Anti-TSLP Fab with improved stability

Abstract

The present disclosure relates to anti-TSLP Fabs with improved stability, nucleic acids encoding the Fabs, host cells and vectors containing the nucleic acids, and methods of using the Fabs in the treatment of TSLP-associated conditions.

Description

[Technical field]

[0001] The present disclosure relates to anti-TSLP Fabs with improved stability, nucleic acids encoding the Fabs, host cells and vectors containing the nucleic acids, and methods of using the Fabs to treat disease. [Background technology]

[0002] Thymic stromal lymphopoietin (TSLP) is a cytokine that signals through a heterodimeric receptor composed of IL-7Ra subunits and TSLP-R, a unique component that resembles the common y receptor-like chain (Pandey et al., Nat. Immunol. 2000, 1(1):59-64). TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine, and tonsils, as well as airway smooth muscle cells, lung fibroblasts, and stromal cells (Edwards, 2008, Drug news & perspectives 21, 312-316; He and Geha, 2010, Annals of the New York Academy of Sciences 1183, 13-24; Reche et al., 2001, Journal of immunology 167, 336-343).

[0003] These cells produce TSLP in response to inflammatory stimuli, and TSLP drives allergic inflammatory responses through its activity on multiple innate immune cells, including dendritic cells (Soumelis et al., 2002, Nature immunology 3, 673-680), monocytes (Reche et al., 2001, Journal of immunology 167, 336-343), and mast cells (Allakhverdi et al., 2007, The Journal of Experimental Medicine 204, 253-258). The cell population known to have the highest expression of both TSLP-R and IL-7Ra is myeloid dendritic cells (Reche et al., 2001, Journal of immunology 167, 336-343).

[0004] TSLP can promote the proliferation of naive T cells and drive their differentiation into Th2 cells that express high levels of IL-4, IL-5, and IL-13 (Omori and Ziegler, 2007, Journal of immunology 178, 1396-1404). High levels of TSLP expression have been found in asthmatic lung epithelial cells and chronic atopic dermatitis lesions, suggesting a role for TSLP in allergic inflammation (Ziegler and Artis, 2010, Nature immunology 11, 289-293). Furthermore, recent evidence has linked TSLP to Th17 cell differentiation and Th17-driven inflammatory processes (Hartgring et al., 2011, Arthritis and rheumatism 63, 1878-1887; Tanaka et al., 2009, Clinical and experimental allergy: Journal of the British Society for Allergy and Clinical Immunology 39, 89-100; Wu et al., 2014, Journal of molecular and cellular cardiology 76, 33-45). Whereas chronic allergic (atopic) asthma is often characterized by Th2-type inflammation, non-allergic asthmatic inflammation is predominantly neutrophilic with a mixed Th1 and Th17 cytokine milieu. Consequences of chronic inflammation in asthma include bronchial hyperresponsiveness (BHR), mucus overproduction, airway wall remodeling, and airway narrowing (Lambrecht and Hammad, 2014, Nature immunology 16, 45-56). TSLP has been shown to be involved in the initiation and maintenance / enhancement of allergic asthmatic responses (Wang et al., 2006, Immunity 24, 827-838).More recently, TSLP signaling has also been shown to be required for the recall response of memory T cells to local antigen challenge (Wang et al., 2015, The Journal of allergy and clinical immunology 135, 781-791 e783).

[0005] Tezepelumab is a human immunoglobulin G2 (lgG2) monoclonal antibody (mAb) that binds TSLP and prevents its interaction with the TSLP receptor complex. A proof-of-concept study in patients with mild atopic asthma demonstrated that tezepelumab inhibited early and late asthmatic responses after inhaled allergen challenge and suppressed biomarkers of Th2 inflammation. A study evaluating tezepelumab in adult and adolescent patients with severe uncontrolled asthma (NCT03347279) was recently completed and achieved its primary endpoint of reduction in annualized asthma exacerbation rate (AERR) [time frame: baseline to week 52].

[0006] CSJ117 is a potent neutralizing antibody fragment against human thymic stromal lymphopoietin (TSLP) and has been formulated for pulmonary delivery via a dry powder inhaler as a PulmoSol™ engineered powder in a hard capsule (Gauvrea et al ERS 2020 56:Suppl.64,3690).

[0007] Therefore, targeting TSLP for the treatment of inflammatory diseases such as asthma has been clinically validated, and given that most asthma patients are accustomed to self-medicating with inhaled drugs, the administration of inhaled TSLP antagonists is of interest.

[0008] The present disclosure aims to build on existing treatment options, particularly those via inhalation, in this emerging pharmaceutical class. Summary of the Invention

[0009] Surprisingly, the present disclosure finds that multiple mutations in the CH1 domain of tezepelumab, converting the molecule into a Fab, reduce aggregation under accelerated stability conditions (2 weeks in 1×PBS at 45° C.). The accelerated stability study in 1×PBS may be used as a rough approximation of in vivo serum stability.

