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  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• Thymic stromal lymphopoietin is an interleukin 7 -like cytokine that plays a critical role in the regulation of immune responses and in the differentiation of hematopoietic cells.
• TSLP is widely expressed in epithelial cells of the lung, skin and gut, HassalTs corpuscles in the thymic medulla, mucosa-associated lymphoid tissues and tonsils (Liu, Soumelis et al.
• High TSLP expression levels are found in the heart, liver, spleen and prostate compared to expression levels in lung, skeletal muscle, kidney, spleen, ovary, small intestine and colon (Quentmeier, Drexler et al. 2001).
• the epithelial cell-derived cytokine is produced in response to environmental and proinflammatory stimuli. It induces the release of T-cell attracting chemokmes from monocytes and enhances maturation of myeloid dendritic cells. Within the thymus, TSLP activates myeloid and plasmacytoid dendritic cells, resulting in production of regulatory T cells. TSLP is important in regulating type 2 (Th2, T2) immunity through its activity on dendritic ceils, T and B ceils, and production of cytokines by antigen-specific Th2 cells (Ziegler, Roan et al. 2013).
• TSLP signals through a heterodimeric receptor complex composed of the thymic stromal lymphopoietin receptor (TSLPR) and the IL-7R alpha-chain (Verstraete, van Seine et al. 2014).
• TSLPR thymic stromal lymphopoietin receptor
• IL-7R alpha-chain IL-7R alpha-chain
• TSLP can activate multiple signal transduction pathways (Zhong, Sharma et al. 2014).
• Stimulation of IL7R/TSLPR complex by TSLP induces the phosphorylation and activation of Janus kinases (JAKs).
• JAKs Janus kinases
• Activated JAKs regulate the activity of multiple transcription (STAT) factors, which include STAT1, STAT3, STAT4, STAT5a, STAT5b and STAT6.
• STAT multiple transcription
• STAT multiple transcription
• Several other proteins such as AKT1, ERK1/2, JNK
• TSLP can regulate phosphorylation of 226 proteins including kinases and protein phosphatases such as S HP-- 1 and SHP-2.
• TSLP is known to be involved in the initiation of an inflammatory cascade and can contribute to development of chronic disease including allergic asthma, atopic dermatitis, allergic rhinitis, ulcerative colitis, and many chronic fibroproliferative disorders (Vannella, Ramalingam et al. 2016). Studies have suggested also that TSLP is involved in immune disorders including infection (Piliponsky et al 2016), cancer (De Monte et al. 2011), autoimmunity (Moret et al. 2011); as well as in innate and adaptive immune responses (Ziegler, Roan, 2013; Headley et al. 2009).
• TSLP is a master regulator of allergic inflammation, particularly type 2 inflammatory pathways, and therefore has been a promising target for treatment of asthma.
• Asthma is a disorder characterized by inflammation of the bronchial tubes with increased production of mucous inside the tubes.
• People with asthma experience symptoms such as coughing, wheezing, shortness of breath, chest tightness, pain or pressure due to chronic airway hyper-responsiveness, typically with eosinophil infiltration (Ohta, Nagase et al. 2017).
• Some patients with severe asthma have frequent exacerbations associated with persistent eosinophilic inflammation despite continuous treatment with high-dose inhaled glucocorticoids with or without oral
• glucocorticoids A certain percentage of refractile asthma patients, those who do not respond to conventional treatment, have severe symptoms due most likely to a non-eosinophil, non-Th2 cell population.
• TSLP expression is higher m the airways of patients with asthma than in those of healthy controls, and its levels correlate with Th2 cytokine and chemokme expression and disease severity (Gacuteau , O'Byrne et al. 2014). More recently, Tezepelumab (AMG 157), a human anti-TSLP IgG 2 antibody that specifically binds human TSLP, is in clinical trials for uncontrolled asthma (Comeau, DESMEDT et al. 2006) (Comeau, Smothers et al. 2012) (Corren, Fames et al. 2017).
• IL-5 IL-5 receptor mAbs directed toward IL-5 have been approved for patients with severe asthma and an eosinophilic phenotype, Mepolizumab and Reslizumab, winch bmd directly to IL-5, and Benralizumab which binds to the IL-5 receptor (Ortega, Liu et al. 2014) (Castro, Zangrilii et al. 2015) (Bfeecker, FitzGerald et al. 2016).
• Dupilumab an antibody that binds to the 11-4 receptor alpha chain and prevents binding of both IL-4 and 1L-13, has been approved for patients with moderate-to-severe eosinophilic asthma (Castro, Corren et al. 2018). Notably, all these therapeutic antibodies appear to be more efficacious in patients with increasing levels of T2 biomarkers relative to those with lower levels.
• TSLP TSLP in systemic sclerosis
• systemic sclerosis an autoimmune disease
• TSLP is highly expressed in perivascular areas and in immune cells of the skin.
• TSLP is upregulated in cutaneous epithelial cells, mast cells, and fibroblasts of patients with systemic sclerosis (Usategui, Criado et al. 2013).
• TSLP and its receptor are strongly expressed in lungs of patients with idiopathic pulmonary fibrosis, a severe fibrotic lung disease (Datta, Alexander et al. 2013).
• idiopathic pulmonary fibrosis a severe fibrotic lung disease (Datta, Alexander et al. 2013).
• dermal fibroblasts produce high levels of TSLP in response to TGFb in keloidal tissues.
• TSLP has a role m the initiation and progression of a variety of tumors. These include solid tumors (such as breast, colon, and pancreatic) as well as hematological tumors (such as B cell acute lymphocytic leukemia (B-ALL)) (Corren, Ziegler 2019). Tumors displaying Th2-type responses generally have a worse prognosis than those tumors with predominantly Th-type responses. Tumor cells secrete II- 1 a and IL-1 b which are required for TSLP expression by the cancer associated fibroblasts, suggesting that the tumor and tumor microenvironment are important in induction of TSLP. Studies with human cervical carcinoma cells, gastric and ovarian cancers also provide evidence that there is TSLP mediated cross-talk between hematopoietic ceils that infiltrate the tumor and stroma, and the tumor itself.
• B-ALL B cell acute lymphocytic leukemia
• TSLP has an important role in cancer, its function as a pro or anti-tumor factor is dependent on the type of tumor.
• TSLP in metastatic breast cancer is an area of intense study, but there have been contradictory findings in mouse models and human breast-tumor cell lines.
• the role of TSLP in skin tumors is dichotomous.
• the role of TSLP in B-ALL is complicated by the presence of genetic lesions with mutations in genes encoding components of the TSLP signaling pathway which occur in 50-60% of patients with poor prognosis (Corren, Ziegler 2019).
• asthma As an example, mortality due to asthma related symptoms is around 250,000 per year globally, and the number is predicted to grow by more than 100 million by 2025 (Pawankar 2014). People with allergic asthma often have other conditions like diabetes, obesity, cardiovascular disease, gastro-oesophageai disease leading to more complicated and worse outcomes. About 70% of asthmatics also have allergies. Allergic diseases include anaphylaxis, food allergies, certain forms of asthma, rhinitis, conjunctivitis, angioedema, urticaria, atopic dermatitis, eczema, eosinophilic disorders, including eosinophilic esophagitis and drug and insect allergies (Pawankar 2014) (Kay 2001).
• the disclosure provides for monoclonal antibodies and antigen-binding fragments thereof that specifically bind and neutralize, inhibit, block, abrogate, reduce, or interfere with, at least one activity of thymic stromal lymphopoietin, TSLP.
• the activity of TSLP that can be neutralized, inhibited, blocked, abrogated, reduced or interfered, by the antibodies or fragments thereof as disclosed herein includes, but is not limited by, neutralization of TSLP activation of its receptor complex, and the like.
• the antibodies are monoclonal antibodies, e.g., mouse monoclonal antibodies or humanized antibodies.
• the antibodies are antigen-binding antibody fragments.
