Recombinant bispecific antibodies targeting TSLP and IL4R

Abstract

The present disclosure provides a recombinant bispecific antibody comprising i) a TSLP-binding domain and ii) an IL4R-binding domain. The present disclosure provides a nucleic acid molecule encoding the recombinant bispecific antibody, a vector comprising the nucleic acid, and a host cell transformed or transfected with the vector or such a nucleic acid molecule. The present disclosure also provides a method for producing the recombinant bispecific antibody, medical uses of the recombinant bispecific antibody, and a kit comprising the recombinant bispecific antibody.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure generally relates to recombinant bispecific antibodies capable of binding to TSLP and IL4R with high affinity and high functionality. The present disclosure also provides nucleic acid molecules encoding the recombinant bispecific antibodies, expression vectors, host cells, and methods for expressing the recombinant bispecific antibodies. Furthermore, the present disclosure provides pharmaceutical compositions that may contain the recombinant bispecific antibodies and methods of treatment using the pharmaceutical compositions. [Background technology]

[0002] TSLP Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine. It is closely related to IL-7 and shares an overlapping but distinct biological profile with IL-7. TSLP binds to a heterodimeric receptor complex composed of the TSLP receptor chain (TSLPR, also known as CRLF2) and the IL-7 receptor α chain (IL-7Rα). TSLP mRNA is expressed primarily by epithelial cells of the thymus, lung, skin, intestine, and tonsils, as well as by stromal cells and mast cells. On the other hand, TSLPR mRNA is found in many immune cell types, including dendritic cells (DCs), T cells, B cells, mast cells, NK cells, and monocytes (Rui He et al., (2010) Annals of the New York Academy of Sciences 1183:13-24; Quentmeier H et al., (2001) Leukemia 15(8):1286-1292; Rimoldi M et al., (2005) Nature Immunology 6(5):507-514).

[0003] TSLP-triggered signaling pathways have been extensively studied. For example, TSLP protein may induce DC polarization to promote T helper (Th)2 cell differentiation and Th2 cytokine production during the induction phase of immune responses. It may also directly promote T cell proliferation and amplify Th2 cytokine secretion. Therefore, TSLP is considered a key regulator of Th2-driven inflammation, and upregulation of TSLP has been linked to the pathogenesis of Th2-related diseases such as atopic dermatitis and asthma (Rui He et al., (2010) supra; Ito T et al., (2005) The Journal of Experimental Medicine 202(9):1213-1223; He R et al., (2008) Proc Natl Acad Sci USA 105(33):11875-11880). TSLP also mediates several immune homeostatic functions in the intestine and thymus. For example, TSLP is upregulated in intestinal epithelial cell lines in a strain-dependent manner by bacterial stimulation and synergizes with transforming growth factor β to promote Treg cell differentiation. TSLP also acts on CD103 + It is also produced by primary human intestinal epithelial cells to condition DCs to a tolerogenic phenotype (Katerina Tsilingiri et al., (2017) Cellular and Molecular Gastroenterology and Hepatology 3(2):174-182; Zeuthen LH et al., (2008) Immunology 123:197-208; Iliev ID et al., (2009) Gut 58:1481-1489).

[0004] Due to the dual role of TSLP in the immune system, two isoforms have been discovered: a long isoform and a short isoform consisting of the terminal 63 amino acid residues of the long isoform. These two isoforms are controlled by different promoter regions and are expressed in different contexts, tissues, and stimuli (Harada M et al. (2011) American Journal of Respiratory Cell and Molecular Biology 44:787-793). The expression patterns of these isoforms have also been studied in several TSLP-related diseases. For example, overexpression of long TSLP has been observed in asthma, ulcerative colitis, atopic dermatitis, and psoriasis, while decreased expression of long TSLP has been observed in celiac disease. Furthermore, expression of short TSLP has been downregulated in Crohn's disease, celiac disease, and atopic dermatitis (Katerina Tsilingiri et al. (2017) supra; Fornasa G et al. (2015) J Allergy Clin Immunol 136:413-422).

[0005] IL-4R Interleukin-4 (IL-4) and IL-13 are central factors in type 2 immunity and are required to drive most of the key features associated with type 2 inflammation, including immunoglobulin E production and recruitment of innate cells to the inflammatory site (Gruning G et al. (1998) Science 282:2261-2263; Rankin JA et al. (1996) Proc Natl Acad Sci USA 93:7821-7825; Wills-Karp M et al. (1998) Science 282:2258-2261).

[0006] These two cytokines bind to cell surface receptors, regulating cellular functions and activating transcriptional machinery. Specifically, IL-4 first binds to the IL-4Rα chain with picomolar affinity and recruits the IL-2Rγc (γc) chain to form the type I IL-4 receptor complex, or recruits the IL-13Rα1 chain to form the type II IL-4 receptor complex. The levels or availability of IL-2Rγ and IL-13Rα1 determine which are recruited in receptor complex formation. Formation of the type II IL-4 receptor complex can also be initiated by IL-13 binding to the IL-13Rα1 chain with nanomolar affinity, resulting in the further recruitment of the IL-4Rα chain.

[0007] Upon assembly of the IL-4 receptor complex, intracellular signaling molecules are activated, such as STAT6 (signal transducer and activator of transcription 6) and IRS (insulin receptor substrate) signaling, which responds to type I IL-4 receptor activation (Heller NM et al., (2008) Sci Signal 1(51):ra17-ra17). STAT6 signaling is important for TH2 cell differentiation and IL-4 production (Gadani SP et al., (2012) J Immunol 189:4213-4219), and IRS molecules activate signaling pathways including PI3K and mTOR (Gadani SP et al., (2012) J Immunol 189:4213-4219).

[0008] Excessive IL-4 / IL-13 signaling has been suggested as a possible cause of allergic diseases, and several therapeutic antibodies have been developed to modify IL-4 and IL-13 signaling. For example, leplikizumab, anrukinzumab, and tralokinumab bind to IL-13, and pascolizumab targets IL-4. Dupilumab is a fully human monoclonal antibody directed against IL-4Rα that inhibits both IL-4 and IL-13 signaling and has been approved for the treatment of patients with type 2 inflammatory diseases, including atopic dermatitis (AD), asthma, and chronic rhinosinusitis with nasal polyposis (CRSwNP) (Haddad, E.B. et al., (2022) Dermatol Ther 12:1501-1533). Furthermore, it has been reported that STAT6 inhibitors suppress the proliferation of prostate cancer cells, suggesting that targeting IL-4 / IL-13 may also be beneficial in cancer therapy (Nappo G et al., (2017) Oncogenesis 2017, 6(5):e342).

[0009] Bispecific or multispecific antibodies Because some monospecific antibodies or proteins have limited therapeutic efficacy, bispecific or trispecific antibodies have been gradually developed in recent years and have shown promising efficacy in preclinical and clinical trials.

[0010] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0011] The inventors of the present application have designed and prepared a recombinant bispecific antibody that targets both TSLP and IL4R. Compared to prior art monoclonal anti-IL4R or anti-TSLP antibodies, such as dupilumab and tezepelumab, the antibody has: i) equivalent binding affinity / activity to TSLP and / or IL4R; ii) equivalent inhibitory activity against IL4-IL4R binding / interaction and / or TSLP-TSLPR / IL7Rα binding / interaction; and / or iii) equivalent or greater inhibitory activity against TSLP- and / or IL4-induced or mediated signal transduction. In particular, the recombinant bispecific antibody of the present disclosure exhibits significantly greater inhibitory activity against TSLP+IL4-induced signal transduction, including CCL-17 production by PBMCs, than monospecific anti-IL4R or anti-TSLP antibodies, and even the combination of dupilumab and tezepelumab.

[0012] The recombinant bispecific antibodies of the present disclosure can be used in in vitro and in vivo assays and for the treatment of diseases associated with TSLP, IL4 and / or IL13 signaling, such as inflammatory diseases.

[0013] In a first aspect, the present disclosure provides a recombinant bispecific antibody that binds to both TSLP and IL4R, which may comprise a TSLP-binding domain and an IL4R-binding domain.

[0014] The TSLP-binding domain can be, for example, an anti-TSLP antibody, such as an IgG antibody, or an antigen-binding site thereof, such as a Fab, Fv, single-chain variable region (scFv), etc. In certain embodiments, the TSLP-binding domain is antagonistic.

[0015] The IL4R-binding domain may be, for example, an anti-IL4R antibody, such as an IgG antibody, or an antigen-binding site thereof, such as a Fab, Fv, or single-chain variable region (scFv). In certain embodiments, the IL4R-binding domain is antagonistic.

[0016] The recombinant bispecific antibodies of the present disclosure comprise: i) an anti-TSLP antibody or antigen-binding portion thereof, comprising an anti-TSLP heavy chain variable region, an anti-TSLP light chain variable region, a heavy chain constant region, and a light chain constant region; ii) anti-IL4R single chain variable region (scFv) and may include:

[0017] The anti-IL4R scFv can be linked to the N-terminus of the anti-TSLP heavy chain variable region, the N-terminus of the anti-TSLP light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region.

