Methods for treating systemic sclerosis

Abstract

The present invention provides methods for treating systemic sclerosis by administering a dual V region bispecific antibody that specifically binds IL-4 and IL13.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 852,941, filed May 24, 2019, European Patent Application No. EP19306309.6, filed October 8, 2019, and U.S. Provisional Application No. 62 / 979,875, filed February 21, 2020, each of which is incorporated by reference herein in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 183952031840SEQLIST.TXT, Recorded: May 21, 2020, Size: 16KB).

[0003] The present invention relates to bispecific anti-IL-4, anti-IL-13 antibodies for the treatment of systemic sclerosis (also called scleroderma). [Background technology]

[0004] Systemic sclerosis (also known as scleroderma) is a chronic, disabling condition characterized by three crucial features: immune dysregulation, small-vessel disease, and fibrosis. The commonly accepted classification of systemic sclerosis (SSc) has two major subgroups: localized cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) (Non-Patent Document 1). In lcSSc, fibrosis is limited to the distal upper and lower extremities and may involve the face. While fibrosis tends to stabilize within the first few years of onset, the condition continues to develop in the viscera, particularly the lungs, and may lead to the development of pulmonary arterial hypertension (PAH), the main cause of lcSSc-related mortality, in later stages. In contrast, dcSSc is a rapidly progressive disorder that goes beyond the limited form and affects larger areas of the skin, potentially accompanied by truncal symptoms. These patients often present with initial visceral involvement and more severe systemic symptoms, such as joint pain, tendon friction rubs, and weight loss. Although skin fibrosis is a prominent feature, pathological changes in the lungs, gastrointestinal tract, kidneys, and heart ultimately determine clinical outcome. However, the extent of skin lesions and their rate of progression can reflect the severity of visceral complications, outcome, and survival rate (Non-Patent Document 2; Non-Patent Document 3). Survival rates for patients with dcSSc have improved over the past several decades; the average 10-year survival rate is currently estimated to be approximately 70% to 80%. Although mortality associated with renal crisis has decreased significantly over the past 20 to 30 years due to the use of angiotensin-converting enzyme (ACE) inhibitors, pulmonary involvement remains the leading cause of death in these patients (Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7).

[0005] The prevalence of SSc in 2014 was estimated to be approximately 120,000 in the United States and EU5 countries (i.e., France, Germany, Italy, Spain, and the United Kingdom), with over 60% of cases being diffuse. Prevalence may increase by as much as 20% in the future using the new American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) 2013 classification criteria (8), which are more accurate than the previous ACR 1980 criteria. Overall, the disease is more frequent in women (3-6:1) and in certain races (e.g., blacks). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] LeRoy EC et al., J.Rheumatol.1988, 15(2):202-5 [Non-patent document 2] Domsic RT et al., Ann.Rheum.Dis.2011, 70(1):104-9 [Non-patent document 3] Cottrell TR et al. Ann.Rheum.Dis.2014, 73(6):1060~6 [Non-patent document 4] Elhai M. et al., Rheumatology (Oxford) 2012, 51(6):1017~26 [Non-Patent Document 5] Nikpour M. and Baron M., Curr. Opin. Rheumatol 2014, 26(2):131-7 [Non-patent document 6] Nihtyanova SI et al. QJM 2010, 103(2):109-15 [Non-Patent Document 7] Winstone TA et al., Chest 2014, 146(2):422-36 [Non-patent document 8] van den Hoogen F. et al., Ann. Rheum. Dis. 2013, 72(11):1747-55 Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, there are no approved therapies for SSc. The general treatment strategy is to address specific SSc symptoms (e.g., Raynaud's phenomenon, digital ulcers, gastrointestinal lesions, PAH, etc.) while controlling the underlying inflammatory process in either the skin or internal organs using effective immunosuppressive therapies (e.g., cyclophosphamide, mycophenolate mofetil, azathioprine, methotrexate, rituximab). Because these immunosuppressive therapies do not target specific pathways related to the fibrotic process, they are not particularly effective and are often associated with significant side effects. Therefore, there is an unmet need to find effective targeted therapies with limited side effect profiles for this disease population.

[0008] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Means for solving the problem]

[0009] In some aspects, the present invention provides a method for treating systemic sclerosis (SSc) in a human subject with SSc, comprising subcutaneously administering to the subject about 200 mg of a dual-V region bispecific antibody or antigen-binding fragment that specifically binds IL-4 and IL-13. In some embodiments, 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days. In some embodiments, treatment is administered for at least about 24 weeks. In some embodiments, the bispecific antibody is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and the pH of the formulation is about 7.0. In some embodiments, the formulation is reconstituted from a lyophilized formulation. In some embodiments, the bispecific antibody is administered in combination with another agent. In some embodiments, the additional agent is administered prior to, concurrently with, or after administration of the bispecific antibody, hi some embodiments, the systemic sclerosis is diffuse cutaneous systemic sclerosis.

[0010] In some embodiments of the invention, the bispecific antibody or bispecific antibody fragment thereof comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8 comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16); hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13); VHhBD4-8 comprises three CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19). hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2; VH hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2, and VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the light chain comprises the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides. In some embodiments, the light chain polypeptides comprise an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptides comprise an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the light chain polypeptides comprise the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO: 23.

[0011] In some aspects, the present invention provides methods for reducing sclerotic plaques in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; wherein the sclerotic plaques are reduced by at least about 20%, 40%, 60%, 80%, or 100% about 24 weeks after the first administration of the bispecific antibody, compared to baseline. In some embodiments, the baseline is determined before treatment. In some embodiments, the baseline is a normal control from an individual without scleroderma. In some embodiments, the baseline is a historical control. In some embodiments, a subset of treated human subjects with SSc have an improved modified Rodnan skin score (mRSS) of at least about 20%, 40%, and 60% about 24 weeks after the first administration of the bispecific antibody, compared to baseline. In some embodiments, the improved mRSS is measured as a least squares mean change from baseline. In some embodiments, the least squares mean change from baseline is greater than about any of -3.00, -3.5, -4.0, -4.5, -5.0, -5.5, or -6.0.

[0012] In some embodiments of the method for reducing sclerotic plaques in a human subject with SSc, the anti-IL4 / anti-IL13 antibody is RKB. In some embodiments, about 200 mg of the anti-IL4 / anti-IL13 antibody is subcutaneously administered to the subject. In some embodiments, 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days. In some embodiments, the treatment is administered for at least about 24 weeks. In some embodiments, the bispecific antibody is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and the pH of the formulation is about 7.0. In some embodiments, the formulation is reconstituted from a lyophilized formulation. In some embodiments, the bispecific antibody is administered in combination with another agent. In some embodiments, the additional agent is administered prior to, concurrently with, or after administration of the bispecific antibody, hi some embodiments, the systemic sclerosis is diffuse cutaneous systemic sclerosis.

[0013] In some embodiments of the method for reducing sclerotic plaque in a human subject with SSc, the bispecific antibody or bispecific antibody fragment thereof comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8 comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16); hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13); VHhBD4-8 comprises three CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19). hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2; VH hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2, and VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the light chain comprises the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides. In some embodiments, the light chain polypeptides comprise an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptides comprise an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the light chain polypeptides comprise the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptides comprise the amino acid sequence of SEQ ID NO: 23. [Brief explanation of the drawings]

[0014] [Figure 1]

[0023] Figure 1 shows an exemplary bispecific anti-IL-4 / anti-IL-13 antibody comprising two light chain polypeptides and two heavy chain polypeptides. The two light chains comprise the moiety N-VLhB-B13-linker-VLhBD4-8-CL-C, and the two heavy chain polypeptides comprise the moiety N-VHhB-B13-linker-VHhBD4-8-CH1-CH2-CH3-C. The linker sequence comprises (G4S)2; i.e., GGGGSGGGGS (SEQ ID NO: 6). [Figure 2] Diagram of a clinical trial study in patients with systemic sclerosis. Patients were randomized to either the treatment arm (top) where they received weekly subcutaneous injections of romilkimab 200 mg (RKB; also known as SAR156597) for 24 weeks or the placebo arm (bottom) where they received weekly subcutaneous injections of placebo for 24 weeks (R). This includes a screening period before the treatment period and a follow-up period after the treatment period. During the screening period, clinic visits were conducted at D1 (Day 1), W2 (Week 2), W4, W8, W12, W24, and W35. Phone calls (indicated in parentheses) were conducted at W6, W16, W18, and W30. [Figure 3] Figure 1 shows graphs depicting least squares mean change in mRSS at each visit. 90% confidence intervals are shown for each estimate. The solid line represents patients receiving placebo, and the dashed line represents patients receiving 200 mg qw RKB. [Figure 4] Figure 1 shows the least squares mean change in HAQ-DI composite score at each visit. 90% confidence intervals are shown for each estimate. The solid line represents patients who received placebo, and the dashed line represents patients who received 200 mg qw RKB. [Figure 5] Figure 1 shows the least squares mean change in FVC(L) at each visit. 90% confidence intervals are shown for each estimate. The solid line represents patients who received placebo, and the dashed line represents patients who received 200 mg qw RKB. [Figure 6]Figure 1 shows the least squares mean change in DLco (mmol / min / kPa) [corrected for hemoglobin] at each visit. 90% confidence intervals are shown for each estimate. The solid line represents patients who received placebo, and the dashed line represents patients who received 200 mg qw RKB. [Figure 7] Graph showing Kaplan-Meier curves of time to first event reflecting progression in the romilkimab and placebo groups. QW, once daily. *Censored = patients who left the study before an event occurred or completed the study before an event occurred. [Figure 8] Figures 8A and 8B are graphs showing the mean change from baseline to week 24 for TARC (Figure 8A) and periostin (Figure 8B) in patients treated with romilkimab versus placebo. QW, once weekly; SE, standard error; TARC, thymus and activation-regulated chemokine. DETAILED DESCRIPTION OF THE INVENTION

[0015] To the extent not inconsistent with the present disclosure, each publication, patent application, patent, and other reference cited herein is expressly incorporated by reference in its entirety.

[0016] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references herein unless the context clearly dictates otherwise.

[0017] It should be noted that for purposes of describing and defining the present invention, the term "substantially" is utilized herein to represent the inherent degree of uncertainty that may result from any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree to which a quantitative representation may vary from the stated reference without resulting in a change in the basic functionality of the subject matter in question.

