promoter. Different approaches to transfection and selection were carried out, namely (I) co-transfecting plasmids G and H simultaneously into CHO-C8TD parental cells and cells selected using the selection markers, hygromycin and puromycin, or (II) transfecting plasmid G in the first round and selecting using hygromycin and / or transfecting plasmid H in the second round and selecting using puromycin, or (III) transfecting plasmid H in the first round and selecting using puromycin and / or transfecting plasmid G in the second round and selecting using hygromycin. Plasmids G and H carry information on leaky stop transmembrane technology (LS-TM) to enable staining and enrichment of highly productive clones during FACS-assisted single cell sorting. To reduce sequence homology and minimize the risk of homologous recombination, the partial phage f1 region between the expression cassettes encoding LC and HC on plasmids G and H was deleted, and the codon optimization of LC and HC is different from each other and from plasmids E and F.

[0311] Figure 5 is a schematic diagram of NVS furin-2A peptide (F2A) plasmid I, in which the combination of protein chains in the expression cassette is different compared to plasmids E and F. On plasmid I, the first expression cassette encodes anti-IL18 lambda LC, anti-IL18 hole HC, and anti-IL13 knob HC, and the second expression cassette encodes anti-IL13 kappa LC. The linearized plasmid I was transfected into CHO-C8TD parental cells. The cells were selected using the selectable markers DHFR and FAR. The partial phage f1 region between the two expression cassettes was deleted, and different codon optimizations of CH were used to reduce sequence homology and minimize the risk of homologous recombination.

[0312] 2. Cell Lines, Cultures, Transfections, and Selections 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 strain was used to prepare the recombinant cell line. The CHO cell line was cultured in suspension in an oscillating flask in a non-humidified shaking cabinet at 150 rpm, 10% CO2, and 36.5 °C in its own chemically defined culture medium. Cell viability and growth rate were monitored by an automated system (ViCell, Beckman Coulter). The cells were passaged into fresh medium two to three times a week and maintained in the logarithmic growth phase.

[0313] The expression plasmid linearized with SwaI encoding the antibody construct was transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). The transfection reaction was performed in a chemically defined culture 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%. 5×10 cells 6Transfection was performed at a density of

[0314] cells per transfection. Immediately after transfection, the cells were transferred to a shake flask containing a chemically defined culture medium. The cell pool was incubated at 36.5 °C and 10% CO2 for 48 hours before starting the selection process.

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

[0316] Depending on the design of the plasmid used for the first transfection and selection round, various possibilities arise for progression. Either a pool was used directly for single cell cloning as described below, or a second selection or supertransfection was performed prior to single cell cloning.

[0317] 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 culture medium. After pool recovery, the cells were frozen in a culture medium supplemented with 7.5% DMSO and a substance prepared for further analysis as described below.

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

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

[0320] The Cytena cell printer contains a disposable dispensing cartridge that contains a microfluidic chip into which the cell suspension is placed. From this cartridge, droplets are ejected through a nozzle into a 96-well plate. During this process, an image of the nozzle region is recorded by a microscope system. An automated image analysis algorithm detects cells on the image and classifies them according to morphological criteria such as size and roundness. Based on the image analysis of the droplet formation region at the nozzle outlet, droplets containing single cells are moved to individual wells of the 96-well plate, while droplets not containing single cells (empty droplets or droplets with multiple cells) are moved to the waste liquid.

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

[0322] Single cell cloning was performed using a Sony Cell Sorter device equipped with a 96-well plate holder and a disposable sorting chip of 100 μm. To ensure that only single cells were sorted, the settings were adjusted to single cell mode and 3-droplet sorting. Using these settings, only droplets containing cells are sorted if both the preceding and subsequent droplets are empty. To improve the likelihood that each droplet contains one or fewer single cells, the cell concentration and flow rate were optimized at the expense of yield. Multiple gates were set to select single, live cells with strong fluorescence.

[0323] After sorting single cells using a Cytena cell printer or Sony Cell Sorter and placing them into individual wells of a 96-well plate, high-resolution microscopic images of each well were acquired to confirm that they were monoclonal and to verify the single cell cloning procedure.