• the disclosure provides for a mouse monoclonal antibody to human TSLP, designated as TAVO202, comprising a heavy chain variable region sequence with amino acid sequence of SEQ ID NO. 1, and a light chain variable region sequence with amino acid sequence of SEQ ID NO. 2.
• the disclosure provides for the heavy chain variable region of TAVO202 comprising three Complementarity Determining Regions (CDRs), designated as lTCDRl, HCDR2 and HCDR3, with ammo acid sequence set forth as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
• CDRs Complementarity Determining Regions
• the disclosure provides for the light chain variable region of TAVO202 comprising three CDRs, designated as LCDR1, LCDR2 and LCDR3, with amino acid sequence set forth as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.
• the disclosure provides for one humanized heavy chain variable region of
• TAVO202 designated as 202H2
• ammo acid sequence set forth as SEQ ID NO. 9.
• the disclosure provides for two humanized light chain variable region of TAVO202, designated as 2021.3 and 2021.4. with amino acid sequences set forth as SEQ ID NO. 10 and SEQ ID NO. 11, respectively.
• the disclosure provides a humanized antibody or antigen-binding fragment thereof that comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NO. 3, SEQ ID NO. 4 SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.
• the humanized antibody comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity ' to SEQ ID NO: 9.
• the humanized antibody comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11.
• the disclosure provides for a first humanized antibody for TAVO202 comprising humanized heavy chain variable region 202IT2 and humanized light chain variable region 202L3 with human IgG2 Fc, designated as TAV06264, comprising heavy chain sequence set forth as SEQ ID NO. 12 and light chain sequence set forth as SEQ ID NO. 13.
• the disclosure provides for a second humanized antibody for TAYO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L4 with human IgG2 Fc, designated as TAV06265, comprising heavy chain sequence set forth as SEQ ID NO. 12 and light chain sequence set forth as SEQ ID NO. 14.
• the disclosure provides for a third humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L3 with human IgGl Fc with L234F, L235E, D265A, F405L mutations, designated as TAV07264, comprising heavy chain sequence set forth as SEQ ID NO. 15 and light chain sequence set forth as SEQ ID NO. 13.
• the disclosure provides for a fourth humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L4 with human IgGl Fc with L234F, L235E, D265A, F405L mutations, designated as TAV07265, comprising heavy chain sequence set forth as SEQ ID NO. 15 and light chain sequence set forth as SEQ ID NO. 14.
• the disclosure provides for a fifth humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L3 with human IgGl Fc with L234A, L235A, M428L, N434S mutations, designated as TAV09764, comprising heavy chain sequence set forth as SEQ ID NO. 16 and light chain sequence set forth as SEQ ID NO. 13.
• the disclosure provides for a sixth humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H 2 and humanized light chain variable region 202L.4 with human IgGl Fc with L234A, L235A, M428L, N434S mutations, designated as TAV09765, comprising heavy chain sequence set forth as SEQ ID NO. 16 and light chain sequence set forth as SEQ ID NO. 14.
• the anti-TSLP monoclonal antibodies can be full length IgGi, IgG 2 , IgG 3 , IgG 4 antibodies or may comprise only an antigen-binding portion including a Fab, F( ab' )2, or scFv fragment that specifically binds (eg, human) TSLP, neutralizes, inhibits, blocks, abrogates, reduces, or interferes with at least one activity of TSLP.
• the antibody backbones may be modified to affect functionality, e.g. to eliminate residual effector functions.
• the disclosure also provides for anti-TSLP monoclonal antibodies with an extended half- life when compared to the wild-type antibody.
• the extension of half-life can be realized by engineering the CH2 and Cm domains of the antibody with any one set of mutations selected from M252Y/S254T/T256E, M428L/N434S, T250Q/M428L, N434A and T307A/E38QA/N434A when compared to a parental wild-type antibody, residue numbering according to the EU Index.
• the disclosure also provides for anti-TSLP monoclonal antibodies with enhanced resistant to proteolytic degradation by a protease that cleaves the wild-type antibody between or at residues 222-237 (EU numbering).
• the resistance to proteolytic degradation can be realized by engineering E233P/L234V/L235A mutations in the hinge region with G236 deleted when compared to a parental wild-type antibody, residue numbering according to the EU index.
• the disclosure also provides for vectors comprising the polynucleotides of the disclosure.
• the disclosure also provides for a method of producing the anti-TSLP monoclonal antibodies of the disclosure, comprising culturing the host cell of the disclosure under conditions that the antibody is expressed, and purifying the antibody.
• the disclosure also provides for a pharmaceutical composition
• a pharmaceutical composition comprising the anti-TSLP antibodies or antigen- binding fragments thereof of the disclosure and a pharmaceutically acceptable carrier.
• the disclosure also provides for methods of detecting the binding of the anti-TSLP antibodies.
• the disclosure also provides for methods of blocking the binding of TSLP to its receptor TLPR, the method comprises contacting the TSLPR with any one of the anti-TSLP antibodies or antigen binding fragments thereof provided herein.
• the disclosure also provides for a method of treating asthma comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• the disclosure also provides for a method of treating COPD and idiopathic pulmonary' fibrosis, comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• the disclosure also provides for a method of treating symptoms of atopic dermatitis, comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• the disclosure also provides for a method of treating symptoms of eczema, comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• the disclosure also provides for a method of treating symptoms of other allergic states such as eosinophilic esophagitis, comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• a method of treating symptoms of inflammatory bowel diseases comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• the disclosure also provides for a method of treating fibrotic conditions such as systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloidal disease, comprising administering to a subject in need a therapeutically effective amount of the provided antibodies to TSLP.
• fibrotic conditions such as systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloidal disease
• the disclosure also provides for a method of treating cancers, i.e., breast, pancreatic, colorectal, lymphoblastic leukemia, head and neck cancer, comprising administering to a subject in need therapeutically effective amounts of the provided antibodies to TSLP.
• cancers i.e., breast, pancreatic, colorectal, lymphoblastic leukemia, head and neck cancer
• FIG. 1 Binding to (A) human and (B) mouse TSLP by mouse monoclonal anti-human TSLP antibody TAVO202.
• FIG. 2 (A). The response of cell proliferation upon stimulation by human TSLP in BAF cells transfected with human TSLP receptor complex. (B). Dose-dependent neutralizing human TSLP- ⁇ driven cell proliferation by TAVO202.
• FIG. 3 (A). Dose-dependent STATS reporter gene expression upon stimulation by human TSLP in a luciferase reporter assay. (B). Dose-dependent neutralization of reporter gene expression by TAVO202 in human TSLP-driven reporter gene expression assay.
• FIG. 4 Sequence alignments of heavy chain and light chain variable regions of
• TAVQ202 with humanized VH and VL variants Humanized antibodies can be formed by pairing the humanized VH variant (202H2) and two humanized VL variants (202L3 and 20214) with different IgG Fc.
• FIG. 5 SDS-PAGE analysis of example humanized anti-TSLP IgG antibodies under (A) non-reduced and (B) reduced conditions.
• C Size Exclusion Chromatography (SEC) analysis of example anti-TSLP IgG antibodies.
• FIG. 6 Binding to (A) human and (B) cynomolgus TSLP by example humanized anti- TSLP IgG antibodies.
• FIG. 7 Neutralizing human TSLP driven reporter gene activation by example humanized anti-TSLP IgG antibodies in STATS reporter gene expression assay.
• FIG. 8 Neutralizing human TSLP driven CCL17 release from dendritic cells isolated from two donors by humanized anti-TSLP IgG antibodies.
• FIG. 9 Binding to mouse FcRn at pH 6.0 by humanized anti-TSLP IgG antibody TAV09765 with half-life extension Fc mutations and TAV07265 lacking such mutations.
• FIG 10 PK profile of anti-TSLP antibody in a cynomolgus monkey.
• Anti-TSLP antibody with Fc mutations for half-life extension (TAV09765) was administered as a 4 mg/kg intravenous dose into a cynomolgus monkey.