[0010] Many therapeutically administered antibodies have a half-life of more than 14 days (2 weeks). Fabs may be more suitable for inhalation delivery, given their smaller size than mAbs and improved biodistribution in the lung after aerosolization. Although administered by inhalation, serum stability remains a significant factor for protein-based antagonists administered to the lung. Therefore, the improved accelerated stability of exemplary Fabs may be advantageous when used to treat TSLP-related conditions.

[0011] Thus, in one embodiment, the disclosure provides a Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0012] In another embodiment, the disclosure provides a Fab comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:2.

[0013] In another aspect, the disclosure provides a Fab comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0014] In another aspect, the disclosure provides a nucleic acid encoding a Fab described herein.

[0015] In another aspect, the disclosure provides a vector comprising a nucleic acid described herein.

[0016] In another aspect, the disclosure provides a host cell comprising a nucleic acid or vector described herein.

[0017] In another aspect, the disclosure provides a method of treatment comprising administering to a subject a therapeutically effective amount of a Fab described herein.

[0018] In another aspect, the disclosure provides a Fab described herein for use in therapy.

[0019] In another aspect, the disclosure provides for the use of the Fabs described herein in therapy.

[0020] In another aspect, the disclosure provides for the use of a Fab described herein in the manufacture of a medicament for use in treatment. [Brief description of the drawings]

[0021] [Figure 1A] FIG. 1 shows a 2-week accelerated stability assay of tezepelumab at approximately 1 mg / ml in 1× PBS at 4° C. The plot shows the HP-SEC traces of tezepelumab after incubation at each temperature for the assigned time periods. [Figure 1B] FIG. 1 shows a 2-week accelerated stability assay of tezepelumab at approximately 1 mg / ml in 1× PBS at 45° C. The plot shows the HP-SEC traces of tezepelumab after incubation at each temperature for the assigned time periods. [Figure 1C] The % monomer loss and % aggregate formation of tezepelumab are shown along with the results for the IgG1 isotype control NIP228. [Figure 2A] Figure 1 shows a 2-week accelerated stability assay of Fab1 at approximately 0.8 mg / ml in 1x PBS at 4°C. The plot shows the HP-SEC traces of Fab1 after incubation at each temperature for the assigned time periods. [Figure 2B] Figure 1 shows a 2-week accelerated stability assay of Fab1 at approximately 0.8 mg / ml in 1x PBS at 45° C. The plot shows the HP-SEC traces of Fab1 after incubation at each temperature for the assigned time periods. [Figure 2C]The % monomer loss and % aggregate formation of Fab1 are shown along with the results for the Fab isotype control R347. [Diagram 3] Binding of Fab1 to human TSLP as measured by KinExA is shown. [Figure 4] Binding of Fab1 to cynomolgus TSLP as measured by KinExA is shown. [Diagram 5] 1 shows competitive binding of Fab1 to human TSLP using an HTRF assay. [Figure 6] We show that Fab1 inhibits CCL17 release from TSLP-challenged PBMCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "an anti-TSLP Fab" is understood to represent one or more anti-TSLP Fabs.

[0023] "Antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (so long as they exhibit the desired antigen-binding activity).

[0024] "Antibody fragments" include antigen-binding portions of antibodies, including, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, tetrabodies, minibodies, linear antibodies, chelating recombinant antibodies, tri- or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single domain antibodies (including camelized antibodies), VHH-containing antibodies, or variants or derivatives thereof, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide (e.g., one, two, three, four, five, or six CDR sequences), provided that the antibody retains the desired biological activity.

[0025] "Fab" refers to an antibody fragment comprising VH-CH1 and VL-CL pairings. The term encompasses Fabs that contain non-canonical sequence variants (e.g., amino acid substitutions, deletions, or insertions) within the Fab outside of sequence regions typically associated with high sequence variability. For example, Fab variants include Fabs that contain non-canonical amino acid or sequence changes in the VH or VL framework regions or in the CH1 or CL domains. Such changes may include the presence of non-canonical cysteines or other derivatizable amino acids, which can be used to conjugate the Fab variants to heterologous moieties. Other such changes include the presence of non-canonical polypeptide linkers (polypeptide sequences that covalently bridge between two domains). For example, Fab variants can include a linker polypeptide that covalently links the CH1 domain to the VL domain or the CL domain to the VH domain so that the Fab can be expressed as a single polypeptide chain.

[0026] "Host cells" refer to cells that harbor a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In describing the process of isolating anti-TSLP Fab from a recombinant host, the terms "cells" and "cell culture" are used interchangeably to indicate the source of anti-TSLP Fab, unless otherwise clearly indicated. In other words, recovery of polypeptide from "cells" can mean either recovery from spun down whole cells or recovery from cell culture containing both medium and suspension cells.

[0027] "Isolated" refers to a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or crude products include those that have been purified to the extent that they are no longer in the form found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0028] A "pharmaceutical composition" refers to a preparation that is in a form that effectively activates the biological activity of an active ingredient (e.g., an anti-TSLP Fab disclosed herein) and that does not contain additional components that are unacceptably toxic to a subject to which the composition is administered. Such compositions may be sterile.

[0029] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0030] "Recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered to be isolated for the purposes of the present invention, being native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized.