[0018] The recombinant bispecific antibodies of the present disclosure comprise: i) an anti-IL4R antibody or antigen-binding portion thereof, which may comprise an anti-IL4R heavy chain variable region, an anti-IL4R light chain variable region, a heavy chain constant region, and a light chain constant region; ii) anti-TSLP single-chain variable domain (scFv) and may include:

[0019] The anti-TSLP scFv can be linked to the N-terminus of the anti-IL4R heavy chain variable region, the N-terminus of the anti-IL4R light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region.

[0020] In certain embodiments, the recombinant bispecific antibody of the present disclosure: i) an antagonistic anti-TSLP antibody comprising an anti-TSLP heavy chain variable region, a heavy chain constant region, an anti-TSLP light chain variable region, and a light chain constant region; ii) an antagonistic anti-IL4R single-chain variable domain (scFv) comprising an anti-IL4R heavy chain variable domain and an anti-IL4R light chain variable domain; may include:

[0021] The anti-IL4R scFv can be linked to the N-terminus of the anti-TSLP heavy chain variable region, the N-terminus of the anti-TSLP light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the anti-TSLP light chain constant region.

[0022] Alternatively, the recombinant bispecific antibody of the present disclosure comprises: i) an antagonistic anti-IL4R antibody comprising an anti-IL4R heavy chain variable region, a heavy chain constant region, an anti-IL4R light chain variable region, and a light chain constant region; and ii) an antagonistic anti-TSLP single-chain variable domain (scFv) comprising an anti-TSLP heavy chain variable domain and an anti-TSLP light chain variable domain; may include:

[0023] The anti-TSLP scFv can be linked to the N-terminus of the anti-IL4R heavy chain variable region, the N-terminus of the anti-IL4R light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the anti-IL4R light chain constant region.

[0024] In certain embodiments, the recombinant bispecific antibody of the present disclosure: i) a first polypeptide chain and a second polypeptide chain comprising an anti-IL4R heavy chain variable region, a heavy chain constant region, and an anti-TSLP scFv, respectively; and ii) a third polypeptide chain and a fourth polypeptide chain comprising an anti-IL4R light chain variable region and a light chain constant region, respectively; i) a first polypeptide chain and a second polypeptide chain comprising an anti-IL4R heavy chain variable region and a heavy chain constant region, respectively; and ii) a third polypeptide chain and a fourth polypeptide chain comprising an anti-IL4R light chain variable region, a light chain constant region, and an anti-TSLP scFv, respectively; i) a first polypeptide chain and a second polypeptide chain comprising an anti-TSLP heavy chain variable region, a heavy chain constant region, and an anti-IL4R scFv, respectively; and ii) a third polypeptide chain and a fourth polypeptide chain comprising an anti-TSLP light chain variable region and a light chain constant region, respectively; or i) a first polypeptide chain and a second polypeptide chain comprising an anti-TSLP heavy chain variable region and a heavy chain constant region, respectively; and ii) a third polypeptide chain and a fourth polypeptide chain comprising an anti-TSLP light chain variable region, a light chain constant region, and an anti-IL4R scFv, respectively; may include the anti-IL4R heavy chain variable region in the first polypeptide chain and the anti-IL4R light chain variable region in the third polypeptide chain bind to form an IL4R-binding domain, and the anti-IL4R heavy chain variable region in the second polypeptide chain and the anti-IL4R light chain variable region in the fourth polypeptide chain bind to form an IL4R-binding domain, the anti-TSLP heavy chain variable region in the first polypeptide chain binds to the anti-TSLP light chain variable region in the third polypeptide chain to form a TSLP-binding domain, and the anti-TSLP heavy chain variable region in the second polypeptide chain binds to the anti-TSLP light chain variable region in the fourth polypeptide chain to form a TSLP-binding domain; The heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are linked to each other.

[0025] In a specific embodiment, the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R scFv, an optional linker, an anti-TSLP heavy chain variable region, and a heavy chain constant region; the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP light chain variable region and a light chain constant region; and the anti-IL4R scFvs of the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R heavy chain variable region, an optional linker, and an anti-IL4R light chain variable region, or an anti-IL4R light chain variable region, an optional linker, and an anti-IL4R heavy chain variable region, respectively.

[0026] In a specific embodiment, the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP heavy chain variable region and a heavy chain constant region; the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R scFv and an optional linker; and the anti-IL4R scFvs of the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R heavy chain variable region, an optional linker, and an anti-IL4R light chain variable region, or an anti-IL4R light chain variable region, an optional linker, and an anti-IL4R heavy chain variable region, respectively.

[0027] In a specific embodiment, the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP heavy chain variable region, a heavy chain constant region, an optional linker, and an anti-IL4R scFv; the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP light chain variable region and a light chain constant region; and the anti-IL4R scFv of the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R heavy chain variable region, an optional linker, and an anti-IL4R light chain variable region, or an anti-IL4R light chain variable region, an optional linker, and an anti-IL4R heavy chain variable region, respectively.

[0028] In a specific embodiment, the first and second polypeptide chains each comprise, from N- to C-terminus, an anti-TSLP scFv, an optional linker, an anti-IL4R heavy chain variable region, and a heavy chain constant region; the third and fourth polypeptide chains each comprise, from N- to C-terminus, an anti-IL4R light chain variable region and a light chain constant region; and the anti-TSLP scFv of the first and second polypeptide chains each comprise, from N- to C-terminus, an anti-TSLP heavy chain variable region, an optional linker, and an anti-TSLP light chain variable region, or an anti-TSLP light chain variable region, an optional linker, and an anti-TSLP heavy chain variable region, respectively.

[0029] In a specific embodiment, the first and second polypeptide chains each comprise, from N-terminus to C-terminus, an anti-IL4R heavy chain variable region and a heavy chain constant region; the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP scFv, an optional linker, an anti-IL4R light chain variable region, and a light chain constant region; and the anti-TSLP scFvs of the third and fourth polypeptide chains each comprise, from N-terminus to C-terminus, an anti-TSLP heavy chain variable region, an optional linker, and an anti-TSLP light chain variable region, or an anti-TSLP light chain variable region, an optional linker, and an anti-TSLP heavy chain variable region, respectively.

[0030] In a specific embodiment, the first and second polypeptide chains each comprise, from N- to C-terminus, an anti-IL4R heavy chain variable region, a heavy chain constant region, an optional linker, and an anti-TSLP scFv; the third and fourth polypeptide chains each comprise, from N- to C-terminus, an anti-IL4R light chain variable region and a light chain constant region; and the anti-TSLP scFv of the first and second polypeptide chains each comprise, from N- to C-terminus, an anti-TSLP heavy chain variable region, an optional linker, and an anti-TSLP light chain variable region, or an anti-TSLP light chain variable region, an optional linker, and an anti-TSLP heavy chain variable region, respectively.

[0031] The anti-TSLP binding domain may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region may comprise a VH CDR1, a VH CDR2, and a VH CDR3, which may comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. The heavy chain variable region may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. The light chain variable region may comprise a VL CDR1, a VL CDR2, and a VL CDR3, which may comprise the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The light chain variable region may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:8.

[0032] The anti-IL4R binding domain may comprise a heavy chain variable region and a light chain variable region. The heavy chain variable region may comprise a VH CDR1, a VH CDR2, and a VH CDR3, which may comprise the amino acid sequences of SEQ ID NOS: 11, 12, and 13, respectively. The heavy chain variable region may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS: 17. The light chain variable region may comprise a VL CDR1, a VL CDR2, and a VL CDR3, which may comprise the amino acid sequences of SEQ ID NOS: 14, 15, and 16, respectively. The light chain variable region may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18.

[0033] The anti-TSLP binding domain and the anti-IL4R binding domain may optionally comprise heavy chain and light chain constant regions, respectively.

[0034] The heavy chain constant region may be an IgG1, IgG2, or IgG4 heavy chain constant region, such as a human IgG1, IgG2, or IgG4 heavy chain constant region, or a functional fragment thereof, such as an Fc fragment. The heavy chain constant region may be naturally occurring or may be engineered to have specific desired properties. In certain embodiments, the heavy chain constant region has reduced or eliminated binding affinity for Fc receptors and / or complement system proteins. In certain embodiments, the heavy chain constant region may comprise the amino acid sequence of SEQ ID NO: 9 or 29. The light chain constant region may be a kappa or lambda light chain constant region. In certain embodiments, the light chain constant region may be, for example, a human kappa light chain constant region having the amino acid sequence of SEQ ID NO: 10.

[0035] The optional linker may be a peptide linker consisting of 5 to 30, 10 to 30, 10 to 20, or 15 amino acids. The linker may be a GS linker such as -(Gly-Gly-Gly-Gly-Ser)- (SEQ ID NO: 19) or -(Gly-Gly-Gly-Gly-Ser)- (SEQ ID NO: 20). In a specific embodiment, the linker in the scFv is -(Gly-Gly-Gly-Gly-Ser)- (SEQ ID NO: 20). In a specific embodiment, the linker between the scFv and the IgG heavy chain / light heavy chain is -(Gly-Gly-Gly-Gly-Ser)- (SEQ ID NO: 19).