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

[0325] 4. Upstream processing After selection, substances were produced by shake flask fed-batch culture. The fed-batch culture was inoculated at a defined cell seeding density, the addition of the proprietary feed solution was started on day 3, and the culture temperature was changed to 33 °C on day 5. During the culture, in-process controls were performed to monitor the concentration of the antibody construct. The individual cultures were incubated for 14 days. At the end of the culture process, the cells were separated from the culture supernatant by centrifugation, followed by sterile filtration, and then further downstream processing was performed to characterize by analysis. In the cell culture supernatant, Protein A HPLC or RP-LC was used to determine the volumetric productivity of the selected pool in order to determine all types of products and related impurities carrying the Fc portion.

[0326] Example 2. LC-MS Screening and Purity Evaluation of IL-13 / IL-18 Bispecific Antibody 100 μg of purified bispecific mAb was diluted to 1 mg / ml in 20 mM Tris-HCl pH 7.5 and deglycosylated at 37 °C for 4 h using 2 μl of PNGaseF enzyme (New England Biolabs). The deglycosylated sample was subjected to an LC-MS system using a Waters ACQUITY UPLC Class equipped with a PLRP-S RP column (3 μm, 2.1 × 150 mm, 300 Å, Agilent) and a TripleTOF 6600 mass spectrometer with a dual spray ion source (Sciex). 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 a 40-min gradient as follows: 0 - 4 min 35% B, 4 - 28 min 35 to 50% B, 28 - 29 min 50 to 80% B, 29 - 34 min 80% B, 34 - 35 min 80 to 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 with Analyst software TF1.7 (ABSciex) and analyzed using BioPharmaView (version 3.0, ABSciex) and PeakView (version 2.2, ABSciex) software. Identification and relative quantification of bbmAb species and mispaired mutants were based on the match between the theoretically predicted mass and the relative mass signal intensity of the deconvoluted mass spectra, and the results are shown in Table 3.

[0327] Multiple appropriate heavy chain heterodimerizations, heavy chain homodimerizations and half-molecules were detected. The desired degree of heterodimerization was high at >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 were not suitable for therapeutic development.

[0328]

Table 23

[0329] Example 3. Evaluation of the thermal stability of the CH2 and Fab domains of an IL-13 / IL-18 bispecific antibody The stability of an antibody greatly affects its performance (i.e., its specificity and affinity). Therefore, stability has become a major issue for researchers and manufacturers, especially as the use of antibodies in therapeutic, diagnostic, and rapid analysis platforms increases. Important 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. The aggregation rate can be predicted from the T m value and the percentage of unfolded state (Robinson et al., 2018).

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

[0331] The melting temperatures of the CH2 and Fab domains of the modified antibodies are listed and shown in Table 4, and the melting curves of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 are shown in Figure 6.

[0332] 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 values in the CH2 domain.

[0333] Compared with the anti-13 Fab domain of bbmAb3 (64 °C), higher melting temperatures (67 - 68 °C) were observed for the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4. Since the CDR sequences in the anti-13 Fab domain of bbmAb3 differ from those of the other candidates, this result indicates that the specific CDR sequences in the anti-13 Fab domains of bbmAb1, bbmAb2, bbmAb5, and bbmAb4 can give rise to more stable molecules with improved thermal stability.

[0334]

Table 24

[0335] Example 4. Affinity for Recombinant Human and Cynomolgus IL-13 and IL-18 Measured by SET Determination of the equilibrium dissociation constant (KD) was achieved by solution equilibrium titration (SET) measurements as described below.

[0336] In sample buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.02% Tween-20), 22 serial 2n dilutions of the antigen were prepared (maximum concentrations: hsIL-18, 20 nM; cyIL-18, 40 nM; hsIL-13, 20 nM), and a constant concentration of the antibody was added (4 pM for hsIL-13 readings, either 10 or 4 pM for hsIL-18 readings, and 5 pM for cyIL-18 readings).

