Multispecific binding agents and uses thereof

Abstract

The present application relates to multispecific binding agents and uses thereof, specifically multispecific binding agents that bind to both interleukin-4 receptor alpha (IL-4Rα) and interleukin 31 (IL-31). The present application further provides compositions (e.g., pharmaceutical compositions) comprising the multispecific binding agents provided herein and uses thereof.

Description

MULTISPECIFIC BINDING AGENTS AND USES THEREOFRELATED APPLICATION

[0001] This application claims priority to Chinese Patent Application No. 202410971584.0, filed on July 19, 2024, U.S. Provisional Application No. 63 / 674,223, filed on July 22, 2024, and Chinese Patent Application No. 202510922251.3, filed on July 3, 2025, the entirety of each of which is incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14832-010-228_SEQLISTING.xml”, was created on June 25, 2025, and is 81,453 bytes in size.1. FIELD

[0003] The present application relates to multispecific binding agents that bind to both interleukin-4 receptor alpha (IL-4Ra) and interleukin 31 (IL-31). The present application further provides compositions (e.g., pharmaceutical compositions) comprising the multispecific binding agents provided herein and methods of use thereof.2. BACKGROUND

[0004] The continued prevalence of allergic diseases is a common public health problem in developed and developing countries. Although a series of allergic diseases, such as atopic dermatitis, asthma, chronic spontaneous urticaria, chronic prurigo, are heterogeneous diseases occurring in different target tissues, they have similar type II inflammatory characteristics (itching and / or high serum IgE concentrations) (Beck LA, Cork MJ, et al. Type 2 Inflammation Contributes to Skin Barrier Dysfunction in Atopic Dermatitis. JID Innov. 2022 Apr 26;2(5): 100131).

[0005] Type II inflammation is mainly driven by Th2 cells. The pathogenic driving factors include cytokines such as TSLP, IL-4, IL-5, IL-13, IL-31 and IgE, as well as mast cells and basophils. Type II inflammation is caused by the action and interaction of these molecules and cells (Galli SJ. & Tsai M. IgE and mast cells in allergic disease. Nat. Med. 2012 May 4;18(5):693-704). Interleukin-4 (IL-4) is secreted by immune cells such as CD4 + T cells, B cells, and mast cells, which can promote the differentiation of helper T cells (Th)into Th2 cells and promote their proliferation. Th2 cells further secrete IL-4 and IL- 13 to promote B cell proliferation and promote their IgM conversion to produce immunoglobulin E (IgE). After the allergen-specific IgE binds to its receptor, it promotes mast cells and basophils to degranulate and release inflammatory mediators (such as histamine, leukotrienes, etc). IL-4 and IL-13 play an important role in skin barrier damage. They have been shown to downregulate filaggrin and chloroalanine, damage skin structure, and reduce the ability of the skin barrier to resist pathogens and allergens (Dubin C, Duca ED, et al. The IL-4, IL-13 and IL-31 pathways in atopic dermatitis. Expert Rev Clin Immunol. 2021 Aug;17(8):835-852). IL- 13 plays a unique role in affecting dermal fibroblasts, increasing the production of collagen and fibrosis, leading to skin remodeling and lichen-like skin formation (Dubin C, Duca ED, et al. The IL-4, IL-13 and IL-31 pathways in atopic dermatitis. Expert Rev Clin Immunol. 2021 Aug;17(8):835-852). Since atopic dermatitis is a chronic disease, patients experience repeated relapses and remissions. Typical pruritus is one of the main factors affecting the quality of life and sleep of patients with skin diseases (Furue M, Yamamura K, Kido-Nakahara M, et al. Emerging role of interleukin-31 and interleukin-31 receptor in pruritus in atopic dermatitis. Allergy. 2018 Jan;73(l):29-36).

[0006] IL-31 is another major Th2 cytokine involved in the pathogenesis of allergic skin diseases. It plays an important role in mediating pruritus and is a key mediator of pruritus in type II skin diseases such as atopic dermatitis and nodular prurigo. IL-4, IL- 13 and IL-31 play an indispensable role in Th2 immune response, contributing to immune activation and participating in skin barrier function destruction and pruritus. IL-4 and IL-13 have also been shown to act directly on pruritic sensory neurons and promote neurogenic itch through IL-31 interaction (Dubin C, Duca ED, et al. The IL-4, IL-13 and IL-31 pathways in atopic dermatitis. Expert Rev Clin Immunol. 2021 Aug;17(8):835-852.) In summary, type II cytokines IL-4, IL-31, and IL- 13 play multiple important and non-redundant roles in type II inflammation and can synergistically regulate type II immune responses.

[0007] Interleukin-4 receptor alpha (IL-4Ra) is a common component of the IL-4 and IL- 13 receptor complexes. Dupilumab (Dupixent®), jointly developed by Sanofi / Regeneron, is a fully human monoclonal antibody targeting IL-4Ra that regulates type II immunity by simultaneously blocking the inflammatory pathways mediated by IL-4 and IL-13.Dupilumab has shown therapeutic effects in a series of type II inflammatory diseases such as severe asthma, atopic dermatitis, prurigo nodularis, chronic sinusitis with nasal polyps, and eosinophilic esophagitis (Blauvelt A, de Bruin-Weller M, Gooderham M, et al. Long-term management of moderate-to-severe atopic dermatitis with dupilumab and concomitant topicalcorticosteroids (LIBERTY AD CHRONOS): a 1-year, randomized, double-blinded, placebo- controlled, phase 3 trial. Lancet. 2017 Jun 10;389(10086):2287-2303; Castro M, Corren J, Pavord ID, et al. Dupilumab Efficacy and Safety in Moderate-to-Severe Uncontrolled Asthma. N Engl J Med. 2018 Jun 28;378(26):2486-2496; Bachert C, Han JK, Desrosiers M, et al. Efficacy and safety of dupilumab in patients with severe chronic rhinosinusitis with nasal polyps (LIBERTY NP SINUS-24 and LIBERTY NP SINUS-52): results from two multicentre, randomized, double-blind, placebo-controlled, parallel-group phase 3 trials. Lancet. 2019 Nov 2;394(10209): 1638-1650; Dellon ES, Rothenberg ME, Collins MH, et al. Dupilumab in Adults and Adolescents with Eosinophilic Esophagitis. N Engl J Med. 2022 Dec 22;387(25):2317-2330; Yosipovitch G, Mollanazar N, Stander S, et al. Dupilumab in patients with prurigo nodularis: two randomized, double-blind, placebo-controlled phase 3 trials. Nat Med. 2023 May;29(5): 1180-1190). However, during clinical research and real- world use of Dupilumab in atopic dermatitis, it was found that, with long-term use, Dupilumab can significantly improve the symptoms of patients; skin lesions, but more than half of the patients are not satisfied with the anti-itching effect. At the same time, during Dupilumab treatment, the onset of effect is relatively slow.

[0008] Nemolizumab, a monoclonal antibody targeting IL-3 IRA developed by Maruho, relieves itching by blocking the binding of IL-31 to IL-3 IRA. Clinically, it has been shown that the itching symptoms of patients treated with Nemolizumab can be rapidly and significantly improved (Kabashima K, Matsumura T, Komazaki H, et al. Trial of Nemolizumab and Topical Agents for Atopic Dermatitis with Pruritus. N Engl J Med. 2020 Jul 9;383(2): 141-150; Kwatra SG, Yosipovitch G, Legat FJ, et al. Phase 3 Trial of Nemolizumab in Patients with Prurigo Nodularis. N Engl J Med. 2023 Oct 26;389(17): 1579- 1589).

[0009] Overall, novel therapeutic agent is needed to improve the treatment of related diseases or disorders.3. SUMMARY OF THE INVENTION

[0010] In one aspect, provided herein are multispecific binding agents. In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain; and(ii) an interleukin 31 (IL-3 l)-binding domain, wherein the first IL-4Ra-binding domain comprises (a) a VHH comprising a complementarity-determining region 1 (CDR1), a complementarity-determining region 2(CDR2), and a complementarity-determining region 3 (CDR3) as set forth in the amino acid sequence of SEQ ID NO: 12; or (b) a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

[0011] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain; and(ii) an interleukin 31 (IL-3 l)-binding domain, wherein the IL-31 -binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NON and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:8.

[0012] In certain embodiments, the multispecific binding agent provided herein further comprises an Fc region.

[0013] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain;(ii) an interleukin 31 (IL-31)-binding domain; and(iii) an Fc region comprising one or more amino acid substitutions as compared to a wildtype Fc region, wherein the one or more amino acid substitutions are capable of depleting Fc- gamma receptor (FcyR) binding or enhancing neonatal Fc receptor (FcRn) binding.

[0014] In certain embodiments, the IL-31-binding domain comprises divalent antibody fragments (F(ab’)2 fragments) comprising the VH and the VL.

[0015] In certain embodiments, the IL-31-binding domain is linked to the Fc region. In certain embodiments, the C-terminus of the IL-31-binding domain is linked to the N-terminus of the Fc region. In certain embodiments, the multispecific binding agent further comprises a second IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL.

[0016] In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region. In certain embodiments, the N-terminus of the VHH or VH of the first or second IL-4Ra-binding domain is linked to the C-terminus of the Fc region. In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO: 13.

[0017] In certain embodiments, the first or second IL-4Ra-binding domain comprises (a) a VHH comprising a complementarity-determining region 1 (CDR1), a complementaritydetermining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) as set forth in the amino acid sequence of SEQ ID NO: 12; or (b) a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

[0018] In certain embodiments, the first or second IL-4Ra-binding domain comprises a VHH or VH comprising:(i) a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55;(ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and(iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57.

[0019] In certain embodiments, the first or second IL-4Ra-binding domain comprises a VHH or VH comprising:(i) a CDR1 comprising the amino acid sequence of SEQ ID NOV, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11;(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50;(iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50;(iv) a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50; or(v) a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0020] In certain embodiments, the first or second IL-4Ra-binding domain comprises a VHH or VH comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the first or second IL-4Ra-binding domain comprises a VHH or VH comprising the amino acid sequence of SEQ ID NO: 12.

[0021] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:4 and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:8.

[0022] In certain embodiments, the IL-31-binding domain comprises:(i) a VH comprising:(a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42;(b) a CDR2 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:43; and(c) a CDR3 comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:35, or SEQ ID NO:44; and(ii) a VL comprising:(a) a CDR1 comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:36, or SEQ ID NO:45;(b) a CDR2 comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:37, or SEQ ID NO:46; and(c) a CDR3 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:47.

[0023] In certain embodiments, the IL-31-binding domain comprises:(i) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(ii) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:33, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(iii) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:38, a CDR2 comprising the amino acid sequence of SEQ ID NO:39, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(iv) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:40, a CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7; or(v) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a CDR2 comprising the amino acid sequence of SEQ ID NO:43, and a CDR3 comprising the amino acid sequence of SEQ ID NO:44; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:47.

[0024] In certain embodiments, the IL-31-binding domain comprises:(i) a VH comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NON; and(ii) a VL comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

[0025] In certain embodiments, the IL-31-binding domain comprises:(i) a VH comprising the amino acid sequence of SEQ ID NON; and(ii) a VL comprising the amino acid sequence of SEQ ID NO:8.

[0026] In certain embodiments, the multispecific binding agent provided herein further comprises a third IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL. In certain embodiments, the multispecific binding agent provided herein further comprises a fourth IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL.

[0027] In certain embodiments, the third or fourth IL-4Ra-binding domain is the same as the first or second IL-4Ra-binding domain. In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain. In certain embodiments, the N-terminus of the VHH of the third or fourth IL-4Ra- binding domain is linked to the C-terminus of the VHH of the first or second IL-4Ra-binding domain. In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO: 13.

[0028] In certain embodiments, the Fc region comprises one or more amino acid substitutions as compared to a wild-type Fc region, wherein the one or more amino acidsubstitutions are capable of depleting Fc-gamma receptor (FcyR) binding or enhancing neonatal Fc receptor (FcRn) binding. In certain embodiments, the Fc region comprises L234A, L235A, G237A, M428L and N434A amino acid substitutions as compared to a wildtype Fc region, wherein numbering is according to the EU numbering system.

[0029] In certain embodiments, the multispecific binding agent provided herein comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, and the VHH of the first or second IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.

[0030] In certain embodiments, the multispecific binding agent provided herein comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, the VHH of the first or second IL-4Ra-binding domain, and the VHH of the third or fourth IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain, and the VHH of the third or fourth IL-4Ra-binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.

[0031] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 14; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0032] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 18;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0033] In certain embodiments, the first polypeptide and the second polypeptide are linked to each other via one or more disulfide bonds, wherein the second polypeptide and the third polypeptide are linked to each other linked via one or more disulfide bonds, and wherein the third polypeptide and the fourth polypeptide are linked to each other linked via one or more disulfide bonds.

[0034] In another aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding the multispecific binding agent or a portion thereof provided herein.

[0035] In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides or a complementary polynucleotide or polynucleotides thereto provided herein.

[0036] In another aspect, provided herein are cells. In certain embodiments, provided herein is a cell comprising the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein.

[0037] In another aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein, and a pharmaceutically acceptable excipient.

[0038] In another aspect, provided herein are kits. In certain embodiments, provided herein is a kit comprising the multispecific binding agent provided herein, the polynucleotideor polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein.

[0039] In yet another aspect, provided herein are methods for treating a disease or disorder in a subject. In certain embodiments, provided herein is a method for treating a disease or disorder in a subject comprising administering to the subject the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein. In certain embodiments, the disease or disorder is atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

[0040] In yet another aspect, provided herein are multispecific binding agents for use in treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

[0041] In yet another aspect, provided herein is use of a multispecific binding agent in the manufacture of a medicament for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGs. 1A-1D are schematic diagrams of exemplary binding agent structures provided herein. FIGs. 1 A-1B depict the structure of anti-IL-3 l / IL-4Ra vHH (FIG. 1 A with three parts annotated; FIG. IB with polypeptides annotated); FIGs. 1C-1D depict the structure of anti-IL-3 l / IL-4Ra vHHs (FIG. 1C with three parts annotated; FIG. ID with polypeptides annotated).

[0043] FIG. 2A depicts the binding curve of exemplary binding agents provided herein to 293F-rhesus IL-4Ra cells.

[0044] FIG. 2B depicts the binding curve of exemplary binding agents provided herein to 293F-rhesus IL-4Ra cells.

[0045] FIG. 3 A depicts the binding curve of exemplary binding agents provided herein and Cynomolgus monkey IL-31 protein.

[0046] FIG. 3B depicts the binding curve of exemplary binding agents provided herein and human IL-31 protein.

[0047] FIG. 4 shows that exemplary binding agents provided herein simultaneously bound to human IL-4Ra and human IL-31 protein.

[0048] FIG. 5 depicts the binding curve of exemplary binding agents provided herein to 293F-huIL-4Ra cells in the presence of human IL-31 protein.

[0049] FIG. 6 shows that exemplary binding agents provided herein blocked the binding of human IL-4 protein to 293F-huIL-4Ra cells.

[0050] FIG. 7 shows that exemplary binding agents provided herein blocked the binding of human IL-31 protein to CHO -huIL-31RA cells.

[0051] FIG. 8 shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT3 in 293T-huIL-31RA / OSMRp cells induced by human IL- 31.

[0052] FIG. 9A shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells induced by human IL-4.

[0053] FIG. 9B shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells induced by human IL-13.