[0036] In certain embodiments, the recombinant bispecific antibodies of the present disclosure are selected from the group consisting of: i) SEQ ID NOs: 21, 21, 39, and 39, respectively; ii) SEQ ID NOs: 22, 22, 39, and 39, respectively; iii) SEQ ID NOs: 23, 23, 40, and 40, respectively; iv) SEQ ID NOs: 24, 24, 40, and 40, respectively; v) SEQ ID NOs: 25, 25, 40, and 40, respectively; vi) SEQ ID NOs: 26, 26, 40, and 40, respectively; vii) SEQ ID NOs: 2 or viii) a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 28, 28, 39, and 39, respectively.

[0037] The present application also provides nucleic acid molecules encoding the recombinant bispecific antibodies of the present disclosure, as well as expression vectors that may contain such nucleic acids, and host cells that may contain such expression vectors or such nucleic acid molecules, or alternatively, have nucleic acids of the present disclosure integrated into their genome. Methods for preparing recombinant bispecific antibodies of the present disclosure using host cells are also provided and may include (i) expressing the recombinant bispecific antibody in the host cell, and (ii) isolating the recombinant bispecific antibody from the host cell or a cell culture thereof.

[0038] The present application also provides pharmaceutical compositions that may comprise the recombinant bispecific antibodies, nucleic acid molecules, expression vectors, or host cells of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions may further comprise an additional agent, such as an anti-inflammatory agent.

[0039] The present application also provides kits that may include the recombinant bispecific antibodies, nucleic acid molecules, expression vectors, or host cells of the present disclosure.

[0040] In a second aspect, the present application provides a method for treating a disease associated with TSLP and / or IL4 / IL13 induced or mediated signaling in a subject in need thereof, which may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0041] The disease may be an inflammatory disease, such as an allergic disease, or an autoimmune disease, including, but not limited to, atopic dermatitis, asthma, chronic rhinosinusitis, nasal polyps, and eosinophilic esophagitis.

[0042] In certain embodiments, the subject is a human.

[0043] The present disclosure also provides methods for reducing or eliminating excessive immune responses associated with TSLP and / or IL4 / IL13-induced or -mediated signaling in a subject in need thereof, which may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0044] The present disclosure also provides methods for reducing or eliminating inflammation associated with TSLP and / or IL4 / IL13-induced or -mediated signaling in a subject in need thereof, which may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0045] In certain embodiments, the subject is a human.

[0046] Also provided are uses of pharmaceutical compositions of the present disclosure in treating diseases associated with TSLP and / or IL4 / IL13-induced signaling, reducing or eliminating excessive immune responses associated with TSLP and / or IL4 / IL13-induced or mediated signaling, and / or reducing or eliminating inflammation associated with TSLP and / or IL4 / IL13-induced or mediated signaling, and in the preparation of medicaments for treating diseases associated with TSLP and / or IL4 / IL13-induced signaling, reducing or eliminating excessive immune responses associated with TSLP and / or IL4 / IL13-induced signaling, and / or reducing or eliminating inflammation associated with TSLP and / or IL4 / IL13-induced signaling.

[0047] Other features and advantages of the present disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0048] The following detailed description is given by way of example, and is not intended to limit the invention to only the particular embodiments as described, and may be best understood in conjunction with the accompanying drawings, in which:

[0049] [Figure 1] FIG. 1 shows a schematic diagram of a recombinant bispecific antibody of the present disclosure. [Figure 2] 2A and 2B show the binding ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to human TSLP in capture ELISA. [Figure 3]3A and 3B show the binding ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to human IL4R in capture ELISA. [Figure 4] 4A to 4B show the binding ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to human IL4Rα-expressing cells in a cell-based binding FACS assay. [Figure 5] 5A-5B show the simultaneous binding ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to human TSLP and human IL4R in double-binding ELISA. [Figure 6] 6A-6B show the ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to block TSLP-TSLPR / IL7R binding in a competitive ELISA. [Figure 7] Figures 7A-7B show the ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to block TSLP-cell surface TSLPR / IL7R binding in a cell-based blocking FACS assay. [Figure 8] 8A-8B show the ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to block IL4-IL4R binding in competitive ELISA. [Figure 9] 9A-9B show the ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 (A) and BSI-502-005 to BSI-502-008 (B) to block IL4-cell surface IL4Rα binding in a cell-based blocking FACS assay. [Figure 10]FIG. 10 shows the ability of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 to block TSLP-induced STAT5 signaling in a cell-based reporter assay. [Figure 11] FIG. 11 shows the activity of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 in inhibiting IL4-induced STAT6 phosphorylation in a cell-based assay. [Figure 12] FIG. 12 shows the activity of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 in inhibiting IL13-induced STAT6 phosphorylation in a cell-based assay. [Figure 13] FIG. 13 shows CCL17 production by human PBMC induced by TSLP and / or IL4. [Figure 14] FIG. 14 shows the ex vivo inhibitory effects of recombinant bispecific antibodies BSI-502-001 to BSI-502-004 on CCL-17 production by PBMCs induced by the combination of TSLP and IL4. DETAILED DESCRIPTION OF THE INVENTION

[0050] The term "TSLP" refers to thymic stromal lymphopoietin. The term "TSLP" includes variants, isoforms, homologs, orthologs, and paralogs. The term "human TSLP" refers to a TSLP protein having an amino acid sequence of human origin, e.g., human TSLP having the amino acid sequence of Genbank Accession No. NP_149024.1 or SEQ ID NO: 32.

[0051] The term "IL4Rα" refers to the interleukin-4 receptor subunit α. The term "IL4Rα" includes variants, isoforms, homologs, orthologs, and paralogs. The term "human IL4Rα" refers to an IL4Rα protein having an amino acid sequence of human origin, for example, the human IL4Rα having the amino acid sequence of Genbank Accession No. NP_001244335.1 or SEQ ID NO: 33.

[0052] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments (i.e., "antigen-binding sites"), or single chains thereof. Full-length antibodies are glycoproteins consisting of two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (V) H The heavy chain constant region is composed of the C H1 , C H2 , and C H3 Each light chain is composed of three domains: a light chain variable region (referred to herein as V L The light chain constant region consists of one domain, C L It consists of: V H Area and V L The region is divided into highly variable regions called complementarity-determining regions (CDRs) and relatively conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. A functional fragment of a heavy chain constant region is a portion of the constant region that retains desired properties, such as the ability to bind to Fc receptors and / or complement system proteins and / or the ability to extend the serum half-life of an antibody or its antigen-binding portion.

[0053] The term "antigen-binding site" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL4Rα or TSLP protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding site" of an antibody include (i) Fab fragments, V L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) a V fragment of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H (v) a dAb fragment consisting of the V domain (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity-determining region (CDR). In addition, the Fv fragment contains two domains, V and V. L and V H are encoded by separate genes, but V L Area and V H The regions can be joined by a synthetic linker that allows them to be produced as a single protein chain that pairs to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies.

[0054] As used herein, an antibody that "specifically binds to human TSLP" refers to an antibody that binds to human TSLP protein (and optionally TSLP proteins from one or more non-human species), but does not substantially bind to non-TSLP proteins. Preferably, the antibody binds to human TSLP protein with "high affinity," i.e., with a binding affinity of 5.0 x 10 -8 M or less, preferably 1.0 × 10 -8 M or less, more preferably 1.0 × 10 -10 K below M D Combine with.

[0055] As used herein, an antibody that "specifically binds to human IL4Rα" is intended to refer to an antibody that binds to human IL4Rα protein (and optionally IL4Rα proteins from one or more non-human species), but does not substantially bind to non-IL4Rα proteins. Preferably, the antibody binds to human IL4Rα protein with "high affinity," i.e., greater than 5.0×10 -8 M or less, preferably 1.0 × 10 -8 M or less, more preferably 5.0 × 10 -9 K below M D Combine with.

[0056] As used herein, the term "K assoc " or "K a " is intended to refer to the association rate of a particular antibody-antigen interaction, while "K dis " or "K d The term "K", as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction. D " as used herein is intended to refer to the dissociation constant, which is K d and K. a The ratio of (i.e., K d / K a ) and expressed as molar concentration (M). D The K value can be determined using methods established in the art. DA preferred method for determining is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore™ system.

[0057] The term “EC 50 ", also known as the half-maximal effective concentration, refers to the concentration of antibody that induces a response halfway between the baseline and maximum response after a specific exposure time.

[0058] The term “IC 50 ", also known as the half-maximal inhibitory concentration, refers to the concentration at which an antibody inhibits a specific biological or biochemical function by 50% compared to the absence of the antibody.

[0059] The term "subject" includes humans and non-human animals. The term "non-human animals" includes all vertebrates, i.e., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals such as non-human primates, sheep, dogs, cats, cows, and horses being preferred.

[0060] The term "therapeutically effective amount" refers to an amount of an antibody, e.g., a recombinant bispecific antibody, of the disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as an inflammatory disease) and / or reduce the severity of the disease or condition. A therapeutically effective amount is understood in the context of the condition being treated, and the actual effective amount is readily determined by one of ordinary skill in the art.

[0061] The term "identity" as used herein refers to the sequence similarity between two polynucleotide sequences or two amino acid sequences. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.

[0062] An "antagonistic" anti-TSLP antibody is an antibody that specifically binds to TSLP, inhibits TSLP-TSLPR binding / interaction, and suppresses TSLP-induced or mediated signal transduction. An "antagonistic" anti-IL4R antibody is an antibody that specifically binds to IL4R, inhibits IL4R formation, or inhibits the binding of IL4R to IL4 or IL13, and suppresses IL4- or IL13-induced or mediated signal transduction.