[0031] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject, except where the subject is defined as a "healthy subject." Mammalian subjects include humans, domestic animals, livestock, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc. Preferably, the subject is a human.

[0032] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliative, and remission (partial or total) of the disease state, both detectable and undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to having the condition or disorder or those in whom the condition or disorder is to be prevented.

[0033] A "therapeutically effective amount" refers to an amount of an anti-TSLP Fab or other drug disclosed herein effective to "treat" a disease or disorder in a subject or mammal.

[0034] "TSLP" refers to thymic stromal lymphopoietin. TSLP is a cytokine that signals through a heterodimeric receptor composed of IL-7Ra subunits and TSLP-R, a unique component that resembles the common y receptor-like chain. (Pandey et al., Nat. Immunol. 2000, 1(1):59-64). TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine, and tonsils, as well as airway smooth muscle cells, lung fibroblasts, and stromal cells (Edwards, 2008, Drug news & perspectives 21, 312-316; He and Geha, 2010, Annals of the New York Academy of Sciences 1183, 13-24; Reche et al., 2001, Journal of immunology 167, 336-343). These cells produce TSLP in response to inflammatory stimuli, and TSLP drives allergic inflammatory responses through its activity on multiple innate immune cells, including dendritic cells (Soumelis et al., 2002, Nature immunology 3, 673-680), monocytes (Reche et al., 2001, Journal of immunology 167, 336-343), and mast cells (Allakhverdi et al., 2007, The Journal of Experimental Medicine 204, 253-258). The cell population known to have the highest expression of both TSLP-R and IL-7Ra is myeloid dendritic cells (Reche et al., 2001, Journal of immunology 167, 336-343).

[0035] TSLP can promote the proliferation of naive T cells and drive their differentiation into Th2 cells that express high levels of IL-4, IL-5, and IL-13 (Omori and Ziegler, 2007, Journal of immunology 178, 1396-1404). High levels of TSLP expression have been found in asthmatic lung epithelial cells and chronic atopic dermatitis lesions, suggesting a role for TSLP in allergic inflammation (Ziegler and Artis, 2010, Nature immunology 11, 289-293). Furthermore, recent evidence has linked TSLP to Th17 cell differentiation and Th17-driven inflammatory processes (Hartgring et al., 2011, Arthritis and rheumatism 63, 1878-1887; Tanaka et al., 2009, Clinical and experimental allergy: Journal of the British Society for Allergy and Clinical Immunology 39, 89-100; Wu et al., 2014, Journal of molecular and cellular cardiology 76, 33-45). Whereas chronic allergic (atopic) asthma is often characterized by Th2-type inflammation, non-allergic asthmatic inflammation is predominantly neutrophilic with a mixed Th1 and Th17 cytokine milieu. Consequences of chronic inflammation in asthma include bronchial hyperresponsiveness (BHR), mucus overproduction, airway wall remodeling, and airway narrowing (Lambrecht and Hammad, 2014, Nature immunology 16, 45-56). TSLP has been shown to be involved in the initiation and maintenance / enhancement of allergic asthma responses (Wang et al., 2006, Immunity 24, 827-838). More recently, TSLP signaling has also been shown to be required for the recall response of memory T cells to local antigen challenge (Wang et al., 2015, The Journal of allergy and clinical immunology 135, 781-791 e783).

[0036] "Vector" refers to a construct capable of delivering, and in some embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0037] Anti-TSLP Fab The present disclosure provides a Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0038] In another embodiment, the disclosure provides a Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0039] In another embodiment, the disclosure provides a Fab comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0040] In the Examples, Fabs with these sequence characteristics are shown to have improved stability under accelerated stability testing conditions compared to full-length antibodies with similar VH and VL domain sequences. An exemplary Fab with improved stability is referred to herein as Fab1.

[0041] In some cases, the disclosure provides a Fab that has equivalent stability to Fab1. In some cases, the equivalent stability is under accelerated stability testing conditions. In some cases, the accelerated stability testing conditions are 1×PBS, 45° C., 2 weeks. In some cases, the concentration of the Fab under the accelerated testing conditions is about 0.8 mg / ml.

[0042] In some cases, a Fab comprises a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some cases, a Fab comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some cases, a Fab comprises a stability equivalent to Fab1.

[0043] In some cases, the Fab comprises a light chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the Fab comprises a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the Fab comprises a stability equivalent to Fab1.

[0044] In some cases, the Fab comprises a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the Fab comprises stability comparable to Fab1.

[0045] In some cases, the Fab comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the Fab comprises a stability equivalent to Fab1.

[0046] In some cases, the disclosure provides an antigen-binding fragment that includes the heavy chain of SEQ ID NO:1.

[0047] In some cases, the disclosure provides an antigen-binding fragment comprising a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some cases, the antigen-binding fragment comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some cases, the antigen-binding fragment comprises a stability equivalent to Fab1.

[0048] In some cases, the antigen-binding fragment comprises a light chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the antigen-binding fragment comprises a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the antigen-binding fragment comprises a stability equivalent to Fab1.