[0018] Moreover, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover, eds., 1985); Oligonucleotide Synthesis (M.J. Gait, eds., 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, eds., 1985); Transcription And Translation (B.D. Hames & S.J. Higgins, eds., 1984); Animal Cell Culture (R.I. Freshney, ed., 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, 1986; See Sons, Inc. (1994).

[0019] The following non-limiting definitions of several terms and phrases are provided to guide those of ordinary skill in the art.

[0020] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as by site-directed mutagenesis, or may be accidental, such as due to mutations of hosts producing the protein or errors resulting from PCR amplification.

[0021] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits, such as phosphoramidates, and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate phosphodiester oligomer. Additionally, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with appropriate primers.

[0022] "Interleukin-4" (IL-4) refers to naturally occurring or endogenous mammalian IL-4 proteins and proteins having an amino acid sequence identical to that of a naturally occurring or endogenous corresponding mammalian IL-4 protein; e.g., recombinant proteins, synthetic proteins (i.e., produced using synthetic organic chemistry methods). Thus, as defined herein, the term includes mature IL-4 proteins, polymorphic or allelic variants, as well as other isoforms of IL-4 and modified or unmodified forms of the foregoing (e.g., lipidated, glycosylated). Naturally occurring or endogenous IL-4 includes wild-type proteins, such as mature IL-4, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be recovered or isolated, for example, from sources that naturally produce IL-4. These proteins and proteins having the same amino acid sequence as a naturally occurring or endogenous corresponding IL-4 are referred to by the name of the corresponding mammal. For example, if the corresponding mammal is a human, the protein is designated human IL-4. Several mutant IL-4 proteins are known in the art, such as those disclosed in WO03 / 038041.

[0023] "Interleukin-13" (IL-13) refers to naturally occurring or endogenous mammalian IL-13 protein, and to proteins (e.g., recombinant proteins, synthetic proteins (i.e., made using synthetic organic chemistry methods)) having an amino acid sequence that is the same as that of a naturally occurring or endogenous corresponding mammalian IL-13 protein. Thus, as defined herein, the term includes mature IL-13 protein, polymorphic or allelic variants, as well as other isoforms of IL-13 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., lipidated, glycosylated). Naturally occurring or endogenous IL-13 includes wild-type proteins, such as mature IL-13, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., humans, non-human primates). For example, as used herein, IL-13 encompasses human IL-13 variants associated with asthma (atopic and nonatopic asthma) in which Arg at position 110 of mature human IL-13 is replaced with Gln (position 110 of mature IL-13 corresponds to position 130 of the precursor protein), as well as other variants of IL-13 (Heinzmann et al., Hum Mol Genet. (2000) 9:549-559). Such proteins can be recovered or isolated, for example, from sources that naturally produce IL-13. These proteins and proteins having the same amino acid sequence as naturally occurring or endogenous corresponding IL-13 are referred to by the name of the corresponding mammal. For example, if the corresponding mammal is a human, the protein is designated human IL-13. Several mutant IL-13 proteins are known in the art, such as those disclosed in WO03 / 035847.

[0024] In some aspects, the present invention relates to the treatment of systemic sclerosis (SSc). In some embodiments, the present invention relates to the treatment of diffuse cutaneous systemic sclerosis (dcSSc). In some embodiments, the present invention relates to the treatment of localized cutaneous SSc (lcSSc). IL-4 and IL-13 are therapeutically important cytokines based on their biological functions and play critical roles in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005, Vol. 5, pp. 161-166). IL-4 has been shown to be able to inhibit autoimmune diseases, and both IL-4 and IL-13 have shown the potential to enhance anti-tumor immune responses. Because both cytokines are involved in the pathogenesis of allergic or fibrotic diseases, inhibitors of these cytokines would offer therapeutic benefits.

[0025] The phrase "substantially identical" with respect to an antibody chain polypeptide sequence can be interpreted as an antibody chain exhibiting at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference polypeptide sequence. With respect to a nucleic acid sequence, the term can be interpreted as a series of nucleotides exhibiting at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference nucleic acid sequence. Identity can be determined by using any bioinformatics tool available to those skilled in the art. For example, the Basic Local Alignment Search Tool (BLAST) is commonly used to determine sequence identity (Altschul et al., J. Mol. Biol. (1990) 215:403-410).

[0026] The term "identity" or "homology" can refer to the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, without considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for alignment are available and well known in the art. Sequence identity can be measured using sequence analysis software.

[0027] "Substitutional" variants are those that have at least one amino acid residue in the native sequence removed and replaced with a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or multiple, where two or more amino acids are substituted in the same molecule. The multiple substitutions may be at contiguous positions. Also, an amino acid may be replaced with multiple residues, in which case the variant includes both substitutions and insertions. "Insertional" variants are those that have one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native sequence. "Immediately adjacent to an amino acid" means that the amino acid is attached to either the α-carboxyl or α-amino functional group of the amino acid. "Deletional" variants are those that have one or more amino acids in the native amino acid sequence removed. Deletional variants usually have one or two amino acids deleted in a specific region of the molecule.

[0028] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antigen-binding antibody fragments, or synthetic polypeptides containing one or more CDRs or CDR-derived sequences, so long as the polypeptide exhibits the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Generally, antibodies are considered to be Igs with defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. The antibodies of the present invention may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1; IgA2, etc.) ("type" and "class," as well as "subtype" and "subclass," are used interchangeably herein). Natural or wild-type antibodies and immunoglobulins, i.e., antibodies and immunoglobulins obtained from non-artificially engineered members of a population, are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. "Non-artificially engineered" means that the antibody has not been engineered to contain or express a foreign antigen-binding molecule. Wild-type antibodies may refer to the most prevalent alleles or species found in a population or antibodies obtained from non-engineered animals, compared to alleles or polymorphisms, or variants or derivatives obtained by forms of manipulation, such as mutagenesis, the use of recombinant methods to change the amino acids of antigen-binding molecules, etc.

[0029] As used herein, "anti-IL-4 antibody" means an antibody or polypeptide (derivative) derived therefrom that specifically binds to IL-4 as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of IL-4 to its receptor or inhibit IL-4 activity.

[0030] As used herein, "anti-IL-13 antibody" means an antibody or polypeptide derived therefrom that specifically binds to IL-13 as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of IL-13 to its receptor or inhibit IL-13 activity.

[0031] As used herein, "anti-IL-4 / anti-IL-13 bispecific antibody" means a bispecific antibody or a polypeptide (derivative) derived therefrom that specifically binds to IL-4 and / or IL-13 as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of IL-4 to its receptor or inhibit IL-4 activity, and / or that substantially reduce the binding of IL-13 to its receptor or inhibit IL-13 activity.

[0032] The term "variable" in the context of antibody variable domains refers to the several portions of the related molecule that vary extensively in sequence between and within antibodies and are used in the specific recognition and binding of a particular antibody to its particular target. However, variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), also known as hypervariable regions, in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework (FR) regions or sequences. Native heavy- and light-chain variable domains each contain four FR regions that primarily adopt a β-sheet configuration, connected by three CDRs that form loops connecting, and in some cases, part of, the β-sheet structure. The CDRs in each chain are often held close together by FR regions, and together with the CDRs from the other chain, contribute to the formation of the target (epitope or determinant) binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, unless otherwise indicated, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al. One CDR can have the ability to specifically bind to its cognate epitope.

[0033] The term "hinge" or "hinge region", as used herein, refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

[0034] Phrases and terms such as "fragment," "functional fragment," "variant," "derivative," or "analog" of an antibody, antigen, or antigen-binding protein, and word forms thereof, refer to compounds or molecules that have qualitative biological activity in common with the full-length antibody or antigen of interest. For example, a functional fragment or analog of an anti-IL-4 antibody is one that can bind to an IL-4 molecule or can block or substantially reduce the ability of a ligand, or an agonist or antagonist antibody, to bind to IL-4. In another example, a functional fragment or analog of an anti-IL-13 antibody is one that can bind to an IL-13 molecule or can block or substantially reduce the ability of a ligand, or an agonist or antagonist antibody, to bind to IL-13. In yet another example, a functional fragment or analog of an anti-IL-4 / anti-IL-13 bispecific antibody is one that can bind to an IL-4 molecule and / or an IL-13 molecule or can block or substantially reduce the ability of a ligand, or an agonist or antagonist antibody, to bind to IL-4 and / or IL-13.

[0035] Furthermore, the terms "fragment" and "antibody fragment" refer to an intact or full-length chain or portion of an antibody, generally the target-binding or variable region. In some examples, the fragment of an antibody fragment of an antibody-like binding molecule comprises an antigen-binding domain. For bispecific antibody-like binding molecules, the molecule comprises two or more antigen-binding domains. For example, fragments or analogs of anti-IL-4 and / or IL-13 antibodies can block or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, "fragment," "functional fragment," and "antibody fragment" generally refer to antibodies that can block or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.

[0036] As used herein, monoclonal antibodies specifically include "chimeric" antibodies in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass (type or subtype), while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity of binding to IL-4 and / or IL-13 or affecting IL-4 and / or IL-13 activity or metabolism (U.S. Pat. No. 4,816,567; and Morrison et al. (1984), Proc Natl Acad Sci USA 81:6851). Thus, CDRs from one class of antibody can be grafted into the FRs of an antibody of a different class or subclass.

[0037] Monoclonal antibodies are highly specific, being directed against a particular target site, epitope, or determinant. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes) on an antigen, each monoclonal antibody is directed against a specific determinant on the target. In addition to their specificity, monoclonal antibodies are advantageously synthesized by a host cell, uncontaminated by other immunoglobulins, providing for the cloning of the relevant gene and mRNA encoding the antibody chain. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. The parent monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method described in Kohler et al. (1975) Nature 256:495, or by recombinant methods well known in the art.

[0038] The term "multivalent antibody" as used herein refers to an antibody that contains two or more antigen-binding sites, which may have the same or different structures, and thus can bind to two or more antigens simultaneously. The term "bivalent" means that the antibody contains two antigen-binding sites. The term "tetravalent" means that the antibody contains four antigen-binding sites.

[0039] The term "antigen-binding site", as used herein, refers to the portion of an antibody that comprises the area that specifically binds to and is complementary to part or all of an antigen. If the antigen is large, the antibody may only bind to a specific portion of the antigen, which portion is called an epitope. An antigen-binding domain is provided by one or more antibody variable domains. Preferably, the antigen-binding domain consists of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0040] The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of being bound by an antibody of the present invention. An antigen may have one or more epitopes. Examples of antigens recognized by the antibodies of the present invention include, but are not limited to, serum proteins, cytokines such as IL-4, IL-5, IL-9, and IL-13, biologically active peptides, cell surface molecules, such as receptors, transporters, ion channels, viral and bacterial proteins.