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

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

[0339] [Table 25]

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

[0341] hsIL-13 reading: After blocking with 50 μl / well of blocking buffer (PBS containing 5% BSA) for at least 1 hour (h) at room temperature (RT) or following the subsequent washing step (TBST, TBS containing 0.05% Tween 20), a streptavidin Multi-Array® 384-well plate (MSD L21SA-5) was coated with 30 μl / well of biotinylated human IL-13 (0.2 μg / ml, PBS) and incubated for at least 1 h at RT on a shaker.

[0342] A 30 μl / well volume of the equilibrated antigen-antibody mixture was transferred from the polypropylene MTP to the coated MSD plate and incubated for 20 minutes at RT. After a further washing step, 30 μl of sulfo-tag-labeled anti-hsIgG detection antibody (0.5 μg / ml) diluted in sample buffer was added to each well and incubated for 30 minutes at RT on a shaker. The MSD plate was washed, 35 μl / well of MSD read buffer was added, and incubated for 5 minutes at RT. An electrochemiluminescence (ECL) signal was generated and measured by an MSD Sector Imager 6000.

[0343] SET data was exported to Xlfit, an MS Excel add-in software. The average ECL-signal was calculated from duplicate measurements within each assay. The data was baseline-adjusted by subtracting the minimum value from all data points and plotting against the corresponding antigen concentration to create a titration curve. The plot was fitted using a 1:1 binding model for the following knob-in-hole bispecific Ab to determine the K D value.

Number

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

[0345]

Table 26

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

[0347] This technique allows for the determination of the binding (k a , association rate constant) and dissociation (k d, the label-free determination of the kinetic rate constant with respect to the dissociation rate constant becomes possible. The equilibrium dissociation constant K D is calculated from the kinetic rate constant.

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

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

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

[0351]

Table 27

[0352] The kinetic traces were evaluated with Biacore (trademark) T200 Control Software v2.0.1. A full set of these traces at increasing concentrations is called a run. The set of traces for a run was fit to a 1:1 binding model (R provided by Biacore T200 Evaluation Software v3.0 maxFit to the set (global). To allow for double referencing, zero-concentration samples (blanks) were included in each run.

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

[0354] [Table 28]

[0355] Example 6. Affinity for Human Fc Receptors Measured by Surface Plasmon Resonance (SPR) Spectroscopy To examine the binding characteristics of the modified IL-13 / IL-18 bispecific antibody to human Fc receptors, a direct binding assay was performed using surface plasmon resonance (SPR) spectroscopy. SPR provides continuous readings of complex formation and dissociation, enabling the analysis of interactions between analytes with solutions and ligands linked to the sensor chip surface. Thus, it is a technique commonly applied to the affinity and kinetic analysis of protein-protein, protein-peptide, protein-DNA, and protein-small molecule interactions.

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

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

[0358] 2. Measurement in the binding of FcRn receptor in vitro The affinity of the human FcRn receptor for the Fc fragment of bbmAb2 (IL18(mAb1)knob YTE IL13(mAb2)hole YTE1+1bsAb) was determined on a Biacore T200 instrument. The affinity of the human FcRn receptor for the Fc fragments of bbmAb5 (bispecific KiH LALA YTE) and bbmAb1 (IL18 hole YTE(mAb1)IL13(mAb2)knob YTE1+1bsAb) was determined on a Biacore 8K instrument. Samples were diluted to 5 μg / ml in 10 mM sodium acetate pH 4.5 and immobilized on a CM5 sensor chip at a density of approximately 750 resonance units on a Biacore T200 instrument applying standard amine coupling procedures. The same procedure was applied on a Biacore 8K instrument, but the immobilization was at a density of approximately 1310 resonance units. On the Biacore T200, flow cell 1 was the immobilized blank and served as a reference. On the Biacore 8K, there were 8 channels in total and the flow cells of each channel were left blank and used as reference surfaces. Subsequently, kinetic binding data were collected on all flow cells at a flow rate of 50 μl / min and a temperature of 25 °C by injecting a 1:2 dilution series of the human FcRn receptor. The FcRn receptor was diluted in two different running buffers to cover a concentration range of 4.88 - 2500 nM to check for pH-dependent binding: PBS pH 5.8 with 0.005% Tween-20 and PBS pH 7.4 with 0.005% Tween-20. The chip surface was regenerated at a flow rate of 50 μl / min for 120 s using PBS pH 7.4 with 0.005% Tween-20. Zero concentration samples (blank runs) were measured to allow for double referencing during data evaluation. Duplicate injections of each sample and buffer blank were run over all surfaces. Data were evaluated using Biacore T200 evaluation software version 3.0 and Biacore 8K evaluation software (v.3.0.12.15655).