[0049] In some cases, the antigen-binding fragment comprises a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the antigen-binding fragment comprises stability comparable to Fab1.

[0050] In some cases, the antigen-binding fragment comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some cases, the antigen-binding fragment comprises stability equivalent to Fab1.

[0051] nucleotide The disclosure also provides nucleic acids (or "polynucleotides") encoding the Fab and antigen-binding fragments disclosed herein.

[0052] The polynucleotides disclosed herein can further comprise additional nucleic acid, for example, encoding a signal peptide that directs the secretion of an encoded polypeptide described herein.

[0053] Polynucleotides can be generated or manufactured by any method known in the art. For example, if the nucleotide sequence of the Fab is known, a polynucleotide encoding an anti-TSLP Fab can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly involves the synthesis of overlapping oligonucleotides that contain a portion of the sequence encoding the anti-TSLP Fab, annealing and ligating these oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0054] Alternatively, a polynucleotide encoding an anti-TSLP Fab may be generated from nucleic acid of a suitable source. If a clone containing a nucleic acid encoding a Fab is not available but the sequence of the Fab is known, the nucleic acid encoding the Fab can be chemically synthesized or obtained from a suitable source, for example, by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for the particular sequence of interest. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0055] Once the nucleotide sequence and corresponding amino acid sequence of a Fab are determined, the nucleotide sequence can be manipulated using methods for the manipulation of nucleotide sequences well known in the art, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY), both of which are incorporated herein by reference in their entireties), to generate Fabs with different amino acid sequences, e.g., to make amino acid substitutions, deletions, and / or insertions.

[0056] A polynucleotide encoding an anti-TSLP Fab may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an anti-TSLP Fab may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, a polynucleotide encoding the Fab may be composed of a triple-stranded region that contains RNA or DNA, or both RNA and DNA. A polynucleotide encoding an anti-TSLP Fab may also contain one or more modified bases or a DNA or RNA backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and exceptional bases such as inosine. Since various modifications can be made to DNA and RNA, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0057] Isolated polynucleotides encoding non-naturally occurring variants of immunoglobulin-derived polypeptides (e.g., immunoglobulin heavy or light chain portions) can be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0058] Manufacturing method A polynucleotide encoding an anti-TSLP Fab or antigen-binding fragment is typically inserted into an expression vector for introduction into a host cell that can be used to produce desired quantities of Fab. Thus, expression vectors containing a polynucleotide encoding a Fab as defined herein, and host cells containing such expression vectors, are encompassed herein.

[0059] Recombinant expression of a Fab or antigen-binding fragment requires construction of an expression vector containing a polynucleotide encoding the Fab. Once a polynucleotide encoding a Fab of the disclosure is obtained, the vector for production of the Fab can be generated by recombinant DNA technology using techniques well known in the art.

[0060] DNA sequences encoding the Fabs can be generated either simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated with consensus constant region primers or with more specific primers based on published DNA and amino acid sequences. PCR can also be used to isolate DNA clones encoding antibody variable light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes (e.g., mouse constant region probes).

[0061] Thus, methods for preparing proteins by expressing polynucleotides containing Fab-encoding nucleotide sequences are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing anti-TSLP Fab-encoding sequences and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present disclosure provides replicable vectors containing nucleotide sequences encoding the Fabs of the present disclosure operably linked to a promoter.

[0062] For the purposes of this disclosure, numerous expression vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses (e.g., bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus). Other vectors involve the use of a polycistronic system with an internal ribosome binding site. Additionally, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. Such elements can include signal sequences, splice signals, as well as transcription promoters, enhancers, and termination signals.

[0063] Any expression vector capable of inducing expression in eukaryotic cells can be used in the present disclosure. Examples of suitable vectors include, but are not limited to, the plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF 1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and the plasmid pCI (available from Promega, Madison, Wis.). Generally, a large number of transformed cells are screened for expressing cells.

[0064] More generally, once a vector or DNA sequence encoding a Fab has been prepared, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. Such techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway (1988) "Mammalian Expression Vectors" (Vectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.) Chapter 24.2, pp. 470-472). Typically, plasmid introduction into the host is accomplished by electroporation. The host cell carrying the expression construct is grown under conditions suitable for the production of anti-TSLP Fab, and protein synthesis is assayed. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0065] The expression vector is transfected into a host cell by conventional techniques, and the transfected cells are cultured by conventional techniques to produce an anti-TSLP Fab for use in the methods described herein. Thus, the present disclosure includes a host cell containing a polynucleotide encoding a Fab of the present disclosure, e.g., a heavy or light chain, or a variable heavy or variable light chain, operably linked to a heterologous promoter.

[0066] In one case, a culture medium is provided that contains the host cells. In one case, a fermentation vessel is provided that contains the culture medium.

[0067] In some cases, the culture medium and fermentation vessels are suitable for carrying out the methods of producing Fabs as defined herein.