[0054] FIG. 10A shows that exemplary binding agents provided herein blocked the proliferation of TF-1 cells induced by human IL-4.

[0055] FIG. 10B shows that exemplary binding agents provided herein blocked the proliferation of TF-1 cells induced by human IL-13.

[0056] FIG. 11 A shows that exemplary binding agents provided herein blocked the expression of CCL2 in BEAS-2B cells induced by human IL-4 and IL-31.

[0057] FIG. 1 IB shows that exemplary binding agents provided herein blocked the expression of CCL2 in BEAS-2B cells induced by human IL-4, IL-13 and IL-31.

[0058] FIG. 12 shows that exemplary binding agents provided herein bound to human IL- 4Ra and human IL-31 protein simultaneously after being placed at 40°C for 0, 14 and 28 days.

[0059] FIG. 13 shows the half-life of exemplary binding agents provided herein in mice.

[0060] FIG. 14A shows pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the OXA-induced mouse dermatitis model.

[0061] FIG. 14B shows pharmacodynamics of exemplary binding agents provided herein on the IgE content in mouse serum in the OXA-induced mouse dermatitis model.

[0062] FIG. 14C shows H&E staining of the effect of exemplary binding agents provided herein on mouse ear skin tissue in the OXA-induced mouse dermatitis model.

[0063] FIG. 15A shows pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the MC903-induced mouse dermatitis model.

[0064] FIG. 15B shows pharmacodynamics of exemplary binding agents provided herein on the IgE content in mouse serum in the MC903 -induced mouse dermatitis model.

[0065] FIG. 15C shows H&E staining of the effect of exemplary binding agents provided herein on mouse ear skin tissue in the MC903-induced mouse dermatitis model.

[0066] FIG. 16A shows the results of scratching times in mice within 3 hours after injection of PBS, human IL-31 protein, human IL-31 protein + anti-IL-3 l-anti-IL-4Ra vHH, or human IL-31 protein + Nemolizumab analogue.

[0067] FIG. 16B shows the results of scratching times in mice within 3 hours after injection of PBS, human IL-31 protein, or human IL-31 protein + anti-muIL-4Ra control.

[0068] FIG. 17A depicts the study timeline in Atopic Dermatitis Model induced by Calcipotriol (MC903) in huIL-4Ra / huIL-4 / huIL-31 / huIL-31RA / huOSMRp KI mice.

[0069] FIGs. 17B-17C show pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the MC903-induced mouse dermatitis model.

[0070] FIG. 17D shows pharmacodynamics of exemplary binding agents provided herein on the IgE content in mouse serum in the MC903 -induced mouse dermatitis model.

[0071] FIG. 17E shows H&E staining of the effect of exemplary binding agents provided herein on mouse ear skin tissue in the MC903-induced mouse dermatitis model.

[0072] FIG. 18A depicts the study timeline in Atopic Dermatitis Model induced by Calcipotriol (MC903) in huIL-4Ra / huIL-4 / huIL-31 / huIL-31RA / huOSMRp KI mice.

[0073] FIGs. 18B-18C show pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the MC903 -induced mouse dermatitis model (*p<0.05, **p<0.01, ***p<0.001, for MC903 + anti-IL-31-anti-IL-4Ra vHH versus MC903, by two-way ANOVA; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, for Vehicle versus MC903, by two-way ANOVA; &p<0.05, for MC903 + Dupixent versus MC903, two-way ANOVA).

[0074] FIG. 18D shows pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the MC903-induced mouse dermatitis model at day 7 (*p<0.05, *** p<0.001 by one-way ANOVA Tukey’s test).

[0075] FIG. 18E shows pharmacodynamics of exemplary binding agents provided herein on the thickness of mouse ears in the MC903-induced mouse dermatitis model at day 9 (*p<0.05, *** p<0.001 by one-way ANOVA Tukey’s test).

[0076] FIG. 18F shows pharmacodynamics of exemplary binding agents provided herein on the IgE content in mouse serum in the MC903 -induced mouse dermatitis model.

[0077] FIG. 18G shows pharmacodynamics of exemplary binding agents provided herein on the CCL17 content in mouse serum in the MC903-induced mouse dermatitis model.

[0078] FIG. 18H shows H&E staining of the effect of exemplary binding agents provided herein on mouse ear skin tissue in the MC903-induced mouse dermatitis model.

[0079] FIG. 19A shows the results of scratching times in mice within 3 hours after injection of human IL-31 protein + PBS, human IL-31 protein + Dupixent, human IL-31 protein + Nemolizumab analogue, or human IL-31 protein + anti -IL-3 l-anti-IL-4Ra vHH (*p<0.05 by two-tailed paired T test).

[0080] FIG. 19B shows the results of scratching times in mice within 3 hours after injection of human IL-31 protein + PBS (*p<0.05 by two-tailed paired T test).

[0081] FIG. 19C shows the results of scratching times in mice within 3 hours after injection of human IL-31 protein + Dupixent (*p<0.05 by two-tailed paired T test).

[0082] FIG. 19D shows the results of scratching times in mice within 3 hours after injection of human IL-31 protein + Nemolizumab analogue.

[0083] FIG. 19E shows the results of scratching times in mice within 3 hours after injection of human IL-31 protein + anti-IL-31 -anti -IL-4Ra vHH.

[0084] FIG. 20A shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells induced by human IL-4.

[0085] FIG. 20B shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT6 in HEK-blue IL-4 / IL-13 cells induced by human IL-13.

[0086] FIG. 21 shows that exemplary binding agents provided herein blocked the binding of human IL-31 protein to CHO -huIL-31RA cells.

[0087] FIG. 22 shows that exemplary binding agents provided herein blocked the phosphorylation signal of STAT3 in 293T-huIL-31RA / OSMRp cells induced by human IL- 31.

[0088] FIG. 23 shows the effect of exemplary binding agents provided herein on IL-4 and IL-31-induced expression of CCL2 in BEAS-2B cells.5. DETAILED DESCRIPTION OF THE INVENTION

[0089] Provided herein are multispecific binding agents and uses thereof. In one aspect, provided herein is a multispecific binding agent or a portion thereof (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof). In certain embodiments,provided herein is a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof. Exemplary multispecific binding agents are described in Section 5.3. In another aspect, also provided herein are polypeptides, vectors and cells related to the multispecific binding agents provided herein (Section 5.4). In another aspect, provided herein are pharmaceutical compositions related to the multispecific binding agents provided herein (Section 5.5). In yet another aspect, provided herein are methods for treating a disease or disorder in a subject related to the multispecific binding agents provided herein and multispecific binding agents for use in treating a diseases or disorder (Section 5.6). In yet another aspect, provided herein is use of a multispecific binding agent in the manufacture of a medicament for treating a disease or disorder (Section 5.6). Also provided herein are methods of making the multispecific binding agent provided herein (Section 5.7). Also provided herein are kits related to the multispecific binding agent provided herein (Section 5.8).

[0090] Without being bound by the theory, a multispecific binding agent as provided herein simultaneously targets IL-4Ra and IL-31, thus is able to block the IL-4 / IL-13 / IL- 4Ra / IL-13Ral and IL-3 l / IL-3 IRA signaling pathways simultaneously. Correspondingly, the multispecific binding agent has the potential to maximize the synergistic therapeutic effects of rapid and effective antipruritic and further improve a subject’s skin lesion symptoms. In short, a multispecific binding agent provided herein can show superior binding affinity and biological activity. Moreover, reduced frequency of administration is also an unmet clinical need for patients having related chronic diseases or disorders. A multispecific binding agent provided herein further contains Fc mutations that have the potential to increase the half-life of the multispecific binding agent and reduce the frequency of administration.5.1 Definitions

[0091] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used in the present descriptionhave the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0092] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. An antibody can be human, humanized, chimeric and / or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy -terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding domain that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be foundin, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Pliickthun and Skerra, 1989, Meth. Enzymol. 178:497- 515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule. Antibodies may be agonistic antibodies or antagonistic antibodies. Antibodies may be neither agonistic nor antagonistic.

[0093] An “antigen” is a structure to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, for example, is present on or in a cell.

[0094] An “intact” antibody is one comprising an antigen-binding domain as well as a CL and at least heavy chain constant regions, CHI, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0095] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0096] The term “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0097] “Single domain antibody” or “sdAb” as used herein refers to a single monomeric variable antibody domain and which is capable of antigen binding (e.g., single domain antibodies that bind to CD 19). Single domain antibodies include VHH (also referred to as “vHH”) domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, a single domainantibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the application. In some embodiments, the single domain antibody (e.g., VHH) provided herein has a structure of FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0098] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding domain on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (koir) to association rate (kon) of a binding molecule (e.g, an antibody) to a monovalent antigen (k0ff / k0n) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both konand koir. The dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding domain does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding domains, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0099] In connection with the binding molecules described herein terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide. A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet®,Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). Typically, a specific or selective reaction is at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In certain embodiments, the extent of binding of a binding molecule or antigen binding domain to a “non-target” protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to IpM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.

[0100] In certain embodiments, the binding molecules or antigen binding domains can comprise “chimeric” sequences in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.. 1984, Proc. Natl. Acad. Sci. USA 81 :6851-55). Chimeric sequences may include humanized sequences.

[0101] In certain embodiments, the binding molecules, binding agents, or antigen binding domains can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodies that include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse,rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature 321 :522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter etal., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Pat. Nos: 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0102] In certain embodiments, the binding molecules, binding agents, or antigen binding domains can comprise portions of a “fully human antibody” or “human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. The binding molecules may comprise a single domain antibody sequence. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising nonhuman antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1 : 755-68 (2006)). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., MonoclonalAntibodies and Cancer Therapy 77 (1985); Boerner et aL, J. Immunol. 147(l):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5): 561 -66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0103] The term “monospecific” when used in reference to a binding agent (e.g., an antibody) means that the binding agent (e.g., an antibody) has one or more binding domains, each of which binds to the same epitope of the same antigen.

[0104] The term “multispecific” when used in reference to a binding agent (e.g., an antibody) means that the binding agent can specifically bind to two or more different epitopes, for example different epitopes on different antigens or different epitopes on the same antigen. A bispecific binding agent (e.g., an antibody) is a multiple specific binding agent that binds to two different epitope (on the same antigen or different antigens), or two different antigens (e.g., IL-4Ra and IL-31). A bispecific binding agent (e.g., an antibody) can have a 1+1 format, where one antigen-binding domain binds to a first antigen or epitope and the other antigen-binding domain binds to a second antigen or epitope. A bispecific binding agent (e.g., a bispecific antibody) can have a 2+1 format or a 1+2 format, wherein one antigen-binding domain binds to a first antigen or a first epitope, and the other two antigenbinding domains bind to a second antigen or second epitope. A bispecific binding agent (e.g., an antibody) can have a 2+2 format, wherein two antigen-binding domains bind to a first antigen or a first epitope, and the other two antigen-binding domains bind to a second antigen or second epitope. A bispecific binding agent (e.g., a bispecific antibody) can have a 4+1 format or a 1+4 format, wherein one antigen-binding domain binds to a first antigen or a first epitope, and the other four antigen-binding domains bind to a second antigen or second epitope.

[0105] In certain embodiments, the binding agents or antigen binding domains can comprise portions of a “recombinant human antibody,” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodiesisolated from an animal (e.g., a mouse or cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. etal., Nucl. Acids Res. 20:6287-6295 (1992)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0106] In certain embodiments, the binding agent or antigen binding domains can comprise a portion of a “monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody typically recognize a single epitope on the antigen. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single hybridoma or other types of cells. The term “monoclonal” is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present application may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991), for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0107] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and a isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding domain. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5thed. 2001).

[0108] The term “Fab” or “Fab region” refers to an antibody region that binds to antigens. A conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions. The VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present application. For example, VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG. Alternatively, VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.

[0109] The term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the aminoterminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the P sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding domain of antibodies (see, e.g., Kabat et al. , Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

[0110] The term “variable region residue numbering according to Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The “EU numbering system” or “EU numbering system” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU numbering system reported in Kabat et al., supra). The “EU numbering system as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0111] The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, 5, and y contain approximately 450 amino acids, while p and a contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.

[0112] The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) or lambda (X) based on the amino acid sequence of the constant domains.

[0113] As used herein, the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably. A “CDR” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.

[0114] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat el al., supra). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modelingsoftware (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra, Chothia and Lesk, supra, Martin, supra, Lefranc el al., supra). The residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.Table 1. Exemplary CDRs According to Various Numbering Systems

[0115] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR isgiven. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR as set forth in a specific VH or VHH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH, VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

[0116] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.

[0117] The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding domain. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0118] The term “framework” or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0119] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art. A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native or wild-type sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native or wild-type sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.

[0120] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope). It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure. In other embodiments, a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0121] A “blocking” antibody or an “antagonist” antibody is one that inhibits or reduces a biological activity of the antigen it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0122] An “agonist” or activating antibody is one that enhances or initiates signaling by the antigen to which it binds. In some embodiments, agonist antibodies cause or activate signaling without the presence of the natural ligand.

[0123] “Percent (%) amino acid sequence identity”, “percent (%) identical to” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0124] The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this application may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.

[0125] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces a binding molecule of the present application may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into whichnucleic acids encoding the antibodies have been introduced. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”

[0126] An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule. Specifically, an “isolated” nucleic acid molecule encoding a CAR or an sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.

[0127] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0128] As used herein, the term “operatively linked,” and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed infunctional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5’ and 3’ UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (ie., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0129] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0130] The term “host” as used herein refers to an animal, such as a mammal (e.g., a human).

[0131] The term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0132] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0133] “Allogeneic” refers to a graft derived from a different individual of the same species.

[0134] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous polynucleotide is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous polynucleotide. The cell includes the primary subject cell and its progeny. In certain embodiments, an exogenously introduced polynucleotide is not a polynucleotide that exits naturally in a cell, e.g., an engineered immune cell.

[0135] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0136] “Excipient” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.

[0137] In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, di saccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990).

[0138] As used herein, the term “pharmaceutically acceptable” when used in reference to a carrier, is intended to mean that the carrier, diluent or excipient is not toxic or otherwise undesirable, (i.e., the material may be administered to a subject without causing any undesirable biological effects), and it is compatible with the other ingredients of the formulation. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions. A saline solution can be a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0139] In some embodiments, excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral compositions, including formulations, can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[0140] Compositions, including pharmaceutical compounds, may contain a binding molecule (e.g., an antibody), for example, in isolated or purified form, together with a suitable amount of excipients.

[0141] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a single domain antibody or a therapeutic molecule comprising an agent and the single domain antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.

[0142] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0143] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0144] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and / or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease. The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.

[0145] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., diabetes or a cancer).

[0146] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0147] As used in the present application and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0148] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0149] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0150] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).5.2 IL-4R«XIL-31 Binding Agents

[0151] In one aspect, provided herein are multispecific binding agents comprising one or more IL-4Ra-binding domains and one or more IL-31 -binding domains. In certain embodiments, provided herein are multispecific binding agents comprising two IL-4Ra- binding domains and one IL-31 -binding domain. In certain embodiments, provided herein are multispecific binding agents comprising four IL-4Ra-binding domains and one IL-31- binding domain.