• the plasma concentration is displayed at time points up to day 35 post-dose.
• Antibodies is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multi-specific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity'.
• “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH?. and Cm).
• Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
• VL light chain variable region
• CL light chain constant region
• the VH and the VL regions may be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
• CDRs are“antigen binding sites” in an antibody.
• CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDRl, HCDR2, HCDR3) and three in the VL (LCDRl, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat 1970) (Rabat, National Institutes of et al. 1991); (ii)“Hypervariable regions,”“HVR,” or“HV,” three in the VH (HI, H2, H3) and three in the VL (Li, L2, L3) refer to the regions of an antibody variable domains which are
• CDR CDR defined by any of the methods described supra, Rabat, Chothia or IMGT, unless otherwise explicitly stated in the specification.
• Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant region amino acid sequence.
• IgA and IgG are further sub-classified as the isotypes IgA 1 , IgA 2 , IgG 1 , IgG 2 , IgG 3 and IgG 4
• Antibody light chains of any vertebrate species may assign to one of two clearly distinct types, namely kappa (K) and lambda (l), based on the amino acid sequences of their constant regions.
• Antibody fragments refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1 , 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL).
• Antibody fragments include well known Fab, F(ab')2., F d and F v fragments as well as domain antibodies (dAb) consisting of one VH domain.
• VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the V H and V L domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Patent Publ. Nos. WO 1998/44001, WO1988/01649, WO1994/13804 and W01992/01047.
• scFv single chain Fv
• “Monoclonal antibody” refers to an antibody population with single ammo acid composition in each heavy and each light chain, except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies typically bind one antigenic epitope, except that bispecific monoclonal antibodies bind two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multi-specific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
• isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding for example TSLP is substantially free of antibodies that specifically bind antigens other than TSLP).
• isolated antibody encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure
• Humanized antibody refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or human immunoglobulin germLine gene sequences.
• Human antibody refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin and is optimized to have minimal immune response when administered to a human subject. If the antibody contains a constant region or a portion of the constant region, the constant region also is derived from sequences of human origin.
• A“variant” of a polypeptide comprises an ammo acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
• Variants include antibodies and fragments thereof that have a recited percent identity to an antibody or fragment provided herein or to an antibody or fragment having a recited DNA or amino acid sequence.
• identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences.
• Perfect identity means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”).
• sequences being compared are typically aligned in a way that gives the largest match between the sequences.
• the constant region sequences of the mammalian IgG heavy chain are designated m sequence as CH 1 -hinge-CH 2 -CH 3
• The“hinge,”“hinge region” or“hinge domain” of an IgG is generally defined as including Glu216 and terminating at Pro230 of human IgG 1 according to the EU Index but functionally, the flexible portion of the chain may be considered to include additional residues termed the upper and lower hinge regions, such as from Glu216 to Gly237 and the lower hmge has been referred to as residues 233 to 239 of the Fc region where FVyR binding was generally attributed.
• Hinge regions of other IgG isotypes may be aligned with the IgG 1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds.
• boundaries may vary slightly, as numbered according to the EU Index, the CHI domain is adjacent to the VH domain and ammo terminal to the hinge region of an
• the immunoglobulin heavy chain molecule includes the first (most amino terminal) constant region of an immunoglobulin heavy chain, e.g., from about EU positions 1 18-215.
• the F c domain extends from amino acid 231 to amino acid 447; the CH2 domain is from about Ala231 to Lys340 or Gly341 and the CH3 from about Gly341 or Gln342 to Lys447.
• the residues of the IgG heavy chain constant region of the CH 1 region terminate at Lys.
• the Fc domain containing molecule comprises at least the CH 2 and the CHI domains of an antibody constant region, and therefore comprises at least a region from about Ala231 to Lys447 of IgG heavy chain constant region.
• the F c domain containing molecule may optionally comprise at least portion of the lunge region.
• Epitope refers to a portion of an antigen to which an antibody specifically binds.
• Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.
• An epitope may be composed of contiguous and/or discontinuous amino acids that form a conformational spatial unit. For a discontinuous epitope, amino acids from differing portions of the linear sequence of the antigen come near in 3-dimensional space through the folding of the protein molecule.
• Antibody“epitope” depends on the methodology used to identify the epitope.
• A“leader sequence” as used herein includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well-known in the art..
• polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
• the terms also include polypeptides that have co- translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.
• a“polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, if the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.
• nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature.
• recombinant as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression from a recombinant
• recombinant refers to a host cell or virus into which a recombinant polynucleotide has been introduced.
• Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g, a cell, a nucleic acid, a protein, or a vector).
• polynucleotide “oligonucleotide,”“nucleic acid” and“nucleic acid molecule” are used interchangeably herein to include a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary' structure of the molecule.
• Vector refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
• Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector.
• the vector polynucleotide may be DNA or RNA molecules, cDNA, or a hybrid of these, single stranded or double stranded.
• “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
• Value refers to the presence of a specified number of binding sites specific for an antigen in a molecule.
• the terms“monovalent,”“bivalent,”“tetravalent,” and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.
• heterologous nucleic acid sequences, proteins or polypeptides means that these molecules are not naturally occurring in the cell from which the heterologous nucleic acid sequence, protein or polypeptide was derived.
• the nucleic acid sequence coding for a human polypeptide that is inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in that context.
• heterologous nucleic acids may be derived from different organism or animal species, such nucleic acid need not be derived from separate organism species to be heterologous.
• a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be heterologous to a cell into which it is introduced in that the cell did not previously contain the synthetic nucleic acid.
• a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be considered heterologous to a human cell, e.g , even if one or more components of the synthetic nucleic acid sequence or a polypeptide encoded therefrom was originally derived from a human cell.
• A“host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
• a nucleic acid e.g., an expression vector that comprises a nucleotide sequence encoding a multimeric polypeptide of the present disclosure
• A“recombinant host cell” (also referred to as a“genetically modified host cell”) is a host ceil into which has been introduced a heterologous nucleic acid, e.g., an expression vector.
• a genetically modified eukaryotic host cell is genetically modified by introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
• Specific binding or“specifically binds” or“binds” refer to an antibody binding to a specific antigen with greater affinity than for other antigens.
• the equilibrium dissociation constant (KD) for binding is about 1 1 if 8 M or less, for example about 1 x 10 -9 M or less, about 1 x10 -10 M or less, about 1 x10 -11 M or less, or about 1 x 10 -12 M or less, typically with the K D that is at least one hundred-fold less than its K D for binding to a non-specific antigen (e.g., B S A, casein).
• the KD may be measured using standard procedures.
• the terms“treatment,”“treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
• Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e. , causing regression of the disease.
• the terms“individual,”“subject,”“host,” and“patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murine (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equine, bovine, ovine, porcine, caprine), etc.
• murine e.g., rats, mice
• lagomorphs e.g., rabbits
• non-human primates humans
• canines felines
• ungulates e.g., equine, bovine, ovine, porcine, caprine
• A“therapeutically effective amount” or“efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.
• The“therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
• composition of anti-TSLP antibody and antigen binding fragment Composition of anti-TSLP antibody and antigen binding fragment
• monoclonal antibodies and antigen-binding fragments thereof that specifically bind and neutralize, inhibit, block, abrogate, reduce, or interfere with, at least one activity of TSLP, e.g., a functional activity of TSLP to its receptor complex.
• the anti-TSLP antibodies and antigen binding fragments can be therapeutically administered to a subject to treat TSLP mediated diseases.
• the disclosure provides for a mouse monoclonal antibody, designated as TAU ⁇ 202, which was identified from a mouse hybridoma screening, that specifically binds and neutralizes, inhibits, blocks, abrogates, reduces, or interferes with, at least one activity of human thymic stromal lymphopoietin (TSLP).