[0068] A variety of host-expression vector systems can be utilized to express the anti-TSLP Fabs described herein. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the molecules of the disclosure in situ. These host expression systems include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli, B. subtilis), transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the coding sequence; yeast (e.g., Saccharomyces, Pichia), transformed with a recombinant yeast expression vector containing the coding sequence; insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the coding sequence; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0069] Bacterial cells such as Escherichia coli, and more preferably eukaryotic cells, are used for the expression of Fab. For example, the combination of mammalian cells such as Chinese hamster ovary cells (CHO) and vectors such as the major intermediate-early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio / Technology 8:2 (1990)).

[0070] Host cell lines used for protein expression are often of mammalian origin. One of skill in the art is credited with the ability to preferentially determine the particular host cell line that is most suitable for the desired gene product to be expressed. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB13 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), VERO, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese Hamster Fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / 0 (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). Typically, host cell lines are available from commercial services, the American Tissue Culture Collection, or published literature.

[0071] In addition, a host cell strain can be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

[0072] In the long term, high yield production, stable expression of recombinant protein is preferred. For example, cell lines can be engineered that stably express anti-TSLP Fab. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA regulated by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. After introduction of the foreign DNA, engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be cloned and expanded into cell lines. This method can be advantageously used to generate cell lines that stably express anti-TSLP Fab.

[0073] A number of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 13:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)), which genes may be used in tk-, hgprt-, and aprt- cells, respectively. Additionally, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1521 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2012 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 52:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB TECH 13(5):155-215 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:141 (1984)).Methods generally known in the art of recombinant DNA technology that may be used are described in Ausubel et al. (1993) Current Protocols in Molecular Biology (John Wiley & Sons, NY); Kriegler (1990) "Gene Transfer and Expression" in A Laboratory Manual (Stockton Press, NY); Dracopoli et al. (eds) (1994) Current Protocols in Human Genetics (John Wiley & Sons, NY) Chapters 12 and 13; Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0074] The expression level of Fab can be increased by vector amplification (for a review, see Bebbington and Hentschel (1987) "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning" (Academic Press, NY) Vol. 3). If the marker of the vector system expressing anti-TSLP Fab is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the anti-TSLP Fab gene, production of anti-TSLP Fab will also increase (Crouse et al., Mol. Cell. Biol. 3:251 (1983)).

[0075] In vitro production allows for scale-up to obtain large amounts of the desired polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogenous suspension culture (e.g., in airlift reactors and continuous stirred reactors) or immobilized or entrapped cell culture (e.g., in hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges). If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods (e.g., gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, or (immuno) affinity chromatography), e.g., after preferential biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein.

[0076] The genes encoding the anti-TSLP Fabs of the present disclosure can also be expressed in non-mammalian cells (e.g., insect, bacterial, yeast, or plant cells). Bacteria that readily take up nucleic acids include Enterobacteriaceae (e.g., Escherichia coli or Salmonella), Bacillaceae (e.g., Bacillus subtilis), Pneumococcus, Streptococcus, and members of Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the heterologous polypeptide typically becomes part of inclusion bodies. The heterologous polypeptide must be isolated, purified, and assembled into a functional molecule. If a tetravalent form of the antibody is desired, the subunits self-assemble into a tetravalent antibody (WO02 / 096948A2).

[0077] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use of the anti-TSLP Fab to be expressed. For example, when producing large quantities of such proteins for the generation of pharmaceutical compositions of the Fab, a vector directing high levels of expression of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)) (the coding sequence can be individually ligated in frame with the lacZ coding region into the vector to produce a fusion protein), pIN vector (Inouye and Inouye, Nucleic Acids Res. iJ:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)), and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0078] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae (i.e., common baker's yeast) is the most commonly used among eukaryotic microorganisms, although a number of other strains (e.g., Pichia patens) are commonly available.

[0079] For expression in Saccharomyces, for example, the plasmid YRp7 (Stinginchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:151 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trpl lesion as a feature of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0080] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The Fab coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0081] Once recombinantly expressed, the Fab of this disclosure can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique in protein purification. Alternatively, a preferred method for increasing the affinity of the antibodies of this disclosure is disclosed in US Patent Application Publication No. 2002 0123057 A1.

[0082] Treatment methods The disclosure also provides methods of treatment, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TSLP Fab or pharmaceutical composition described herein, in some cases for the treatment of a TSLP-associated condition.

[0083] The disclosure also provides an anti-TSLP Fab or pharmaceutical composition described herein for use in therapy. In some cases, the therapy is treatment of a TSLP-associated condition.

[0084] The disclosure also provides for the use of an anti-TSLP Fab or pharmaceutical composition in the manufacture of a medicament for use in treating a disease. In some cases, the disease is a TSLP-associated condition.

[0085] The disclosure also provides for the use of an anti-TSLP Fab or pharmaceutical composition in therapy. In some cases, the therapy is treatment of a TSLP-associated condition.

[0086] In some cases, the TSLP-associated condition is a TSLP-associated inflammatory condition. In some cases, the TSLP-associated inflammatory condition is selected from asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis, eosinophilic esophagitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), asthma, COPD overlap syndrome (ACOS), chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, vasculitis, GvHD, uveitis, chronic idiopathic urticaria, sinusitis, or pancreatitis.