[0359] The raw data was double-referenced, i.e., the reaction of the measurement flow cell was corrected against the reaction of the reference flow cell, and the reaction of the blank injection was subtracted at the second stage. The obtained sensorgram was fitted using either a steady-state model or a 1:1 Langmuir model to calculate the equilibrium dissociation constant (K D ).

[0360] 3. Measurement of C1q binding in vitro The affinity of human C1q for bbmAb2 was determined on a Biacore T200 instrument. The affinities of human C1q for bbmAb5 and bbmAb1 were determined on a Biacore 8K instrument. Samples were diluted to 50 μg / ml in 10 mM sodium acetate buffer pH 4.5 and immobilized at a density of approximately 8900 resonance units on a CM5 sensor chip by applying standard amine coupling procedures on the Biacore T200 instrument. Similar procedures were applied on the Biacore 8K instrument, but the immobilization was at a density of approximately 9400 resonance units. On the Biacore T200, flow cell 1 was the immobilized blank and served as the reference. There were a total of 8 channels on the Biacore 8K, and the flow cells of each channel were left blank and used as the reference surface. Kinetic binding data were collected by subsequently injecting a 1:2 dilution series of human C1q on all flow cells at a flow rate of 30 μl / min and a temperature of 25 °C. Human C1q was diluted in running buffer (HBS-EP+ pH 7.4) in the concentration range of 0.49 nM to 250 nM. After each measurement cycle, the chip surface was regenerated using 50 mM NaOH for 30 s at a flow rate of 30 μl / min including a 60 s stabilization period. Zero-concentration samples (blank runs) were measured to enable double referencing during data evaluation. Duplicate injections of each sample and buffer blank were flowed across all surfaces. The data were evaluated using Biacore T200 evaluation software version 3.0 and Biacore 8K evaluation software (v.3.0.12.15655).

[0361] The raw data was double-referenced, i.e., the reaction of the measurement flow cell was corrected against the reaction of the reference flow cell, and the reaction of the blank injection was subtracted at the second stage. The resulting sensorgram was fitted using any of the steady-state models to calculate the equilibrium dissociation constant (K D ).

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

[0363]

Table 29

[0364] Example 7. PK study of IL-13 / IL-18 bispecific antibody PK study conducted in hFcRn transgenic mice Mouse experiments were performed using Tg276 B6.Cg-Fcgrttm1DcrTg(CAG-FCGRT)276Dcr / DcrJ hemizygous mice derived from C57BL / 6 mice and purchased from Jackson Laboratory (USA). FcRn− / − hFcRn (line 276) Tg mice carry a null mutation for the transgene expressing the mouse gene and the hFcRn α-chain under the control of the ubiquitous CAG promoter. All mice were treatment-naive male, 8 - 12 weeks old at the start of the study. For dosing, the antibody was prepared in phosphate-buffered saline (1× PBS pH 7.3) and administered as a single intravenous dose of 10 mg / kg at a volume of 5 mL / kg into the left lateral tail vein. Over a 29-day study period, a continuous sampling approach (before dosing, 1 h, 1 d, 2 d, 3 d, 6 d, 9 d, 17 d, and 29 d) was utilized to evaluate a total of 3 animal replicates for each antibody. Blood samples (30 - 50 μL) were collected into serum separation tubes and allowed to clot at room temperature for 20 - 30 minutes. The samples were then processed to obtain serum by centrifugation (2000g, room temperature, 10 minutes). The resulting serum was stored at -80 °C until analysis.