[0152] A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application, including by RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by OCTET®, using, for example, an OCTET®Red96 system, or by BIACORE®, using, for example, a BIACORE®™-2000 or a BIACORE®™-3000. An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the OCTET®Red96, the BIACORE®™-2000, the BIACORE®™-3000 system, the BIACORE®™-8K, or the BIACORE®™-8K+ system.

[0153] In certain embodiments, provided herein are multispecific binding agents comprising a first interleukin-4 receptor alpha (IL-4Ra)-binding domain and an interleukin 31 (IL-31)-binding domain. In certain embodiments, the multispecific binding agent provided herein further comprises an Fc region. In certain embodiments, the IL-31-bindingdomain is linked to the Fc region. In certain embodiments, the C-terminus of the IL-31- binding domain is linked to the N-terminus of the Fc region.

[0154] In certain embodiments, the multispecific binding agent further comprises a second IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL. In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region. In certain embodiments, the N-terminus of the VHH or VH of the first or second IL-4Ra-binding domain is linked to the C-terminus of the Fc region. In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the structure of the multispecific binding agent is as depicted in FIGs. 1 A-1B.

[0155] In certain embodiments, the multispecific binding agent provided herein further comprises a third IL-4Ra-binding domain comprising a VHH. In certain embodiments, the multispecific binding agent provided herein further comprises a third IL-4Ra-binding domain comprising a VH and a VL. In certain embodiments, the multispecific binding agent provided herein further comprises a fourth IL-4Ra-binding domain comprising a VHH. In certain embodiments, the multispecific binding agent provided herein further comprises a fourth IL-4Ra-binding domain comprising a VH and a VL.

[0156] In certain embodiments, the third or fourth IL-4Ra-binding domain is the same as the first or second IL-4Ra-binding domain. In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain. In certain embodiments, the N-terminus of the VHH of the third or fourth IL-4Ra- binding domain is linked to the C-terminus of the VHH of the first or second IL-4Ra-binding domain In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the structure of the multispecific binding agent is as depicted in FIGs. 1C-1D.

[0157] In certain embodiments, the multispecific binding agent provided herein comprises one or more linkers. In certain embodiments, the linker links peptide fragments together to form the multispecific binding agent. In certain embodiments, the linker links an IL-4Ra-binding domain to the Fc region. In certain embodiments, the linker links two IL-4Ra-binding domains to each other. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:59. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:60. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:61. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:62. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:63. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:64. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:65. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:66. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:67. In certain embodiments, the multispecific binding agent provided herein comprises a linker comprising the amino acid sequence of SEQ ID NO:68. In certain embodiments, the multispecific binding agent provided herein comprises one or more linkers known in the art.

[0158] In certain embodiments, the multispecific binding agent provided herein comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, and the VHH of the first or second IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL;wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.

[0159] In certain embodiments, the multispecific binding agent provided herein comprises:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, the VHH of the first or second IL-4Ra-binding domain, and the VHH of the third or fourth IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain, and the VHH of the third or fourth IL-4Ra-binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.5.2.1 Interleukin-4 receptor alpha (IL-4Ra)-binding domain of the multispecific binding agent

[0160] As used herein, IL-4Ra refers to an IL-4Ra polypeptide, an IL-4Ra polypeptide fragment, an IL-4Ra peptide, or an IL-4Ra epitope. In certain embodiments, an IL-4Ra- binding domain is derived from a human or humanized antibody (e.g., comprising human framework regions) that binds IL-4Ra, including an IL-4Ra polypeptide, an IL-4Ra polypeptide fragment, an IL-4Ra peptide, or an IL-4Ra epitope. In certain embodiments, the IL-4Ra-binding domain binds to IL-4Ra, such as human IL-4Ra, or a portion thereof.

[0161] In certain embodiments, the multispecific binding agent comprises one, two, three, four or more IL-4Ra-binding domains. In certain embodiments, the multispecific binding agent comprises two IL-4Ra-binding domains. In certain embodiments, the multispecific binding agent comprises four IL-4Ra-binding domains. The embodiments of the one or more IL-4Ra-binding domains of the multispecific binding agent provided herein are provided in this section. Exemplary amino acid sequences of the one or more IL-4Ra-binding domains of the multispecific binding agent provided herein are listed in Table 2.Table 2. Exemplary amino acid sequences of the one or more IL-4Ra-binding domains of the multispecific binding agent(i) IL-4Ra-binding domain comprising a VHH

[0162] In certain embodiments, the IL-4Ra-binding domain comprises a VHH. In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12.

[0163] In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57.

[0164] In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, the IL-4Ra- binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, theIL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO:57.

[0165] In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the IL-4Ra-binding domain comprises a VHH comprising the amino acid sequence of SEQ ID NO: 12.

[0166] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12.

[0167] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57.

[0168] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54,and a CDR3 comprising the amino acid sequence of SEQ ID NO:50. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57.

[0169] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising the amino acid sequence of SEQ ID NO: 12.(ii) IL-4Ra-binding domain comprising a VH and a VL

[0170] In certain embodiments, the IL-4Ra-binding domain comprises a VH and a VL. In certain embodiments, the IL-4Ra-binding domain comprises divalent antibody fragments (F(ab’)2 fragments) comprising a VH and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a single-chain Fv (scFv) comprising a VH and a VL.

[0171] In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

[0172] In certain embodiments, the IL-4Ra-binding domain comprises (i) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57; and (ii) a VL.

[0173] In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11 and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ IDN0:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO:57 and a VL.

[0174] In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12 and a VL. In certain embodiments, the IL-4Ra-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL.

[0175] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

[0176] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises (i) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57; and (ii) a VL.

[0177] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NOV, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11 and a VL. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the aminoacid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50 and a VL. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 57 and a VL.

[0178] In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12 and a VL. In certain embodiments, the first, second, third or fourth IL-4Ra-binding domain comprises a VHH comprising the amino acid sequence of SEQ ID NO: 12 and a VL.5.2.2 Interleukin 31 (IL-31)-binding domain of the multispecific binding agent

[0179] As used herein, IL-31 refers to an IL-31 polypeptide, an IL-31 polypeptide fragment, an IL-31 peptide, or an IL-31 epitope. In certain embodiments, an IL-31 -binding domain is derived from a human or humanized antibody (e.g., comprising human framework regions) that binds IL-31, including an IL-31 polypeptide, an IL-31 polypeptide fragment, an IL-31 peptide, or an IL-31 epitope. In certain embodiments, the IL-31 -binding domain binds to IL-31, such as human IL-31, or a portion thereof.

[0180] In certain embodiments, the multispecific binding agent comprises one, two, three, four or more IL-31 -binding domains. In certain embodiments, the multispecific binding agent comprises two IL-31 -binding domains. The embodiments of the one or more IL-31- binding domains of the multispecific binding agent provided herein are provided in this section. Exemplary amino acid sequences of the one or more IL-31 -binding domains of the multispecific binding agent provided herein are listed in Table 3.Table 3. Exemplary amino acid sequences of the one or more IL-31-binding domains of the multispecific binding agent

[0181] In certain embodiments, the IL-31-binding domain comprises divalent antibody fragments (F(ab’)2 fragments) comprising a VH and a VL. In certain embodiments, the IL- 31 -binding domain comprises a single-chain Fv (scFv) comprising a VH and a VL.

[0182] In certain embodiments, the IL-31 -binding domain of the multispecific binding agent provided herein comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:4 and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 8.

[0183] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NON and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:8.

[0184] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42; a CDR2 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NON, SEQ ID NO:35, or SEQ ID NO:44. In certain embodiments, the IL-31-binding domain comprises a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NON, SEQ ID NO:36, or SEQ IDNO:45; a CDR2 comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:37, or SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:47.

[0185] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0186] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:33, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO:37, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0187] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:38, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO:37, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0188] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:40, a CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO:37, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7.

[0189] In certain embodiments, the IL-31-binding domain comprises a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a CDR2 comprising the amino acid sequence of SEQ ID NO:43, and a CDR3 comprising the amino acid sequence of SEQ ID NO:44; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:47.

[0190] In certain embodiments, the IL-31-binding domain comprises a VH comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4; and a VL comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:8.

[0191] In certain embodiments, the IL-31-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:4; and a VL comprising the amino acid sequence of SEQ ID NO:8.5.2.3 Fc region of the multispecific binding agent

[0192] In certain embodiments, the multispecific binding agent provided herein further comprises an Fc region.

[0193] In certain embodiments, the Fc region comprises two polypeptides, where each polypeptides comprises one or more amino acid substitutions provided herein. In certain embodiments, the first polypeptide and the second polypeptides comprise the same one or more amino acid substitutions provided herein. In certain embodiments, the first polypeptide and the second polypeptides comprise different one or more amino acid substitutions disclosed herein. Reference to amino acid substitutions in an Fc region disclosed herein is by EU numbering by Kabat. EU numbering is known and is according to the updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information System®) and the EU index as reported in Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NTH publication No. 91-3242 (1991).

[0194] In certain embodiments, the Fc region of the multispecific binding agent provided herein comprises one or more amino acid substitutions as compared to a wild-type Fc region, wherein the one or more amino acid substitutions are capable of increasing the half-life of the multispecific binding agent.

[0195] In certain embodiments, the Fc region of the multispecific binding agent provided herein comprises one or more amino acid substitutions as compared to a wild-type Fc region, wherein the one or more amino acid substitutions are capable of depleting Fc-gamma receptor (FcyR) binding. In certain embodiments, the Fc region of the multispecific binding agent provided herein comprises one or more amino acid substitutions as compared to a wildtype Fc region, wherein the one or more amino acid substitutions are capable of enhancingneonatal Fc receptor (FcRn) binding. In certain embodiments, the wild-type Fc region is an Fc region of a human IgGl antibody.

[0196] In certain embodiments, the Fc region comprises L234A amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises L235A amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises G237A amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises M428L amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises N434A amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system.

[0197] In certain embodiments, the Fc region comprises M252Y / S254T / T256E (YTE) amino acid substitutions as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises M428L / Q331R amino acid substitution as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises M428L / N434S amino acid substitutions as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises T250E / M428L amino acid substitutions as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system. In certain embodiments, the Fc region comprises N343A amino acid substitution as compared to a wildtype Fc region, wherein numbering is according to the EU numbering system.5.3 Exemplary IL-4RaxIL-31 Binding Agents

[0198] In certain embodiments, provided herein is a multispecific binding agent comprising a polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, provided herein is a multispecific binding agent comprising a polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14.

[0199] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14;(iii) a third polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14; and(iv) a fourth polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15.

[0200] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 14; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0201] In certain embodiments, the first polypeptide and the second polypeptide are linked to each other via one or more disulfide bonds, wherein the second polypeptide and the third polypeptide are linked to each other linked via one or more disulfide bonds, and wherein the third polypeptide and the fourth polypeptide are linked to each other linked via one or more disulfide bonds.

[0202] In certain embodiments, provided herein is a multispecific binding agent comprising a polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, provided herein is a multispecific binding agent comprising a polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18.

[0203] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18;(iii) a third polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18; and(iv) a fourth polypeptide comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15.

[0204] In certain embodiments, provided herein is a multispecific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 18;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0205] In certain embodiments, the first polypeptide and the second polypeptide are linked to each other via one or more disulfide bonds, wherein the second polypeptide and the third polypeptide are linked to each other linked via one or more disulfide bonds, and wherein the third polypeptide and the fourth polypeptide are linked to each other linked via one or more disulfide bonds.5.3.1 Multispecific binding agent “anti-IL-3 l / LL-4Ra vHH”

[0206] In certain embodiments, provided herein is a multispecific binding agent that is referred to herein as “anti-IL-3 l / IL-4Ra vHH,” “ anti -IL-31 -anti -IL-4Ra vHH,” or “anti-IL- 3 l / anti-IL-4Ra vHH.” The structure of the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” is as depicted in FIGs. 1 A and IB. There are four polypeptides in the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH.” As depicted on the very left in FIGs. 1 A and IB, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 15, which comprises, from N-terminus to C-terminus, a first VL of an IL-31 -binding domain and a first CL. Asdepicted second to the left in FIGs. 1 A and IB, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 14, which comprises, from N-terminus to C-terminus, a first VH of the IL-31 -binding domain, a first CHI, a first CH2, a first CH3, and the VHH of the first or second IL-4Ra-binding domain. As depicted second to the right in FIGs. 1 A and IB, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 14, which comprises, from N-terminus to C-terminus, a second VH of the IL-31-binding domain, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain. As depicted on the very right in FIGs. 1 A and IB, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 15, which comprises, from N-terminus to C-terminus, a second VL of an IL-31 -binding domain and a second CL. In the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH,” the first VL, the first VH, the second VH, and the second VL of the IL-31 -binding domain form the IL-31 -binding domain, and the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region. In certain embodiments, the IL-31 -binding domain and the Fc region are collectively referred to as “anti-IL-31 mAb” (FIG. 1A). In certain embodiments, the constant region of the light chains, which are the first and fourth polypeptides, are of human Kappa chain. The amino acid sequences of the polypeptides in “anti-IL-3 l / IL-4Ra vHH” are provided below:The first and fourth polypeptides in anti-IL-31 / IL-4Ra vHH ([anti-IL-31 VL]-CL; the anti-IL-31 VL sequence is shown underlined):DIVLTOSPGTLSLSPGERATLSCRASORVSTNYLAWYOQKPGOAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCEQYGSSPWTFGOGTKVDIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15)The second and third polypeptides in anti-IL-3 l / IL-4Ra vHH ([anti-IL-31 VH]-CH1- CH2-CH3-[anti-IL-4Ra VHH]; the anti-IL-31 VH sequence is shown underlined; the linker between the CH3 and the anti-IL-4Ra vHH is shown in italic; the anti-IL-4Ra vHH is shown in bold):EVQLLESGGGLVKPGGSLRLSCVDSGFTFSDYSMNWVROAPGKGLEWVSYISSSSST IYYADSVKGRFTISRDNGKNSLYLOMNSLRAQDTAVYYCARRTTMVRGIGAFDIWG OGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPGGG GGHGGGGHGGGGnGGGGHEVQLVESGGGLVQPGGSLRLSCSASGFTFDYYTIGW FRQAPGKEREGVSCISSRAINRYYSDSVKGRFTISRDNSKATVYLQMNSLTPGDT AVYYCAARTRDTTVQIMCDMRAPIPYWGQGTQVTVSS (SEQ ID NO: 14)5.3.2 Multispecific binding agent “anti-IL-3 l / LL-4Ra vHHs”