[0087] In some cases, the TSLP-associated inflammatory condition is asthma.

[0088] Asthma is a complex, heterogeneous inflammatory disease of the airways, characterized by variable and recurrent symptoms, reversible airflow obstruction, and bronchospasm.

[0089] Symptoms of asthma can include wheezing, coughing, chest tightness, and shortness of breath. Symptoms can be caused by exposure to allergens or irritants. Asthma can be classified as atopic (extrinsic) and non-atopic (intrinsic) based on whether symptoms are triggered (atopic) or not (non-atopic) by allergens. An acute asthma exacerbation is commonly referred to as an "asthma attack." Additional signs that may occur during an asthma attack include use of accessory respiratory muscles (sternocleidomastoid and scalene muscles in the neck), and pulsus paradoxus (a pulse that is weak on inspiration and strong on expiration) and hyperinflation of the chest may be observed. Blue skin and nails may result from lack of oxygen. In addition to these positive therapeutic responses, subjects treated with anti-TSLP Fab may experience beneficial effects or improvements in one or more of these symptoms associated with the disease.

[0090] Clinical response can be assessed using screening techniques such as magnetic resonance imaging (MRI) scans, X-ray imaging, computed tomography (CT) scans, flow cytometry or fluorescence activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry (including but not limited to changes detectable by ELISA, RIA, chromatography, etc.).

[0091] The Fabs disclosed herein can be used in combination with any known therapy for inflammatory diseases, including any agent or combination of agents known to be, or that has been, or is currently being used to treat inflammatory diseases (e.g., asthma or COPD). Exemplary active agents that can be administered in combination with the Fabs described herein include, but are not limited to, inhaled corticosteroids (ICS), bronchodilators (including long-acting beta agonists (LABAs), long-acting antimuscarinic agonists (LAMAs), short-acting beta agonists (SABAs), and muscarinic beta2 agonists (MABAs)), antihistamines, anti-leukotrienes, PDE-4 inhibitors, Janus kinase inhibitors, and phosphoinositide 3-kinase inhibitors.

[0092] The term "combination" refers to either a fixed combination in one dosage unit form, or a combined administration in which the anti-TSLP Fab and a combination partner (e.g., another drug (also referred to as a "therapeutic agent" or "co-agent")) can be administered simultaneously and independently or separately within a time interval (e.g., when such time interval allows the combination partners to exhibit a synergistic effect (e.g., synergistic effect)). The single components may be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration. As used herein, terms such as "co-administration" or "combined administration" are intended to include the inclusion of administration of selected combination partners to a single subject (e.g., patient) in need thereof, and include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. As used herein, the term "pharmaceutical combination" refers to the product resulting from the mixing or combination of multiple therapeutic agents, and includes both fixed and non-fixed combinations of therapeutic agents. The term "fixed combination" means that both a therapeutic agent (e.g., an anti-TSLP Fab) and a combination partner are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that both a therapeutic agent (e.g., an anti-TSLP Fab) and a combination partner are administered to a patient as separate entities simultaneously, concurrently, or sequentially without specific time limitations, such administration providing therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy (e.g., administration of three or more therapeutic agents).

[0093] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration includes the co-administration of these therapeutic agents substantially simultaneously, for example in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes the co-administration of multiple or separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration also includes the sequential use of each type of therapeutic agent, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the condition or disorder described herein.

[0094] composition The anti-TSLP Fab in the medical uses and methods disclosed herein can be administered to a subject in the form of a pharmaceutical composition.

[0095] In some cases, any reference herein to an "anti-TSLP Fab" may refer to a pharmaceutical composition that includes a / that anti-TSLP Fab.

[0096] In some cases, an anti-TSLP Fab or a pharmaceutical composition thereof can be administered to a human or other animal in an amount sufficient to produce a therapeutic effect according to the aforementioned methods of treatment / medical uses.

[0097] In some cases, the anti-TSLP Fab or pharmaceutical composition thereof can be administered to such a human or other animal in a conventional dosage form prepared by combining the anti-TSLP Fab with a conventional pharma- ceutically acceptable carrier or diluent according to known techniques.

[0098] One of ordinary skill in the art will recognize that the form and character of the pharma- ceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables.

[0099] In some cases, the pharmaceutical compositions are formulated to include a pharma- ceutically acceptable, non-toxic, sterile carrier (e.g., saline, non-toxic buffers, preservatives, etc.). In some cases, the pharmaceutical compositions can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0100] In some cases, the route of administration of the anti-TSLP Fab or pharmaceutical composition thereof is, for example, oral, parenteral, inhalation, or topical. In some cases, the term parenteral administration as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0101] In some cases, an anti-TSLP Fab or a pharmaceutical composition thereof can be administered by nasal aerosol or inhalation.

[0102] In some cases, the components recited herein for preparing the pharmaceutical compositions can be packaged and sold in the form of a kit, in some cases bearing a label or package insert indicating that the associated pharmaceutical composition is useful for treating a subject suffering from or susceptible to a disease or disorder. EXAMPLES

[0103] Example 1 Stability studies on tezepelumab were performed in 1×PBS at 4° C. or 45° C. The solution containing 1 mg / ml tezepelumab was stored for 2 weeks under conditions of absence.