[0063] The recombinant bispecific antibodies of the present disclosure have comparable, if not greater, binding affinity / activity to TSLP and / or IL4R, comparable blocking activity against IL4-IL4R binding / interaction and / or TSLP-TSLPR / IL7Rα binding / interaction, and / or blocking activity against TSLP- and / or IL4-induced or mediated signaling compared to prior art monospecific anti-IL4R or anti-TSLP antibodies, such as dupilumab or tezepelumab, or combinations thereof.

[0064] The recombinant bispecific antibody of the present disclosure can comprise: i) an anti-TSLP IgG antibody comprising a heavy chain and a light chain; and ii) an anti-IL4R scFv linked to the N-terminus or C-terminus of the heavy chain or light chain of the anti-TSLP IgG antibody.

[0065] The anti-IL4R scFv can be linked to the N-terminus of the heavy or light chain of the anti-TSLP IgG antibody. The anti-IL4R scFv can be linked to the N-terminus of each heavy chain or each light chain. The anti-IL4R scFv may be linked to the N-terminus of each heavy chain. The anti-IL4R scFv may be linked to the N-terminus of each light chain.

[0066] Alternatively, a recombinant bispecific antibody of the present disclosure may comprise: i) an anti-IL4R IgG antibody comprising a heavy chain and a light chain; and ii) an anti-TSLP scFv linked to the N-terminus or C-terminus of the heavy chain or light chain of the anti-IL4R IgG antibody.

[0067] The anti-TSLP scFv can be linked to the N-terminus of the heavy or light chain of the anti-IL4R IgG antibody. The anti-TSLP scFv can be linked to the N-terminus of each heavy chain or each light chain. The anti-TSLP scFv can be linked to the N-terminus of each heavy chain. The anti-TSLP scFv can be bound to the N-terminus of each light chain.

[0068] The TSLP-binding domain may be an anti-TSLP antibody or antigen-binding portion thereof described in WO2021 / 043221, and the IL4R-binding domain may be an anti-IL4R antibody or antigen-binding portion thereof described in WO2021 / 170020. The heavy chain variable region CDRs and light chain variable region CDRs in these TSLP- and IL4R-binding domains are defined according to the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined based on the heavy / light chain variable region sequences using other systems, such as the Chothia, IMGT, AbM, or Contact numbering systems / methods.

[0069] The scFv may be linked to the IgG antibody via a linker. The heavy chain variable region of the scFv may be linked to the light chain variable region via a linker. The linker functions primarily as a spacer between the TSLP-binding domain and the IL4R-binding domain, or between the heavy chain variable region and the light chain variable region of the scFv. The linker may be composed of amino acids linked by peptide bonds, preferably 5 to 30 amino acids, 10 to 30 amino acids, 10 to 20 amino acids, or 15 amino acids linked by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids. One or more of these amino acids may be glycosylated, as understood by those skilled in the art. In one embodiment, the 5 to 30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker may be composed primarily of sterically less hindered amino acids, such as glycine and alanine. Exemplary linkers are polyglycine (particularly (Glys, poly(Gly-Ala)) and polyalanine, e.g., -GGGGSGGGS- (SEQ ID NO: 19) and GGGGSGGGSGGGS- (SEQ ID NO: 20). The linker may also be a non-peptide linker, e.g., -NH-, -(CH2) Alkyl linkers such as sC(O)-, where s=2 to 20, can be used. These alkyl linkers can further include a lower alkyl (e.g., C 1-4 ), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, or any other non-sterically hindering group.

[0070] The recombinant bispecific antibodies of the present disclosure may be modified in one or both variable regions (i.e., V H and / or V L), for example, one or more residues in one or more CDR regions and / or one or more framework regions. Additionally, bispecific antibodies can be engineered by modifying residues in the constant region(s), for example, to alter the effector function(s) of the bispecific antibody.

[0071] For example, bispecific antibodies of the disclosure can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the bispecific antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, bispecific antibodies of the disclosure can be modified chemically (e.g., one or more chemical moieties can be added to the antibody) or to alter glycosylation to alter one or more functional properties.

[0072] In one embodiment, C H1 The hinge region of is modified to change, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is described in more detail in U.S. Pat. No. 5,677,477. H1 The number of cysteine ​​residues in the hinge region of the antibody may be altered to, for example, facilitate association of the light and heavy chains or to increase or decrease the stability of the antibody.

[0073] In another embodiment, the Fc hinge region of the bispecific antibody of the present disclosure is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are made to the C of the Fc hinge fragment. H2 -C H3 The Fc-hinge domain is introduced into the domain interface region to reduce the antibody's ability to bind to Staphylococcal protein A (SpA) compared to native SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,775.

[0074] In yet another embodiment, the glycosylation of the bispecific antibody is modified. For example, a glycosylated bispecific antibody can be generated (i.e., the bispecific antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the anti-CD40 antibody, or its antigen-binding site, for an antigen.

[0075] Another modification of the bispecific antibody herein contemplated by the present disclosure is pegylation. The antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate the antibody, the antibody or its fragment is usually reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that allow one or more PEG groups to bind to the antibody or its antigen-binding site.

[0076] In another aspect, the present disclosure provides a nucleic acid molecule encoding a bispecific antibody of this disclosure or a portion thereof. For example, the nucleic acid molecule of this disclosure can encode an anti-IL4R heavy chain variable region-heavy chain constant region-anti-TSLP scFv chain, an anti-TSLP scFv-anti-IL4R heavy chain variable region-heavy chain constant region chain, a TSLP heavy chain variable region-heavy chain constant region-anti-IL4R scFv chain, or an anti-IL4R scFv-anti-TSLP heavy chain variable region-heavy chain constant region chain.

[0077] Nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. By "isolated" or "substantially purified" nucleic acid, we mean that it has been purified by standard techniques from other cellular components and other impurities, such as other cellular nucleic acids and proteins. Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a DNA molecule.

[0078] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, the nucleic acid molecules of the present disclosure can be chemically synthesized.

[0079] The present disclosure also provides expression vectors comprising the nucleic acid molecules of the present disclosure. Examples of vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformation-adapted artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and human artificial episomal chromosomes (HAECs). The present disclosure further provides host cells comprising the expression vectors of the present disclosure or having the nucleic acid molecules integrated into their genomes. The host cells can be transformed or transfected with the expression vectors. Suitable host cells include E. coli, yeast, and other eukaryotes. In one embodiment, DNA encoding the polypeptide chains forming each bispecific antibody of the present disclosure is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the coding nucleotides are ligated to the vector such that the transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the antibody genes.

[0080] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of nucleotides. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), and adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma virus enhancer. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter and β-globin promoter, can be used. Furthermore, the SRα promoter system contains the SV40 early promoter and the long terminal repeat sequence of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

[0081] In addition to the bispecific antibody-encoding nucleotides and regulatory sequences, the expression vectors of the present disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0082] To express the peptide chains that make up the bispecific antibody, expression vectors encoding the peptide chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the bispecific antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression in eukaryotic cells, particularly mammalian host cells, is most preferred, as such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active bispecific antibodies.

[0083] Preferred mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selectable marker as described by RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When the recombinant expression vector is introduced into mammalian host cells, the bispecific antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the bispecific antibody in the host cells, or more preferably, secretion of the bispecific antibody into the medium in which the host cells are grown. Bispecific antibodies can be recovered from the culture medium using standard protein purification methods.

[0084] In another aspect, the present disclosure provides pharmaceutical compositions that may include a recombinant bispecific antibody, nucleic acid molecule, expression vector, and / or host cell of the present disclosure formulated with a pharmaceutically acceptable carrier. The bispecific antibody, nucleic acid molecule, expression vector, and / or host cell can be administered separately when the composition includes multiple antibodies, nucleic acid molecules, expression vectors, or host cells. The composition can optionally include one or more additional pharmaceutically active ingredients, such as another antibody or a drug, such as an anti-tumor drug.

[0085] Pharmaceutical compositions may contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorings, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is described in Remington: The Science and Practice of Pharmacy, 20th Ed., edited by Gennaro (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0086] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from inactivation by the action of acids and other natural conditions. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, spinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the antibodies of the present disclosure can be administered via a parenteral route, e.g., a topical, epidermal, or mucosal administration route, e.g., a nasal, oral, vaginal, rectal, sublingual, or topical administration route.

[0087] The pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions, or can be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentrations.

[0088] The amount of active ingredient which can be combined with a carrier material to produce a single dose will vary depending on the subject being treated and the particular mode of administration, but will generally be that amount of the composition which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0089] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations, in which case less frequent administration is required.

[0090] When administering the composition, the dosage can range from about 0.0001 to 100 mg / kg.

[0091] A "therapeutically effective dose" of a bispecific antibody, nucleic acid molecule, expression vector, or host cell of the disclosure preferably results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disease or injury. For example, for the treatment of a subject with an inflammatory disease, a "therapeutically effective dose" preferably eliminates inflammation by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to an untreated subject.

[0092] The pharmaceutical compositions can be in the form of controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0093] Pharmaceutical compositions that may include the bispecific antibodies, nucleic acid molecules, expression vectors, or host cells of the disclosure have many in vitro and in vivo utilities, such as treating diseases caused by TSLP and / or IL4 / IL13-induced or mediated signaling.