[0041] The term "monospecific" as used in the present invention means that a multivalent antibody of the present invention recognizes only one antigen, all antigen-binding sites being identical.

[0042] The term "bispecific" as used herein means that the multivalent antibody of the invention recognizes two different epitopes on the same or two different antigens.

[0043] The term "bispecific antibody" (BsAb) refers to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. Thus, bispecific antibodies can simultaneously bind two different antigens. In addition to diagnostic applications, BsAbs open up new therapeutic applications by redirecting potent effector systems to disease sites or by increasing the neutralizing or stimulatory activity of antibodies.

[0044] It is of interest to create bispecific antibodies (BsAbs), which combine the antigen-binding sites of two antibodies within a single molecule. Thus, such molecules can simultaneously bind two different antigens. In addition to diagnostic applications, they open new therapeutic avenues, for example, by redirecting potent effector systems toward disease sites (such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)—cancer cells often develop mechanisms to suppress normal immune responses elicited by monoclonal antibodies) or by increasing the neutralizing or stimulatory activity of antibodies. The first attempts to combine the binding specificities of two whole antibodies for different target antigens for therapeutic purposes utilized chemically fused heteroconjugate molecules (Staerz et al. (1985), Nature 314:628-631).

[0045] Bispecific antibodies were originally generated by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein and Cuello, 1983, 1984), but the complexity of species (up to 10 different species) that arise in cell culture makes purification difficult and expensive (George and Huston, 1997). Despite promising results obtained using heteroconjugates or bispecific antibodies produced from the cell fusions listed above, several factors have made them impractical for large-scale therapeutic application. These factors include: the rapid clearance of heteroconjugates in vivo, the laboratory-intensive techniques required to produce both forms of the molecule, the need for extensive purification of heteroconjugates away from homoconjugates or monospecific antibodies, and the generally low yields.

[0046] Genetic engineering is being used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with desired sets of binding properties and effector functions. A variety of recombinant methods have been developed for the efficient production of BsAbs, both as antibody fragments (Carter et al. (1995) J. Hematotherapy 4:463-470; Pluckthun et al. (1997) Immunotechology 3:83-105; Todorovska et al. (2001) J. Immunol. Methods 248:47-66) and in full-length IgG format (Carter (2001) J. Immunol. Methods 248:7-15).

[0047] Abbott describes a murine dual variable domain IgG (DVD-IgG) bispecific antibody in US Patent US7612181, which is based on the dual Fv format described in Unilever Patent (US5989830). A humanized bispecific format is described in WO2009 / 052081 (TBTI), the entire contents of which are incorporated herein by reference. Addition of constant domains to each chain of the dual Fv (CHI-Fc to the heavy chain and kappa or lambda constant domains to the light chain) resulted in a functional bispecific dual V region antibody-like binding protein.

[0048] The term "multispecific" as used herein means that the multivalent antibody of the invention recognizes several different epitopes on the same or several different antigens.

[0049] The term "linker," as used herein, refers to a peptide adapted to link the variable domains of the antibody constructs of the present invention. Peptide linkers can contain any amino acid, with the amino acids glycine (G) and serine (S) being preferred. Linkers can be equal to or different from each other between and within heavy and light chain polypeptides. Furthermore, linkers can have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptide linker unit for the heavy and light chain domains is GGGGS. The number of linker units in the heavy and light chains can be equal to each other (symmetric order) or different from each other (asymmetric order). In some embodiments, the peptide linker includes two units for the heavy and light chain domains (e.g., GGGGSGGGGS; SEQ ID NO: 6).

[0050] The peptide linker is preferably long enough to allow proper protein folding, to prevent the antibody portions from interfering with each other's activity, e.g., by steric hindrance, and, if necessary, to provide a sufficient degree of flexibility to allow the antibody molecule to interact with two or more, possibly widely spaced, receptors on the same cell; yet it is preferably short enough to allow the antibody portions to remain stable in the cell. Thus, the length, composition, and / or conformation of the peptide linker can be readily selected by one of skill in the art to optimize the desired properties of the multivalent antibody.

[0051] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain sequences derived from non-human immunoglobulins, compared to human antibodies. Generally, humanized antibodies contain substantially all of one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin template. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a selected human immunoglobulin template. Generally, the goal is to have an antibody molecule that is minimally immunogenic in humans. Thus, one or more amino acids in one or more CDRs may be altered to be less immunogenic in a human host without substantially minimizing the specific binding function of one or more CDRs to IL-4 and / or IL-13. Alternatively, the FRs may be non-human, but the most immunogenic amino acids are replaced with less immunogenic ones. Nevertheless, as mentioned above, CDR grafting is not the only method for obtaining a humanized antibody. For example, modifying only the CDR region may be insufficient, since framework residues often play a role in determining the three-dimensional structure of the CDR loop and the overall affinity of the antibody for its ligand. Therefore, any means can be implemented to modify a non-human parent antibody molecule to make it less immunogenic to humans, and comprehensive sequence identity with a human antibody is not necessarily required. Thus, humanization can be achieved, for example, by simply replacing only a few residues, particularly those that are exposed on the antibody molecule and not buried within the molecule, and therefore not easily accessible to the host immune system. Such methods are taught herein for replacing "mobile" or "flexible" residues on an antibody molecule, with the goal of reducing or blunting the immunogenicity of the resulting molecule without compromising the specificity of the antibody to its epitope or determinant.See, for example, Studnicka et al., Prot Eng 7(6) pp. 805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO2006 / 042333 and U.S. Patent No. 5,869,619.

[0052] "Antibody homolog" or "homologue," when used in reference to IL-4 and / or IL-13, refers to any molecule that specifically binds IL-4 and / or IL-13 as taught herein. Antibody homologs therefore include naturally occurring or recombinant antibodies, portions of antibodies such as Fab or Fv molecules, single-chain antibodies, polypeptides containing one or more CDR regions, and the like, whether modified or not, that retain the desired biological properties, such as binding to IL-4 or IL-13. The amino acid sequence of a homolog need not be identical to that of a naturally occurring antibody, and can be altered or modified to have substituted amino acids, inserted amino acids, deleted amino acids, amino acids other than the 20 normally found in proteins, and the like, to obtain a polypeptide with enhanced or other advantageous properties.

[0053] An antibody with a homologous sequence is an antibody having an amino acid sequence that has sequence homology with the amino acid sequence of IL-4, IL-13, or the bispecific IL-4 / IL-13 antibody of the invention. Preferably, the homology is with the amino acid sequence of the variable region of an antibody of the invention. As used herein, "sequence homology" as applied to an amino acid sequence is defined as a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with another amino acid sequence, as determined, for example, by the FASTA search method according to Pearson & Lipman, Proc Natl Acad Sci USA 85, pp. 2444-2448 (1988).

[0054] Chimeric antibodies have different portions of antibodies derived from different sources, such as different antibodies, different classes of antibodies, or different animal species, for example, antibodies with variable regions derived from mouse monoclonal antibodies paired with human immunoglobulin constant regions. Thus, humanized antibodies are a type of chimeric antibody. Methods for producing chimeric antibodies are known in the art, see, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397.

[0055] Functional equivalents of the subject antibodies are also included within the scope of the present invention. The term "functional equivalent" includes antibodies with homologous sequences, antibody homologs, chimeric antibodies, artificial antibodies, and modified antibodies, each defined, for example, by its ability to bind to IL-4 and / or IL-13, inhibit IL-4 and / or IL-13 signaling ability or function, or inhibit IL-4 and / or IL-13 binding to its receptor. Those skilled in the art will appreciate that there is overlap between the group of molecules referred to as "antibody fragments" and the group referred to as "functional equivalents." Methods for producing functional equivalents that retain IL-4 and / or IL-13 binding ability are known to those skilled in the art and are disclosed, for example, in WO 93 / 21319, EPO Ser. No. 239,400, WO 89 / 09622, EPO Ser. No. 338,745, and EPO Ser. No. 332,424.

[0056] The functional equivalents of the present application also include modified antibodies, e.g., antibodies modified by the covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies that have been modified by, e.g., glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand, linkage to a toxin or cytotoxic moiety or other protein, etc. Covalent attachment does not necessarily result in an antibody that is free from generating an anti-idiotypic response. Modifications can be achieved by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis, etc. Additionally, modified antibodies may contain one or more non-classical amino acids.

[0057] As used herein, "treatment" refers to an approach for obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stable (e.g., not worsening) state of disease, prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0058] As used herein, the term "prophylactic treatment" refers to treatment in which an individual is known to have, suspected of having, or at risk of having a disorder, but does not exhibit symptoms of the disorder, or exhibits minimal symptoms. An individual receiving prophylactic treatment is treated before the onset of symptoms.

[0059] An "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source or culture medium from which the protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of antibody in which the polypeptide / protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of contaminating proteins. If the antibody is recombinantly produced, it is preferably also substantially free of culture medium, i.e., the culture medium represents less than about 20%, 10%, 5%, 2.5%, or 1% of the volume of the protein preparation. If the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e., the antibody of interest has been separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations of antibodies have less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In some embodiments of the invention, the antibodies are isolated or purified. In some embodiments, the invention provides compositions comprising an anti-IL-4 / anti-IL-13 bispecific antibody, wherein greater than about 95%, 96%, 97%, 98%, or 99% of the polypeptides in the composition are anti-IL-4 / anti-IL-13 bispecific antibody.

[0060] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent that can be used in the treatment, management, or amelioration of a disease, disorder, condition, etc. associated with aberrant IL-4 and / or IL-13 metabolism and activity.

[0061] As used herein, "dose" refers to the amount of any agent that can be used in the treatment, management, or amelioration of a disease, disorder, condition, etc. associated with aberrant IL-4 and / or IL-13 metabolism and activity.