[0365] For the measurement of total antibodies in unknown mouse serum specimens, calibration standards (Cs) and quality control samples (QC), a quantitative sequential electrochemiluminescence immunoassay (ECLIA) was used. By using both targets for antibody capture and detection, detection of the total amount of bispecific therapeutic antibodies by this assay using their binding sites was enabled. Thus, antibodies were captured by biotinylated human IL-18 immobilized on SA-coated and blocked MSD plates. Rhesus IL-13 labeled with ruthenium (II) tris-bipyridine-(4-methylsulfonate) NHS ester (MSD sulfo-TAG™) was added and bound for detection, which is based on electrochemiluminescence (ECL) and was read on an ECL sector imager from MSD. The resulting ECL values are proportional to the amount of drug present in the initial sample when determined through 5PL regression analysis using a weighting factor of 1 / Y2 (where Y represents the ECL value of the corresponding calibration standard).

[0366] The above experimental approach was used in a humanized mouse model to examine bbmAb1 and bbmAb2 as well as the Fc-silent mutants bbmAb6, bbmAb7, bbmAb8, and bbmAb9. bbmAb7 and bbmAb8 are Fc-silent mutants of bbmAb1 containing the L235C mutation (EU numbering). bbmAb6 is an Fc-silent mutant of bbmAb1 containing the L235C mutation and not containing the YTE mutation (EU numbering). bbmAb9 is an Fc-silent mutant of bbmAb1 containing the L235C / G236C mutation (EU numbering).

[0367] All of the above-mentioned antibodies, after intravenous administration, showed a typical PK curve shape in systemic circulation with a short and good exposure and distribution phase, typical of antibodies with a high C max and an extended half-life, and a long elimination phase. The half-lives of the antibodies examined herein are very similar and can be estimated from the terminal phase of the PK curve in the range of approximately 10 to 16 days (as shown in Figure 7).

[0368] Example 7 Simultaneous inhibition of IL-13 / IL-18 Human peripheral blood mononuclear cells (pBMC) or human keratinocytes were each treated with IL-18 or IL-13, and the differences in gene expression were analyzed. Overlap between the gene signature induced by IL-13 and the gene signature induced by IL-18 was observed only very slightly (data not shown), while both of these were shown to be elevated in published lesional skin compared to non-lesional skin from an AD patient gene expression dataset (He et al. 2020: skin biopsies from 5 AD (lesional and non-lesional) and 7 HV subjects; scRNA-seq data).

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

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

[0371] 4mm skin biopsies were collected from 10 patients with atopic dermatitis. 4mm skin biopsies from 8 healthy volunteers were collected as controls. Each biopsy was cut into 4 small pieces and cultured ex vivo for 24 hours in 100 μl of medium with either + / -α-IL-18, α-IL-13 or both (150 μg / mL each). The cell culture supernatant was centrifuged at low speed to remove the cells without lysing the cells. The Olink assay was used to evaluate changes in protein expression. A decrease in protein expression in the supernatant of the lesional biopsy samples compared to the control samples showed a measurable decrease in the samples treated with anti-IL-13 compared to the control, a decrease in the samples treated with anti-IL-18 compared to the control, and a further decrease in the samples treated with anti-IL-18 / anti-IL-13 compared to the control and compared to the single-treatment samples (data not shown).

[0372] Example 8 Simultaneous inhibition of IL-13 / IL-18 with bispecific antibodies reveals synergistic suppression of the AD-like transcriptome Method Eight-millimeter full-thickness skin biopsies were obtained from surgical waste from 5 individual donors and cultured in IMDM medium containing 1% Pen / Strep and 10% knockout serum replacement in tissue culture inserts for 12-well plates. On day 0, the biopsies were injected with a control (30 μL PBS) or activated with a mixture of anti-CD28 and anti-CD3 antibodies in 30 μL of PBS at a final concentration of 500 ng each. To induce differentiation into the AD-like transcriptome, the biopsies (except for the control) were incubated in IMDM medium with the following cytokines, 50 ng / mL each cytokine: IL-4, IL-13, IL-33, TSLP, IL18, and IL-31, with medium changes on days 2, 4, and 5 and incubated for 6 days. During the 6-day induction period, the induced biopsy samples were treated with a 1 μM IgG1 isotype control antibody with LALA silencing 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, the supernatants were collected and the biopsies were aliquoted for histological and transcriptome analysis (Ampliseq whole transcriptome protocol).