• TAU ⁇ 202 mouse monoclonal antibody
• TAVO202 comprises a heavy chain variable region sequence with amino acid sequence of SEQ ID NO. 1, and a light chain variable region sequence with amino acid sequence of SEQ ID No. 2.
• the heavy chain variable region of TAVO202 comprises three Complementarity Determining Regions, designated as HCDRI, HCDR2 and HCDR3, with amino acid sequence set forth as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
• the light chain variable region of TAVO202 comprises three Complementarity Determining Regions, designated as LCDR1, LCDR2 and LCDR3, with amino acid sequence set forth as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.
• HCDRI sequence of Anti -human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO: 3) GYTFSSYWVN
• HCDR3 sequence of Anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO: 5)
• the mouse anti- human TSLP antibody TAVO202 can be humanized by grafting of mouse CDRs onto human germLine scaffolds. A few key mouse residues are preserved by back mutations to achieve higher stability and better expression while minimizing immunogenicity.
• one humanized VH variant (202H2) is designed based on IGHV1 -69*02 and two humanized VL variants are designed with 202L3 based on IGKV1-9*01 and 202L4 based on IGKV6-21*01 with a couple of back mutations in framework.
• the disclosure provides for one humanized heavy chain variable region of TAVO202, designated as 202H2, with amino acid sequence set forth as SEQ ID NO. 9.
• Humanized heavy chain variable region 202H2 sequence (SEQ ID NO: 9)
• the sequences of three CDRs of humanized heavy chain are underlined.
• the disclosure provides for two humanized light chain variable region of TAVO202, designated as 202L3 and 202L4, with ammo acid sequences set forth as SEQ ID NO. 10 and SEQ ID NO. 1 1 , respectively.
• Humanized light chain variable region 202L3 sequence (SEQ ID NO: 10)
• Humanized light chain variable region 202L4 sequence (SEQ ID NO: 1 1)
• the disclosure provides for a first humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L3 with human IgG2 Fc, designated as TAV06264, comprising heavy chain sequence set forth as SEQ ID NO 12 and light chain sequence set forth as SEQ ID NO. 13
• the disclosure provides for a second humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L4 with human IgG2 Fc, designated as TAV06265, comprising heavy chain sequence set forth as SEQ ID NO. 12 and light chain sequence set forth as SEQ ID NO 14.
• the disclosure provides for a third humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L3 with human IgGl Fc with L234F, L235E, D265A, F405L mutations, designated as TAV07264, comprising heavy chain sequence set forth as SEQ ID NO. 15 and light chain sequence set forth as SEQ ID NO. 13.
• the disclosure provides for a fourth humanized antibody for TAVO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L4 with human IgGl Fc with L234F, L235E, D265A, F405L mutations, designated as TAVO7265, comprising heavy chain sequence set forth as SEQ ID NO. 15 and light chain sequence set forth as SEQ ID NO. 14.
• the disclosure provides for a fifth humanized antibody for TAYO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L3 with human IgGl Fc with L234A, L235A, M428L, N434S mutations, designated as TAV09764, comprising heavy chain sequence set forth as SEQ ID NO. 16 and light chain sequence set forth as SEQ ID NO. 13.
• the disclosure provides for a sixth humanized antibody for TAYO202 comprising humanized heavy chain variable region 202H2 and humanized light chain variable region 202L4 with human IgGl Fc with L234A, L235A, M428L, N434S mutations, designated as TAVO9765, comprising heavy chain sequence set forth as SEQ ID NO. 16 and light chain sequence set forth as SEQ ID NO. 14.
• Anti-TSLP humanized antibody Heavy Chain based on 202H2 with igG2 Fc (SEQ ID NO: 12)
• variable domain of heavy chain is underlined.
• Anti-TSLP humanized antibody Light Chain based on 202L3 (SEQ ID NO: 13)
• variable domain of light chain is underlined.
• Anti-TSLP humanized antibody Light Chain based on 202L4 (SEQ ID NO: 14)
• Anti-TSLP humanized antibody Heavy Chain based on 202H2 with IgGl Fc with L234F,
• variable domain of heavy chain is underlined.
• L234F, L235E, D265A, F405L mutations are bolded.
• Anti-TSLP humanized antibody Heavy Chain based on 202H2 with IgGl Fc with L234A, L235A, M428L, N434S mutations (SEQ ID NO: 16)
• variable domain of heavy chain is underlined.
• L234A, L235A, M428L, N434S mutations are bolded.
• the disclosure also provides for preparation of a bispecific antibody or multi specific antibody that can include the engagement of any two, three, or four TSLP epitopes by having Fab or scFv domains comprising the one humanized heavy chain variable region of TAVO202, designated as 202H2, with ammo acid sequence set forth as SEQ ID NO. 9 with one of the two humanized light chain variable region of TAVO202, designated as 202L3 and 202L4, with amino acid sequences set forth as SEQ ID NO. 10 and SEQ ID NO. 11, respectively.
• this can be a bispecific antibody that has Fab or scFv domains 202112 with 202L3 and 202H2 with 202L4, or any other permutation of domains that can engagement two, three, or four epitopes.
• the TSLP binding antibodies and fragments of the present disclosure encompass antigen binding fragments that retain sufficient ability to specifically bind to TSLP.
• the TSLP binding fragments as used herein may include any 3 or more contiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more, or even 10 or more contiguous amino acids) of the antibody and encompasses Fab, Fab’, F(ab’)2, and F(v) fragments, or the individual light or heavy chain variable regions or portions thereof. These fragments lack the F c fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody. These fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab’)2 fragments).
• TSLP binding antibodies and fragments of the present disclosure may also encompass diabodies, which are bivalent antibodies in which VH and VI domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
• the TSLP binding antibodies and fragments of the present disclosure may also encompass single-chain antibody fragments (scFv) that bind to TSLP.
• An scFv comprises an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds TSLP.
• VH antibody heavy chain variable region
• VL antibody light chain variable region
• Such TSLP binding fragments can be prepared by methods known in the art such as, for example, the synthesis or PCR mediated amplification of the variable portions of the heavy and light chains of an antibody molecule and a flexible protein linker composed of the amino acids Gly and Ser. The resulting DNA fragment is cloned for expression in E. cob or mammalian cells.
• the expressed TSLP binding fragments are purified from the host cells.
• the TSLP binding antibodies and fragments of the present disclosure encompass full length antibody comprising two heavy chains and two light chains.
• Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA, and IgD antibodies.
• the present antibodies can be of any class (IgG, IgM, IgE, IgGA, IgD, etc.) or isotype.
• a human antibody can comprise an IgG Fc domain, such as at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
• an IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, ammo acid sequence identity to an IgGl Fc sequence as SEQ ID NO: 17.
• an IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, ammo acid sequence identity to an IgG2 Fc sequence as SEQ ID NO: 18.
• an IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, ammo acid sequence identity to an IgG3 Fc sequence as SEQ ID NO: 19.
• an IgG Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to an IgG4 Fc sequence as SEQ ID NO: 20.
• a S228P mutation may be made into IgG4 antibodies to enhance IgG4 stability.
• IgGi Fc SEQ ID NO: 17:
• IgGz Fc SEQ ID NO: 18:
• the present anti-TSLP antibodies may comprise a modified F c region, wherein the modified F c region comprises at least one ammo acid modification relative to a wild-type F c region.
• the present anti-TSLP antibodies are provided with a modified F c region where a naturally-occurring F c region is modified to extend the half-life of the antibody when compared to the parental wild-type antibody in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
• Exemplary mutations that may be made singularly or in combinations are T250Q, M252Y, 1253 A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations, or a fusion of an albumin-binding peptide onto the C terminus of the Fc) with enhanced circulating antibody half-life relati ve to a control without said Fc mutation.
• the extension of half-life can be realized by engineering the M252Y/S254T/T256E mutations in IgGl Fc as SEQ ID NO: 21 , residue numbering according to the EU Index (Dall'Acqua, Kiener et al. 2006).