[0062] As used herein, "safe dose" refers to any agent or dose of any agent that can be used in the treatment, management, or amelioration of diseases, disorders, conditions, etc. associated with aberrant IL-4 and / or IL-13 metabolism and activity while maintaining a clinically acceptable benefit / risk profile. A safe dose of a dual V-region antibody-like binding protein or fragment thereof disclosed herein is selected from the group consisting of 10 mg, 20 mg, 40 mg, 50 mg, 80 mg, 100 mg, 150 mg, 200 mg, and 300 mg. An embodiment of a safe dose is about 10 mg to about 300 mg. Further embodiments of a safe dose are 200 mg, about 200 mg, up to 200 mg, or any dose not exceeding about 200 mg. In other embodiments, the safe dose is about 50 mg, about 100 mg, or about 200 mg. In some embodiments, the safe dose is administered once a week. In some embodiments, the safety dose is administered once every 7±2 days (i.e., every 5-9 days). In some embodiments, the safety dose is administered every other week (i.e., once every 2 weeks). In some embodiments, the safety dose is administered subcutaneously (SC). In some embodiments, the safety dose is administered subcutaneously (SC) for a period of at least about 24 weeks. In some embodiments, 200 mg of the bispecific antibody is administered once per week. In some embodiments, 200 mg of the bispecific antibody is administered once every 7±2 days (i.e., every 5-9 days). In some embodiments, 200 mg of the bispecific antibody is administered every other week (i.e., once every 2 weeks). In some embodiments, 200 mg of the bispecific antibody is administered subcutaneously (SC). In some embodiments, 200 mg of the bispecific antibody is administered subcutaneously (SC) once per week for a period of at least about 24 weeks.

[0063] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X." The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range that includes the stated numerical value, having a lower limit that is 5%, 10%, or 15% less than the stated numerical value, and an upper limit that is 5%, 10%, or 15% greater than the stated numerical value.

[0064] Anti-IL4-anti-IL13 bispecific antibody In some aspects, the present invention provides methods for treating SSc by administering a bispecific antibody that binds IL-4 and IL-13. Bispecific, dual-variable region (dual-V region) antibody-like binding proteins having four binding sites that specifically bind IL-4 and IL-13 have been reported in WO2009 / 052081, WO2012 / 125775, WO2015 / 121318, WO2014 / 177568, and WO2015 / 198146, each of which is incorporated herein by reference in its entirety.

[0065] An embodiment of the present invention is a method for treating SSc using a bispecific antibody that has been engineered to contain a dual V-region antibody-like protein or fragment thereof that specifically binds to two different epitopes on the same or two different antigens.

[0066] In some embodiments, the light chain variable region (VL) and heavy chain variable region (VH) of the dual V-region antibody-like binding molecule have the following sequences (CDR sequences are shown in bold):

[0067] VL hB-B13 [ka]

[0068] VH hB-B13 [ka]

[0069] VL h8D4-8 [ka]

[0070] VH h8D4-8 [ka] or

[0071] [ka]

[0072] In some aspects, the present invention provides a method for treating SSc by administering to a subject a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, wherein the bispecific antibody or bispecific antigen-binding antibody fragment comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8 comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16); VH hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13); VH hBD4-8comprises three CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18) and LKEYGNYDSFYFDV (SEQ ID NO: 19) or the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO: 19), A method is provided.

[0073] In some embodiments, the present invention provides a method for treating SSc by administering to a subject a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, wherein the bispecific antibody or bispecific antigen-binding antibody fragment comprises a light chain variable domain, VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), and VLhB-B13 comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), and VLhBD4-8 comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), and VHhB-B13 comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2; VH hBD4-8comprises CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18) and LKEYGNYDSFYFDV (SEQ ID NO: 19), and VHhBD4-8 comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4; or VH hBD4-8 comprises CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO: 19), and VHhBD4-8 comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 5; A method is provided.

[0074] In some embodiments, the present invention provides a method for treating SSc by administering to a subject a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, wherein the bispecific antibody or bispecific antigen-binding antibody fragment comprises a light chain variable domain, VL hB-B13 and the light chain variable domain VL hBD4-8 a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1, VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3, VH hB-B13 comprises the amino acid sequence of SEQ ID NO:2, VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, A method is provided.

[0075] In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the light chain polypeptide comprises the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:6.

[0076] In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the light chain polypeptide comprises the following sequence: DIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGQSYMHWY QQKAGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID PVQAEDAATY YCQQNAEDSR TFGGGTKLEI KGGGGSGGGG SDIQMTQSPA SLSVSVGDTI TLTCHASQNI DVWLSWFQQK PGNIPKLLIY KASNLHTGVP SRFSGSGSGT GFTLTISSLQ PEDIATYYCQ QAHSYPFTFG GGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC (SEQ ID NO: 22)

[0077] In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the heavy chain polypeptide comprises the following sequence: EVQLKESGPG LVAPGGSLSI TCTVSGFSLT DSSINWVRQP PGKGLEWLGM IWGDGRIDYA DALKSRLSIS KDSSKSQVFL EMTSLRTDDT ATYYCARDGY FPYAMDFWGQ GTSVTVSSGG GGSGGGGSQV QLQQSGPELV KPGASVKISC KASGYSFTSY WIHWIKQRPG QGLEWIGMID PSDGETRLNQ RFQGRATLTV DESTSTAYMQ LRSPTSEDSA VYYCTRLKEY GNYDSFYFDV WGAGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFEGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG (SEQ ID NO: 23)

[0078] In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the light chain polypeptide has the structure N-VL hBD4-8 -Linker-VL hB-B13 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hBD4-8 -Linker-VH hB-B13 In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the light chain polypeptide comprises the structure N-VL hBD4-8 -Linker-VL hB-B13 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hBD4-8 -Linker-VH hB-B13In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:6.

[0079] In some embodiments of the above-described bispecific antibodies or bispecific antigen-binding antibody fragments thereof, the bispecific antibodies or bispecific antigen-binding antibody fragments thereof comprise two light chains and two heavy chains. In some embodiments, the bispecific antibodies or bispecific antigen-binding antibody fragments are derived from an IgG4 antibody.

[0080] In some embodiments, the present invention provides a method for treating SSc by administering to a subject an antibody antigen-binding fragment thereof that specifically binds to IL-13, wherein the antibody or antibody fragment thereof comprises a light chain variable domain comprising CDRs having the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10), and a heavy chain variable domain comprising CDRs having the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13). In some embodiments, the light chain variable domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and the heavy chain variable domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 1, and the heavy chain domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody competes with the above-mentioned antibody or antibody fragment for binding to IL-13. In some embodiments, the antibody binds to the same epitope as the above-mentioned antibody or antibody fragment. In some embodiments, the antibody is a bispecific antibody or a bispecific antibody fragment thereof. In some embodiments, the antibody specifically binds to IL-13 and IL-4.

[0081] In some embodiments, the invention provides a method for treating SSc by administering to a subject an antibody antigen-binding fragment thereof that specifically binds IL-4, wherein the antibody or antibody fragment thereof comprises a light chain variable domain comprising CDRs having the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), and a heavy chain variable domain comprising CDRs having the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19) or GYSFTSYWIH (SEQ ID NO: 17), IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO: 19). In some embodiments, the light chain variable domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 3, and the heavy chain variable domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:3, and the heavy chain domain comprises the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the antibody competes with the above-mentioned antibody or antibody fragment for binding to IL-4. In some embodiments, the antibody binds to the same epitope as the above-mentioned antibody or antibody fragment. In some embodiments, the antibody is a bispecific antibody or bispecific antibody fragment thereof. In some embodiments, the antibody specifically binds to IL-4 and IL-13.

[0082] In some embodiments of the bispecific antibody or bispecific antigen-binding antibody fragment thereof, the bispecific antibody or bispecific antigen-binding antibody fragment competes with the bispecific antibody or bispecific antigen-binding antibody fragment thereof for binding to IL-13 and / or IL-4. In some embodiments of the bispecific antibody or bispecific antigen-binding antibody fragment thereof, the bispecific antibody or bispecific antigen-binding antibody fragment thereof binds to the same epitope as the bispecific antibody or bispecific antigen-binding antibody fragment thereof.

[0083] In some aspects, the present invention provides methods for treating SSc by administering to a subject a pharmaceutical composition comprising a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, as described above. In some embodiments, the pharmaceutical composition comprises a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, as described above, and a pharmaceutically acceptable carrier.

[0084] In some aspects, the present invention provides a composition for treating SSc, comprising a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, as described above. In some embodiments, the composition is formulated to provide a subject with a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof. In some embodiments, the composition is formulated to provide a subject with a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof once per week. In some embodiments, the composition is formulated to provide a subject with a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof once every other week. In some embodiments, the composition is formulated to provide a subject with a 200 mg dose of the bispecific antibody or bispecific antigen-binding antibody fragment thereof once every 7 days ± 2 days. In some embodiments, the composition is formulated to provide a subject with a 200 mg dose of the bispecific antibody or bispecific antigen-binding antibody fragment thereof once every 5 to 9 days. In some embodiments, the composition is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once per week, hi some embodiments, the composition is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof subcutaneously to a subject once per week for at least about 24 weeks.

[0085] In some embodiments, the invention provides use of a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds IL-13 and IL-4, as described above, in the manufacture of a medicament for treating SSc in a subject. In some embodiments, the composition is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject. In some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once per week. In some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once every other week. In some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once every other week. In some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once per week for at least about 24 weeks. In some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof to a subject once per week, hi some embodiments, the medicament is formulated to provide a dose of about 200 mg of the bispecific antibody or bispecific antigen-binding antibody fragment thereof subcutaneously to a subject once per week for at least about 24 weeks.

[0086] In some embodiments, the present invention provides a method for treating SSc by administering to a subject with SSc huTBTI3_2_1 or SAR156597 or romilkimab (RKB), which comprises a bispecific antibody or bispecific antigen-binding antibody fragment thereof that specifically binds to IL-13 and IL-4, wherein the bispecific antibody or bispecific antigen-binding antibody fragment comprises: (a) a light chain polypeptide comprising two variable light chain domains, one variable light chain domain comprising the amino acid sequence of SEQ ID NO: 1 and one variable light chain domain comprising the amino acid sequence of SEQ ID NO: 3; (b) a heavy chain polypeptide comprising two variable heavy chain domains, one variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 2 and one variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 4; (c) a peptide linker linking SEQ ID NOs: 1 to 3, and a peptide linker linking SEQ ID NOs: 2 to 4, the peptide linker having the amino acid sequence of SEQ ID NO: 6; and (d) a constant region domain.

[0087] Various techniques can be used to extend the serum circulation of antibodies in vivo. For example, inert polymer molecules such as high-molecular-weight polyethylene glycol (PEG) can be attached to antibodies by site-specific conjugation of PEG to the N- or C-terminus of the antibody, or through the ε-amino group present in lysine residues, with or without a multifunctional linker. Linear or branched polymer derivatization can be used, which minimizes loss of biological activity. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or ion exchange chromatography. PEG-derivatized antibodies can be tested for binding activity and in vivo efficacy using methods known to those skilled in the art, for example, by the immunoassays described herein.