[0094] The present disclosure provides methods for treating a disease associated with TSLP and / or IL4 / IL13-induced or mediated signaling in a subject in need thereof, which may comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0095] The disease is an inflammatory disease such as an allergic disease, or an autoimmune disease. The disease includes, but is not limited to, atopic dermatitis, asthma, ulcerative colitis, psoriasis, nasal polyps, rhinosinusitis, etc. In certain embodiments, the subject is a human.

[0096] The present disclosure also provides methods for reducing or eliminating excessive immune responses associated with TSLP and / or IL4 / IL13-induced or -mediated signaling in a subject in need thereof, which may include administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.The present disclosure also provides methods for reducing or eliminating inflammation associated with TSLP and / or IL4 / IL13-induced or -mediated signaling in a subject in need thereof, which may include administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0097] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0098] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures, and all references, Genbank sequences, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference. [Example]

[0099] Example 1. Construction and Expression of Exemplary Recombinant Bispecific Antibodies Bispecific antibodies, including BSI-502-001, BSI-502-002, BSI-502-007, and BSI-502-008, were constructed by linking an anti-TSLP scFv to the N- or C-terminus of the heavy chain of the humanized anti-IL4R antibody huC2C1A1-V15 via a linker. The antibody huC2C1A1-V15 was described in WO 2021 / 170020 and contained a human IgG heavy chain constant region. The anti-TSLP scFv contained the heavy and light chain variable regions of the humanized anti-TSLP antibody hu1C5F12E9-V8, described in WO 2021 / 043221.

[0100] The bispecific antibodies BSI-502-003, BSI-502-004, BSI-502-005, and BSI-502-006 were constructed by linking, via a linker, an anti-IL4R scFv carrying the heavy and light chain variable regions from huC2C1A1-V15 to the N- or C-terminus of the heavy chain of hu1C5F12E9-V8, which carries the human IgG heavy chain constant region.

[0101] See Table 1 and Figure 1 for detailed structures and sequences of these bispecific antibodies.

[0102] The monospecific antibodies, i.e., anti-TSLP hu1C5F12E9-V8 and anti-IL4R huC2C1A1-V15, were prepared as full-length IgG1 or IgG4 antibodies and used as controls in the following assays. Specifically, hu1C5F12E9-V8 contains a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region, and has the amino acid sequences of SEQ ID NOS: 7, 8, 9 (IgG1), 29 (IgG4), and 10, respectively. huC2C1A1-V15 contains a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region, and has the amino acid sequences of SEQ ID NOS: 17, 18, 9 (IgG1), 29 (IgG4), and 10, respectively.

[0103] For both bispecific and monospecific antibodies, the heavy chain constant regions were prepared by introducing L234A, L235Q, and K322Q (abbreviated as AQQ) to reduce Fc effector function and M252Y, S254T, and T256E (abbreviated as YTE) to extend half-life. [Table 1]

[0104] Nucleic acids encoding the polypeptide chains constituting the bispecific antibody were synthesized and inserted into pTT5 plasmid, respectively. The resulting plasmids were then transfected into CHO cells, and the exemplary bispecific antibodies of the present disclosure were expressed and secreted in the CHO cells and subsequently purified using a protein A sepharose column.

[0105] Example 2. Determining the binding affinity of representative bispecific antibodies using BIACORE surface plasmon resonance technology The purified bispecific antibodies were evaluated for binding affinity and kinetics using a Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA). huC2C1A1A1-V15 (IgG1), dupilumab (anti-IL4Rα antibody), hu1C5F12E9-V8 (IgG1), and tezepelumab (anti-TSLP antibody, prepared in-house using heavy and light chains of SEQ ID NOs: 30 and 31) were used as positive controls.

[0106] Goat anti-human IgG (GE Healthcare, Cat#BR100839, Human Antibody Capture Kit) was covalently coupled to a CM5 chip (carboxymethyl dextran-coated chip) via primary amines using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Unreacted sites on the biosensor surface were blocked with ethanolamine. Next, purified bispecific antibodies of the present disclosure at a concentration of 66.67 nM and a control at 10 μg / mL were flowed over the chip at a flow rate of 10 μL / min. Subsequently, serially diluted recombinant human IL4Rα-his (prepared in-house, IL4Rα with a C-terminal His tag, SEQ ID NO: 33) in HBS EP buffer (provided by Biacore) was flowed over the chip at a flow rate of 30 μL / min. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore evaluation software. D , K. a , K. d The values ​​of were determined and are shown in Table 2-1 below. [Table 2] TIFF2025532685000003.tif85152

[0107] Biosion's in-house synthesized recombinant human TSLP-his (TSLP of SEQ ID NO: 32, with a C-terminal His tag) was covalently coupled at a concentration of 10 μg / mL to a CM5 chip (carboxymethyl dextran-coated chip from GE healthcare #BR100530) via primary amines using a standard amine coupling kit provided by Biacore (GE healthcare, Pittsburgh, PA, USA). Unreacted sites on the biosensor surface were blocked with ethanolamine. Serially diluted purified bispecific antibodies and a control (HBS-EP) were then coupled to the CM5 chip. + Antigen-antibody association kinetics was monitored for 4 minutes, and dissociation kinetics was monitored for 13 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore software, and the K D , K. a , K. d The values ​​were calculated and are shown in Table 2-2.

[0108] All of the bispecific antibodies of the present disclosure specifically bind to human TSLP and human IL4R proteins with high binding affinity.

[0109] Example 3. Binding activity of exemplary bispecific antibodies The binding activity of exemplary bispecific antibodies of the disclosure to human TSLP or human IL4Rα was determined by capture ELISA and flow cytometry (FACS).

[0110] 3.1 Capture ELISA for TSLP binding Briefly, 100 μL of 2 μg / mL AffiniPure F(ab')2 fragment goat anti-human IgG (Fcγ fragment specific, Jackson Immuno Research, Cat#109-006-008) in PBS was added to a 96-well microplate and incubated overnight at 4°C. The plate was washed four times with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with 200 μL / well of blocking buffer (5% w / v nonfat milk, PBST) for 2 hours at 37°C. The plate was washed again, and 100 μL of a bispecific antibody of the present disclosure, a positive control, or hIgG (Hualan Biological Engineering Inc.), serially diluted 5-fold from 60 nM in 2.5% nonfat milk in PBST, was added to each well. The plate was incubated for 40 minutes at 37°C and washed four times again. To the plates containing the captured antibody, 100 μL of biotin-labeled human TSLP-His protein (an in-house preparation of TSLP with a C-terminal His tag, SEQ ID NO: 32, at 35 ng / mL in 2.5% nonfat milk in PBST) was added, incubated for 40 minutes at 37°C, and washed four times. Subsequently, streptavidin-conjugated HRP (1:10,000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 μL / well) was added and incubated for 40 minutes at 37°C. After the final wash, the plates were incubated with 100 μL / well of TMB (Innoreagents). After 15 minutes at room temperature, the reaction was stopped with 50 μL / well of 1M H2SO4. The absorbance of each well was read using a microplate reader in dual-wavelength mode, with TMB at 450 nm and a reference wavelength of 630 nm. OD(450-630) values ​​were plotted against antibody concentration. Data were analyzed using Graphpad Prism, and EC 50 The values ​​were reported.

[0111] As shown in Figures 2A and 2B, the bispecific antibodies of the present disclosure specifically bind to human TSLP, exhibiting Bmax (maximum binding) and EC 50 It can be seen that the results were equivalent to those of the positive control, except for BSI-502-002.

[0112] 3.2 Capture ELISA for IL4R binding Briefly, 96-well plates were coated with 2 μg / mL AffiniPure F(ab')2 fragment goat anti-human IgG antibody, Fcγ fragment specific (Jackson Immuno Research, Cat#109-006-008) at 100 μL / well and incubated overnight at 4°C. The plates were washed once with wash buffer (PBS + 0.05% (w / v) Tween-20, PBST) and then blocked with 200 μL / well of blocking buffer containing 5% (w / v) nonfat milk in PBST for 2 hours at 37°C. After washing again, 100 μL / well of a bispecific antibody of the present disclosure, a positive control antibody, or a negative control antibody, hIgG (human immunoglobulin for intravenous use, pH 4, Hualan Biological Engineering Inc.), diluted 5-fold in 2.5% (w / v) nonfat milk in PBST starting at 20 nM, was added and incubated at 37°C for 40 minutes, followed by four washes. 100 μL / well of biotin-labeled human IL4Rα-His protein (IL4Rα of SEQ ID NO: 33 with a C-terminal His tag attached, 6.8 ng / mL in 2.5% (w / v) nonfat milk in PBST) was added to the plate with the captured antibody, incubated at 37°C for 40 minutes, and then washed four times. Next, 100 μL / well of streptavidin-conjugated HRP (diluted 1:10,000 in PBST, Jackson Immuno Research, Cat. #016-030-084) was added and incubated at 37°C for 40 minutes. After a final wash, 100 μL / well of ELISA substrate TMB (Innoreagents, Cat. #TMB-S-002) was added and incubated at 25°C for 10 minutes. The reaction was then stopped by adding 50 μL / well of 1M H2SO4. The absorbance of each well was read using a microplate reader in dual wavelength mode, with TMB at 450 nm and a reference wavelength of 630 nm. Data were analyzed using Graphpad Prism, and EC 50 The values ​​were reported.