[0373] The Ampliseq normalized values were imported into Qlucore Omics Explorer 3.8, and variable elements with values less than 0.5 were removed if they fit in 90% of the samples, resulting in 16,719 variable elements. The threshold was set to 0.25 and the values were log2-transformed. In the donor-corrected samples, with q < 0.1 and FC > 2, the "AD isotype" samples were compared to the control to generate a "disease transcriptome", resulting in 1,485 differentially expressed "disease genes". 507 of them were upregulated in the AD isotype samples. Gene set variation analysis (GSVA) was performed in unimodal mode on this gene set.

[0374] Results As shown in Figure 8, GSVA showed ineffective inhibition against all anti-IL18-treated samples. Anti-IL13 inhibition was partially effective in 2 out of 5 samples. On the other hand, the anti-IL18 / IL13 bispecific antibody bbmAb1 showed definite suppression of the AD-like disease transcriptome in 4 out of 5 samples, indicating a synergistic effect on combined cytokine blockade.

[0375] As shown in Figure 9, t-SNE analysis (perplexity 5) of 507 upregulated genes showed a clear disease effect using the control and AD+ isotype samples that were farthest apart on the X-axis. Four out of five samples treated with the anti-IL13 / 18 bispecific antibody bbmAb1 clustered near the control samples, suggesting that cells from these samples have the minimal AD-like transcriptome of the induced cells. In contrast, only 2 anti-IL13-treated samples showed a similar effect, and the anti-IL18-treated samples could not be distinguished from the AD+ isotype samples, indicating no therapeutic effect. These results were further supported by t-SNE analysis (perplexity 5) of 1485 differentially expressed genes (AD+ isotype vs. control) illustrated in Figure 10. However, Figure 10 suggests a moderate therapeutic effect against blockade of IL-18 alone. Without wishing to be bound by theory, the inventors hypothesize from the data in Figures 8-10 that IL-18 blockade (e.g., using an anti-IL18 antibody) may show a therapeutic effect in patients with AD, but that simultaneous blockade of IL13 / 18 (e.g., by simultaneous or sequential administration of IL-13 and IL-18 antagonists) may be an unexpectedly superior treatment compared to blockade of IL-13 or IL-18.

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

[0377] Example 10. Treatment of Atopic Dermatitis with an Anti-IL-13 / IL-18 Bispecific Antibody A subject having atopic dermatitis is administered a bispecific antibody described herein. At week 16, the subject achieves a greater reduction in one or more signs and / or symptoms of atopic dermatitis compared to placebo-treated subjects.