[0088] Antibodies with increased in vivo half-lives can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into an IgG constant domain, or an FCR-binding fragment thereof (such as an Fc or hinge-Fc domain fragment); see, e.g., WO98 / 23289; WO97 / 34631; and U.S. Pat. No. 6,277,375.

[0089] Furthermore, antibody can be conjugated to albumin to make the antibody more stable in vivo or have a longer half-life in vivo.This technique is known in the art, for example, see WO93 / 15199, WO93 / 15200 and WO01 / 77137; and EPO413,622.Antibody can also be modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc.

[0090] The antibodies of the present invention may also be described or specified in terms of their binding affinity to IL-4 and / or IL-13. -7 Less than M, about 10 -6 Less than M or about 10 -5 K less than M D It can be combined with about 10 -8 ~about 10 -15 M, about 10 -8 ~about 10 -12 M, about 10 -9 ~about 10 -11 M, or about 10 -8 ~about 10 -10 Equilibrium dissociation constant or K DHigher binding affinity of the antibody of interest may be beneficial, such as one having a higher binding affinity than the antibody of interest. The present invention also provides antibodies that competitively inhibit the binding of an antibody to an epitope of the invention, as determined by any method known in the art for determining competitive binding, e.g., the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 60%, or at least about 50%.

[0091] The antibody of the present invention can be administered and / or formulated together with one or more additional therapeutic or active agents.When the ligand is administered together with an additional therapeutic agent, the ligand can be administered before, simultaneously with, or after the administration of the additional agent.Generally, the ligand and the additional agent are administered in a manner that provides overlapping therapeutic effects.The additional agent that can be administered or formulated together with the ligand of the present invention can include various immunotherapeutic agents, such as, for example, cyclosporine, methotrexate, adriamycin, or cisplatin, antibiotics, antifungals, antivirals, and antitoxins.For example, when an antagonist is administered to prevent, suppress, or treat pulmonary inflammation or respiratory disease (e.g., asthma), it may be administered in combination with other antagonists, such as phosphodiesterase inhibitors (e.g., inhibitors of phosphodiesterase 4), bronchodilators (e.g., β2-agonists, anticholinergics, theophylline), short-acting β-agonists (e.g., albuterol, salbutamol, bambuterol, fenoterol [sigma]l, isoetamol, benzodiazepines, benzocaine ... anticholinergics (e.g., ipratropium bromide and oxitropium bromide), long-acting anticholinergics (e.g., tiotropium), theophylline (e.g., short-acting anticholinergics (e.g., thiazolinone ... short-acting preparations, long-acting preparations), inhaled steroids (e.g., beclomethasone, beclometasone, budesonide, flunisolide, fluticasone propionate, and triamcinolone), oral steroids (e.g., methylprednisolone, prednisolone, prednisolone, and prednisone), short-acting beta-agonists, and They are administered in combination with anticholinergics (e.g., albuterol / salbutamol / ipratropium and fenoterol / ipratropium), long-acting beta-agonist and inhaled steroid combinations (e.g., salmeterol / fluticasone and formoterol / budesonide), and mucolytics (e.g., erdosteine, acetylcysteine, bromhexine, carbocysteine, guaifenesin, and iodized glycerin).

[0092] Other suitable co-therapeutic agents that can be administered with the antibodies of the invention to prevent, inhibit, or treat asthma (e.g., allergic asthma) include corticosteroids (e.g., beclomethasone, budesonide, fluticasone), cromoglycate, nedocromil, beta-agonists (e.g., salbutamol, terbutaline, bambuterol, fenoterol, reproterol, tulobuterol, salmeterol, fomtero), zafirlukast, salmeterol, prednisone, prednisolone, theophylline, zileutron, montelukast, and leukotriene modifiers. The ligands of the present invention can be co-administered with a variety of co-therapeutic agents suitable for treating diseases (e.g., SSc, Th-2-mediated diseases, YL-A-mediated diseases, IL-13-mediated diseases, and IL-4-mediated diseases), including cytokines, analgesics / antipyretics, antiemetics, and chemotherapeutic agents.

[0093] The antibodies of the invention are provided in pharmaceutically acceptable compositions known in the art or described herein. Terms such as "physiologically acceptable," "pharmacologically acceptable," and the like mean approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0094] Bispecific anti-IL-4 / IL-13 antibodies are administered to mammals, particularly humans, to treat SSc by any acceptable route. Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes, including intralesional administration if immunosuppressive treatment is desired. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial, or intraperitoneal administration. The antibodies or compositions may be administered by any conventional route, e.g., infusion or bolus injection, via absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), or coadministered with other bioactive agents. Administration may be systemic or local. Furthermore, it may be desirable to introduce therapeutic antibodies or compositions of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Moreover, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. In some embodiments, the bispecific anti-IL-4 / IL-13 antibody is administered subcutaneously to a human subject.

[0095] Therapeutic formulations of polypeptides or antibodies can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired purity with optional "pharmaceutically acceptable" carriers, diluents, excipients, or stabilizers typically utilized in the art, i.e., buffers, stabilizers, preservatives, isotonifiers, non-ionic surfactants, antioxidants, and various other additives; see Remington's Pharmaceutical Sciences, 16th ed., Osol ed. (1980). Such additives are generally nontoxic to recipients at the dosages and concentrations utilized; thus, the excipients, diluents, carriers, etc., are pharmaceutically acceptable.

[0096] Examples of formulations of dual V-region bispecific antibody-like molecules that bind to IL-4 and IL-13 are provided in WO 2014 / 177568, which is incorporated herein by reference in its entirety. Highly stable anti-IL-4 / anti-IL-13 bispecific antibody formulations have surprisingly been found in the form of liquids and lyophilized powders containing an anti-IL-4 / anti-IL-13 bispecific antibody and a buffer system, with the formulation having a pH of about pH 7 and a low salt concentration to reduce the ionic strength of the formulation. The formulations may optionally further contain non-ionic surfactants, sugars, and / or non-ionic stabilizers. These formulations offer an improvement over conventional formulations, which often result in molecular aggregation (HMW) of the antibody and the formation of visible and subvisible particles as the antibody concentration in the formulation increases. In particular, the formulations of the present invention exhibit good stability with respect to visible and subvisible particles, low molecular weight proteins, and high molecular weight proteins.

[0097] In some embodiments, the invention provides a stable antibody formulation comprising a bispecific anti-IL-4 / anti-IL-13 antibody, or antigen-binding portion thereof, comprising a light chain of the formula VL1-linker-VL2 and a heavy chain of the formula VH1-linker-VH2, wherein VL1 and VH1 form an IL-13 antigen-binding domain, and VL2 and VH2 form an IL-4 antigen-binding domain; and a buffer system suitable for maintaining the pH of the formulation at about pH 7, wherein the formulation has a low salt concentration to reduce the ionic strength of the formulation. In some embodiments, VL1 comprises the three CDR sequences of SEQ ID NO: 1; VH1 comprises the three CDR sequences of SEQ ID NO: 2; VL2 comprises the three CDR sequences of SEQ ID NO: 3; and VH2 comprises the CDR sequences of SEQ ID NO: 4 or 5. In alternative specific embodiments, VL1 comprises the amino acid sequence of SEQ ID NO: 1; VH1 comprises the amino acid sequence of SEQ ID NO: 2; VL2 comprises the amino acid sequence of SEQ ID NO: 3; and VH2 comprises the amino acid sequence of SEQ ID NO: 4 or 5. In some embodiments, the light chain comprises the formula N-VL1-linker-VL2-CL, where CL is the light chain constant domain of the antibody, and the heavy chain comprises the formula N-VH1-linker-VH2-CH1-CH2-CH3, where CH2-CH3 corresponds to the Fc domain of the antibody. In specific embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region domain. In some embodiments, the constant region domain is selected from the group consisting of CH1, CH2, CH3, and CL. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a humanized IgG4 bispecific antibody or antigen-binding fragment thereof.

[0098] In some embodiments, the concentration of the antibody or antigen-binding fragment thereof in any of the above formulations is about 100 mg / mL.

[0099] In some embodiments of the present invention, the buffer system of any of the above formulations comprises at least two buffers. In certain embodiments, the concentration of the buffer system is about 10 mM. In certain embodiments, the buffer system comprises a Tris buffer and a phosphate buffer. In certain embodiments, the concentration of the Tris buffer is about 3.7 mM. In certain embodiments, the concentration of the phosphate buffer is about 6.3 mM. In certain embodiments, the concentration of the Tris buffer is about 3.7 mM and the concentration of the phosphate buffer is about 6.3 M.

[0100] In some embodiments of the invention, any of the above formulations further comprises a non-ionic surfactant. In certain embodiments, the concentration of the non-ionic surfactant is about 0.05% to about 0.2% (w / v). In certain embodiments, the non-ionic surfactant is a polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the concentration of polysorbate 80 is about 0.05% to about 0.2% (w / v). In certain embodiments, the concentration of polysorbate 80 is about 0.2% (w / v).

[0101] In some embodiments of the present invention, any of the above formulations further comprises a sugar. In certain embodiments, the concentration of the sugar is about 5% (w / v). In certain embodiments, the sugar is a disaccharide. In certain embodiments, the disaccharide is sucrose. In certain embodiments, the concentration of sucrose is about 5% (w / v).

[0102] In some embodiments of the present invention, the formulation further comprises a non-ionic stabilizer. In certain embodiments, the concentration of the non-ionic stabilizer is about 1% to about 3% (w / v). In certain embodiments, the non-ionic stabilizer is either an amino acid or a sugar. In certain embodiments, the amino acid is proline. In certain embodiments, the sugar is mannitol. In certain embodiments, the concentration of proline is about 1% to about 3% (w / v). In certain embodiments, the concentration of proline is about 3% (w / v). In certain embodiments, the concentration of mannitol is about 3% (w / v).

[0103] In some embodiments of the invention, the formulation is a lyophilized formulation.In some embodiments of the invention, the formulation is a reconstituted lyophilized formulation.

[0104] In some embodiments of the present invention, the formulations exhibit good stability with respect to visible particles, sub-visible particles, low molecular weight proteins, and high molecular weight proteins.