[0207] In certain embodiments, provided herein is a multispecific binding agent that is referred to herein as “anti-IL-3 l / IL-4Ra vHHs,” “anti-IL-3 l-anti-IL-4Ra vHHs,” or “anti-IL- 3 l / anti-IL-4Ra vHHs.” The structure of the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” is as depicted in FIGs. 1C-1D. There are four polypeptides in the multispecific binding agent “anti -IL-3 l / IL-4Ra vHHs.” As depicted on the very left in FIGs. 1C-1D, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 15, which comprises, from N-terminus to C-terminus, a first VL of an IL-31 -binding domain and a first CL. As depicted second to the left in FIGs. 1C-1D, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18, which comprises, from N-terminus to C-terminus, a first VH of the IL-31 -binding domain, a first CHI, a first CH2, a first CH3, the VHH of the first or second IL-4Ra-binding domain, and the VHH of the third or fourth IL-4Ra-binding domain. As depicted second to the right in FIGs. 1C-1D, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 18, which comprises, from N-terminus to C-terminus, a second VH of the IL-31 -binding domain, a second CHI, a second CH2, a second CH3, the VHH of the first or second IL-4Ra-binding domain, and the VHH of the third or fourth IL-4Ra- binding domain. As depicted on the very right in FIGs. 1C-1D, the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 15, which comprises, from N-terminus to C-terminus, a second VL of an IL-31 -binding domain and a second CL. In the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs,” the first VL, the first VH, the second VH, and the second VL of the IL-31 -binding domain form the IL-31 -binding domain, and the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region. In certain embodiments, the IL-31 -binding domain and the Fc region are collectively referred to as “anti-IL-31 mAb” (FIG. 1C). The amino acid sequences of the polypeptides in “anti-IL- 3 l / IL-4Ra vHHs” are provided below:The first and fourth polypeptides in anti-IL-31 / IL-4Ra vHHs ([anti-IL-31 VL]-CL; the anti-IL-31 VL sequence is shown underlined):DIVLTOSPGTLSLSPGERATLSCRASORVSTNYLAWYOQKPGOAPRLLIYGASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCEQYGSSPWTFGOGTKVDIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 15)The second and third polypeptides in anti-IL-3 l / IL-4Ra vHHs ([anti-IL-31 VH]-CH1- CH2-CH3-[anti-IL-4Ra VHH]-[anti-IL-4Ra VHH]; the anti-IL-31 VH sequence is shown underlined; the linker between the CH3 and the anti-IL-4Ra vHH; the linker between the two anti-IL-4Ra vHHs is shown in italic; the anti-IL-4Ra vHHs are shown in bold):EVOLLESGGGLVKPGGSLRLSCVDSGFTFSDYSMNWVRQAPGKGLEWVSYISSSSST IYYADSVKGRFTISRDNGKNSLYLOMNSLRAQDTAVYYCARRTTMVRGIGAFDIWG OGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPGGG GG^GGGG^GGGG^GGGG^EVQLVESGGGLVQPGGSLRLSCSASGFTFDYYTIGW FRQAPGKEREGVSCISSRAINRYYSDSVKGRFTISRDNSKATVYLQMNSLTPGDT AVYYCAARTRDTTVQIMCDMRAPIPYWGQGTQVTVSSGGGG^GGGG^GGGG^G GGG / iEVQLVESGGGLVQPGGSLRLSCSASGFTFDYYTIGWFRQAPGKEREGVSC ISSRAINRYYSDSVKGRFTISRDNSKATVYLQMNSLTPGDTAVYYCAARTRDTTV QIMCDMRAPIPYWGQGTQVTVSS (SEQ ID NO: 18)5.4 Polynucleotides, Vectors and Cells

[0208] In another aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding the multispecific binding agent or a portion thereof provided herein. In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding an IL-4Ra*IL-31 binding agent as described in Section 5.2 or a portion thereof. In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof.

[0209] In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, provided herein is a polynucleotide or polynucleotides encoding multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.

[0210] In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides or a complementary polynucleotide or polynucleotides thereto provided herein. In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides encoding the multispecific binding agent provided herein or a complementary polynucleotide or polynucleotides thereto.

[0211] In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides encoding an IL-4Ra*IL-31 binding agent as described in Section 5.2 or a portion thereof or a complementary polynucleotide or polynucleotides thereto provided herein. In certain embodiments, provided herein is a vector or vectorscomprising the polynucleotide or polynucleotides encoding a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof or a complementary polynucleotide or polynucleotides thereto provided herein.

[0212] In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides encoding multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, provided herein is a vector or vectors comprising the polynucleotide or polynucleotides encoding multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.

[0213] In another aspect, provided herein are cells. In certain embodiments, provided herein is a cell comprising the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein.

[0214] In another aspect, provided herein are cells. In certain embodiments, provided herein is a cell comprising an IL-4Ra*IL-31 binding agent as described in Section 5.2 or a portion thereof. In certain embodiments, provided herein is a cell comprising a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL- 31 -binding domain as described in Section 5.2.2 or a portion thereof. In certain embodiments, provided herein is a cell comprising multispecific binding agent “anti-IL- 31 / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, provided herein is a cell comprising multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.

[0215] In certain embodiments, the cell expresses an IL-4Ra*IL-31 binding agent as described in Section 5.2 or a portion thereof. In certain embodiments, the cell expresses a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof. In certain embodiments, the cell expresses multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, the cell expresses multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2. In certain embodiments, the cell replicates the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein.

[0216] As used herein, a polynucleotide encoding a multispecific binding agent (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof) may encompass two or more polynucleotides, each encoding a portion of the binding agent (e.g., a polypeptide of the binding agent). For example, a polynucleotide encoding a multispecificbinding agent (e.g., the fL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof) can encompass a single polynucleotide that encodes two or more polypeptides comprised in the multispecific binding agent (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof) as well as two or more polynucleotides that each encodes one polypeptide comprised in the multispecific binding agent, where the combination of the two or more polypeptides form the multispecific binding agent. In certain embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In various aspects, the isolated polynucleotide comprises a nucleotide sequence that encodes a VHH, a VH and / or a VL, wherein the VHH, the VH and the VL comprise complementarity determining regions (CDRs) identical to CDRs disclosed herein.

[0217] In certain embodiments, provided are the materials for generating multispecific binding agents and portions thereof. For example, an isolated cell may produce a multispecific binding agent (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof). In this regard, a cell (e.g., an isolated cell) may produce a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof. In certain embodiments, a cell (e.g., an isolated cell) may produce multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, a cell (e.g., an isolated cell) may produce multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.

[0218] In certain embodiments, one or more vectors (e.g., expression vectors) may comprise one or more polynucleotides for expression of the one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying the polynucleotides in host cells to create useful quantities thereof, and for expressing binding agents, such as antibodies or antibody fragments, using recombinant techniques.

[0219] The polynucleotides provided herein can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In certain embodiments, the polynucleotide is in the form of cDNA. In certain embodiments, the polynucleotide is a synthetic polynucleotide.

[0220] Any suitable vectors can be used to introduce one or more polynucleotides that encode an antibody or fragment thereof into the host. Exemplary vectors that have been described include replication deficient retroviral vectors, including but not limited to lentivirus vectors; parvoviral vectors, such as adeno-associated viral (AAV) vectors;adenoviral (AV); an adenoviral adeno-associated viral chimeric or a vaccinia viral or a herpesviral vector; lipofectin mediated gene transfer (BRL); liposomal vectors; and combinations thereof. Optionally, viral vectors are rendered replication-deficient by, e.g., deleting or disrupting select genes required for viral replication.

[0221] Other non-viral delivery mechanisms contemplated include calcium phosphate precipitation, electroporation, direct microinjection, DNA-loaded liposomes, gene bombardment using high velocity microprojectiles.

[0222] A cell may comprise one or more polynucleotides or one or more vectors, e.g., the cell is transformed or transfected with one or more polynucleotides encoding a binding agent (e.g., an antibody) or the one or more vectors comprising the one or more polynucleotides. The cells can be prokaryotic cells, such as Escherichia co . or eukaryotic cells, such as an animal cell (e.g., a myeloma cell, Chinese Hamster Ovary (CHO) cell, or hybridoma cell), yeast (e.g., Saccharomyces cerevisiae), an insect cell, or a plant cell. Use of mammalian host cells may provide for translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on recombinant expression products. Similarly, polypeptides (e.g., multispecific binding agents (e.g., bispecific antibodies)) can be glycosylated or non-glycosylated and / or have been covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0223] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. A host cell can be isolated and / or purified. A host cell also can be a cell transformed in vivo to cause transient or permanent expression of the polypeptide in vivo. A host cell may also be an isolated cell transformed ex vivo and introduced post-transformation, e.g., to produce the polypeptide in vivo for therapeutic purposes. The definition of host cell explicitly excludes a transgenic human being.5.5 Pharmaceutical Compositions

[0224] In another aspect, provided herein are compositions, such as pharmaceutical compositions. In certain embodiments, provided herein is a composition, such as a pharmaceutical composition, comprising the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein, and a pharmaceutically acceptable excipient.

[0225] In certain embodiments, the pharmaceutical composition comprises a multispecific binding agent (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof) and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1 or a portion thereof and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2 or a portion thereof and a pharmaceutically acceptable excipient.

[0226] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a polynucleotide or polypeptides provided herein (such as a polynucleotide or polypeptides encoding a multispecific binding agent provided herein) and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a vector or vectors provided herein (such as a vector or vectors comprising a polynucleotide disclosed herein) and a pharmaceutically acceptable excipient. In certain embodiments, a pharmaceutical composition comprises a therapeutically effective amount of a cell provided herein (such as a cell comprising a polynucleotide disclosed herein) and a pharmaceutically acceptable excipient.

[0227] In certain embodiments, a pharmaceutical composition provided herein comprises a first means for binding to IL-4Ra expressed on a cell, a second means for binding to IL-31 expressed on the cell, and a pharmaceutically acceptable carrier. In certain embodiments, the first means and the second means are an 4L-4Ra*IL-31 binding agent (e.g., an IL-4Ra*IL-31 multispecific antibody as described in Section 5.2).

[0228] In certain embodiments, pharmaceutical compositions provided herein are prepared for storage by mixing the binding agents, polynucleotides, vectors, or cells provided herein having the desired degree of purity with optional pharmaceutically acceptable excipients (see, e.g., Remington, Remington’s Pharmaceutical Sciences (18th ed. 1980)) in the form of aqueous solutions or lyophilized or other dried forms. The binding agents, polynucleotides, vectors, or cells of the present application can be formulated in any suitableform for delivery to a target cell / tissue as described in Remington, Remington’s Pharmaceutical Sciences (18th ed. 1980).5.6 Methods of treatment and Use of the multispecific binding agents

[0229] In yet another aspect, provided herein are methods for treating a disease or disorder in a subject. In certain embodiments, provided herein is a method for treating a disease or disorder in a subject comprising administering to the subject the multispecific binding agent (e.g., an IL-4RaxIL-31 multispecific antibody as described in Section 5.2) provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein. In certain embodiments, the disease or disorder is atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

[0230] In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the multispecific binding agent (e.g., an IL-4Ra><IL-31 multispecific antibody as described in Section 5.2) provided herein. In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof. In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the multispecific binding agent “anti-IL- 31 / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.

[0231] In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the polynucleotide or polynucleotides provided herein.

[0232] In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the vector or vectors provided herein.

[0233] In certain embodiments, provided herein is a method for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma in a subject comprising administering to the subject the cell provided herein.

[0234] In yet another aspect, provided herein are multispecific binding agents (e.g., an IL-4Ra*IL-31 multispecific antibody as described in Section 5.2) for use in treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In certain embodiments, provided herein is a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31 -binding domain as described in Section 5.2.2 or a portion thereof for use in treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In certain embodiments, provided herein is the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1 for use in treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In certain embodiments, provided herein is the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2. for use in treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

[0235] In yet another aspect, provided herein is use of a multispecific binding agent (e.g., an ZL-4Ra*IL-31 multispecific antibody as described in Section 5.2) in the manufacture of a medicament for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In certain embodiments, provided herein is use of a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof in the manufacture of a medicament for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In yet another aspect, provided herein is use of the multispecific binding agent “anti- IL-3 l / IL-4Ra vHH” as described in Section 5.3.1 in the manufacture of a medicament for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma. In yet another aspect, provided herein is use of the multispecific binding agent “anti -IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2 in the manufacture of a medicament for treating atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.5.7 Methods of making the multispecific binding agents

[0236] The present application further provides a method of making a multispecific binding agent e.g., an fL-4Ra*IL-31 multispecific antibody as described in Section 5.2) provided herein. In certain embodiments, provided herein is a method of making a multispecific binding agent provided herein, wherein the method comprises:(i) culturing a host cell under conditions suitable for expression of the multispecific binding agent, wherein the host cell comprises a polynucleotide or polynucleotides provided herein or a vector or vectors provided herein; and(ii) isolating and purifying the multispecific binding agent.

[0237] In certain embodiments, the method comprises culturing a host cell under conditions suitable for expression of the multispecific binding agent. In certain embodiments, the host cell comprises a polynucleotide or polynucleotides encoding the multispecific binding agent or a portion thereof provided herein. In certain embodiments, the host cell comprises a vector or vectors comprising a polynucleotide or polynucleotidesencoding the multispecific binding agent or a portion thereof provided herein. In certain embodiments, the method comprises isolating and purifying the multispecific binding agent. Exemplary methods of making the multispecific binding agent are described in Section 6.

[0238] In certain embodiments, the method comprises culturing a host cell under conditions suitable for expression of the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH.” In certain embodiments, the host cell comprises a polynucleotide or polynucleotides encoding the multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3.1 or a portion thereof. In certain embodiments, the host cell comprises a polynucleotide or polynucleotides encoding the amino acid sequence of SEQ ID NO: 14 and / or SEQ ID NO: 15. In certain embodiments, the host cell comprises a vector or vectors comprising a polynucleotide or polynucleotides encoding the multispecific binding agent “anti-IL-31 / IL- 4Ra vHH” as described in Section 5.3.1 or a portion thereof provided herein. In certain embodiments, the host cell comprises a vector or vectors comprising a polynucleotide or polynucleotides encoding the amino acid sequence of SEQ ID NO: 14 and / or SEQ ID NO: 15. In certain embodiments, the method comprises isolating and purifying the multispecific binding agent “anti -IL-3 l / IL-4Ra vHH” as described in Section 5.3.1.

[0239] In certain embodiments, the method comprises culturing a host cell under conditions suitable for expression of the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2. In certain embodiments, the host cell comprises a polynucleotide or polynucleotides encoding the multispecific binding agent “anti-IL-31 / IL- 4Ra vHHs” as described in Section 5.3.2 or a portion thereof. In certain embodiments, the host cell comprises a polynucleotide or polynucleotides encoding the amino acid sequence of SEQ ID NO: 18 and / or SEQ ID NO: 15. In certain embodiments, the host cell comprises a vector or vectors comprising a polynucleotide or polynucleotides encoding the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2 or a portion thereof provided herein. In certain embodiments, the host cell comprises a vector or vectors comprising a polynucleotide or polynucleotides encoding the amino acid sequence of SEQ ID NO: 18 and / or SEQ ID NO: 15. In certain embodiments, the method comprises isolating and purifying the multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section5.3.2.

[0240] In certain embodiments, provided are the materials for making multispecific binding agents and portions thereof. For example, an isolated cell or host cell may produce a multispecific binding agent (e.g., the IL-4Ra*IL-31 binding agent as described in Section5.2, or a portion thereof). In this regard, a cell (e.g., an isolated cell or host cell) may producea multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof. In certain embodiments, a cell (e.g., an isolated cell or a host cell) may produce multispecific binding agent “ anti -IL-3 l / IL-4Ra vHH” as described in Section 5.3.1. In certain embodiments, a cell (e.g., an isolated cell or host cell) may produce multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2.5.8 Kits

[0241] In another aspect, provided herein are kits. In certain embodiments, provided herein is a kit comprising the multispecific binding agent provided herein, the polynucleotide or polynucleotides provided herein, or the vector or vectors provided herein, or the cell provided herein, packaged into suitable packaging material.