[0113] 3A and 3B, the bispecific antibodies of the present disclosure specifically bound to human IL4R. Bmax and EC 50 The Bmax and EC of the positive control, except for BSI-502-005 and BSI-502-006, are 50 It was close to.

[0114] 3.3 Cell-based coupled FACS The binding activity of the bispecific antibodies of the present disclosure to human IL4Rα was assessed by flow cytometry (FACS) using IL4Rα expressed on the surface of 293F-IL4Rα cells. Briefly, 293F cells (Thermo Fisher Inc., Cat #11625019) were transfected with a pCMV-TP plasmid construct containing nucleotides encoding human IL4Rα (amino acid residues 1-825 of Uniprot #P24394-1) between EcoRI and XbaI. The resulting stable cell pool, "293F-IL4Rα," was used for subsequent cell-based binding assays and ligand inhibition FACS assays. 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Next, 2 × 10 cells were plated per well in a 96-well plate. 5 Cells were incubated on ice for 40 minutes with serially diluted bispecific antibodies, positive controls, or hIgG (starting at 100 nM and serially diluted 5-fold) in FACS buffer at 100 μL / well. Cells were washed twice with FACS buffer, and 100 μL / well of R-phycoerythrin-labeled goat anti-human IgG antibody, Fcγ fragment specific (1:1000 dilution in FACS buffer, Jackson Immunoresearch, Cat# 109-115-098) was added. After 40 minutes of incubation at 4°C in the dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism, and EC 50 The values ​​were reported.

[0115] According to Figures 4A and 4B, the bispecific antibody of the present disclosure specifically binds to human IL4R expressed on the cell surface and inhibits EC 50 were comparable to those of the positive controls, except for BSI-502-005 and BSI-502-006.

[0116] 3.4 Double bond ELISA For the dual binding ELISA, a 96-well ELISA plate was first coated with 100 μL / well of 1 μg / mL human IL4Rα-His protein (IL4Rα of SEQ ID NO: 33, C-terminal His-tagged, prepared in-house) in PBS and incubated overnight at 4°C. The ELISA plate was washed four times with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with 200 μL / well of blocking buffer (5% w / v non-fat milk in PBST) for 2 hours at 37°C. The plate was washed again, and a bispecific antibody of the present disclosure, a positive control antibody, or hIgG was added at 100 μL / well at a 5-fold serial dilution in 2.5% non-fat milk in PBST (starting concentration 100 nM) and incubated at 37°C for 40 minutes. The ELISA plate was washed four times again and incubated with biotin-labeled human TSLP-his solution (TSLP with a C-terminal His tag, SEQ ID NO: 32, diluted 1:2000, final concentration 0.175 μg / mL in PBST buffer, 100 μL / well) for 40 minutes at 37°C. The ELISA plate was washed again and incubated with SA-HRP (100 μL / well) for 40 minutes at 37°C. After the final wash, 100 μL / well of TMB (Innoreagents) was added. The reaction was stopped after 15 minutes at room temperature with 50 μL / well of 1M H2SO4. Absorbance was read on a microplate reader in dual wavelength mode, with TMB at 450 nm and a reference wavelength of 630 nm. OD (450-630) values ​​were plotted against antibody concentration. Data were analyzed using Graphpad Prism, and EC 50 The values ​​were reported and the results are shown in Figures 5A and 5B.

[0117] As a result, the bispecific antibody of the present disclosure simultaneously bound to IL4R and TSLP, whereas the monospecific antibody dupilumab showed no binding signal.

[0118] Example 4. Blocking Activity of Exemplary Bispecific Antibodies for IL4R-IL4 or TSLPR / IL7Ra-TSLP Binding 4.1 Inhibitory effect on TSLPR / IL7Ra-TSLP binding in ligand-blocking ELISA The ability of the bispecific antibodies to block TSLP-TSLPR / IL7Ra binding was measured using a competitive ELISA assay. Briefly, 100 μL of human TSLPR-Fc protein (SEQ ID NO: 34 to SEQ ID NO: 38 fusion, prepared in-house) at 1 μg / mL in PBS and 100 μL of human IL7Ra-Fc protein (SEQ ID NO: 35 to SEQ ID NO: 38 fusion, prepared in-house) in PBS were coated onto a 96-well microplate overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with 5% w / v nonfat milk in PBST for 2 hours at 37°C. The plate was then washed again with wash buffer.

[0119] A bispecific antibody or control of the present disclosure was diluted with biotin-labeled human TSLP-his (TSLP of SEQ ID NO: 32 with a C-terminal His tag, prepared in-house, at 17 ng / mL in 2.5% nonfat milk in PBST) in 5-fold serial dilutions starting from 100 nM and incubated for 40 minutes at room temperature. Then, 100 μL of the antibody / TSLP-his mixture was added to the TSLPR / IL7Ra-coated plate. After incubation at 37°C for 40 minutes, the plate was washed four times with wash buffer. Streptavidin-conjugated HRP was then added and incubated at 37°C for 40 minutes to detect biotin-labeled human TSLP-his bound to TSLPR / IL7Ra. The plate was washed again with wash buffer. Finally, TMB was added, and the reaction was stopped with 1 M H2SO4. Absorbance was read on a microplate reader in dual wavelength mode with TMB at 450 nm and a reference wavelength of 630 nm. OD (450-630) values ​​were plotted against antibody concentration. Data were analyzed using Graphpad Prism and IC values ​​were calculated. 50The values ​​were reported and the results are shown in Figures 6A and 6B.

[0120] As can be seen from Figures 6A and 6B, all bispecific antibodies of the present disclosure were able to block the binding of human TSLP to human TSLPR / IL7Ra with activity comparable to the positive control.

[0121] 4.2 Inhibitory effect of cell-based ligand blocking on TSLPR / IL7Ra-TSLP binding in FACS The activity of the bispecific antibody in blocking binding of TSLP-Fc protein to cell surface human TSLPR / human IL7Ra was assessed in a flow cytometry (FACS) assay using HEK293T-TSLPR / IL7R / STAT5-Luc 5C5 cells expressing cell surface human TSLPR (SEQ ID NO: 36) and human IL7Ra (SEQ ID NO: 37). Cells were prepared according to the Lipofectamine 3000 transfection reagent (Thermo Fisher) instructions by transfecting HEK293T cells (ATCC® CRL-11268) with pCMV-TP plasmid, which contains the TSLPR coding sequence inserted between the EcoRI and XbaI sites, and pCMV3-SP plasmid, which contains the IL7Ra coding sequence inserted between the HindIII and XbaI sites, as well as pGL4.52[luc2P / STAT5RE / Hygro] (Promega).

[0122] Briefly, bispecific antibodies of the present disclosure, positive control, or negative control hIgG (human immunoglobulin for intravenous injection, pH 4, Hualan Biological Engineering Inc.) were diluted with biotin-labeled human TSLP-Fc solution (SEQ ID NO: 32 to SEQ ID NO: 38 fusion, prepared in-house, 0.29 μg / mL in FACS buffer) in 5-fold serial dilutions starting at 100 nM and incubated at room temperature for 40 minutes. Cells were harvested from cell culture flasks, washed twice, and resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). Next, 1 x 10 per well of a 96-well plate was plated.5 HEK293T-TSLPR / IL7R / STAT5-Luc 5C5 cells were incubated with 100 μL / well of antibody / TSLP-Fc-biotin mixture at 4°C for 40 minutes. After washing the cells twice with FACS buffer, 100 μL / well of R-phycoerythrin-conjugated streptavidin (1:500 dilution in FACS buffer, Jackson Immunoresearch, Cat#016-110-084) was added and incubated in the dark at 4°C for 40 minutes. The cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism, and IC 50 The values ​​were reported.

[0123] As shown in Figures 7A and 7B, the bispecific antibodies of the present disclosure had similar IC 50 completely inhibited TSLP-TSLPR / IL7Ra binding, while the inhibitory effect of BSI-502-002 was slightly lower than that of the positive control.

[0124] 4.3 Inhibitory effect on IL4-IL4R binding in ligand-blocking ELISA The ability of the bispecific antibodies of the present disclosure to block IL4-IL4Rα interaction was measured in a competitive ELISA assay. Briefly, 100 μL of human IL4Rα-his protein (IL4Rα with SEQ ID NO: 33 and a C-terminal His tag, prepared in-house) was coated onto a 96-well microplate at 2 μg / mL in PBS overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% w / v Tween-20, PBST) and blocked with 5% w / v nonfat milk in PBST at 37°C for 2 hours. The plate was then washed again, and serially diluted bispecific antibodies or controls (starting at 100 nM in 5-fold serial dilutions) in 2.5% w / v nonfat milk (PBST) were added to the IL4Rα-bound plate at 100 μL per well and incubated at 37°C for 40 minutes. After washing the plate four times with wash buffer, 100 μL / well of 0.3 μg / mL biotin-labeled human IL4 protein (Sino biological inc., Cat#11846-HNAE) was added and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Then, 100 μL / well of streptavidin-conjugated HRP (1:10,000 diluted in PBST buffer, Jackson Immunoresearch, Cat#016-030-084) was added and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added, the reaction was stopped with 1M H2SO4, and the absorbance was measured at 450 nm. Data were analyzed using Graphpad Prism, and IC 50 The values ​​were reported and the results are shown in Figures 8A and 8B.