[0105]

[0010] Embodiments of the present invention provide a method of treating SSc in a subject, comprising administering a bispecific anti-IL4 / anti-IL-13 antibody or antigen-binding fragment thereof, wherein the antibody is: a bispecific antibody or antigen-binding fragment thereof at 100 mg / mL, the antibody or antigen-binding fragment thereof comprising a heavy chain polypeptide comprising a variable region comprising the amino acid sequence of SEQ ID NO:2 and the variable region comprising the amino acid sequence of SEQ ID NO:4, and a light chain polypeptide comprising a variable region comprising the amino acid sequence of SEQ ID NO:1 and the variable region comprising the amino acid sequence of SEQ ID NO:3; in a stable antibody formulation comprising an approximately 10 mM buffer system, the buffer system comprising a Tris buffer concentration of about 3.7 mM and a phosphate buffer concentration of about 6.3 mM; about 0.2% (w / v) polysorbate 80; about 5% (w / v) sucrose; and about 3% (w / v) proline; and the pH of the formulation is about pH 7.

[0106] An embodiment of the present invention provides a method of treating SSc in a subject, comprising administering a bispecific anti-IL4 / anti-IL-13 antibody or antigen-binding fragment thereof, wherein the antibody is in a stable lyophilized antibody formulation comprising: about 100 mg / mL of a bispecific antibody or antigen-binding fragment thereof described herein; about 10 mM of a buffer system comprising a Tris buffer concentration of about 3.7 mM and a phosphate buffer concentration of about 6.3 mM; about 0.2% (w / v) polysorbate 80; about 5% (w / v) sucrose; and about 3% (w / v) mannitol; and the pH of the formulation is about pH 7.

[0107] In some embodiments, the antibody of the present invention is conjugated to various effector molecules such as heterologous polypeptides, drugs, radionucleotides or toxins, see, for example, WO92 / 08495; WO91 / 14438; WO89 / 12624; U.S. Patent No. 5,314,995; and EPO396,387. The antibody or its fragment is conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive metal ion (e.g., an alpha emitter, such as 213Bi). A cytotoxin or cytotoxic agent includes any agent that is toxic to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin, daunomycin, and doxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0108] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, for example, Arnon et al., in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56, Alan R. Liss (1985); Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al. (eds.), pp. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16, Academic Press (1985); and Thorpe et al., Immunol Rev 62:119 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate, such as a bifunctional antibody; see, eg, US Pat. No. 4,676,980.

[0109] The conjugates of the present invention are used to modify a given biological response, and the therapeutic agent or drug moiety should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator; apoptotic agents such as TNF-α, TNF-β, AIMI (WO 97 / 33899), AIMII (WO 97 / 34911), Fas ligand (Takahashi et al., Int. Immunol, 6:1567 (1994)), VEGF (WO99 / 23105); thrombotic agents; anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (GCSF), or other growth factors.

[0110] Preparations to be used for in vivo administration must be sterile. This can be achieved, for example, by filtration through a sterile membrane. For example, the liquid preparations of the present invention are sterilized by filtration using a 0.2 μm to 0.22 μm filter.

[0111] Treatment method In some aspects, the present invention provides a method for treating SSc in a human subject with SSc, comprising subcutaneously administering to the subject about 200 mg of a dual V region bispecific antibody or antigen-binding fragment that specifically binds IL-4 and IL-13 as described herein. In some embodiments, the SSc is diffuse cutaneous systemic sclerosis (dcSSc). In some embodiments, the SSc is localized cutaneous systemic sclerosis (lcSSc). In some embodiments, about 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5-9 days. In some embodiments, the bispecific antibody is administered once every 7±2 days (i.e., every 5-9 days). In some embodiments, the bispecific antibody is administered every other week (i.e., once every 2 weeks). In some embodiments, the bispecific antibody is administered subcutaneously (SC). In some embodiments, the bispecific antibody is administered subcutaneously (SC) for a period of at least about 24 weeks.

[0112] In some embodiments, the bispecific antibody is in a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, wherein the pH of the formulation is about 7.0. In some embodiments, the formulation is reconstituted from a lyophilized formulation.

[0113] In some embodiments of any of the methods of treatment described herein, the bispecific antibody or bispecific antibody fragment thereof comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), and a VH hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), and a VH hBD4-8 comprises three CDRs comprising the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19). hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and VL hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, and hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, and hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1, and VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3, and VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2, and VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 In some embodiments, the light chain comprises the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain comprises the structure N-VH hB-B13 -Linker-VH hBD4-8-CH1-CH2-CH3-C. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides. In some embodiments, the light chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-IL4 / anti-IL13 bispecific antibody is RKB.

[0114] In some embodiments, the methods of treating scleroderma described herein with specific antibodies or bispecific antibody fragments thereof are added to background therapy.

[0115] In some aspects, the invention provides methods for reducing sclerotic plaques in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; and sclerotic plaques are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to baseline at about 4, 8, 12, 24, 36, 48 weeks, or more than 48 weeks after the initial administration of the bispecific antibody. In some embodiments, sclerotic plaques are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to baseline at about 24 weeks after the initial administration of the bispecific antibody. In some embodiments, sclerotic plaques are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 12 weeks after the first administration of the bispecific antibody. In some embodiments, sclerotic plaques are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 8 weeks after the first administration of the bispecific antibody. In some embodiments, sclerotic plaques are reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 4 weeks after the first administration of the bispecific antibody. In some embodiments, the baseline is determined for a human subject with SSc prior to therapeutic administration of the bispecific antibody. In some embodiments, the baseline is the level in a human subject without SSc. In some embodiments, a portion of the human subjects with treated SSCs have an improved modified Rodnan skin score (mRSS) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% at about 4, 8, 12, 24, 36, 48 weeks or more than 48 weeks after the first administration of the bispecific antibody compared to baseline. In some embodiments, a portion of the human subjects with treated SSCs have an improved mRSS of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% at about 24 weeks after the first administration of the bispecific antibody compared to baseline.In some embodiments, a portion of the human subjects with treated SSCs have an mRSS that is improved by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 12 weeks after the first administration of the bispecific antibody. In some embodiments, a portion of the human subjects with treated SSCs have an mRSS that is improved by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 8 weeks after the first administration of the bispecific antibody. In some embodiments, a portion of the human subjects with treated SSCs have an mRSS that is improved by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to baseline about 4 weeks after the first administration of the bispecific antibody. The mRSS is performed by palpation of the skin in 17 regions of the body (fingers, hands, forearms, arms, feet, legs and thighs, face, chest, and abdomen) using a scale of 0 to 3 (0 = normal, 1 = mild thickening, 2 = moderate thickening, and 3 = severe thickening). The total skin score can range from 0 (no thickening) to 51 (severe thickening in all 17 regions).

[0116] In some embodiments, the present invention provides a method for improving the Health Assessment Questionnaire-Disability Index (HAQ-DI) in a human subject with scleroderma, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; the improvement in HAQ-DI, compared to baseline, is at about 4, 8, 12, 24, 36, 45, or more than 45 weeks after the first administration of the bispecific antibody. In some embodiments, the HAQ-DI is measured by the Scleroderma Health Assessment Questionnaire (SHAQ). The SHAQ, which includes the standard HAQ-DI and five SSc-specific VAS assessments for measuring functional disability, is completed by the patient at baseline and during treatment. The SHAQ is a standard, validated, and accepted health assessment questionnaire in patients with SSc for assessing physical / functional disability associated with skin and systemic fibrosis.

[0117] The HAQ-DI includes eight activity domains (dressing, standing, eating, walking, hygiene, stretching, grip strength, and general daily activities), each with at least two questions, for a total of 20 items. For each item, patients report the degree of difficulty they experienced performing the activity. There are four possible responses for each item, ranging from 0 (no difficulty) to 3 (unable to do). For each of the eight domains included in the HAQ-DI, the score is a single response within the domain with the highest score. If an aid or device is used and the highest score is 0 or 1, the score is increased to 2; if the highest score is 2 or 3, the score remains the same. The HAQ-DI composite score is then calculated as the average of the scores for the eight domains. If one or two domains are missing, the HAQ-DI composite score is obtained by dividing the sum of the domains by the number of domains answered. If three or more domains are missing, the HAQ-DI composite score is missing. The composite score is reported on an ordinal scale ranging from 0 to 3. Scores are interpreted as 0 (no impairment) to 3 (maximum impairment).

[0118] The HAQ-DI also includes a VAS that patients use to report the degree of pain they have experienced over the past week. The VAS is a 10 cm line that is converted to a continuous scale from 0 to 3, with 1 cm corresponding to 0.3 points. The VAS anchors range from 0 (no pain) to 100 (very severe pain). To obtain the patient's score, a metric ruler was used to measure the distance in centimeters from the left anchor to the patient's mark and then multiplied by 0.3. The VAS pain score is not incorporated into the HAQ-DI composite score.

[0119] In some embodiments, the invention provides methods of improving respiratory function in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; the improvement in respiratory function is measured by predicted forced vital capacity (FVC) and / or predicted diffusing capacity of the lung for carbon monoxide (DLco) at about 4, 8, 12, 24, 36, 48 weeks, or more than 48 weeks after the first administration of the bispecific antibody, compared to baseline. In some embodiments, the baseline is determined for the human subject with SSc before therapeutic administration of the bispecific antibody. In some embodiments, the baseline is the level in a human subject without SSc.

[0120] Pulmonary function testing is a secondary endpoint that evaluates the change in respiratory function as measured by observed FVC and observed DLco (corrected for hemoglobin) from baseline to the time of sampling. Absolute changes in observed and predicted % change in FVC and DLco from baseline to the time of sampling are evaluated. Manual correction of DLco for hemoglobin, unless automatically corrected during measurement, is based on the following equation: 1) For male patients: DLco 観測 / (係数) , where coefficient = (1.7 x Hb) / (10.22 + Hb); 2) For female patients: DLco 観測 / (係数) , where coefficient = (1.7 x Hb) / (9.38 + Hb). Hb refers to hemoglobin and the value is taken from the same visit when DLco is performed.

[0121] In some embodiments, the invention provides methods for reducing pain, improving vascular function, improving gastrointestinal function, reducing Raynaud's phenomenon, and / or reducing digital ulcers in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody.

[0122] In some embodiments, improvement in gastrointestinal function is measured by the UCLA Scleroderma Clinical Trial Consortium Gastrointestinal Tract 2.0 score at about 4, 8, 12, 24, 36, 48 weeks, or more than 48 weeks after the first administration of the bispecific antibody, compared to baseline. In some embodiments, the UCLA Scleroderma Clinical Trial Consortium Gastrointestinal Tract 2.0 score is improved compared to baseline about 24 weeks after the first administration of the bispecific antibody. The UCLA SCTC GIT 2.0 instrument is a validated self-report questionnaire used to assess quality of life (QOL) related to gastrointestinal function in patients with SSc (Khanna D. et al., Arthritis Rheum. 2009, 61:1257-63). It utilizes seven multi-item scales in the areas of reflux, bloating / distention, diarrhea, fecal soilage, constipation, emotional well-being, and social functioning.