[0242] In certain embodiments, provided herein is a kit comprising a multispecific binding agent or a portion thereof provided herein (e.g., the IL-4Ra*IL-31 binding agent as described in Section 5.2, or a portion thereof), packaged into suitable packaging material. In certain embodiments, provided herein is a kit comprising a multispecific binding agent comprising an IL-4Ra-binding domain as described in Section 5.2.1 and an IL-31-binding domain as described in Section 5.2.2 or a portion thereof, packaged into suitable packaging material. In certain embodiments, provided herein is a kit comprising a multispecific binding agent “anti-IL-3 l / IL-4Ra vHH” as described in Section 5.3. lor a portion thereof, packaged into suitable packaging material. In certain embodiments, provided herein is a kit comprising a multispecific binding agent “anti-IL-3 l / IL-4Ra vHHs” as described in Section 5.3.2 or a portion thereof, packaged into suitable packaging material.

[0243] In certain embodiments, provided herein is a kit comprising the polynucleotide or polynucleotides provided herein, packaged into suitable packaging material. In certain embodiments, provided herein is a kit comprising the vector or vectors provided herein, or the cell provided herein, packaged into suitable packaging material.

[0244] A kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.

[0245] The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc).

[0246] Kits provided herein can include labels or inserts. Labels or inserts include“printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH media, or memory type cards. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date.6. EXAMPLES6.1 Example 1: Design and Preparation of Anti-IL-31-anti-IL-4Ra Bispecific Antibodies

[0247] The variable region (vHH) of the anti-IL-4Ra single domain antibody HZ-8 (SEQ ID NO: 16) was linked to the C-terminus of the heavy chain of the anti-IL-31 monoclonal antibody PM061-106-1 through a GGGGAGGGGAGGGGAGGGGA (SEQ ID NO: 13) linker (FIGs. 1A-1B), forming an fL-4RaxIL-31 binding agent, named “anti-IL-3 l / IL-4Ra vHH.” The variable region (vHHs) of the anti-IL-4Ra single domain antibody combination HZ-88 (SEQ ID NO: 17) was concatenated to the C-terminus of the heavy chain of the anti- IL-31 monoclonal antibody PM061-106-1 through a GGGGAGGGGAGGGGAGGGGA (SEQ ID NO: 13) linker (FIGs. 1C-1D), forming an IL-4RaxIL-31 binding agent, named “anti-IL-3 l / IL-4Ra vHHs.” The antibody amino acid sequences are provided herein, for example, in Section 8.

[0248] The antibody peptide sequences were constructed into the pcDNA3.1 expression frame by molecular cloning technology. The pcDNA3.1 vector carrying polypeptide chain #3 (SEQ ID NO: 14) and polypeptide chain #4 (SEQ ID NO: 15) of anti-IL-3 l-anti-IL-4Ra vHH was transferred into HEK293 cells by chemical transfection to prepare anti-IL-31 / anti- IL-4Ra vHH bispecific antibody. The pcDNA3.1 vector carrying polypeptide chain #5 (SEQ ID NO: 18) and polypeptide chain #4 (SEQ ID NO: 15) of anti-IL-3 l / anti-IL-4Ra vHHs bispecific antibody was transferred into HEK293 cells to prepare anti-IL-3 l / anti-IL-4Ra vHHs bispecific antibody. After 5 days of cell culture, the supernatant was collected and the target protein was purified by protein A magnetic beads (purchased from GenScript). The magnetic beads were washed with an appropriate volume of binding buffer (PBS) and then lysed (0.1% Tween 20, pH 7.4) and resuspended (1-4 times the volume of magnetic beads) and added to the sample to be purified. Samples were incubated at room temperature for 1hour with gentle shaking. The samples were placed on a magnetic rack, the supernatant was discarded, and beads were washed 3 times with binding buffer. Elution buffer (0.1 M sodium citrate, pH 3.2) was added and samples were incubated at room temperature for 5-10 minutes. Samples were then placed on the magnetic rack, elution buffer was collected and transferred to the neutralization buffer. The purified anti-IL-3 l / anti-IL-4Ra vHH and anti-IL-31 / anti-IL- 4Ra vHHs dual antibodies were obtained. The molecular weight was confirmed by LC-MS for subsequent in vitro and in vivo activity detection.6.2 Example 2: Antibody Affinity Detection

[0249] ForteBio affinity determination was performed according to existing methods (Estep P et al., Determination of antibody-antigen affinity and epitope binding based on high- throughput methods. MAbs, 2013.5(2): p.270-8). Briefly, the sensor was equilibrated offline in the assay buffer for 30 minutes, then online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM antigen for 5 minutes, and then the sensor was transferred to PBS for dissociation for 5 minutes. The kinetic analysis was performed using a 1 : 1 binding model.

[0250] The affinity of anti-IL-3 l / IL-4Ra vHHs bispecific antibody and anti-IL-31 / IL- 4Ra vHH bispecific antibody molecules to human IL-4Ra, cynomolgus monkey IL-4Ra, human IL-31 and cynomolgus monkey IL-31 proteins was determined by Forte Bio Octet. The data fitting results are shown in Tables 2-1 and 2-2. The two bispecific antibodies had good binding activity to human IL-4Ra, cynomolgus monkey IL-4Ra, human IL-31 and cynomolgus monkey IL-31 proteins, and the affinity was comparable to that of IL-4Ra or IL- 31 single-terminal antibodies.Table 2-1. Affinity detection of anti- IL-31 / IL-4Ra bispecific antibodies to IL-4RaTable 2-2. Affinity detection of anti- IL-31 / IL-4Ra bispecific antibodies to IL-316.3 Example 3: Binding Activity of Anti-IL-31-anti-IL-4Ra Bispecific Antibodies to IL-4Ra Cells

[0251] Specifically, the nucleotide sequences encoding the full-length human IL-4Ra amino acid sequence (SEQ ID NO: 31) and the full-length rhesus monkey IL-4Ra amino acid sequence (SEQ ID NO:32) were inserted into the multiple cloning site of the PLVX-puro vector (purchased from Addgene) by double digestion with EcoR I and Hind III, respectively. 293T cells were used to package lentivirus and infect 293F cells, and 293F cells stably overexpressing human IL-4Ra (293F-huIL-4Ra cells) and 293F cells overexpressing rhesus monkey IL-4Ra (293F-rhesusIL-4Ra cells) were obtained by pressure screening by adding puromycin (purchased from Invitrogen). The expanded overexpressing cells were adjusted to an appropriate cell density and added to a 96-well flow plate. After centrifugation, the gradient diluted samples to be tested were added and incubated at 4°C for 30 minutes. Samples were washed twice with 1 *PBS, fluorescent secondary antibody diluted to appropriate concentration was added, and samples were incubated at 4°C for 30 minutes, and then washed twice with 1 *PBS. The cells were resuspended in 1 *PBS, and detected on CytoFlex flow cytometer to calculate the corresponding MFI. The results are shown in Table 3-1 and FIGs. 2A-2B.

[0252] Table 3-1 and FIGs. 2A-2B show that all anti -IL-31 -anti -IL-4Ra bispecific antibodies had binding activity to human and rhesus monkey IL-4Ra overexpressing cells. And the binding activity to rhesus monkey IL-4Ra overexpressing cells was comparable to that of control antibodies Dupilumab analogue (SEQ ID NO: 19, SEQ ID NO:20) and NM26- 2198 (SEQ ID NO:27, SEQ ID NO:28).Table 3-1. Binding activities of the anti-IL-31-anti-IL-4Ra bispecific antibodies to human and rhesus monkey IL-4Ra cells and the anti-IL-31-anti-IL-4Ra bispecific antibodies’ blocking of binding of human IL-4 protein to 293F-huIL-4Ra cells6.4 Example 4: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies ELISA Protein Level Binding Test

[0253] Next, we used ELISA to analyze the binding ability of anti-IL-3 l-anti-IL-4Ra bispecific antibody to human and cynomolgus monkey IL-31 protein. Specifically, 1 x ELISA coating working solution was used to dilute human and cynomolgus monkey IL-31 protein samples to 1 pg / mL, and 100 pL / well was coated on a 96-well ELISA plate at 4°C overnight. The coated ELISA plate was taken out, the coating solution was discarded, 250 pL / well of ELISA washing solution was added, and it was allowed to stand for 30 seconds. The washing solution was discarded, and the process was repeated 3 times. 200 pL / well of ELISA blocking solution was added, and the plate was incubated at room temperature for 2 hours. The antibody was diluted with ELISA working solution, starting from 100 nM, with a 3 -fold gradient dilution for a total of 12 points, and 100 pL / well was added to the blocked ELISA plate, and the reaction was allowed to react at room temperature for 1 hour. HRP anti-huIgG, was diluted to HRP working solution with ELISA working solution, and set aside. For the ELISA plate that had completed the binding reaction, the reaction solution was discarded, ELISA washing solution at 250 pL / well was added, and plate was incubated for 30 seconds, the washing solution was discarded. Washing steps were repeated 3 times. HRP working solution was added at 100 pL / well, and incubated at room temperature for 1 hour. For the ELISA plate that had completed the reaction with HRP working solution, the reaction solution was discarded, ELISA washing solution at 250 pL / well was added, and plate was incubated for 30 seconds, the washing solution was discarded. Washing steps were repeated 3 times. TMB single-component color development solution was added at 100 pL / well, and left to react at room temperature for 3 minutes. For the ELISA plate that had completed the color development, ELISA stop solution was added at 50 pL / well to terminate the reaction. The absorbance value at 450 nm of the ELISA plate that had terminated the reaction was read within 5 minutes after termination, and recorded for data processing. The results are shown in Table 4-1 and FIGs. 3A-3B.

[0254] Table 4-1 and FIGs. 3A-3B show that all anti -IL-31 -anti -IL-4Ra bispecific antibodies had binding activity to both human and cynomolgus monkey IL-31 proteins, andthe binding ability was comparable to that of the control antibody BMS-981164 analogue (SEQ ID NO:25, SEQ ID NO:26) and the single-terminal antibody.Table 4-1. Binding activities of the anti-IL-31-anti- IL-4Ra bispecific antibodies to human and cynomolgus IL-31 proteins and the anti-IL-31-anti-IL-4Ra bispecific antibodies’ blocking activities of CHO cells overexpressing human IL-31RA and downstream pSTAT3 signaling in IL-31R 293T cells induced by IL-316.5 Example 5: Co-binding Activity of Anti-IL-31-anti-IL-4Ra Bispecific Antibodies with IL-4Ra and IL-31 Protein

[0255] As a bispecific antibody specifically targeting IL-4Ra and IL-31, we further used ELISA detection to verify the ability of anti-IL-31 -anti -IL-4Ra bispecific antibody to simultaneously bind IL-4Ra and IL-31. Specifically, human IL-4Ra protein was dissolved according to the instructions, diluted to 1 pg / mL with ELISA coating solution, and 100 pL / well was coated on the ELISA plate, overnight at 4°C, washed 3 times with PBST, and 200 pL / well was added to the ELISA blocking solution for 2 hours at room temperature. The coating solution was discarded, and 100 pL / well of the bispecific antibody diluted in ELISA working solution was added, and incubated at room temperature for 2 hours. The reaction solution was discarded, washed 3 times with PBST, and 100 pL / well of biotin-labeled human IL-31 protein (in house) diluted with ELISA working solution was added, and incubated at room temperature for 1 hour. The reaction solution was discarded, samples were washed 3 times with PBST, 100 pL / well of SA-HRP diluted with ELISA working solution was added, and samples were incubated at room temperature for half an hour. The reaction solution was discarded, samples were washed 3 times with PBST, 100 pL / well of ELISA colorimetricsolution was added, samples were left to react at room temperature for 3 minutes, 50 pL / well of ELISA stop solution was added, and the absorbance value was read at 450 nm.

[0256] As shown in FIG. 4, the two anti -IL-31 -anti -IL-4Ra bispecific antibodies were able to bind to human IL-4Ra and IL-31 proteins simultaneously, and the binding activities were comparable.6.6 Example 6: Effect of Human IL-31 Protein on the Binding Activity of Anti-IL- 31-anti-IL-4Ra Bispecific Antibodies to 293F-huIL-4Ra Cells

[0257] The anti-IL-3 l-anti-IL-4Ra bispecific antibody was able to bind to human IL-4Ra and human IL-31 protein at the same time. We further used flow cytometry to detect whether the presence of human IL-31 protein affects the binding of the anti- IL-31 -anti -IL-4Ra bispecific antibody to 293F-huIL-4Ra cells. Specifically, the density of expanded cultured 293F-huIL-4Ra cells was adjusted to 2 * 106cells / mL, 100 pL / well was added to a 96-well flow plate, and centrifuged for later use; the antibody to be tested was diluted with PBS, 3- fold gradient dilution starting from 100 nM for a total of 12 points, 60 pL / well was added to a 96-well sample plate, and 60 pL / well of human IL-31 protein (Kactus) or PBS diluted with PBS was added to the above sample plate, with a final concentration of 100 ng / mL; then 100 pL / well was added to the above cell sample wells, incubated at 4°C for 30 minutes; washed twice with PBS, added the corresponding fluorescent secondary antibody diluted to the appropriate concentration, incubated at 4°C for 20 minutes, and washed twice with PBS; PBS was added to resuspend the cells, and the detection was performed on the CytoFlex flow cytometer and the corresponding MFI was calculated.

[0258] As shown in FIG. 5, the two anti -IL-31 -anti -IL-4Ra bispecific antibodies showed comparable binding activity to 293F-huIL-4Ra cells in the presence or absence of human IL- 31 protein, and their binding activity was not affected by human IL-31 protein.6.7 Example 7: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked the Binding Activity of Human IL-4 to 293F-huIL-4Ra Cells

[0259] After showing that the anti-IL-3 l-anti-IL-4Ra bispecific antibody was able to specifically bind to human IL-4Ra, we measured the ability of the bispecific antibody molecules to block the receptor ligand. First, flow cytometry was used to detect the activity of the anti -IL-3 l-anti-IL-4Ra bispecific antibody in blocking the binding of human IL-4 protein to 293F-huIL-4Ra cells. Specifically, the cell density of the expanded cultured 293F- huIL-4Ra cells was adjusted to 2 * 106cells / mL, and 100 pL / well was added to a 96-well flow plate and centrifuged for use. The purified bispecific antibody was diluted with 1 *PBS,and 3 -fold dilutions were made starting from 400 nM for a total of 12 points. 60 pL / well of the diluted sample was added to the above 96-well flow plate with cells and incubated at 4°C for 30 minutes. Then 60 pL / well of the biotinylated human IL-4 protein (Aero) diluted with 1 *PBS was added and incubated at 4°C for 30 minutes. Samples were washed twice with I xPBS, 100 pL / well of SAPE antibody diluted 150 times with I xPBS was added, and samples were incubated at 4°C for 20 minutes. Samples were washed twice with 1 xPBS, 100 pL / well of 1 xPBS was added to resuspend the cells, and the detection was performed on the CytoFlex flow cytometer and the corresponding MFI was calculated.