• the extension of half-life can also be realized by engineering the M428L/N434S mutations in IgGl Fc as SEQ ID NO: 22 (Zalevsky, Chamberlain et al. 2010). In certain embodiments, the extension of half-life can also be realized by engineering the T25QQ/M428L mutations m IgGi Fc as SEQ ID NO: 23 (Hinton, Xiong et al. 2006).
• the extension of half-life can also be realized by engineering the N434A mutations in IgGi Fc as SEQ ID NO: 24 (Shields, Namenuk et al. 2001).
• the extension of half-life can also be realized by engineering the T307A/E380A/N434A mutations in IgGi Fc as SEQ ID NO: 25 (Petkova, Akilesh et al. 2006).
• IgGi Fc with M252Y/S254T/T256E mutations (SEQ ID NO: 21)
• the present anti-TSLP antibodies are provided with a modified F c region where a naturally-occurring F c region is modified to enhance the antibody resistance to proteolytic degradation by a protease that cleaves the wild-type antibody between or at residues 222-237 (EU numbering).
• the antibody or antigen binding fragment thereof further comprises a mutation in the lower hinge region of Fc (e.g., equivalent to residues 222-237 of IgGi Fc) and allowing for increased protease resistance (e.g., resistance to degradation by MMP- 3, MMP-7, MMP-12, MMP-13, cathepsin G, pepsin, IdeS, or GluV8) relative to the native IgGi antibody in environments with endogenous proteolytic activity.
• Fc e.g., equivalent to residues 222-237 of IgGi Fc
• protease resistance e.g., resistance to degradation by MMP- 3, MMP-7, MMP-12, MMP-13, cathepsin G, pepsin, IdeS, or GluV8
• the resistance to proteolytic degradation can be realized by engineering E233P/L234V/L235A mutations in the hinge region with G236 deleted when compared to a parental wild-type antibody as SEQ ID NO: 26, residue numbering according to the EU Index (Kinder, Greenplate et al. 2013).
• the antibodies of the disclosure may further be engineered to introduce at least one mutation m the antibody F c that reduces binding of the antibody to an activating F c y receptor (F c yR) and/or reduces F c effector functions such as Cl q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell- mediated cytotoxicity (ADCC) or phagocytosis (ADCP).
• F c yR activating F c y receptor
• F c effector functions such as Cl q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell- mediated cytotoxicity (ADCC) or phagocytosis (ADCP).
• Fc positions that may be mutated to reduce binding of the antibody to the activating F c yR and subsequently to reduce effector functions are those described for example in (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993) (Chu, Vostiar et al. 2008) (Shields, Namenuk et al. 2001). Fc mutations with minimal ADCC, ADCP, CDC, Fc medicated cellular activation have been described also as sigma mutations for IgGl, XgG2 and IgG4 (Tam, McCarthy et al. 2017).
• Exemplary mutations that may be made singularly or in combinations are K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331 S mutations on IgGi, IgG2, IgGb or IgGr.
• Exemplary combination mutations that may be made to reduce ADCC are L234A/L235A on IgGi, V 234 A/G237A/P238 S/H268 A/V309L/A33 OS /P331 S on IgG 2 , F234A/L235A on IgCi4, S228P/F234A/L235A on IgGi, N297A on IgGi, IgGi, IgGs or IgGi, V234A/G237A on !gG 2 ,
• Hybrid IgG 2/4 F c domains may also be used, such as F c with residues 117-260 from IgGz and residues 261 -447 from IgGi.
• the present anti-TSLP antibodies are provided with a modified F c region where a naturally-occurring F c region is modified to facilitate the generation of bispecific antibody by F c heterodimerization.
• the Fc heterodimerization can be realized by engineering F405L and K409R mutations on two parental antibodies and the generation of bispecific antibody in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014).
• the Fc heterodimerization can also be realized by Fc mutations to facilitate Knob-in-FIole strategy (see, e.g., Inti. Publ. No. WO 2006/028936).
• An ammo acid with a small side chain (hole) is introduced into one F c domain and an amino acid with a large side chain (knob) is introduced into the other F c domain.
• a heterodimer is formed as a result of the preferential interaction of the heavy chain with a“hole” with the heavy chain with a“knob” (Ridgway, Presta et al. 1996).
• Exemplary F c mutation pairs forming a knob and a hole are: T366Y/F405A,
• the Fc heterodimerization can also be realized by Fc mutations to facilitate the electrostatically-matched interactions strategy (Gunasekaran, Pentony et al.
• Mutations can be engineered to generate positively charged residues at one Fc domain and negatively charged residues at the other Fc domain as described in US Patent Publ. No.
• Heavy chain heterodimerization can be formed by electrostatically-matched interactions between two mutated Fc.
• Constant modifications refer to ammo acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the ammo acid sequences. Conservative modifications include amino acid substitutions, additions and deletions.
• amino acids with acidic side chains e.g., aspartic acid, glutamic acid
• basic side chains e.g., lysine, arginine, histidine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, pro line, phenylalanine, methionine
• uncharged polar side chains e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan
• aromatic side chains e.g., phenylalanine, tryptophan, histidine, tyrosine
• aliphatic side chains e.g., glycine, alanine, valine, leucine, isoleucme, serine, threon
• any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis ((MacLennan, Rice et ai. 1998); (Sasaki and Sutoh 1998)).
• Amino acid substitutions to the antibodies of the disclosure may be made by known methods for example by PCR mutagenesis (US Patent No. 4,683,195).
• libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp).
• the resulting antibody variants may be tested for their characteristics using assays described herein.
• the antibodies of the disclosure may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifi cations may occur m vivo or in vitro.
• the antibodies of the disclosure may be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation may be carried out by techniques known to those skilled in the art.
• Antibodies of the disclosure may be modified to improve stability, selectivity, cross- reactivity, affinity, immunogen! city or other desirable biological or biophysical property are within the scope of the disclosure.
• Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein/protein interface interactions that have impact upon the HC and LC pairing, (3) burial of polar and charged residues, (4) H-bonding network for polar and charged residues; and (5) surface charge and polar residue distribution among other intra- and inter- molecular forces (Worn and PJuckthun 2001).
• Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modelling in certain cases, and the effect of the residues on antibody stability may be tested by generating and evaluating variants harboring mutations in the identified residues.
• One of the ways to increase antibody stability is to raise the thermal transition midpoint (T m ) as measured by differential scanning calorimetry (DSC).
• T m i the protein T m i s correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation m solution and the degradation processes that depend on the tendency of the protein to unfold (Remmele and Gombotz 2000).
• Antibodies of the disclosure may have ammo acid substitutions in the F c region that improve manufacturing and drug stability.
• An example for IgGi is H224S (or H224Q) in the hinge 221-DKTHTC-226 (Eu numbering) which blocks radically induced cleavage (Yates, Gunasekaran et al. 2010); and for IgGr, the S228P mutation blocks half-antibody exchange (Angal, King et al. 1993, Labrijn, Buijsse et al. 2009).
• the anti-TSLP antibodies and fragments of the disclosure can be encoded by a single nucleic acid (e.g , a single nucleic acid comprising nucleotide sequences that encode the light and heavy chain polypeptides of the antibody), or by two or more separate nucleic acids, each of which encode a different part of the antibody or antibody fragment.
• a single nucleic acid e.g , a single nucleic acid comprising nucleotide sequences that encode the light and heavy chain polypeptides of the antibody
• two or more separate nucleic acids each of which encode a different part of the antibody or antibody fragment.
• the disclosure provides nucleic acid sequence as SEQ ID NO: 27 which encodes anti-TSLP humanized antibody heavy chain based on 202H2 with IgG2 Fc, nucleic acid sequence as SEQ ID NO: 28 which encodes the anti-TSLP antibody light chain sequence based on 202L3, and nucleic acid sequence as SEQ ID NO: 29 which encodes the anti- TSLP antibody light chain sequence based on 2021,4.