[0125] As can be seen from Figures 8A and 8B, the bispecific antibodies of the present disclosure were able to inhibit the binding of human IL4 to human IL4R with activity comparable to that of the positive control.

[0126] 4.4 Inhibitory effect of cell-based ligand blocking on IL4R-IL4 binding in FACS The activity of the bispecific antibodies to block the binding of IL4 protein to cell surface human IL4Rα was assessed by flow cytometry (FACS) using the 293F-IL4Rα cells described above.

[0127] Briefly, 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in PBS containing 2% v / v fetal bovine serum (FACS buffer) at 1 × 10 cells per well in a 96-well plate. 5 Cells were incubated on ice for 40 minutes with 100 μL of serially diluted bispecific antibodies, positive controls, or negative controls in FACS buffer (starting at 100 nM in a 5-fold serial dilution). The plate was washed twice with FACS buffer, and 100 μL / well of 0.3 μg / mL biotin-labeled human IL4 protein (Sino biological inc., Cat#11846-HNAE) was added and incubated in the dark at 4°C for 40 minutes. After washing the plate twice with FACS buffer, 100 μL / well of R-phycoerythrin-labeled streptavidin (1:500 dilution in FACS buffer, Jackson Immunoresearch, Cat#016-110-084) was added and incubated in the dark at 4°C for 40 minutes. Cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism and IC 50 The values ​​were reported.

[0128] As shown in Figures 9A and 9B, all bispecific antibodies of the present disclosure were able to block IL4 binding to cell surface IL4R, demonstrating blocking abilities similar to the positive control.

[0129] Example 5. Cell-based functional assays of exemplary bispecific antibodies 5.1 Cell-based reporter assays A cell-based reporter assay was performed to assess the neutralizing activity of the bispecific antibodies against TSLP-induced cellular STAT5-Luc reporter gene expression using the reporter cell line HEK293T-TSLPR / IL7R / STAT5-Luc 5C5 described in Example 4, which expresses cell surface human TSLPR (SEQ ID NO: 36) and human IL7Ra (SEQ ID NO: 37).

[0130] Briefly, HEK293T-TSLPR / IL7R / STAT5-Luc 5C5 cells were harvested from cell culture flasks and then cultured in 5 × 10 ml of 100 μL DMEM medium (Gibco, Cat# 10566-016) supplemented with 10% FBS (Gibco, Cat# 10099-141). 4 Cells were plated in 96-well cell culture plates. Meanwhile, 60 μL of human TSLP-his (TSLP with a C-terminal His tag, SEQ ID NO: 32, 0.3 μg / mL in DMEM medium supplemented with 10% FBS) was mixed with 60 μL of serially diluted bispecific antibodies and a control containing an in-house produced anti-CD22 antibody (starting from 333 nM, diluted 3-fold in DMEM medium supplemented with 10% FBS). The resulting mixture was incubated at room temperature for 30 minutes. Next, 100 μL of the bispecific antibody / TSLP-his mixture was added to the cell-coated plate at 100 μL per well and incubated at 37°C in a CO2 incubator for 16–18 hours. Then, 100 μL of the supernatant was discarded and luciferase detection reagent (50 μL per well, Vazyme, Cat# DD1201-02) was added. After 10 minutes, the plate was analyzed using a Tecan infinite 200Pro plate reader. The luminescence signal was analyzed using Graphpad prism, and IC 50 The values ​​were reported and the results are shown in Figure 10.

[0131] Bispecific antibodies, including BSI-502-001, BSI-502-002, BSI-502-003, and BSI-502-004, effectively inhibited the TSLP-induced STAT5 signaling pathway, whereas the isotype control anti-CD22 antibody had no effect, demonstrating the specific inhibitory activity of the bispecific antibodies. The inhibitory activity of the bispecific antibodies was comparable to that of the parent anti-TSLP antibody hu1C5F12E9-V8 (IgG4) and significantly superior to that of tezepelumab.

[0132] 5.2 Cell-based STAT6 phosphorylation assay It has been reported that IL4 and IL13 bind to plasma membrane human IL4Rα and induce STAT6 phosphorylation in HEK293T-IL4R / STAT6 / STAT6-Luc LB2 cells, suggesting that STAT6 phosphorylation is important for the IL4 / IL13 signaling pathway.

[0133] HEK293T-IL4R / STAT6 / STAT6-Luc LB2 cells were prepared in-house. Briefly, HEK293T cells (ATCC CRL-11268) naturally expressing IL13Rα1 were stably transfected with the pcDNA3.1-Puro plasmid (YouBio Biological Inc., Cat#VT9222) containing a nucleotide sequence encoding human IL4Rα inserted between BamHI and XhoI, the STAT6 plasmid (Sino Biological Inc., Cat#HG13190-NH) containing a nucleotide sequence encoding human STAT6 inserted between KpnI and XbaI, and the STAT6 luciferase reporter plasmid STAT6-Luc (Yeasen Biological Inc., Cat#11588ES03). Single-cell clones, LB2, were then selected for subsequent functional assays.

[0134] Briefly, logarithmic-phase HEK293T-IL4R / STAT6 / STAT6-Luc LB2 cells were plated in a 96-well plate in 100 μL of medium (RPMI1640 + 10% FBS), 2 × 10 5 Cells were seeded at 1000 μL / well. Next, 50 μL of serially diluted bispecific antibodies or controls (starting at 100 nM, 5-fold serial dilutions) were added to the plate and incubated at 37°C for 30 minutes. Next, 50 μL of human IL4 protein (600 pg / mL, Sino biological inc., Cat#11846-HNAE) or human IL13 protein (60 ng / mL, Sino biological inc., Cat#10369-HNAC) was added to the plate and incubated at 37°C for 20 minutes. The plate was centrifuged and washed twice with staining buffer (DPBS + 0.5% w / v BSA + 2 mM EDTA), after which 50 μL / well of fixation buffer (BD biosciences inc., Cat#5545655) was added and incubated at 4°C for 30 minutes. The cells were washed twice, permeabilization buffer (250 μl / well, BD biosciences inc., Cat# 558050) was added, and the cells were incubated on ice for 30 minutes. The plate was washed twice with staining buffer, and anti-pSTAT6 antibody (Alexa Fluor® 647 anti-STAT6 phospho Tyr641 at a 50-fold dilution, Biolegend, Cat# 686012) was added and the cells were incubated on ice for 60 minutes. Finally, the plate was washed twice and resuspended in staining buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism, and IC 50 The values ​​were reported.

[0135] The results are shown in Table 3 and Figures 11 and 12. [Table 3]

[0136] The bispecific antibodies of the present disclosure were able to inhibit IL4- or IL13-induced STAT6 phosphorylation in HEK293T-IL4R / STAT6 / STAT6-Luc LB2 cells, whereas the isotype control anti-CD22 had no effect, demonstrating the specificity of the inhibitory effect of the bispecific antibodies. The inhibitory activity of the bispecific antibodies was comparable to that of huC2C1A1A1-V15 (IgG4) and dupilumab.

[0137] Example 6. Ex vivo inhibitory effects of exemplary bispecific antibodies on TSLP- and / or IL4-induced CCL-17 production in human PBMCs 6.1 Inducible CCL-17 production in human PBMCs ex vivo with TSLP and / or IL4 CCL-17 (CC motif chemokine ligand 17), also known as TARC (thymus and activation-regulated chemokine), is a CC chemokine that plays an important role in allergic diseases such as atopic dermatitis and bronchial asthma. High serum CCL-17 concentrations were observed in patients with atopic dermatitis, closely associated with disease activity (Umeda, M., et al., (2020) Sci Rep 10(1):6010).

[0138] The inducing activity of TSLP and / or IL-4 on CCL-17 production was assessed in a bioassay based on human peripheral blood mononuclear cells (PBMCs). Briefly, human PBMCs (Milestone, P122011103C) were collected by centrifugation at 300 g for 8 minutes at room temperature and resuspended in 3 mL of RPMI 1640 (Gibco, Cat# A10491-01) containing 10% FBS. Subsequently, 120,000 cells / well were added to a 96-well plate in 100 μL of RPMI 1640 containing 10% FBS and incubated at 37°C in a 5% CO2 incubator for 2 hours. To each well, add 100 μL of: i) human IL-4 (Stemcell, Cat#78045.1) and human TSLP-his (TSLP with a C-terminal His tag, SEQ ID NO: 32) at a final concentration of 10 ng / mL in RPMI 1640 containing 10% FBS; ii) 100 μL of human TSLP-his at a final concentration of 10 ng / mL in RPMI 1640 containing 10% FBS; iii) 100 μL of human IL-4 at a final concentration of 10 ng / mL in RPMI 1640 containing 10% FBS; and vi) 100 μL of RPMI 1640 medium per well. After 48 hours of incubation at 37°C in a 5% CO incubator, the cell supernatant was collected and CCL17 levels were measured using an ELISA kit (Elabscience, Cat#E-EL-H0026C). The ELISA plate was read using a PERLONG #DNM-9602 microplate reader at OD (450-630). The data was analyzed using Graphpad Prism, and the results are shown in Figure 13.