[0260] The results are shown in FIG. 6 and Table 3-1. Both anti-IL-3 l-anti-IL-4Ra bispecific antibodies were able to completely block the binding of human IL-4 protein to 293F-huIL-4Ra cells, and the blocking activity was equivalent to that of their IL-4Ra singleterminal antibodies.6.8 Example 8: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked the Binding Activity of Human IL-31 to CHO-huIL-31RA Cells

[0261] After showing that the anti-IL-3 l-anti-IL-4Ra bispecific antibody was able to specifically bind to human IL-31, we further used flow cytometry to measure the activity of the anti-IL-3 l-anti-IL-4Ra bispecific antibody in blocking huIL-3 l / huIL-3 IRA binding. Specifically, the nucleotide sequence encoding the full-length human IL-3 IRA amino acid sequence (SEQ ID NO:29) was inserted into the multiple cloning site of the PLVX-puro vector (purchased from Addgene) by double digestion with EcoRI and Hindlll. The lentiviral supernatant was packaged by 293T cells and infected CHO and 293T cells. The transfected cells were screened by adding puromycin (purchased from Invitrogen) to obtain CHO cells (CHO-huIL-31RA cells) and 293T cells (293T-huIL-31RA cells) that stably overexpressed human IL-3 IRA. The purified bispecific antibody was diluted with 1 xPBS, and a total of 12 points were obtained by 3 -fold gradient dilution starting from 400 nM. 60 pL / well of the diluted sample was added to a new 96-well flow plate. Then 60 pL / well of biotinylated human IL-31 protein (in house) diluted to an appropriate concentration with 1 xPBS was added, mixed and incubated at 4°C in the dark for 30 minutes. The density of the expanded CHO-huIL-3 IRA cells was adjusted to 2 x io6cells / mL, and 100 pL / well was added to the 96-well flow plate, centrifuged and the supernatant was removed. 100 pL / well of the incubated sample was added to the cell well after centrifugation, mixed and incubated at 4°C in the dark for 30 minutes. Samples were washed twice with 1 xPBS, and 100 pL / well of SAPE antibody diluted 150 times with 1 xPBS was added, and incubated at 4°C for 30minutes. The cells were washed twice with 1 *PBS, and 100 pL / well of 1 *PBS was added to resuspend the cells. The cells were detected on a CytoFlex flow cytometer and the corresponding MFI was calculated.

[0262] The results are shown in FIG. 7 and Table 4-1. Both anti-IL-3 l-anti-IL-4Ra bispecific antibodies were able to completely block the binding of human IL-31 protein to CHO-huIL-3 IRA cells, and the blocking activity was comparable to that of their IL-31 monoclonal antibodies and superior to the bispecific control antibody NM26-2198.6.9 Example 9: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked Human IL-31- induced STAT3 Phosphorylation in 293T-huIL-31RA / OSMR|J Cells

[0263] The blocking effect of the anti-IL-31 -anti-IL-4Ra bispecific antibody on IL-31 / IL- 3 IRA binding was shown at the cellular level. We further analyzed the blocking activity of the bispecific antibody from a functional perspective. Specifically, the nucleotide sequence encoding the full-length human OSMR P amino acid sequence (SEQ ID NO:30) was inserted into the multiple cloning site of the PMS061-2-h-hygro vector by double digestion with Agel and Nhel, respectively. The lentiviral supernatant was packaged in 293T cells and infected with 293T-huIL-3 IRA cells. The transfected cells were screened by adding hygromycin (purchased from Invivogen) to obtain 293T cells (293T-huIL-31RA / OSMRp cells) that stably overexpressed human IL-3 IRA / OSMRp. The purified bispecific antibody was diluted with 1 *PBS, and 3-fold dilutions were made starting from 400 nM for a total of 10 points. Then, human IL-31 protein (Kactus) diluted with 1 *PBS was added to the above plate and incubated at 4°C for 30 minutes. 293T-huIL-3 IRA / OSMRp cells were removed, and the cell density was adjusted to 2.0 * 106cells / mL with culture medium. 100 pL / well was added to the 96-well flow plate and centrifuged for use. 100 pL / well of the incubated sample was added to the above 96-well flow plate with cells and incubated at 4°C for 10 minutes.Samples were washed once with pre-cooled 1 *PBS, 4% paraformaldehyde was added to 100 pL / well, and samples were incubated at room temperature for 10 minutes. Samples were washed once with pre-cooled 1 *PBS, 100 pL / well of pre-cooled methanol was added, and samples were incubated at 4°C for 30 minutes. 200 pL of pre-cooled lx wash buffer was added to each well to wash twice, and 100 pL / well of pSTAT3 flow cytometry antibody (1 :200) diluted in pre-cooled lx wash buffer was added, and incubated at 4°C for 20 minutes. 200 pL / well of pre-cooled lx wash buffer was added to wash twice, and 100 pL / well of precooled lx wash buffer was added to resuspend the cells, and the cells were detected on the CytoFlex flow cytometer and the corresponding MFI was calculated.

[0264] The results are shown in FIG. 8 and Table 4-1. Both IL-3 l / IL-4Ra bispecific antibodies were able to completely inhibit IL-31-induced STAT3 phosphorylation in 293T- huIL-31RA / OSMRP cells, and the blocking activity was comparable to that of their IL-31 single-terminal antibodies and the control antibody BMS-981164, and was superior to the bispecific control antibody NM26-2198.6.10 Example 10: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked IL-4 / LL-13- induced Downstream Signal STAT6 Phosphorylation

[0265] The blocking effect of the anti-IL-31 -anti-IL-4Ra bispecific antibody on IL-4 / IL- 4Ra binding has been shown at the cellular level. We further analyzed the blocking activity of the bispecific antibody functionally. HEK-Blue IL-4 / IL-13 reporter cells expressing the receptor IL-4Ra / IL-13Ral on the surface were stably transfected with human STAT6 and SEAP genes. When cells are stimulated by IL-4 / IL-13, the phosphorylation signal of STAT6 downstream of the receptor is activated and induces the expression of SEAP reporter gene. We used HEK-Blue IL-4 / IL-13 reporter cells to detect the ability of anti -IL-3 l-anti-IL-4Ra bispecific antibody to block downstream signals induced by IL-4 / IL-13. Specifically, HEK- blue IL-4 / IL-13 reporter gene cells (Invivogen) were trypsinized, washed once with 50 mL 1 *PBS, resuspended in DMEM medium containing 10% inactivated FBS, the cell density was adjusted to 5 * 105cells / mL, and 100 pL / well were added to a 96-well flat-bottom culture plate. The antibody to be tested was diluted to 24 nM with DMEM medium containing 10% inactivated FBS, 3-fold gradient dilution for a total of 9 points, and 50 pL / well was added to the above 96-well flat-bottom culture plate. Human IL- 13 protein (Aero) and human IL-4 protein (Aero) were diluted to appropriate concentrations with DMEM medium containing 10% inactivated FBS, and 50 pL / well was added to the above 96- well flat-bottom culture plate. Cells were cultured overnight at 37°C, 5% CO2 incubator. On the second day, a new 96-well flat-bottom plate was prepared, 180 pL of QUANTI-Blue Solution (Invivogen) was added to each well, and 20 pL / well of the supernatant from the overnight culture was added. Plate was incubated at 37°C in the dark for 4 hours, and the absorbance was read at 620-655 nm on a microplate reader.

[0266] Results are shown in FIGs. 9A-9B. The anti-IL-3 l-anti-IL-4Ra bispecific antibodies were able to completely inhibit IL-4 / IL- 13 -induced intracellular signal transduction, and the inhibitory activity was equivalent to that of the IL-4Ra single-terminal antibody.6.11 Example 11: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked IL-4 / IL-13- induced TF-1 Cell Proliferation

[0267] The growth of TF-1 cells is completely dependent on GM-CSF or IL-3. They express multiple receptors on their surface. Cytokines such as IL-4, IL- 13, and IL-5 can stimulate the proliferation of TF-1 cells. We used growth medium without GM-CSF or IL-3 to detect the inhibitory activity of two anti-IL-3 l-anti-IL-4Ra bispecific antibodies on the proliferation of TF-1 cells induced by IL-4 and IL-13. Specifically, TF-1 cells (ATCC) were taken, the supernatant was removed after centrifugation, and then washed twice with 50 mL 1 *PBS, resuspended in RPMI1640 containing 10% FBS, and the cell density was adjusted to 2 * 105cells / mL, and 100 pL / well was added to a 96-well cell culture flat-bottom plate. The antibody was diluted with RPMI1640 medium containing 10% FBS, and the purified antibody with gradient dilution was added to the above 96-well cell plate at 50 pL / well. Human IL-4 protein (Aero) and human IL- 13 protein (Novoprotein) were diluted in RPMI1640 medium containing 10% FBS, and 50 pL / well was added to the above 96-well cell plate. Plates were incubated in a 37°C, 5% CO2 incubator for 2-3 days. 20 pL of CCK-8 solution (Dojindo) was added to each well, incubated at room temperature for 8 hours, and then the absorbance was measured at 450 nm.

[0268] The results are shown in FIGs. 10A-10B. Both anti -IL-3 l-anti-IL-4Ra bispecific antibodies were able to completely inhibit IL-4 / IL- 13 -induced TF-1 cell proliferation, and in the human IL-13-induced TF-1 cell proliferation experiment, both anti- IL-31-anti-IL-4Ra bispecific antibodies were superior to the bispecific control antibody NM26-2198.6.12 Example 12: Anti-IL-31-anti-IL-4Ra Bispecific Antibodies Blocked IL-4 and IL- 31, as well as IL-4, IL-13 and IL-31-induced CCL2 Expression in BEAS-2 B Cells

[0269] BEAS-2B cells are healthy adult bronchial epithelial cells that express multiple receptors on their surface. Human IL-4, IL-31, and IL-13 can all induce the expression of CCL2. We used ELISA to detect the expression of CCL2 secreted by BEAS-2B to show the ability of two anti-IL-31 -anti -IL-4Ra bispecific antibodies to simultaneously block IL-4 and IL-31 -mediated signaling pathways, as well as the ability to simultaneously block IL-4, IL- 13, and IL-31 -mediated signaling pathways. Specifically, BEAS-2B cells (ATCC) were trypsinized, resuspended in culture medium, and the cell density was adjusted to 3 x 105cells / mL. 100 pL / well was added to a 96-well cell culture flat-bottom plate overnight. The cell plate was washed twice with PBS for later use; the antibody was diluted to 40 nM with culture medium, and 100 pL / well was added to the 96-well cell plate, and 3 replicates weremade; IL-4, IL-31, and IL-13 were diluted to 4 ng / mL, 50 ng / mL, and 100 ng / mL with culture medium, and 100 pL / well was added to the 96-well cell plate, and 3 replicates were made, and Blank and positive control wells were set at the same time. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours; the CCL2 content in the supernatant was detected using a human CCL2 ELISA kit.

[0270] The results are shown in FIG. 11 A. Both anti-IL-3 l-anti-IL-4Ra bispecific antibodies were able to completely inhibit the production of CCL2 induced by IL-4 and IL- 31. The inhibitory activity was similar to that of the anti-IL-4Ra antibody and the combination of anti -IL-31 antibodies was equivalent, and the ability of the two anti- IL-31- anti-IL-4Ra bispecific antibodies to inhibit CCL2 expression in BEAS-2B cells was significantly stronger than that of their single-ended antibodies (anti-IL-31 monoclonal antibody and HZ-8) and control monoclonal antibodies (Dupilumab analogue and Nemolizumab analogue (SEQ ID NO:23, SEQ ID NO:24)).

[0271] The results are shown in FIG. 1 IB. Both anti -IL-31 -anti -IL-4Ra bispecific antibodies were able to completely inhibit the production of CCL2 induced by IL-4, IL- 13 and IL-31. The inhibitory activity of the anti-IL-3 l-anti-IL-4Ra bispecific antibodies was similar to that of the combination of anti-IL-4Ra antibodies and anti-IL-31 antibodies. The ability of the two anti -IL-3 l-anti-IL-4Ra bispecific antibodies to inhibit CCL2 expression in BEAS-2B cells was significantly stronger than that of their single-ended antibodies (anti-IL- 31 monoclonal antibody and HZ-8) and control monoclonal antibodies Dupilumab analogue and Nemolizumab analogue.6.13 Example 13: Analysis of the Physicochemical Properties of Anti-IL-31-anti-IL- 4Ra Bispecific Antibodies

[0272] The anti-IL-3 l-anti-IL-4Ra bispecific antibody showed good in vitro activity, whereas the physicochemical properties of the antibody are also crucial. Specifically, we used DSC (Differential Scanning Calorimetry), HIC (Hydrophobic Interaction Chromatography), and SEC (Size Exclusion Chromatography) methods to detect the thermal stability, hydrophobic interaction, and aggregation analysis of the bispecific antibody.

[0273] The results are shown in Table 13-1. The anti-IL-3 l-anti-IL-4Ra bispecific antibodies showed good relevant physicochemical properties.Table 13-1. Physicochemical properties of anti -IL-31-anti-IL-4Ra bispecific antibodies6.14 Example 14: Accelerated Stability Testing of Anti-IL-31-anti-IL-4Ra Bispecific Antibodies

[0274] Based on the good physical and chemical properties of the above bispecific antibodies, we subsequently evaluated the accelerated stability sample purity test and activity assay of the antibody. Specifically, we used the SEC method to determine the purity of the target antibody after being placed at 40°C for 0, 14 and 28 days, and used the ELISA method to detect the co-binding activity of the accelerated stability sample and human IL-4Ra and human IL-31 protein, thereby evaluating the long-term thermal stability of the antibody.

[0275] The results are shown in Table 14-1 and FIG. 12, where anti -IL-3 l-anti-IL-4Ra at a concentration of 1 to 2 mg / mL. The purity of the vHH molecule did not change significantly, and the co-binding activity was basically the same after 0, 14, and 28 days at 40°C, indicating that the anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody molecules had relatively good thermal stability.Table 14-1. Monomer ratio of anti-IL-31-anti-IL-4Ra vHH bispecific antibodies accelerated stability samples6.15 Example 15: Half-life of Anti-IL-31-anti-IL-4Ra Bispecific Antibody in Mice

[0276] The present invention performed M428L / N434A mutation modifications on theFc fragment of the anti-IL-31 -anti -IL-4Ra bispecific antibody, and we performed PKdetection in human FcRn KI mice. Specifically, we used tail vein injection, blood collection at different time points, and ELISA method to detect the half-life of the anti-IL-31-anti-IL- 4Ra bispecific antibody in human FcRn KI mice.

[0277] The results are shown in FIG. 13. The half-life of anti-IL-3 l-anti-IL-4Ra vHH in human FcRn KI mice was approximately 239.2 hours, and the half-life of Dupilumab analogue in human FcRn KI mice was approximately 211.9 hours, indicating that the anti-IL- 31 -anti-IL-4Ra vHH exhibited a longer half-life and was superior to the control antibody Dupilumab analogue.6.16 Example 16: Efficacy of Anti-IL-31-anti-IL-4Ra Bispecific Antibody in a Mouse Atopic Dermatitis Model

[0278] Based on the fact that anti -IL-31 -anti -IL-4Ra vHH showed good in vitro activity and drugability in the early stage. We used B-hIL-4 / hIL-4Ra KI C57BL / 6 mice to construct an OXA-induced mouse atopic dermatitis model to evaluate the efficacy of the anti-IL-31- anti-IL-4Ra vHH bispecific antibody.