Claims

**Claim 1** A multispecific antibody comprising: a. a first portion comprising a first light chain variable domain (VL1) and a first heavy chain variable domain (VH1) that specifically binds to interleukin-18 (IL-18); b. a second portion comprising a second light chain variable domain (VL2) and a second heavy chain variable domain (VH2) that specifically binds to interleukin-13 (IL-13). The multispecific antibody according to claim 1. **Claim 2** The multispecific antibody according to claim 1, which is a bispecific antibody. **Claim 3** The VH1 and VH2 each comprise complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL1 and VL2 each comprise LCDR1, LCDR2, and LCDR3, e. the VH1 domain comprises (for example, in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 32, the HCDR2 having the amino acid sequence SEQ ID NO: 33, and the HCDR3 having the amino acid sequence SEQ ID NO: 34, or ii. the HCDR1 having the amino acid sequence SEQ ID NO: 35, the HCDR2 having the amino acid sequence SEQ ID NO: 36, and the HCDR3 having the amino acid sequence SEQ ID NO: 37, or iii. the HCDR1 having the amino acid sequence SEQ ID NO: 38, the HCDR2 having the amino acid sequence SEQ ID NO: 39, and the HCDR3 having the amino acid sequence SEQ ID NO: 40, and f. the VL1 domain comprises (for example, in order): i. the LCDR1 having the amino acid sequence SEQ ID NO: 4, the LCDR2 having the amino acid sequence SEQ ID NO: 5, and the LCDR3 having the amino acid sequence SEQ ID NO: 6, or ii. the LCDR1 having the amino acid sequence SEQ ID NO: 7, the LCDR2 having the amino acid sequence SEQ ID NO: 8, and the LCDR3 having the amino acid sequence SEQ ID NO: 9, or iii. the LCDR1 having the amino acid sequence SEQ ID NO: 10, the LCDR2 having the amino acid sequence SEQ ID NO: 11, and the LCDR3 having the amino acid sequence SEQ ID NO: 12, and g. the VH2 domain comprises (for example, in order): i. the HCDR1 having the amino acid sequence SEQ ID NO: 46, the HCDR2 having the amino acid sequence SEQ ID NO: 47, and the HCDR3 having the amino acid sequence SEQ ID NO: 48, or ii. the HCDR1 having the amino acid sequence SEQ ID NO: 49, the HCDR2 having the amino acid sequence SEQ ID NO: 50, and the HCDR3 having the amino acid sequence SEQ ID NO: 51, or iii. The above-mentioned HCDR1 having the amino acid sequence SEQ ID NO: 52, the above-mentioned HCDR2 having the amino acid sequence SEQ ID NO: 53, and the above-mentioned HCDR3 having the amino acid sequence SEQ ID NO: 54 comprising, and h. The above-mentioned VL2 domain is (for example, in order) i. The above-mentioned LCDR1 having the amino acid sequence SEQ ID NO: 18, the above-mentioned LCDR2 having the amino acid sequence SEQ ID NO: 19, and the above-mentioned LCDR3 having the amino acid sequence SEQ ID NO: 20, or ii. The above-mentioned LCDR1 having the amino acid sequence SEQ ID NO: 21, the above-mentioned LCDR2 having the amino acid sequence SEQ ID NO: 22, and the above-mentioned LCDR3 having the amino acid sequence SEQ ID NO: 23, or iii. The above-mentioned LCDR1 having the amino acid sequence SEQ ID NO: 24, the above-mentioned LCDR2 having the amino acid sequence SEQ ID NO: 25, and the above-mentioned LCDR3 having the amino acid sequence SEQ ID NO: 26 The multispecific antibody according to claim 1 or 2, comprising.

4. The multispecific antibody according to any one of claims 1 to 3, comprising a first lambda light chain and a second kappa light chain.

5. The multispecific antibody according to claim 4, wherein the first light chain is of the lambda 1 type and the second light chain is of the kappa 4 type.

6. (a) The above-mentioned VL1 domain comprises the amino acid sequence SEQ ID NO: 13, and (b) The above-mentioned VL2 domain comprises the amino acid sequence SEQ ID NO: 27, The multispecific antibody according to any one of claims 1 to 5.

7. a. The above-mentioned VH1 domain comprises the amino acid sequence SEQ ID NO: 41, and b. The above-mentioned VL1 domain comprises the amino acid sequence SEQ ID NO: 13, and c. The above-mentioned VH2 domain comprises the amino acid sequence SEQ ID NO: 55, and d. The above-mentioned VL2 domain comprises the amino acid sequence SEQ ID NO: 27, The multispecific antibody according to any one of claims 1 to 6.

8. The multispecific antibody according to any one of claims 1 to 7, comprising a first light chain comprising the amino acid sequence represented by SEQ ID NO: 14 and a second light chain comprising the amino acid sequence represented by SEQ ID NO:

28.

9. The multispecific antibody according to any one of claims 1 to 8, comprising a first heavy chain comprising a heterodimerization modification and a second heavy chain comprising a heterodimerization modification complementary to the heterodimerization modification of the first heavy chain.

10. The first and second heavy chains are human IgG1, and a) The heterodimerization modification of the first heavy chain includes serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the second immunoglobulin heavy chain includes tryptophan at position 366, or b) The heterodimerization modification of the second heavy chain includes serine at position 366, alanine at position 368, and valine at position 407, and the heterodimerization modification of the first immunoglobulin heavy chain includes tryptophan at position 366, The multispecific antibody according to claim 9, wherein the amino acid residues are numbered according to EU numbering. **Claim 11** A bispecific antibody, which is a bispecific antibody comprising a mutation that enhances the half-life of the bispecific antibody via enhanced FcRn binding, according to any one of claims 1 to 10. **Claim 12** The bispecific antibody according to claim 11, wherein the mutation that enhances the half-life of the bispecific antibody is M252Y / S254T / T256E (YTE), and the amino acid residues are numbered according to EU numbering. **Claim 13** The multispecific antibody according to any one of claims 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 42, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:

56. **Claim 14** The multispecific antibody according to any one of claims 1 to 12, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 57, and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:

58. **Claim 15** The multispecific antibody according to any one of claims 1 to 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 acid sequence shown in SEQ ID NO: 56, and the second light chain comprises the amino acid sequence shown in SEQ ID NO:

28. **Claim 16** The multispecific antibody according to any one of claims 1 to 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 acid sequence shown in SEQ ID NO: 58, and the second light chain comprises the amino acid sequence shown in SEQ ID NO:

28. **Claim 17** A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 16 in combination with one or more pharma- ceutically acceptable excipients, diluents or carriers.

18. 20. The pharmaceutical composition of claim 17, further comprising one or more additional active agents.

19. An isolated nucleic acid molecule encoding a multispecific antibody according to any one of claims 1 to 16.

20. A cloning or expression vector comprising one or more nucleic acid sequences according to claim 19, suitable for the recombinant production of a multispecific antibody according to any one of claims 1 to 16.

21. 21. A host cell comprising one or more cloning or expression vectors according to claim 20.

22. 27. A process for the production of a multispecific antibody according to any one of claims 1 to 16, comprising culturing a host cell according to claim 21 under conditions sufficient to express said multispecific antibody, and subsequently purifying and recovering said multispecific antibody from the host cell culture.

23. 19. A kit comprising the multispecific antibody of any one of claims 1 to 16 or the pharmaceutical composition of claim 17 or 18, said kit additionally comprising instructions for use and a pharmaceutical delivery device for administering said multispecific antibody or said pharmaceutical composition to a subject in need thereof.

24. 24. The kit of claim 23, wherein the pharmaceutical delivery device for administration comprises a syringe, an autoinjector, an injection pen, a vial and syringe, an infusion pump, a patch, or an infusion bag and needle.

25. 17. A method for simultaneously inhibiting the activity of IL-13 and IL-18, comprising contacting a plurality of mammalian cells with an effective amount of a multispecific antibody according to any one of claims 1 to 16.

26. 17. A method for simultaneously inhibiting the activity of IL-13 and IL-18 in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 16.

27. 17. A method of treating an IL-13 and / or IL-18 mediated disorder in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 16.

28. A method for preventing or treating an inflammatory or immune condition, comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 16.

29. A multispecific antibody according to any one of claims 1 to 16 for use in preventing or treating an inflammatory or immune condition in a subject in need thereof.

30. The method or use according to claim 28 or 29, wherein the inflammatory or immune condition is a skin condition.

31. The method or use according to claim 30, wherein the skin condition is atopic dermatitis.

32. The method or use according to claim 31, 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.

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

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

35. The method or use according to any one of claims 31 to 34, wherein the atopic dermatitis is inadequately controlled with topical corticosteroids.

36. A method for treating an inflammatory or immune condition in a subject in need thereof, comprising administering to the subject an IL-13 and an IL-18 antagonist simultaneously or sequentially.

37. The method according to claim 36, comprising administering a first antagonist selected from an IL-13 and an IL-18 antagonist and a second antagonist selected from an IL-13 and an IL-18 antagonist, wherein the first and second antagonists are structurally different molecules.

38. The method according to claim 36, comprising administering an anti-IL18 antagonist antibody and an anti-IL13 antagonist antibody.

39. The method according to claim 36, comprising administering an anti-IL13 / 18 bispecific antibody.

40. The method according to claim 36, wherein the inflammatory or immune condition is a skin condition.

41. The method according to claim 36, wherein the inflammation or immune state is atopic dermatitis, for example moderate to severe atopic dermatitis or atopic dermatitis that is inadequately controlled with, for example, topical corticosteroids.

42. The method according to claim 41, which is an improved treatment as compared to treatment with a single specific anti-IL-13 antagonist and / or a single specific anti-IL-18 antagonist.

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

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