[0104] As a result, 10% monomer loss was observed after 2 weeks at 45°C in D-PBS (Figure 1). The % monomer loss, % aggregation, and % fragmentation were calculated by quantifying the area under the curve (AUC) from the HP-SEC chromatograms. Based on the elution volume, peaks were assigned as monomer, aggregate, and fragmentation products. The AUC can be calculated using standard analysis tools provided with the HP SEC analysis software.

[0105] A Fab was constructed containing the heavy and light chain complementarity determining regions (CDRs) of tezepelumab and with multiple mutations in the CH1 domain. This Fab is referred to herein as Fab1. The stability of Fab1 was analyzed as described above for tezepelumab. The results are shown in Figure 2.

[0106] Surprisingly, Fab1 showed improved stability properties under accelerated stability testing conditions (1×PBS at 45° C.) when stored at 0.8 mg / ml for 2 weeks.

[0107] Example 2: Fab1 binds human and cynomolgus TSLP with pM affinity Affinity of Fab1 binding to TSLP quantified by BIAcore The specificity and affinity of Fab1 for recombinant mammalian cell-expressed human and cynomolgus TSLP was quantified using a Biacore 8K SPR instrument (GE Healthcare, Little Chalfont, Bucks, UK).

[0108] S Series C1 biosensor chips, amine coupling kit, hepes-buffered saline base buffer, and regeneration buffer were obtained from GE Healthcare and used according to the manufacturer's instructions. Streptavidin surfaces were prepared using lyophilized streptavidin reconstituted in D-PBS. Briefly, streptavidin was diluted to 4 μg mL-1 in 10 mM sodium acetate pH 4.5 and covalently immobilized onto two flow cell surfaces of an S Series C1 biosensor chip using standard amine coupling methods. Ultimately, a streptavidin surface of 170 response units (RU) was achieved. A control blank surface with no immobilized streptavidin was also prepared using amine coupling reagents to serve as a reference surface in each flow cell. N-terminally tagged biotinylated TSLP (human and cynomolgus monkey) was then titrated onto each streptavidin surface to achieve <100 RU of Fab1 binding at saturation (Rmax). The low level of analyte binding ensured that artifacts caused by mass transport were minimized, especially when combined with the relatively fast assay flow rate of 50 μL min-1 used during the kinetic measurement step. Dilutions of monomerized Fab1 (2-fold dilutions in HBS-EP+ buffer ranging from 1.25 to 20 nM) (Multi-Cycle Kinetics) were injected at an assay flow rate of 50 μL min-1, with association times of 2 min and dissociation times of 10 min. Multiple injections of buffer alone were performed under the same conditions throughout the experiment to allow for double referencing of the final set of sensorgrams.

[0109] The chip surface was fully regenerated by two 30-s pulses of 10 mM glycine (pH 1.7). Binding affinity and kinetics were quantified using a 1:1 Langmuir model.

[0110] The results, shown in Table 1, demonstrate that Fab1 binds immobilized human and cynomolgus TSLP with similar affinity (within 2-fold; 46 pM and 88 pM, respectively). [Table 1]

[0111] Quantification of binding affinity by kinetic exclusion assay (KinExA). The solution-phase binding affinity (K D ) were also quantified and the resulting data were processed using KinExA Pro software version 4.1.11. KinExA methodology has been reviewed ( Darling and Brault, 2004 ).

[0112] Fab1 was premixed with varying concentrations of human and cynomolgus TSLP until equilibrium was reached (at least 12 concentrations of human and cynomolgus TSLP were prepared using two-fold serial dilutions). The amount of free Fab1 was then measured using the KinExA instrument by capturing free Fab with human TSLP-coated beads, washing away unbound material, and fluorescently detecting bound Fab1 with a commercially available species-specific antibody (Alexa Fluor 647-labeled mouse anti-human heavy and light chain specific antibody (Jackson Immunoresearch 209-605-088)). The K D was extracted by performing a global 1:1 fit to three data sets derived from titration of human TSLP into fixed Fab1 concentration solutions of 1000 pM (filled diamonds), 500 pM (filled inverted triangles), or 40 pM (open squares) (Figure 3). D was extracted by performing a global 1:1 fit to two data sets derived from titrations of cynomolgus TSLP into fixed Fab1 concentration solutions of 1000 pM (filled diamonds) or 40 pM (open squares) (Figure 4).

[0113] The amount of free Fab1 detected at each human and cynomolgus TSLP concentration was plotted against the titrated concentration of TSLP (Figures 3 and 4, respectively). The equilibrium dissociation constant (KD) was calculated using KinExA software. The results, shown in Table 2, indicate that Fab1 binds human TSLP with 1.7-fold higher affinity than it binds to cynomolgus TSLP in free solution. [Table 2]

[0114] Example 3: Fab1 and tezepelumab bind to TSLP with similar binding properties The binding properties of Fab1 to human TSLP were directly compared with tezepelumab.