[0279] Specifically, acetone:olive oil = 4: 1 was used as solvent to prepare OXA at concentrations of 0.8% and 0.4%, respectively. On day 0, 0.8% OXA was applied to the right ear and back for sensitization. On days 7, 9, 11, 14, 16, and 17, 0.4% OXA was applied to the right ear and back once a day. Before applying OXA on days 7, 11, and 14, different antibodies of equal volume and molar mass were injected intraperitoneally. On day 17, the mice were anesthetized, and the mouse blood was collected by orbital bleeding. After centrifugation, the mouse serum was collected and stored at -80°C for later use. At the same time, the right ear and back tissues of the mice were collected, half of which was fixed with 4% paraformaldehyde, and H&E tissue staining was performed after fixation. The remaining half was frozen in a -80°C refrigerator for later use for protein and RNA extraction. The collected mouse serum was used to determine the IgE content in the mouse serum using an ELISA detection kit (Biolegend).

[0280] As shown in FIG. 14A-14C, compared with the blank control group, the mouse ear thickness and serum IgE concentration in the OXA modeling group were significantly increased. H&E staining showed a significant increase in inflammatory cell infiltration in the ear tissue and thickening of the epidermis. This indicates that the OXA-induced B-hlL- 4 / hIL-4RA KI C57BL / 6 mouse atopic dermatitis model was successfully established.

[0281] As shown in FIG. 14A mouse ear thickness results, compared with the OXA modeling group, anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody high- and low-concentration administration groups had significantly improved ear thickness, and the improvement in ear thickness was greater than that of the control antibody Dupilumab analogue.

[0282] As shown in FIG. 14B ELISA test results compared with the OXA modeling group, the concentration of total IgE in mouse serum in the anti -IL-31 -anti -IL-4Ra vHH double antibody administration group was significantly lower, and the IgE content was comparable to the control antibody Dupilumab analogue.

[0283] As shown in the H&E pathological staining results in FIG. 14C, compared with the OXA modeling group, the anti-IL-3 l-anti-IL-4Ra vHH dual-antibody group showed a significant decrease in inflammatory cell infiltration in the ear tissue of mice, and a certain improvement in epidermal thickening. The vHH dual antibody administration group had better effects on the pathological changes related to the mouse ear tissue than the control antibody Dupilumab analogue low concentration group, and was comparable to the control antibody Dupilumab analogue high concentration group.6.17 Example 17: Efficacy of Anti-IL-31-anti-IL-4Ra Bispecific Antibody in a Mouse Atopic Dermatitis Model Induced by Calcipotriol (MC903)

[0284] We used B-hIL-4 / hIL-4Ra KI C57BL / 6 mice to establish a MC903-induced mouse atopic dermatitis model to evaluate the efficacy of anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody drug. Specifically, 1 nM MC903 solution was prepared with anhydrous ethanol. Different antibodies of equal volume and molar mass were intraperitoneally injected before the first sensitization in the first and second weeks. The right ear was smeared with 1 nM MC903, 5 times a week, for a total of two weeks. On the 11th day, the mice were anesthetized, and the mouse blood was collected by orbital bleeding. After centrifugation, the mouse serum was collected and stored at -80°C for later use. At the same time, the right ear of the mouse was collected, half of which was fixed with 4% paraformaldehyde, and H&E tissue staining was performed after fixation, and the remaining half was frozen in a - 80°C refrigerator for use for RNA extraction. The collected mouse serum was used to determine the IgE content in the mouse serum using an ELISA detection kit (Biolegend).

[0285] As shown in FIG. 15A-15C, compared with the blank control group, the mouse ear thickness and serum IgE concentration in the MC903 model group were significantly increased. H&E staining showed a significant increase in inflammatory cell infiltration in the ear tissue and epidermal hyperplasia. Thick, indicating that the MC903-induced B-hIL4 / hIL- 4RA KI C57BL / 6 mouse atopic dermatitis model was successfully established.

[0286] As shown in FIG. 15A mouse ear thickness results, compared with the MC903 modeling group, anti-IL-31 -anti- IL-4Ra vHH bispecific antibody high- and low- concentration administration groups had significantly improved ear thickness, and the improvement in ear thickness was greater than that of the control antibody Dupilumab analogue.

[0287] As shown in FIG. 15B ELISA test results, compared with the MC903 model group, the concentration of total IgE in mouse serum in the anti-IL-31 -anti- IL-4R a vHH double antibody administration group was significantly lower, and the IgE content in the high-concentration group was comparable to the control antibody Dupilumab analogue.

[0288] As shown in FIG. 15C H&E pathological staining results, compared with the MC903 model group, in the anti -IL-31 -anti- IL-4Ra vHH dual antibody administration group, the inflammatory cell infiltration in the ear tissue of the mice in the administration group was improved. From the staining of pathological tissue, it can be seen that the anti-IL- 31-anti-IL-4Ra vHH dual-antibody administration group had better pathological changes in mouse ear tissue than the control antibody Dupilumab analogue group.6.18 Example 18: Efficacy of Anti-IL-31-anti-IL-4Ra Bispecific Antibody in the Human IL-31-induced Pruritus Model in Mice

[0289] We used B-hIL-3 l / hIL-3 IRA / OSMRp KI C57BL / 6 mice to establish a human IL- 31 -induced mouse itch model to evaluate the efficacy of anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody drugs. Specifically, on the first day, mice were intradermally injected with PBS and the scratching monitoring device was used to record the number of scratches and rounds of the mice themselves for 3 hours, and the number of scratches and rounds of the mice themselves were recorded as the baseline; on the second day, the mice were intradermally injected with human IL-31 protein, and the scratching monitoring device was used to record the number of scratches and rounds of the mice mediated by IL-31 protein, and the recording was continued for 3 hours. After the recording was completed, the mice were intraperitoneally injected with bispecific antibodies or control antibodies ; on the third day, the mice were intradermally injected with human IL-31 protein, and the scratching monitoring device was used to record half an hour before the injection of human IL-31 protein to exclude abnormal itching of the mice, and then the scratching monitoring device was used to record the number of scratches and rounds of the mice after the injection of human IL-31 protein, and the recording was continued for 3 hours. The data was then sorted and statistically analyzed to reflect the itch inhibition effect of the antibody.

[0290] As shown in FIGs. 16A-16B, compared with the baseline of subcutaneous injection of blank PBS, the number of mouse scratches in the subcutaneous injection of human IL-31 model group increased significantly, indicating that the human IL-31 induced B-hIL-31 / hIL-31RA / OSMRp KI C57BL / 6 mouse itch model was successfully established.

[0291] As shown in FIG. 16 A, the results of scratching times in mice within 3 hours after injection of human IL-31 protein, compared with the model group, the number of mice scratching in the anti-IL-3 l-anti-IL-4R a vHH bispecific antibody administration group was significantly reduced, and the degree of improvement in itching was comparable to that of the control antibody Nemolizumab analogue.

[0292] As shown in FIG. 16B, the number of scratches in mice within 3 hours after the injection of human IL-31 protein is shown. Anti-mouse IL-4Ra (SEQ ID NO:21, SEQ ID NO:22) monoclonal antibodies were injected intraperitoneally. Afterwards, there was no significant difference in the number of scratches in mice, indicating that the effect of inhibiting itching and scratching in mice was completely dependent on anti -IL-31 -anti -IL-4R a vHH Blocking effect of anti -IL-31 end of the bispecific antibody.6.19 Example 19: Efficacy of Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody in a Mouse Atopic Dermatitis Model Induced by Calcipotriol (MC903) in huIL-4Ra / huIL- 4 / huIL-31 / huIL-31RA / huOSMRp KI Mice

[0293] We used huIL-4Ra / huIL-4 / huIL-3 l / huIL-3 IRA / huOSMRp KI C57BL / 6 mice to establish a MC903-induced mouse atopic dermatitis model to evaluate the efficacy of anti-IL- 3 l-anti-IL-4Ra vHH bispecific antibody drug. Specifically, 1 nM MC903 solution was prepared with anhydrous ethanol. Different doses of the antibodies of equal volume (3 mg / kg, 10 mg / kg, and 30 mg / kg) were intraperitoneally injected before the first sensitization on day 0 and day 6. The right ear was smeared with 1 nM MC903, on day 0, 1, 2, 3, 4, 7, 8, 9 (FIG. 17A). Ear thickness was measured daily. On day 9, the mice were anesthetized, and the mouse blood was collected by orbital bleeding. After centrifugation, the mouse serum was collected and stored at -80°C for later use. At the same time, the right ears of the mice were collected, half of which was fixed with 4% paraformaldehyde, and H&E tissue staining was performed after fixation. The collected mouse serum was used to determine the IgE content in the mouse serum using an ELISA detection kit (Biolegend).

[0294] As shown in FIGs. 17B, 17C, and 17E, the anti-IL-31 -anti- IL-4Ra vHH bispecific antibody has shown dose-dependent ear thickness reduction induced by MC903 in huIL-4 / huIL-4RA / huIL-31 / huIL-31RA / huOSMRp KI C57 mice.

[0295] As shown in FIG. 17D, the anti-IL-31 -anti- IL-4Ra vHH bispecific antibody has shown dose-dependent serum total IgG reduction induced by MC903 in huIL-4 / huIL- 4RA / huIL-3 l / huIL-3 IRA / huOSMRp KI C57 mice.6.20 Example 20: Efficacy of Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody in a Mouse Atopic Dermatitis Model Induced by Calcipotriol (MC903) in huIL-4Ra / huIL- 4 / huIL-31 / huIL-31RA / huOSMRp KI Mice

[0296] We used huIL-4Ra / huIL-4 / huIL-3 l / huIL-3 IRA / huOSMRp KI C57BL / 6 mice to establish a MC903-induced mouse atopic dermatitis model to evaluate the efficacy of anti-IL- 3 l-anti-IL-4Ra vHH bispecific antibody drug. Specifically, 1 nM MC903 solution was prepared with anhydrous ethanol. The mice were intraperitoneally injected with the anti -IL- 31 -anti-IL-4Ra vHH bispecific antibodies (30 mg / kg) or control antibodies (Dupixent, Nemolizumab analogue, or NM26-2198) before the first sensitization on day 0 and day 6 (Table 20-1). The right ear was smeared with 1 nM MC903, on day 0, 1, 2, 3, 4, 7, 8, 9 (FIG. 18 A). Ear thickness was measured daily. On day 9, the mice were anesthetized, and the mouse blood was collected by orbital bleeding. After centrifugation, the mouse serum was collected and stored at -80°C for later use. At the same time, the right ears of the mice were collected, half of which was fixed with 4% paraformaldehyde, and H&E tissue staining was performed after fixation. The collected mouse serum was used to determine the IgE content in the mouse serum using an ELISA detection kit (Biolegend). Serum TARC (also known as CCL17) level was determined using an ELISA detection kit (Invitrogen).Table 20-1. Treatment Groups and Dosages

[0297] As shown in FIGs. 18B-18E, the anti -IL-31 -anti -IL-4Ra vHH bispecific antibody significantly reduced ear thickness induced by MC903, outperforming other comparators.

[0298] As shown in FIGs. 18F-18G, the anti -IL-3 l-anti-IL-4Ra vHH bispecific antibody reduced MC903-induced serum IgE and TARC (CCL17) levels, similarly to Dupixent and outperforming Nemolizumab and NM26-2198.

[0299] As shown in FIG. 18H, the anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody reduced MC903-induced ear inflammation, outperforming other competitors.6.21 Example 21: Efficacy of Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody in the Human IL-31-induced Pruritus Model in huIL4 / IL4RA / IL-31 / IL-31RA / OSMRp KI Mice

[0300] We used huIL4 / IL4RA / IL-3 l / IL-3 IRA / OSMRp KI C57BL / 6 mice to establish a human IL-31 -induced mouse itch model to evaluate the efficacy of anti -IL-3 l-anti-IL-4Ra vHH bispecific antibody. Specifically, to establish the baseline, mice were intradermally injected with PBS and the scratching monitoring device was used to record the number of scratches and rounds of the mice themselves for 3 hours, and the number of scratches and rounds of the mice themselves were recorded. Next, the mice were intraperitoneally injected with the anti-IL-3 l-anti-IL-4Ra vHH bispecific antibodies or control antibodies (Dupixent or Nemolizumab analogue). On the following day, the mice were intradermally injected with human IL-31 protein, and the scratching monitoring device was used to record half an hour before the injection of human IL-31 protein to exclude abnormal itching of the mice, and then the scratching monitoring device was used to record the number of scratches and rounds of the mice after the injection of human IL-31 protein, and the recording was continued for 3 hours. The data was then sorted and statistically analyzed to reflect the itch inhibition effect of the antibody.

[0301] As shown in FIGs. 19A-19E, the anti-IL-31 -anti -IL-4Ra vHH bispecific antibody and Nemolizumab analogue, but not Dupixent antibody, reduced IL-31 -induced scratching in huIL4 / IL4RA / IL-3 l / IL-3 IRA / OSMRp KI mice.6.22 Example 22: Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody Demonstrated Comparable Ability to NM26-2198 on Inhibiting IL-4 or IL-13-Induced Downstream STAT6 Phosphorylation in a Dose-Dependent Manner

[0302] The HEK-Blue IL-4 / IL-13 reporter cells (InvivoGen) stably express the IL- 4Ra / IL-13Ral receptor complex on their surface along with human STAT6 and SEAP genes. Upon stimulation with IL-4 / IL-13, activation of the STAT6 phosphorylation signal downstream of the receptor leads to the induction of the SEAP reporter gene expression. For the assay, HEK-blue IL-4 / IL-13 reporter gene cells were trypsinized, washed once with1 *PBS, resuspended in DMEM medium supplemented with 10% inactivated FBS, adjusted to a density of 5 x io5cells / mL, and 100 pL / well was seeded into a 96-well flat-bottom culture plate. Testing antibodies were diluted to an initial concentration of 24 nM with DMEM medium containing 10% FBS and subjected to a 3 -fold gradient dilution across 9 points, with 50 pL / well added to the 96-well flat-bottom culture plate. Human IL- 13 protein (Aero) or human IL-4 protein (Aero) were diluted to specific concentrations (IL-4: 0.4 ng / mL, IL-13: 80 ng / mL) in DMEM medium with 10% inactivated FBS, and 50 pL / well was added to the plate. The cells were then incubated for 16-20 hours at 37°C in a 5% CO2 incubator. Post-incubation, a new 96-well flat-bottom plate was prepared with 180 pL of QUANTLBlue Solution (InvivoGen) in each well, followed by the addition of 20 pL of the cell supernatant from the incubated cultures. This plate was incubated at 37°C in the dark for 4-6 hours, after which the absorbance was measured at 620-655 nm using a SpectraMax Plate Reader.

[0303] As shown in FIGs. 20A-20B, the anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody demonstrated comparable ability to NM26-2198 on inhibiting IL-4 or IL- 13 induced downstream STAT6 phosphorylation in a dose- dependent manner.6.23 Example 23: Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody Blocked IL- 31 / IL-31RA Protein Interaction in Comparison with NM26-2198

[0304] The nucleotide sequence encoding the full-length human IL-3 IRA amino acid sequence (UniProt Entry: Q8NI17) was cloned into the PLVX-puro vector (acquired from Addgene) using double enzyme digestion approach with EcoRI and Hindlll. Lentiviral particles were packaged in 293T cells and utilized to infect CHO cells. Post-transfection, the cells were subjected to selection pressure with puromycin (procured from Invitrogen) to isolate and expand stably overexpressing human IL-3 IRA CHO cells, designated as CHO- huIL-3 IRA cells. For the antibody testing, antibodies were diluted in PBS with a 3-fold gradient dilution starting from 400 nM, across a total of 12 points. A volume of 60 pL / well was added to a 96-well sample plate, and 60 pL / well of biotin-labeled human IL-31 protein (in-house) diluted in PBS was added to the same plate. The plate was incubated at room temperature in dark for 30 minutes. Concurrently, the density of CHO-huIL-3 IRA cells cultured in expansion was adjusted to 2 x io6cells / mL, and 100 pL / well was aliquoted into a 96-well flow plate and centrifuged for subsequent use. Following incubation, 100 pL / well of the pre-incubated samples were transferred to the cell-containing wells and incubated at 4°C for an additional 30 minutes. The wells were then washed twice with PBS, and theappropriate concentration of fluorescent secondary antibody was added, followed by a 20- minute incubation at 4°C. After two further washes with PBS, the cells were resuspended in PBS, and the samples were analyzed using the CytoFlex flow cytometer. The mean fluorescence intensity (MFI) was subsequently calculated to quantify the binding signal.