[0123] In some embodiments, pain reduction is measured by change in tender joint count 28 (TJC28) about 24 weeks after the first administration of the bispecific antibody. TJC28 is an assessment of global joint pain based on examination of 28 major joints. It is a reliable and validated method of assessing general joint pain. The 28 joints that are part of the assessment include: shoulder (2 joints), elbow (2 joints), wrist (2 joints), metacarpophalangeal joints (10 joints), proximal interphalangeal joints (10 joints), and knee (2 joints).

[0124] In some embodiments, the reduction in digital ulcers is measured by the number of digital ulcers at about 4, 8, 12, 24, 36, 48 weeks, or more than 48 weeks after the first administration of the bispecific antibody. In some embodiments, the reduction in digital ulcers is measured by the number of digital ulcers at about 24, 48 weeks after the first administration of the bispecific antibody. The digital ulcer count obtains the number of active open wounds (or digital ulcers) on the fingertips secondary to SSc (and not secondary to local trauma or injury). In some embodiments, it does not include cracks, fissures, or further skin defects associated with calcification.

[0125] In some embodiments, the present invention provides a method for improving the Composite Response Index for Diffuse Cutaneous Systemic Sclerosis (CRISS) in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; the improvement in CRISS, compared to baseline, occurs at about 4, 8, 12, 24, 36, 48, or more than 48 weeks after the first administration of the bispecific antibody. In some embodiments, the improvement in CRISS, compared to baseline, occurs at about 24 weeks after the first administration of the bispecific antibody. The CRISS tool summarizes changes in clinical and patient-reported outcomes using a single composite score that reflects the probability that a patient with dcSSc will improve (Khanna D. et al., Arthritis Care Res. (Hoboken) 2016, 68(2):167-78). Regarding effective therapeutic agents for dcSSc, CRISS can summarize a higher probability of improvement in subjects treated with an anti-IL4 / anti-IL13 bispecific antibody (e.g., RKB) versus an ineffective agent or background. CRISS is a two-step process as described below.

[0126] Step 1: Patients who develop new or worsening cardiopulmonary and / or renal impairment due to SSc are considered unimproved (regardless of improvement in other primary outcomes) and are assigned a probability of improvement equal to 0.0. Specifically, subjects experience any of the following: a new scleroderma renal crisis; a 15% or greater (relative) decline in predicted FVC% and a predicted FVC% of less than 80% predicted, confirmed by another FVC% within 1 month on high-resolution computed tomography (HRCT) to confirm ILD (if a previous chest HRCT did not show ILD); new onset of left ventricular failure (defined as a left ventricular ejection fraction of 45% or less) requiring treatment; or new onset of PAH (attributable to SSc) on right heart catheterization requiring treatment (PAH is defined as a mean pulmonary artery pressure of 25 mmHg or greater at rest, an end-expiratory pulmonary artery wedge pressure of 15 mmHg or less, and a pulmonary vascular resistance of more than 3 Wood units).

[0127] Step 2: For the remaining patients, step 2 involves computing the predicted probability of improvement for each subject using the following equation (the equation derived the predicted probability from a logistic regression model):

number

[0128] Patient and physician global assessments of overall health are used in calculating Step 2 of the CRISS. These two assessments are based on a Likert scale ranging from 0 (very good) to 10 (very bad) (Khanna D. et al., Arthritis Care Res. 2016, 68(2):167-78).

[0129] In some embodiments, the present invention provides a method for improving the composite response index for the European Quality of Life-5 Dimension-5 Level (EQ-5D-5L) index in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody; the improvement in EQ-5D-5L, compared to baseline, is about 4, 8, 12, 24, 36, 48 weeks, or more than 48 weeks after the first administration of the bispecific antibody. In some embodiments, the improvement in EQ-5D-5L, compared to baseline, is about 24 weeks after the first administration of the bispecific antibody. The EQ-5D-5L questionnaire is a standardized measure of health status developed by the EuroQol group to provide a simple, general measure of health for clinical and economic evaluation. The EQ-5D-5L is designed for self-administration by patients.

[0130] The EQ-5D includes two separate scales: the EQ-5D Descriptive System and the EQ Visual Analogue Scale (EQ VAS). The EQ-5D Descriptive System has five items, each measuring one aspect of health: mobility, self-care, daily activities, pain / discomfort, and anxiety / depression. Each aspect / item has a five-level Likert-type response scale: no problems, mild problems, moderate problems, severe problems, and extreme problems. Responses to the five aspects can be combined into a single five-digit number describing the respondent's health profile and converted into a single index value for calculating quality-adjusted life years (QALYs) and informing economic evaluations of health interventions. The EQ VAS provides a quantitative measure of health as judged by individual respondents on a vertical visual analogue scale. The EQ VAS "thermometer" has endpoints ranging from 100 ("the best health you can imagine") to 0 ("the worst health you can imagine").

[0131] Intracellular signaling following ligation of IL-4 and IL-13 to their cell surface receptors is mediated in part by phosphorylation of the signaling molecule signal transducer and activator of transcription 6 (Stat6).

[0132] Chemokine (CC motif) ligand 17 (CCL17) is a small cytokine belonging to the CC chemokine family. CCL17 is also known as thymus and activation-regulated chemokine (TARC). TARC is induced by IL-4 and / or IL-13 through State phosphorylation (Wirnsberger et al., (2006) Eur J Immunol. 36: 1882-91; Liddiard et al., (2006) BMC Mol Biol. 29: 7: 45; Monick et al., (2007) J Immunol. 179: 1648-58). Therefore, for example, the inhibition of IL-4 and / or IL-13-mediated signal transduction by IL-4 / IL-13-binding antibody-like proteins correlates with the inhibition of TARC induction. In some embodiments, the methods disclosed herein include methods for detecting binding of an antibody or antibody-like binding protein, or fragment thereof, administered to a subject to IL-4 and / or IL-13, the method comprising: (a) administering to the subject an antibody or antibody-like binding protein, or fragment thereof; and (b) determining the amount of CCL17 / TARC in a blood, serum, or plasma sample taken from the subject, wherein a decrease in the amount of CCL17 / TARC in the sample compared to a sample taken from the subject before administration of the antibody or antibody-like binding protein, or fragment thereof, indicates binding of the antibody or antibody-like binding protein, or fragment thereof, to IL-4 and / or IL-13. In some embodiments, the subject is a human subject. In some embodiments, the antibody or antibody-like binding protein, or fragment thereof, is a dual V-region antibody-like binding protein, or fragment thereof. In some embodiments, the dual V-region antibody-like binding protein, or fragment thereof, is specific for IL-4 or IL-13, or is bispecific for IL-4 and IL-13.In some embodiments, step (c) further comprises increasing the dose if the reduction in TARC / CCL17 measured in step (b) is below a threshold (i.e., the TARC / CCL17 level is not sufficiently reduced), or decreasing the dose if the reduction in TARC / CCL17 measured in step (b) exceeds a threshold (i.e., the TARC / CCL17 level is reduced excessively). In some embodiments, the threshold in step (c) is about a 10%, or about a 15%, or about a 20%, or about a 25%, or about a 30%, or about a 35%, or about a 40%, or about a 45%, or about a 50%, or about a 55%, or about a 60%, or about a 65% reduction in the amount of TARC / CCL17 compared to the amount of TARC / CCL17 in the subject measured before the dose is administered. In some embodiments, the threshold is about a 20% to about 60% decrease, or about a 40% to about 50% decrease, in the amount of TARC / CCL17 compared to the amount of TARC / CCL17 measured in the subject before the dose is administered. In some embodiments, the threshold is about a 43% decrease in the amount of TARC / CCL17 compared to the amount of TARC / CCL17 measured in the subject before the dose is administered. For example, a 43% decrease for a 200 mg dose indicates binding of a 200 mg dose of a bispecific anti-IL-4 / IL-13 dual V-region antibody-like binding protein to IL-4 / IL-13.

[0133] In some embodiments, protein biomarkers associated with disease activity (cartilage oligomeric matrix protein [COMP], chemokine CC motif ligand 2 [CCL2]) and protein biomarkers associated with the IL-4 / IL-13 pathway (TARC, periostin, and eotaxin-3) are measured to monitor treatment. In some embodiments, the presence of anti-drug antibodies (ADA) is used to monitor treatment.

[0134] In certain embodiments, a formulation of the invention is administered in combination with one or more therapeutic agents (e.g., a therapeutic agent other than a formulation of the invention that is currently administered to prevent, treat, manage, and / or ameliorate an IL-4 and / or IL-13 mediated disease (e.g., SSc)). The use of the term "in combination" does not restrict the order in which therapeutic agents are administered to a subject. The first therapeutic agent is administered prior to (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently with, or after (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) administration of the second therapeutic agent to a subject who had, has, or is susceptible to an IL-4 and / or IL-13-mediated disease (e.g., SSc). In some embodiments, the anti-IL-4 / anti-IL13 antibody is administered in combination with a therapeutic agent used to treat SSc. In some embodiments, the anti-IL-4 / anti-IL13 antibody is administered in combination with pirfenidone or nintedanib. Any additional therapeutic agent may be administered in any order with other additional therapeutic agents. Non-limiting examples of therapeutic agents that can be administered in combination with the antibodies of the present invention include approved anti-inflammatory agents listed in the United States Pharmacopoeia and / or Physician's Desk Reference.

[0135] Illustrative Embodiments 1. A method for treating systemic sclerosis (SSc) in a human subject having SSc, the method comprising subcutaneously administering to the subject about 200 mg of a dual V region bispecific antibody or antigen-binding fragment that specifically binds IL-4 and IL-13.

[0136] 2. The method of embodiment 1, wherein 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days.

[0137] 3. The method of embodiment 1 or 2, wherein treatment is administered for at least about 24 weeks.

[0138] 4. The method of any one of embodiments 1 to 3, wherein the bispecific antibody is in a pharmaceutical formulation.

[0139] 5. The method of embodiment 4, wherein the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and wherein the pH of the formulation is about 7.0.

[0140] 6. The method of embodiment 5, wherein the formulation is reconstituted from a lyophilized formulation.