• Nucleotide sequence for anti-TSLP humanized antibody heavy chain based on 202H2 with IgG2 Fc (SEQ ID NO: 27)
• the nucleic acids can be inserted into vectors, e.g , nucleic acid expression vectors and/or targeting vectors.
• vectors can be used in various ways, e.g., for the expression of anti- TSLP binding antibody or antibody fragment in a cell or transgenic animal.
• Vectors are typically selected to be functional in the host cell in which the vector will be used.
• a nucleic acid molecule encoding anti-TSLP binding antibody or fragment may be amplified / expressed m prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells.
• Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete mtron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
• a promoter typically contains one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete mtron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
• a leader or signal sequence is engineered at the N-terminus of the anti- TSLP antibodies or fragments to guide its secretion.
• the secretion of anti-TSLP antibodies or fragments from a host cell will result in the removal of the signal peptide from the antibody or fragment.
• the mature antibody or fragment will lack any leader or signal sequence.
• the disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the disclosure.
• the cell can be any type of ceil capable of being transformed with the nucleic acid or vector of the disclosure so as to produce a polypeptide encoded thereby.
• DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
• Methods of introducing nucleic acids and vectors into isolated cells and the culture and selection of transformed host cells in vitro include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran- mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.
• the cell After introducing the nucleic acid or vector of the disclosure into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence.
• the antibody, antigen binding fragment, or portion of the antibody then can be isolated from the cell.
• two or more vectors that together encode anti-TSLP binding antibodies, or antigen binding fragments thereof can be introduced into the cell.
• Purificati on of anti-TSLP binding antibodies or fragments which have been secreted into the cell media can be accomplished using a variety of techniques including affinity,
• antibodies comprising a F c region may be purified by affinity chromatography with Protein A, which selectively binds the F c region.
• Modified forms of an antibody or antigen binding fragment may be prepared with affinity tags, such as hexahisddine or other small peptide such as FLAG (Eastman Kodak Go., New Haven, Conn.) or rnyc (Invitrogen) at either its carboxyl or ammo terminus and purified by a one-step affinity column.
• affinity tags such as hexahisddine or other small peptide such as FLAG (Eastman Kodak Go., New Haven, Conn.) or rnyc (Invitrogen) at either its carboxyl or ammo terminus
• affinity tags such as hexahisddine or other small peptide such as FLAG (Eastman Kodak Go., New Haven, Conn.) or rnyc (Invitrogen) at either its carboxyl or ammo terminus
• polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen® nickel columns)
• Modified forms of an antibody or antigen binding fragment may be prepared with affinity tags, such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl or amino terminus and purified by a one-step affinity column.
• affinity tags such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl or amino terminus
• polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen® nickel columns) can be used for purification of polyhistidine- tagged selective binding agents. In some instances, more than one purification step may be employed.
• Thymic stromal lymphopoietin is an epithelial cell derived protein belonging to a cytokine family that is implicated in allergic inflammation (Cianferoni and Spergel 2014). It is produced mainly by non-hematopoietie cells such as fibroblasts, epithelial cells and different - like cells, and plays an important role in the maturation of T cell populations through activation of antigen presenting cells. TSLP is produced in response to proinflammatory stimuli and drives allergic inflammatory responses mainly by influence on dendritic cells for T helper (Th) 2 cytokine production.
• Th T helper
• TSLP Human TSLP has two isoforms.
• the long form of TSLP, lfTSLP, is expressed in several tissues including heart, liver and prostate and induces release of T-cell attracting chemokines from monocytes and enhances the maturation of CD1 lc-dendritic cells. It can induce allergic inflammation by directly activating mast ceils (Reche, Soumelis et al. 2001 , Zhang and Zhou 2012).
• Isoform 2 is the short form of TSLP, sfTSLP, and is the predominant form in
• keratinocytes of oral mucosa, skin and in salivary glands may function as an antimicrobial peptide (Bjerkan, Sonesson et al. 2016) .
• Human TSLP is mapped to chromosome 5q22.1
• amino acid sequence of lfTSLP is provided as SEQ ID No. 30, of which the first 28 amino acids constitute the signal peptide and Y29 to Q159 is the mature form.
• the ammo acid sequence of sfTSLP is provided as SEQ ID No. 31.
• TSLP receptor binds TSLP with low affinity and is a member of the hematopoietin receptor family (Park, Martin et al. 2000).
• a combination of IL-7 receptor alpha chain (IL-7Ra) and TSLPR is required for high affinity binding of TSLP to form the ternary complex (Verstraete, van Schie et al. 2014).
• the ternary complex is necessary for cell proliferation and signalling.
• mouse and human TSLP The homology between mouse and human TSLP is 43% and 39% for the TSLPR, with no cross reactivity between the species.
• human TSLP and TLPLR are orthologs to mouse TSLP and TSLPR.
• mouse models have been used for various biological studies, including use of a chimeric human TSLP and human TSLPR transgenic mouse strain (Francis, Milford et al. 2016).
• TSLP expression is linked to many disease states including asthma (Ying, O’Connor et al. 2005), inflammatory arthritis (Koyama, Ozawa et al. 2007), inflammatory bowel disease (Park, Jeong et al. 2017), atopic dermatitis (Ebner, Nguyen et al. 2007), eczema , eosinophilic esophagitis and other allergic states (Soumelis and Liu 2004), (Soumelis, Reche et al. 2002). Understanding the mechanism of TSLP production and those potential substances that block the production may enable better methods to prevent or treat these conditions. Thus, targeting TSLP may inhibit multiple biologic pathways involved in asthma and Th2 cytokine mediated inflammatory disorders (Galuteau , O'Byrne et a ⁇ . 2014).
• the present disclosure encompasses anti-TSLP monoclonal antibodies or antibody fragments that bind selectively to TSLP with greater affinity than to other antigens.
• the anti- TSLP monoclonal antibodies and fragments may bind selectively to human TSLP, but also bind detectably to non-human TSLP.
• TSLP binding antibodies and antibody fragments may have the same or substantially the same potency against recombinant human TSLP and endogenous human TSLP.
• the binding of TSLP to their receptors may be determined by immobilizing a TSLP binding antibody, sequestering TSLP with the immobilized antibody and determining whether the TSLP is bound to the antibody, and contacting a soluble form of receptor with the bound TSLP/antibody complex and determining whether the soluble receptor is bound to the complex.
• the protocol may also include contacting the soluble receptors with the immobilized antibody before the contact with TSLP antibody, to confirm that the soluble receptor does not bind to the immobilized antibody. This protocol can be performed using a Biacore® instrument for kinetic analysis of binding interactions. Such a protocol can also be employed to determine whether an antibody or other molecule permits or blocks the binding of TSLP to their receptors.
• the permitting or blocking of TSLP binding to its receptor may be determined by comparing the binding of TSLP to its receptor in the presence or absence of TSLP antibodies or TSLP binding fragments thereof. Blocking is identified in the assay readout as a designated reduction of TSLP binding to its receptor in the presence of anti- TSLP antibodies or TSLP binding fragments thereof, as compared to a control sample that contains the corresponding buffer or diluent but not an TSLP antibody or TSLP binding fragment thereof.
• the assay readout may be qualitatively viewed as indicating the presence or absence of blocking or may be quantitatively viewed as indicating a percent or fold reduction in binding due to the presence of the antibody or fragment.
• the TSLP binding to its receptor is reduced by at least 10-fold, alternatively at least about 20-fold, alternatively at least about 50- fold, alternatively at least about 100-fold, alternatively at least about 1000-fold, alternatively at least about 10000-fold, or more, compared to binding of the same concentrations of TSLP and its receptor in the absence of the antibody or fragment.
• Preferred anti- TSLP antibodies for use in accordance with the disclosure generally bind to human TSLP with high affinity (e.g., as determined with BIACQRE), such as for example with an equilibrium binding dissociation constant (KD) for TSLP of about 10 iiM or less, about 5 iiM or less, about 1 nM or less, about 500 pM or less, or more preferably about 250 pM or less, about 100 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, or about 0.3 pM or less.