[0305] As shown in FIG. 21, both the anti-IL-3 l-anti-IL-4Ra vHH bispecific antibody and NM26-2198 demonstrated dose-dependent inhibition of IL-31 binding to CHO-huIL- 3 IRA cells, and NM26-2198 was less effective, unable to reduce the binding to baseline levels even at the highest doses tested.6.24 Example 24: Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody Inhibited IL-31- Induced Downstream STAT3 Phosphorylation in Comparison with NM26-2198

[0306] The full-length human IL-3 IRA sequence (UniProt: Q8NI17) was cloned into the PLVX-puro vector via EcoRI and Hindlll digestion, packaged into lentiviral particles using 293T cells, and used to transduce 293T cells. Puromycin selection yielded stable IL-31RA- expressing cells (293T-huIL-31RA). Additionally, the full-length human OSMRP sequence (UniProt: Q99650) was inserted into the PMS061-2-h-hygro vector via Agel and Nhel digestion. Lentivirus produced in 293T cells was used to infect 293T-huIL-3 IRA cells. Hygromycin selection generated stable double-expressing 293T-huIL-31RA / OSMRp cells. For the bispecific antibody analysis, the purified antibody was diluted in 1 *PBS with a 3-fold dilution series from 400 nM for a total of 10 points. Human IL-31 protein (Kactus) diluted in 1 *PBS was added to the plate and incubated at room temperature for 30 minutes. 293T-huIL- 3 IRA / OSMRp cells were adjusted to a density of 2.0 * 106cells / mL in culture medium, and 100 pL / well was added to a 96-well flow plate. 100 pL / well of the incubated sample was added to the cell-containing wells and incubated at 4°C for 10 minutes. After wash with I xPBS, 4% paraformaldehyde was added to 100 pL / well and incubated at room temperature for 10 minutes. Following l x wash with I xPBS, 100 pL / well of pre-cooled methanol was added and incubated at 4°C for 30 minutes. After washes with 1 x wash buffer, 100 pL / well of pSTAT3 flow cytometry antibody (1 :200 dilution) in 1 x wash buffer was added and incubated at 4°C for 20 minutes. After final washes with 1 x wash buffer, the cells were resuspended in 100 pL / well of 1 x wash buffer and analyzed on the CytoFlex flow cytometer, with the corresponding mean fluorescence intensity (MFI) calculated.

[0307] As shown in FIG. 22, the IL-3 l / IL-4Ra vHH bispecific antibody exhibited dosedependent inhibition of IL-31 -induced STAT3 activation (phosphorylation) to the baselinelevel, and NM26-2198 was not able to achieve inhibition to baseline levels even at high doses.6.25 Example 25: Anti-IL-31-anti-IL-4Ra vHH Bispecific Antibody Inhibited IL-4 Plus IL-31-Induced CCL2 Expression in Comparison with NM26-2198

[0308] BEAS-2B cells (ATCC; CRL-3588) were first trypsinized to detach them from the culture vessel. They were then resuspended in culture medium and adjusted to a concentration of 3.0 x 105cells / Ml. Next, 100 pL of this cell suspension was aliquoted into each well of a 96-well flat-bottom cell culture plate and incubated overnight to allow the cells to adhere. Testing antibodies were prepared by diluting them in culture medium to initiate a 3-fold dilution series starting at 200 nM, spanning a total of 9 concentration points. Then, 100 pL of each diluted antibody solution was added to the corresponding wells of the 96-well plate. Additionally, human IL-4 and human IL-31 proteins were diluted in culture medium to concentrations of 4 ng / mL and 50 ng / mL, respectively. These cytokines, 100 pL each, were added to the wells to reach a final volume of 200 pL per well. The plate was then incubated in a 37°C, 5% CO2 incubator for 24 hours. Following the incubation period, the supernatant from each well was carefully collected, and the CCL2 content was quantified using a human CCL2 ELISA kit, according to the manufacturer's instructions (InvitroGen).

[0309] As shown in FIG. 23, the IL-3 l / IL-4Ra vHH bispecific antibody demonstrated dose-dependent inhibition of IL-4 plus IL-31 induced CCL2 expression in BEAS-2B cells, and outperformed NM26-2198 in reducing CCL2 expression levels induced by IL-4 and IL- 31 in BEAS-2B cells.7. EQUIVALENTS

[0310] The binding agents, nucleic acids, methods, host cells, and compositions disclosed herein are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the binding agents, nucleic acids, methods, host cells, and compositions in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0311] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.8. SEQUENCES

Claims

1. WHAT IS CLAIMED:

1. A multi specific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain; and(ii) an interleukin 31 (IL-3 l)-binding domain, wherein the first IL-4Ra-binding domain comprises (a) a VHH comprising a complementarity-determining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) as set forth in the amino acid sequence of SEQ ID NO: 12; or (b) a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

2. A multi specific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain; and(ii) an interleukin 31 (IL-3 l)-binding domain, wherein the IL-31 -binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:4 and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO:8.

3. A multi specific binding agent comprising:(i) a first interleukin-4 receptor alpha (IL-4Ra)-binding domain;(ii) an interleukin 31 (IL-31)-binding domain; and(iii) an Fc region comprising one or more amino acid substitutions as compared to a wildtype Fc region, wherein the one or more amino acid substitutions are capable of depleting Fc- gamma receptor (FcyR) binding or enhancing neonatal Fc receptor (FcRn) binding.

4. The multispecific binding agent of claim 1 or 2 further comprising an Fc region.

5. The multispecific binding agent of any one of claims 2 to 4, wherein the IL-31- binding domain comprises divalent antibody fragments (F(ab’)2 fragments) comprising the VH and the VL.

6. The multispecific binding agent of any one of claims 3 to 5, wherein the IL-31- binding domain is linked to the Fc region.

7. The multispecific binding agent of any one of claims 3 to 6, wherein the C-terminus of the IL-31-binding domain is linked to the N-terminus of the Fc region.

8. The multispecific binding agent of any one of claims 1 to 7, wherein the multispecific binding agent further comprises a second IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL.

9. The multispecific binding agent of any one of claims 3 to 8, wherein the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region.

10. The multispecific binding agent of any one of claims 3 to 9, wherein the N-terminus of the VHH or VH of the first or second IL-4Ra-binding domain is linked to the C-terminus of the Fc region.

11. The multispecific binding agent of claim 3 to 10, wherein the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO: 58.

12. The multispecific binding agent of any one of claims 3 to 11, wherein the VHH or VH of the first or second IL-4Ra-binding domain is linked to the Fc region via a linker comprising the amino acid sequence of SEQ ID NO: 13.

13. The multispecific binding agent of any one of claims 1 to 12, wherein the first or second IL-4Ra-binding domain comprises (a) a VHH comprising a complementaritydetermining region 1 (CDR1), a complementarity-determining region 2 (CDR2), and a complementarity-determining region 3 (CDR3) as set forth in the amino acid sequence of SEQ ID NO: 12; or (b) a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 12 and a VL.

14. The multispecific binding agent any one of claims 1 to 13, wherein the first or second IL-4Ra-binding domain comprises a VHH or VH comprising:(i) a CDR1 comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO:48, SEQ ID N0:51, SEQ ID NO:53, or SEQ ID NO:55;(ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO:49, SEQID NO:52, SEQ ID NO:54, or SEQ ID NO:56; and(iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO:50, or SEQ ID NO:57.

15. The multispecific binding agent of any one of claims 1 to 14, wherein the first or second IL-4Ra-binding domain comprises a VHH or VH comprising:(i) a CDR1 comprising the amino acid sequence of SEQ ID NO:9, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11;(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:48, a CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50;(iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:51, a CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50;(iv) a CDR1 comprising the amino acid sequence of SEQ ID NO:53, a CDR2 comprising the amino acid sequence of SEQ ID NO:54, and a CDR3 comprising the amino acid sequence of SEQ ID NO:50; or(v) a CDR1 comprising the amino acid sequence of SEQ ID NO:55, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO:57.

16. The multispecific binding agent of any one of claims 1 to 15, wherein the first or second IL-4Ra-binding domain comprises a VHH or VH comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 12.

17. The multispecific binding agent of any one of claims 1 to 16, wherein the first or second IL-4Ra-binding domain comprises a VHH or VH comprising the amino acid sequence of SEQ ID NO: 12.

18. The multispecific binding agent of any one of claims 1 to 17, wherein the IL-31- binding domain comprises a VH comprising a CDR1, a CDR2, and a CDR3 as set forth in theamino acid sequence of SEQ ID NON and a VL comprising a CDR1, a CDR2, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 8.

19. The multispecific binding agent any one of claims 1 to 18, wherein the IL-31 -binding domain comprises:(i) a VH comprising:(a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:40, or SEQ ID NO:42;(b) a CDR2 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:34, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:43; and(c) a CDR3 comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:35, or SEQ ID NO:44; and(ii) a VL comprising:(a) a CDR1 comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:36, or SEQ ID NO:45;(b) a CDR2 comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:37, or SEQ ID NO:46; and(c) a CDR3 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:47.

20. The multispecific binding agent of any one of claims 1 to 19, wherein the IL-31- binding domain comprises:(i) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(ii) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:33, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(iii) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:38, a CDR2 comprising the amino acid sequence of SEQ ID NO:39, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the aminoacid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7;(iv) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:40, a CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7; or(v) a VH comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:42, a CDR2 comprising the amino acid sequence of SEQ ID NO:43, and a CDR3 comprising the amino acid sequence of SEQ ID NO:44; and a VL comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:47.

21. The multispecific binding agent of any one of claims 1 to 20, wherein the IL-31- binding domain comprises:(i) a VH comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NON; and(ii) a VL comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 8.

22. The multispecific binding agent of any one of claims 1 to 21, wherein the IL-31- binding domain comprises:(i) a VH comprising the amino acid sequence of SEQ ID NON; and(ii) a VL comprising the amino acid sequence of SEQ ID NO:8.

23. The multispecific binding agent of any one of claims 1 to 22 further comprising a third IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL.

24. The multispecific binding agent of any one of claims 1 to 23 further comprising a fourth IL-4Ra-binding domain comprising (a) a VHH or (b) a VH and a VL.

25. The multispecific binding agent of claim 23 or 24, wherein the third or fourth IL-4Ra- binding domain is the same as the first or second IL-4Ra-binding domain.

26. The multispecific binding agent of any one of claims 23 to 25, wherein the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL- 4Ra-binding domain.

27. The multispecific binding agent of any one of claims 23 to 26, wherein the N- terminus of the VHH of the third or forth IL-4Ra-binding domain is linked to the C-terminus of the VHH of the first or second IL-4Ra-binding domain28. The multispecific binding agent of any one of claims 23 to 27, wherein the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL- 4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO:58.

29. The multispecific binding agent of any one of claims 23 to 28, the VHH of the third or fourth IL-4Ra-binding domain is linked to the VHH of the first or second IL-4Ra-binding domain via a linker comprising the amino acid sequence of SEQ ID NO: 13.

30. The multispecific binding agent of any one of claims 4 to 29, wherein the Fc region comprises one or more amino acid substitutions as compared to a wild-type Fc region, wherein the one or more amino acid substitutions are capable of depleting Fc-gamma receptor (FcyR) binding or enhancing neonatal Fc receptor (FcRn) binding.

31. The multispecific binding agent of any one of claims 3 to 30, wherein the Fc region comprises L234A, L235A, G237A, M428L and N434A amino acid substitutions as compared to a wild-type Fc region, wherein numbering is according to the EU numbering system.

32. The multispecific binding agent of any one of claims 8 to 31 comprising:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, and the VHH of the first or second IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.

33. The multispecific binding agent of any one of claims 23 to 32 comprising:(i) a first polypeptide comprising, from N-terminus to C-terminus, a first VL and a first CL;(ii) a second polypeptide comprising, from N-terminus to C-terminus, a first VH, a first CHI, a first CH2, a first CH3, the VHH of the first or second IL-4Ra-binding domain, and the VHH of the third or fourth IL-4Ra-binding domain;(iii) a third polypeptide comprising, from N-terminus to C-terminus, a second VH, a second CHI, a second CH2, a second CH3, and the VHH of the first or second IL-4Ra- binding domain, and the VHH of the third or fourth IL-4Ra-binding domain; and(iv) a fourth polypeptide comprising, from N-terminus to C-terminus, a second VL and a second CL; wherein the first VL, the first VH, the second VH, and the second VL form the IL-31 -binding domain, and wherein the first CH2, the second CH2, the first CH3, and the second CH3 form the Fc region.

34. A multi specific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 14; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

35. A multi specific binding agent comprising:(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15;(ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 18;(iii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 18; and(iv) a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

36. The multispecific binding agent of claim 34 or 35, wherein the first polypeptide and the second polypeptide are linked to each other via one or more disulfide bonds, wherein the second polypeptide and the third polypeptide are linked to each other linked via one or more disulfide bonds, and wherein the third polypeptide and the fourth polypeptide are linked to each other linked via one or more disulfide bonds.

37. A polynucleotide or polynucleotides encoding the multispecific binding agent or a portion thereof of any one of claims 1 to 36.

38. A vector or vectors comprising the polynucleotide or polynucleotides of claim 37 or a complementary polynucleotide or polynucleotides thereto.

39. A cell comprising the multispecific binding agent of any one of claims 1 to 36, the polynucleotide or polynucleotides of claim 37, or the vector or vectors of claim 38.

40. A pharmaceutical composition comprising the multispecific binding agent of any one of claims 1 to 36, the polynucleotide or polynucleotides of claim 37, or the vector or vectors of claim 38, or the cell of claim 39, and a pharmaceutically acceptable excipient.

41. A kit comprising the multispecific binding agent of any one of claims 1 to 36, the polynucleotide or polynucleotides of claim 37, or the vector or vectors of claim 38, or the cell of claim 39.

42. A method for treating a disease or disorder in a subject comprising administering to the subject the multispecific binding agent of any one of claims 1 to 36, the polynucleotide or polynucleotides of claim 37, or the vector or vectors of claim 38, or the cell of claim 39.

43. The method of claim 42, wherein the disease or disorder is atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

44. The multispecific binding agent of any one of claims 1 to 36 for use in treating a disease or disorder, wherein the disease or disorder is atopic dermatitis (AD), prurigonodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

45. The use of the multispecific binding agent of any one of claims 1 to 36 in the manufacture of a medicament for treating a disease or disorder, wherein the disease or disorder is atopic dermatitis (AD), prurigo nodularis (PN), chronic spontaneous urticaria (CSU), bullous pemphigoid (BP), acute allergic contact dermatitis (ACD), eczema, alopecia areate (AA), dermatomyositis, or atopic asthma.

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