[0139] FIG. 13 shows that both IL4 and TSLP alone can induce CCL17 production by PBMCs, and the combination of IL4 and TSLP induced higher levels of CCL17 production.

[0140] 6.2 Ex vivo effects of exemplary bispecific antibodies on human CCL-17 production Human PBMCs were harvested according to the protocol described above and incubated in a 96-well plate at 37°C in a 5% CO2 incubator for 2 hours. 50 μL of the disclosed bispecific antibodies, monospecific anti-TSLP mAbs, anti-IL4R mAbs, or a combination of tezepelumab and dupilumab, diluted 10-fold starting at 100 nM in RPMI 1640 containing 10% FBS, was added to the plate (combinations started at 100 nM tezepelumab and 100 nM dupilumab). After 48 hours of incubation at 37°C in a 5% CO2 incubator, cell supernatants were harvested and CCL17 concentrations were measured using an ELISA kit (Elabscience, Cat#E-EL-H0026C). The ELISA plates were read using a PERLONG#DNM-9602 microplate reader at OD (450-630). The data was analyzed using GraphPad Prism and the results are shown in FIG.

[0141] The bispecific antibodies of the present disclosure effectively inhibited TSLP+IL4-induced CCL-17 production by human PBMCs with significantly greater activity than either monospecific antibody alone or the monospecific antibody combination, suggesting that targeting TSLP may synergize with targeting IL4R in inhibiting CCL-17 production.

[0142] While the present disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the specification is intended to cover all alternatives, modifications, and equivalents that may be included within the spirit and scope of the appended claims. All references cited herein are further incorporated by reference in their entirety.

[0143] The sequences in this application are summarized below. TIFF2025532685000005.tif194156TIFF2025532685000006.tif205149TIFF2025532685000007.tif205152TIFF2025532685000008.tif206150TIFF2025532685000009.tif203149TIFF2025532685000010.tif53148

Claims

1. A recombinant antibody comprising: i) an anti-TSLP antibody or antigen-binding portion thereof, and ii) anti-IL4R single chain variable region (scFv); or i) an anti-IL4R antibody or an antigen-binding portion thereof, and ii) Anti-TSLP single chain variable region (scFv) A recombinant antibody comprising:

2. 2. The recombinant antibody of claim 1, i) an antagonistic anti-TSLP antibody comprising an anti-TSLP heavy chain variable region, a heavy chain constant region, an anti-TSLP light chain variable region, and a light chain constant region; and ii) an antagonistic anti-IL4R single chain variable region (scFv) comprising an anti-IL4R heavy chain variable region and an anti-IL4R light chain variable region; or i) an antagonistic anti-IL4R antibody comprising an anti-IL4R heavy chain variable region, a heavy chain constant region, an anti-IL4R light chain variable region, and a light chain constant region; and ii) an antagonistic anti-TSLP single chain variable domain (scFv) comprising an anti-TSLP heavy chain variable domain and an anti-TSLP light chain variable domain; A recombinant antibody comprising:

3. The recombinant antibody of claim 2, the anti-IL4R scFv is linked to the N-terminus of the anti-TSLP heavy chain variable region, the N-terminus of the anti-TSLP light chain variable region, the C-terminus of the anti-TSLP heavy chain constant region, or the C-terminus of the anti-TSLP light chain constant region; or the anti-TSLP scFv is linked to the N-terminus of the anti-IL4R heavy chain variable region, the N-terminus of the anti-IL4R light chain variable region, the C-terminus of the anti-IL4R heavy chain constant region, or the C-terminus of the anti-IL4R light chain constant region; Recombinant antibodies.

4. 3. The recombinant antibody of claim 2, wherein the anti-TSLP heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 1, 2 and 3, respectively, and the anti-TSLP light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 5 and 6, respectively.

5. 5. The recombinant antibody of claim 4, wherein the anti-TSLP heavy chain variable region and the anti-TSLP light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 7 and 8, respectively.

6. 3. The recombinant antibody of claim 2, wherein the anti-IL4R heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOS: 11, 12, and 13, respectively, and the anti-IL4R light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOS: 14, 15, and 16, respectively.

7. 7. The recombinant antibody of claim 6, wherein the anti-IL4R heavy chain variable region and the anti-IL4R light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 17 and 18, respectively.

8. 3. The recombinant antibody of claim 2, wherein the heavy chain constant region is a human IgG1, IgG2 or IgG4 constant region.

9. 3. The recombinant antibody of claim 2, i) first and second polypeptide chains comprising, in order from N-terminus to C-terminus, the anti-IL4R scFv, an optional linker, the anti-TSLP heavy chain variable region, and the heavy chain constant region, respectively; and third and fourth polypeptide chains comprising, in N-terminal to C-terminal order, the anti-TSLP light chain variable region and the light chain constant region, respectively; ii) first and second polypeptide chains comprising, in N-terminal to C-terminal order, the anti-TSLP heavy chain variable region and the heavy chain constant region, respectively; and third and fourth polypeptide chains comprising, in N-terminal to C-terminal order, the anti-IL4R scFv, an optional linker, the anti-TSLP light chain variable region, and the light chain constant region, respectively; iii) first and second polypeptide chains comprising, in N-terminal to C-terminal order, the anti-TSLP heavy chain variable region, the heavy chain constant region, an optional linker, and the anti-IL4R scFv, respectively; and third and fourth polypeptide chains comprising, in N-terminal to C-terminal order, the anti-TSLP light chain variable region and the light chain constant region, respectively; iv) first and second polypeptide chains comprising, in N-terminal to C-terminal order, the anti-TSLP scFv, an optional linker, the anti-IL4R heavy chain variable region, and the heavy chain constant region, respectively; and third and fourth polypeptide chains comprising, in N-terminal to C-terminal order, the anti-IL4R light chain variable region and the light chain constant region, respectively; v) first and second polypeptide chains comprising, in N-terminal to C-terminal order, the anti-IL4R heavy chain variable region and the heavy chain constant region, respectively; and third and fourth polypeptide chains comprising, in N-terminus to C-terminus order, the anti-TSLP scFv, an optional linker, the anti-IL4R light chain variable region, and the light chain constant region, respectively; or vi) first and second polypeptide chains comprising, in N-terminal to C-terminal order, the anti-IL4R heavy chain variable region, the heavy chain constant region, an optional linker, and the anti-TSLP scFv, respectively; and third and fourth polypeptide chains comprising, in N-terminal to C-terminal order, the anti-IL4R light chain variable region and the light chain constant region, respectively; Including, the anti-IL4R heavy chain variable region in the first polypeptide chain and the anti-IL4R light chain variable region in the third polypeptide chain bind to form an IL4R-binding domain, and the anti-IL4R heavy chain variable region in the second polypeptide chain and the anti-IL4R light chain variable region in the fourth polypeptide chain bind to form an IL4R-binding domain, the anti-TSLP heavy chain variable region in the first polypeptide chain and the anti-TSLP light chain variable region in the third polypeptide chain bind to form a TSLP-binding domain, and the anti-TSLP heavy chain variable region in the second polypeptide chain and the anti-TSLP light chain variable region in the fourth polypeptide chain bind to form a TSLP-binding domain; the anti-IL4R scFv comprises, in order from N-terminus to C-terminus, the anti-IL4R heavy chain variable region, an optional linker, and the anti-IL4R light chain variable region, or the anti-IL4R light chain variable region, an optional linker, and the anti-IL4R heavy chain variable region; the anti-TSLP scFv comprises, in order from N-terminus to C-terminus, the anti-TSLP heavy chain variable region, an optional linker and the anti-TSLP light chain variable region, or the anti-TSLP light chain variable region, an optional linker and the anti-TSLP heavy chain variable region; the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the second polypeptide chain are linked to each other; Recombinant antibodies.

10. 10. The recombinant antibody of claim 9, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain are: i) SEQ ID NOs: 21, 21, 39 and 39, respectively; ii) SEQ ID NOs: 22, 22, 39 and 39, respectively; iii) SEQ ID NOs: 23, 23, 40 and 40, respectively; iv) SEQ ID NOs: 24, 24, 40 and 40, respectively; v) SEQ ID NOs: 25, 25, 40 and 40, respectively; vi) SEQ ID NOs: 26, 26, 40 and 40, respectively; vii) SEQ ID NOs: 27, 27, 39 and 39, respectively; or viii) SEQ ID NOs: 28, 28, 39 and 39, respectively; 1. A recombinant antibody comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to

11. A nucleic acid molecule encoding the recombinant antibody of claim 1 or 2.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising an expression vector according to claim 12 or having the nucleic acid molecule according to claim 11 integrated into its genome.

14. A pharmaceutical composition comprising the recombinant antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.

15. 15. A method for treating a disease associated with TSLP or IL4 / IL13 in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 14.

16. 16. The method of claim 15, wherein the disease is an inflammatory disease.

17. 17. The method of claim 16, wherein the disease is atopic dermatitis, asthma, chronic rhinosinusitis, nasal polyps, or eosinophilic esophagitis.

18. A kit comprising the recombinant antibody of claim 1 or 2.

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