[0115] A homogeneous fluorescence resonance energy transfer (FRET) homogeneous time-resolved fluorescence (HTRF®, Cisbio International)-based TSLP:mAb binding assay was used to quantify the in vitro binding potency of Fab1. Streptavidin cryptate was used for detection of biotinylated TSLP. Briefly, samples of unlabeled Fab1 were titrated into the HTRF assay to compete with DyLight-labeled tezepelumab for binding to biotinylated His-Avi human TSLP. A competition assay was also performed using unlabeled tezepelumab and DyLight-labeled tezepelumab as positive controls.

[0116] The results show that Fab1 competes with tezepalumab for binding to human TSLP and binds to human TSLP with a similar potency as tezepalumab (IC50: Fab1: 0.38 nM; tezepalumab: 0.23 nM (Figure 5)).

[0117] Example 4: Fab1 neutralizes TSLP activity in peripheral blood mononuclear cell (PBMC) assays Next, we determined whether Fab1 that binds TSLP has functional blocking activity in a primary cell assay by measuring TSLP-induced CCL17 release from PBMCs upon treatment with Fab1.

[0118] Blood was obtained from healthy donors under an established blood donation program at MedImmune (Cambridge, UK). Peripheral blood mononuclear cells were isolated by standard procedures using a Ficoll gradient. Briefly, 20 ml of blood diluted in PBS (10 ml blood: 30 ml PBS) was layered on 15 ml of Ficoll. The tubes were centrifuged at 400 g for 40 min at room temperature without brake. The PBMC layer was collected and cells were washed twice with 50 ml of PBS. PBMCs were counted using a hemocytometer and trypan blue to exclude dead cells, and were resuspended in culture medium (RPMI with 10% fetal bovine serum and 1% penicillin / streptomycin) before plating in 96-well plates. Cells were stimulated with TSLP (0.5 ng / ml) for 48 h in the presence of the TSLP-binding antibody fragment Fab1. The assay was also performed using the TSLP-binding antibody tezepelumab as a positive control. After 48 hours, supernatants were removed and assayed for CCL17 production using the R&D DuoSet ELISA according to the manufacturer's protocol. Experiments were performed in three independent experiments with six donors.

[0119] As a result, Fab1 had an IC of 1.39 nM. 50 It was shown that IL-17 inhibited CCL17 production from PBMCs (Figure 6).

[0120] array SEQ ID NO:1 (Fab1 heavy chain) QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK SEQ ID NO:2 (Fab1 light chain) SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Claims

1. A kit comprising a Fab, wherein the Fab comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

2.

2. A kit comprising a Fab, wherein the Fab comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:1 and a light chain having the amino acid sequence set forth in SEQ ID NO:

2.

3. A kit comprising a Fab, wherein the Fab comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:

2.

4. The kit according to any one of claims 1 to 3, wherein the Fab is an IgG1 Fab.

5. The kit of any one of claims 1 to 3, wherein the Fab is stable at 45°C for at least 2 weeks.

6. The kit of claim 5, wherein the Fab is stable in 1x PBS at a concentration of 0.8 mg / ml.

7. The kit of any of claims 1 to 3, wherein the Fab shows less than 10% monomer loss after incubation in 1x PBS at 45°C for 2 weeks, and the monomer is quantified by HP-SEC.

8. A kit comprising a pharmaceutical composition comprising a Fab, wherein the Fab comprises a heavy chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:1, and a light chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:2, or consisting of the amino acid sequence set forth in SEQ ID NO:

2.

9. A vector comprising a nucleic acid encoding a Fab, wherein the Fab comprises a heavy chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:1, and a light chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:2, or consisting of the amino acid sequence set forth in SEQ ID NO:

2.

10. A host cell comprising the vector described in claim 9.

11. A method for producing a Fab, comprising culturing a host cell according to claim 10, expressing the Fab, and purifying the Fab.

12. A kit according to any one of claims 1 to 3 for use in therapy.

13. A kit according to any one of claims 1 to 3 for use in the treatment of a TSLP-associated condition.

14. 14. The kit of claim 13, wherein the TSLP-associated condition is asthma.

15. A pharmaceutical composition comprising a Fab, wherein the Fab comprises a heavy chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:2, wherein the pharmaceutical composition is for use in the treatment of asthma, sepsis, septic shock, rheumatoid arthritis, asthma-COPD overlap syndrome (ACOS), chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, vasculitis, GvHD, uveitis, chronic idiopathic urticaria, sinusitis, or pancreatitis.

16. The pharmaceutical composition of claim 15, wherein the Fab is an IgG1 Fab.

17. Use of a Fab, wherein the Fab comprises a heavy chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:1 and a light chain comprising, having, or consisting of the amino acid sequence set forth in SEQ ID NO:2, said use in the manufacture of a pharmaceutical for the treatment of asthma, sepsis, septic shock, rheumatoid arthritis, asthma-COPD overlap syndrome (ACOS), chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, vasculitis, GvHD, uveitis, chronic idiopathic urticaria, sinusitis, or pancreatitis.

18. The use of claim 17, wherein the Fab is an IgG1 Fab.

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