• KD equilibrium binding dissociation constant
• Antibodies or fragments of the present disclosure may, for example, bind to TSLP with an EC50 of about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nM or less, as determined by enzyme linked immunosorbent assay (ELISA).
• ELISA enzyme linked immunosorbent assay
• the antibody or antibody fragment of the present disclosure does not cross- react with any target other than TSLP.
• the present antibodies and fragments may bind to TSLP, but do not detectably bind to, or have at least about 100 times (e.g., at least about 150 times, at least about 200 times, or even at least about 250 times) greater selectivity in its binding of TSLP relative to its binding to other proteins.
• the key amino acid residues (epitope) bound by the TSLP binding antibody or fragment described in this disclosure may be determined using a peptide array, such as for example, a PepSpotTM peptide array (JPT Peptide Technologies, Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanning the entire TSLP ammo acid sequence, each peptide overlapping by 11 ammo acid to the previous one, is synthesized directly on a membrane.
• a peptide array such as for example, a PepSpotTM peptide array (JPT Peptide Technologies, Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanning the entire TSLP ammo acid sequence, each peptide overlapping by 11 ammo acid to the previous one, is synthesized directly on a membrane.
• antibody competition experiments may be performed and such assays are well known in the art.
• the present disclosure also encompasses neutralizing antibodies or neutralizing fragments thereof which bind to TSLP so as to neutralize its biological activity.
• Neutralization of biological activity of TSLP can be assessed by assays for one or more indicators of biological activity, such as TSLP stimulated reporter gene expression in a reporter assay.
• Neutralization of biological activity of TSLP can also be assessed in vivo by animal models of asthma or atopic dermatitis.
• the TSLP binding antibodies and fragments of the present disclosure neutralize the biological activity of TSLP connected with the signalling function of their receptors bound by the TSLP.
• the present antibodies or fragments may be neutralizing antibodies or fragments which bind specifically to TSLP epitopes that affects biological activity of TSLP.
• the present antibodies or fragments can bind to neutralization-sensitive epitopes of TSLP. When a neutralization-sensitive epitope of TSLP is bound by one of the present antibodies or fragments, the result is a loss of biological activity containing the epitope.
• an antibody or fragment thereof of the present disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with, an activity of TSLP by binding to an epitope of TSLP that is directly involved in the targeted activity of TSLP.
• an antibody or fragment thereof of the disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with, an activity of TSLP by binding to an epitope of TSLP that is not directly involved in the targeted activity of TSLP, but the antibody or fragment binding thereto sterically or conformationally inhibits, blocks, abrogates, reduces or interferes with, the targeted activity of TSLP.
• an antibody or fragment thereof of the disclosure binds to an epitope of TSLP that is not directly involved in the targeted activity of TSLP (i.e., a non-blocking antibody), but the antibody or fragment binding thereto results in the enhancement of the clearance of TSLP.
• the antibody or antigen-binding fragment thereof : a) inhibits binding of TSLP and TSLPR; b) reduces the expression level of an TSLP-dependent gene; c) inhibits TSLP-induced cell proliferation; d) inhibits TSLP-mduced STATS activation; and/or e) inhibits TSLP-induced CCL17 production from primary human dendritic cells.
• TSLP bmdrag antibodies and antibody fragments for use according to the present disclosure can be formulated in compositions, especially pharmaceutical compositions, for use in the methods herein.
• Such compositions comprise a therapeutically or prophyiactically effective amount of an TSLP binding antibody or antibody fragment of the disclosure in admixture with a suitable carrier, e.g., a pharmaceutically acceptable agent.
• a suitable carrier e.g., a pharmaceutically acceptable agent.
• TSLP binding antibodies and antibody fragments of the disclosure are sufficiently purified for administration to an animal before formulation in a pharmaceutical composition.
• Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
• the composition can be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents.
• compositions can be suitable for parenteral administration.
• Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (mtraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial,
• compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) sustained release and/or increased stability or half-life in a particular local environment.
• product e.g., bolus, depot effect
• the present antibodies and fragments are useful for the prophylaxis and treatment of TSLP-mediated diseases or medical conditions, e.g., asthma, allergic rhinitis, atopic dermatitis, allergies, and certain cancers.
• One aspect of the disclosure provides a method of treating moderate to severe uncontrolled asthma, including asthma that is not Th2 driven, by administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibody or antigen binding fragment thereof and the subject pharmaceutical composition.
• Another aspect of the disclosure provides a method of treating a fibrotic disease and related condition, including systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloidal disease, by administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibody or antigen binding fragment thereof and the subject pharmaceutical composition.
• Another aspect of the disclosure provides a method of treating chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, and refractory asthma, by administering to a subject in need of a therapeutically effective amount of any one of the subject antibody or antigen binding fragment thereof and the subject pharmaceutical composition.
• chronic obstructive pulmonary disease such as emphysema, chronic bronchitis, and refractory asthma
• Another aspect of the disclosure provides a method of treating disorders associated with inflammatory diseases, including rhinitis, atopic dermatitis, eczema, food allergies, and inflammatory bowel disease: the method comprising administering to a subject in need of a therapeutically effective amount of any one of the subject antibody or antigen-binding fragments thereof and the subject pharmaceutical composition.
• inflammatory diseases including rhinitis, atopic dermatitis, eczema, food allergies, and inflammatory bowel disease
• Another aspect of the disclosure provides a method of treating certain cancers, including lung, breast, pancreatic, colorectal, lymphoblastic leukemia, head and neck carcinomas; the method comprising administering to a subject in need of a therapeutically effective amount of any one of the subject antibody or antigen-binding fragments thereof and the subject pharmaceutical composition.
• An aspect of the disclosure also provides uses of any of the antibodies or antigen binding fragments thereof provided herein, in a the manufacture of a medicament for blocking the binding of TSLP to its receptor TLPR, and/or a medicament for the prophylaxis and treatment of TSLP-mediated diseases or medical conditions such as those provided herein.
• An aspect of the disclosure also provides for use of the provided antibodies or antigen binding fragments thereof for blocking the binding of TSLP to its receptor TLPR and/or for in methods for the prophylaxis and treatment of TSLP-mediated diseases or medical conditions such as those provided herein.
• the disclosure provides uses the antibodies provided herein in methods for treating asthma, COPD, idiopathic pulmonary fibrosis, atopic dermatitis, eczema, eosinophilic esophagitis, inflammatory bowel disease, systemic sclerosis, systemic idiopathic pulmonary fibrosis, keloidal disease, or cancer.
• the present antibodies and fragments can be used in diagnostic methods to detect TSLP (for example, in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (Rizos C.V.), an radioimmunoassay (RIA) or tissue immunohistochemistry.
• a method for detecting TSLP in a biological sample can comprise the steps of contacting a biological sample with one or more of the present antibodies or fragments and detecting either the antibody or fragment bound to TSLP or unbound antibody or fragment, to thereby detect TSLP in the biological sample.
• the antibody or fragment can be directly or indirectly labelled with a detectable substance to facilitate detection of the bound or unbound antibody.
• Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
• Example 1 TSLP binding affinity for mouse anti-TSLP antibody TAVO202
• TAVO202 was identified as a mouse anti-human TSLP antibody by hybridoma screening.
• ELISA-based binding assay was employed to evaluate TAVO202 binding to recombinant human TSLP.
• 1 gg/niL recombinant human TSLP R&D Systems
• Increasing concentrations of TAVO202 antibodies were applied on the plate and their binding to the recombinant human TSLP were detected by HRP-conjugated anti-mouse secondary antibody. It was observed that TAVO202 dose-dependently bound recombinant human TSLP with EC50 at 2.4 ng/mL ( Figure 1 A).
• TAVO202 The binding of TAVO202 to mouse TSLP was also evaluated in similar ELIS A assay by coating the plate with mouse TSLP (R&D Systems). TAVO202 did not show significant binding affinity to mouse TSLP ( Figure IB).
• Example 2 in vitro assays for TSLP neutralization by TAVO202
• a TSLP-mediated cell proliferation assay was developed to assess functional activity of TAVO202.
• human IL-7 receptor alpha (IL-7Ra) and TSLP receptor (TSLPR) were co-transfected into BAF3 mouse pro-B cells.
• Recombinant human TSLP was added to the transfected cells and cell proliferation was quantitated with a cell proliferation assay two days later. It was observed that recombinant human TSLP could stimulate the proliferation of the transfected BAF3 cell line in a dose-dependent manner with EC50 at 0.18 ng/mL ( Figure 2A).
• TAVO202 along with 0.5 ng/mL human TSLP were applied with BAF3 ceils co-transfected with IL-7Ra and TSLPR. It was observed that TAVO202 could dose-dependently neutralize TSLP activity in stimulating transfected BAF3 cell proliferation with IC50 at 6.6 ng/mL ( Figure 2B).
• TSLP-driven reporter gene expression assay was also developed to assess the functional activity of TAVO202.
• STATS activation is a downstream event that occurs after TSLP binds to and activates its receptor complex
• TSLPR IL-7Ra
• plasmids expressing TSLPR, IL-7Ra and a STAT5 luciferase reporter construct were transiently transfected into HEK293T ceils.
• recombinant human TSLP was added and TSLP-driven luciferase reporter gene expression was quantitated 24 hours later. It was observed that human TSLP could dose-dependently drive reporter gene expression with EC50 at 1.4 ng/mL ( Figure 3 A).
• TAVO202 along with 10 ng/mL human TSLP were applied with HEK293T cells co transfected with IL ⁇ 7Ra, TSLPR and STATS luciferase reporter gene. It was observed that TAVO202 dose-dependently neutralized TSLP activity in stimulating reporter gene expression with IC50 of 20 ng/mL ( Figure 3B ).
• the mouse anti- human TSLP antibody TAVO202 was humanized by grafting of mouse CDRs onto human gemiLine scaffolds. A few key mouse residues were preserved by back mutations to achieve higher stability and better expression while minimizing immunogenieity.
• TAVO202 one humanized VH variant (202H2) was designed based on 1GHV1 -69*02 and two humanized VL variants were designed with 202L3 and 202L4 based on IGKV1-9*01 and IGKV6-21 *01 respectively with a couple of back mutations (Figure 4).
• two humanized TAVO202 antibodies with IgG2 Fc designated as TAV06264 and TAV06265
• TAV06264 and TAV06265 were generated with 202H2 variant pairing with 202L3 and 202L4 respectively
• TAVQ202 antibodies with IgGl Fc with L234F, L235E, D265A, F405L mutations designated as TAV07264 and TAV07265
• TAV07264 and TAV07265 were also generated with 202H2 variant pairing with 202L3 and 202L4 respectively.
• TAV09764 and TAVQ9765 two humanized TAVO202 antibodies with IgGl Fc with L234A, L235A, M428L, N434S mutations, designated as TAV09764 and TAVQ9765, were also generated with 202H2 variant pairing with 202L3 and 202L4 respectively ( Figure 4).
• TAV07264, TAV07265, TAV09764, TAV09765 were co-transfected into Expi293F ceils following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 pm filter. The purification of expressed antibodies m supernatant was carried out by affinity chromatography over protein A agarose column (GE Healthcare Life Sciences). The purified antibodies were buffer-exchanged into DPBS, pH7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.
• the purified humanized TAVO202 antibodies were subjected to SDS-PAGE analysis and an example for TAV07264 and TAV07265 was shown in Figure 5A and 5B. Under the reduced condition, all four antibodies showed heavy chains and light chains with the expected molecular weight. Under the non-reduced condition, all four antibodies migrated as a major protein band with a molecular weight around 150 kDa.
• the purified humanized TAVO202 antibodies were also assessed by Size Exclusion Chromatography (SEC).
• Figure 5C revealed SEC profiles of example antibodies TAV07264 and TAV07265.
• Example 5 Binding to TSLP by humanized TAVO202 ELISA-based binding assays were employed to evaluate humanized TAVO202 antibodies binding to recombinant human TSLP antigen.
• 1 gg/mL recombinant human TSLP R&D Systems
• HRP-conjugated anti-mouse secondary antibody HRP-conjugated anti-mouse secondary antibody.
• SPR surface plasmon resonance
• Example 6 In vitro assay for TSLP neutralization by humanized TAVO202 antibodies The TSLP-driven STATS luciferase reporter gene expression assay was employed to assess the functional activity of humanized TAVO202 antibodies. Increasing concentrations of TAY06264 and TAV06265 along with 10 ng/mL human TSLP were applied with HEK293T cells co-transfected with IL-7Ra, TSLPR and STATS luciferase reporter gene. It was observed that both TAV06264 and TAY06265 could dose-dependently neutralize TSLP activity in stimulating reporter gene expression with IC50 comparable to TAVO202 ( Figure 7).
• Example 7 Inhibition of TSLP-mediated dendritic cell aetivation by humanized
• TSLP can stimulate the activation of primary human CDlc ⁇ blood dendritic ceils with the increased secretion of CCL17 chemokine.
• An ex vivo assay was set up to evaluate the humanized TAVO202 antibodies in neutralizing TSLP-driven CCL17 release from activated dendritic cells.
• CDl c + dendritic cells were isolated from donor peripheral blood mononuclear cells (PBMC) by anti-CD l c + antibody coated magnetic beads selection (Miltenyi Biotec). The isolated cells were incubated with recombinant human TSLP and the release of CCL17 was quantitated by ELISA.
• Example 8 F c engineering of humanized antibodies for extended half-life and rednced effeetor functions
• F c mutations can be introduced to IgGl antibody to extend antibody half-life.
• M428L/N434S mutations have been demonstrated to extend antibody half-life by increasing FcRn binding affinity (Booth,
• TAV09764 and TAV09765 were generated and designated as TAV09764 and TAV09765, respectively ( Figure 4).
• the binding by TAL ⁇ 9765 and TAV07265 to mouse FcRn were assessed in ELISA-based binding assay. 1 gg/rnl, recombinant mouse FcRn (R&D Systems) were coated on ELISA plate.
• TAV09765 and TAV07265 antibodies were applied on the plate and their binding to the recombinant FcRn under pH 6.0 were detected by FIRP-conjugated anti- human secondary antibody. It was observed that TAV09765, which has the Fc M428L/N434S mutations, could bind FcRn with about 10-fold better potency than TAV07265 which is lacking such half-life extension mutations (Figure 9).
• TAV09765 was tested in a cynomolgus monkey PK model. TAV09765 was administered as an intravenously infusion at 4 mg/kg into a male naive cynomolgus monkey at a volume of 1.0 mL/kg for 3 minutes based on the body weight on day 0. Whole blood was collected into EDTA-K2 collection tubes at pre-dose, and at lh, 2h, and on days 2, 5, 9, 12, 16, 21, 24, 26, 29, 32, 35 post-dose.
• PK parameters for TAV09765 were as follows: Co : 131405 ng mL -1 , half-life: 26.131 d, AUCo-t : 1268621 ng-d-mL -1 , AUCo-mf : 2128728 ng-d-mL 1 , clearance : 1.879 mL-kg -1 d -1 , Vd : 71.049 mL-kg -1 .
• TSLP an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic ceil maturation.
• Vafa () , G. L. Gilliland, R. J. Brezski, B. Strake, T. Wilkinson, E. R. Lacy, B. Scallon, A.
• Verstraete K., L. van Scliie, L. Vyncke, Y. Bloch, J. Tavernier, E. Pauweis, F. Peelman and S.
• TSLP signaling pathway map a platform for analysis of TSLP-mediated signaling.
• Database the journal of biological databases and curation 2014: bau007-bau007.
• TSLP thymic stromal lymphopoietin

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