[0141] 7. The method of any one of embodiments 1-6, wherein the bispecific antibody is administered in combination with another agent.

[0142] 8. The method of embodiment 7, wherein the other agent is administered prior to, simultaneously with, or after administration of the bispecific antibody.

[0143] 9. The method of any one of embodiments 1-8, wherein the systemic sclerosis is diffuse cutaneous systemic sclerosis.

[0144] 10. The bispecific antibody or bispecific antibody fragment thereof comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), VH hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), VH hBD4-8 comprises three CDRs containing the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18) and LKEYGNYDSFYFDV (SEQ ID NO: 19), 10. The method according to any one of embodiments 1 to 9.

[0145] 11.VL hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, VL hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, VH hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, VH hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, 11. The method of embodiment 10.

[0146] 12.VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1, VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3, VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2, VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4, 12. The method of embodiment 10 or 11.

[0147] 13. The light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VHhB-B13 -Linker-VH hBD4-8 12. The method of any one of embodiments 9 to 11, comprising -CH1-C.

[0148] 14. The light chain has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 14. The method of any one of embodiments 10 to 13, comprising -CH1-CH2-CH3-C.

[0149] 15. The method of embodiment 13 or 14, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.

[0150] 16. The method of any one of embodiments 10 to 15, wherein the bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides.

[0151] 17. The method of any one of embodiments 10-16, wherein the light chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 23.

[0152] 18. The method of any one of embodiments 10 to 17, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 22 and the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

[0153] 19. A method for reducing sclerotic plaques in a human subject with SSc, comprising administering to the subject an effective amount of an anti-IL4 / anti-IL13 bispecific antibody, wherein sclerotic plaques are reduced by at least about 20%, 40%, 60%, 80% or 100% compared to baseline about 24 weeks after the first administration of the bispecific antibody.

[0154] 20. The method of embodiment 19, wherein some of the treated human subjects with SSC have at least about a 20%, 40%, and 60% improved modified Rodnan skin score (mRSS) about 24 weeks after the first administration of the bispecific antibody compared to baseline.

[0155] 21. The method of embodiment 20, wherein the improved mRSS is measured as a least squares mean change from baseline.

[0156] 22. The method of embodiment 20 or 21, wherein the least squares mean change from baseline is greater than about any of -3.00, -3.5, -4.0, -4.5, -5.0, -5.5, or -6.0.

[0157] 23. The method of any one of embodiments 19-22, wherein the anti-IL4 / anti-IL13 antibody is RKB.

[0158] 24. The method of any one of embodiments 19-23, wherein about 200 mg of anti-IL4 / anti-IL13 antibody is administered subcutaneously to the subject.

[0159] 25. The method of embodiment 24, wherein 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days.

[0160] 26. The method of embodiment 24 or 25, wherein treatment is carried out for at least about 24 weeks.

[0161] 27. The method of any one of embodiments 19 to 26, wherein the bispecific antibody is in a pharmaceutical formulation.

[0162] 28. The method of embodiment 27, wherein the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and wherein the pH of the formulation is about 7.0.

[0163] 29. The method of embodiment 27, wherein the formulation is reconstituted from a lyophilized formulation.

[0164] 30. The method of any one of embodiments 19-29, wherein the bispecific antibody is administered in combination with another agent.

[0165] 31. The method of embodiment 30, wherein the other agent is administered before, simultaneously with, or after administration of the bispecific antibody.

[0166] 32. The method of any one of embodiments 19-31, wherein the systemic sclerosis is diffuse cutaneous systemic sclerosis.

[0167] 33. The bispecific antibody or bispecific antibody fragment thereof comprises a light chain variable domain VL hB-B13 and the light chain variable domain VL hBD4-8 and a light chain polypeptide comprising a heavy chain variable domain VH hB-B13 and the heavy chain variable domain VH hBD4-8 a heavy chain polypeptide comprising: VL hB-B13 comprises three CDRs comprising the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNAEDSRT (SEQ ID NO: 10); VL hBD4-8 comprises three CDRs comprising the amino acid sequences HASQNIDVWLS (SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), VH hB-B13 comprises three CDRs comprising the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and DGYFPYAMDF (SEQ ID NO: 13), VH hBD4-8 comprises three CDRs containing the amino acid sequences GYSFTSYWIH (SEQ ID NO: 17), IDPSDGETR (SEQ ID NO: 18) and LKEYGNYDSFYFDV (SEQ ID NO: 19), The method according to any one of embodiments 19 to 32.

[0168] 34.VL hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, VL hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, VH hB-B13 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, VH hBD4-8 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, 34. The method of embodiment 33.

[0169] 35.VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1, VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3, VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2, VH hBD4-8 comprises the amino acid sequence of SEQ ID NO: 4, The method of embodiment 32 or 33.

[0170] 36. The light chain polypeptide has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain polypeptide comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 36. The method of any one of embodiments 33-35, comprising -CH1-C.

[0171] 37. The light chain has the structure N-VL hB-B13 -Linker-VL hBD4-8 -CL-C, and the heavy chain comprises the structure N-VH hB-B13 -Linker-VH hBD4-8 37. The method of any one of embodiments 33-36, comprising -CH1-CH2-CH3-C.

[0172] 38. The method of embodiment 36 or 37, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.

[0173] 39. The method of any one of embodiments 33 to 38, wherein the bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides.

[0174] 40. The method of any one of embodiments 33-39, wherein the light chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO: 23.

[0175] 41. The method of any one of embodiments 33-40, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 22, and the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 23. [Example]

[0176] The following examples are illustrative of specific embodiments of the present disclosure and various uses thereof. They are set forth for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Example]

[0177] Efficacy and safety of the humanized anti-IL-4 / IL-13 bispecific antibody, RKB, in the treatment of subjects with diffuse systemic sclerosis RKB was evaluated in a Phase 2 study (NCT02921971) for efficacy against dermal fibrosis in subjects with diffuse systemic sclerosis (dcSSc) compared to placebo when administered subcutaneously for 24 weeks of treatment.

[0178] method A multinational, randomized, double-blind, placebo-controlled, two-group, proof-of-concept, phase 2 study investigated the efficacy and safety of RKB 200 mg administered subcutaneously once weekly for 24 weeks in subjects with diffuse SSc. Approximately 94 patients were randomized 1:1 to two treatment groups: 1) RKB group (N=47) receiving weekly subcutaneous RKB 200 mg; and 2) placebo group (N=47) receiving weekly subcutaneous placebo. Randomization was stratified based on the patient's history of SSc interstitial lung disease (SSc-ILD; yes or no). The study design is shown in Figure 2.

[0179] Study population Prior to randomization into cohorts, subjects were screened to assess their eligibility to participate in the study within 28 days prior to Day 1 (D-28, Figure 2).

[0180] Inclusion criteria for eligible subjects were: systemic sclerosis according to the ACR / EULAR 2013 criteria (van den Hoogen F. et al., Ann. Rheum. Dis. 2013, 72(11):1747-55); and diffuse cutaneous form of SSc according to the Leroy criteria.

[0181] Exclusion criteria included the following: age under 18 years; disease duration >36 months from the time of first non-Raynaud's phenomenon episode; modified Rodnan skin score (mRSS) <10 or >35 at screening and baseline visits; history of vasculitis (active or in remission); diagnosis of connective tissue disease (other than SSc) or overlap syndrome (e.g., polymyositis / SSc); positive human immunodeficiency virus (HIV) serology or known history of HIV infection (active or in remission); abnormal hepatitis B and / or hepatitis C tests indicating active or chronic infection; positive or two confirmed and indeterminate QuantiFERON-TB tests at screening. Gold test (regardless of previous treatment status); severe infection within 4 weeks of screening (e.g., pneumonia, pyelonephritis), infection requiring hospitalization or intravenous antibiotics within 4 weeks of screening, or chronic bacterial infection (e.g., osteomyelitis); history of anaphylaxis to any biologic therapy; any clinically significant, severe, or unstable, acutely or chronically progressing, uncontrolled infection or medical condition (e.g., brain, cardiac, pulmonary, renal, hepatic, gastrointestinal, or neurological other than SSc or SSc-ILD) or previous active or ongoing surgical disorder, or, in the investigator's judgment, Evidence of any condition that could affect patient safety in the setting of the study; forced vital capacity (FVC)% ≤75% predicted and hemoglobin-corrected diffusing capacity for carbon monoxide (DLCO)% ≤40% predicted at screening; history of heart failure (including acute decompensation in the setting of preserved ejection fraction), left ventricular ejection fraction (LVEF) ≤45%, coronary artery disease, angina pectoris, myocardial infarction, ischemic cardiomyopathy, and / or hypertrophic cardiomyopathy; history of any prior malignancy or active malignancy, including lymphoproliferative disorders, within 5 years prior to baseline (excluding successfully treated in situ carcinoma of the neck, non-metastatic squamous cell or basal cell carcinoma of the skin);Ischemic ECG changes (except those not supported by findings from a left heart catheterization performed within the last year within screening) and / or other clinically significant ECG findings at screening (including but not limited to second-degree heart block, third-degree heart block, symptomatic long QT interval, sick sinus syndrome, complete left bundle branch block, complete right bundle branch block, uncontrolled atrial fibrillation, uncontrolled atrial flutter, Wolff-Parkinson-White syndrome, intra-atrioventricular nodal reentrant tachycardia, and ventricular tachycardia, ventricular fibrillation, torsades de pointes, etc.) high-dose steroids (greater than 10 mg / day of prednisone or equivalent) or a change in steroid dose within 4 weeks prior to randomization (or baseline visit) or an expected change during the course of the study; previous treatment with rituximab within 12 months prior to screening; bone marrow transplant, total body lymph node irradiation, or ablation; previous treatment with ultra-high-dose cyclophosphamide; high-dose immunosuppressants (e.g., >1 mg / kg orally / day or >750 mg / day) within 3 months of screening IV / month; cyclophosphamide >100 mg / day; azathioprine >15 mg / week; methotrexate >2 g / day; mycophenolate mofetil >2 g / day) or a change in dose within 4 weeks prior to randomization (or baseline visit) or a projected change in dose during the course of the study; treatment with etanercept, cyclosporine A, intravenous immunoglobulin (IVIG), rapamycin, D-penicillamine, tyrosine kinase inhibitors within 4 weeks of screening, or antithymocyte globulin within 6 months of screening; treatment with infliximab, certolizumab, golimumab, abatacept, or adalimumab, tocilizumab within 8 weeks of screening, or anakinra within 1 week of screening; treatment with any investigational drug within 1 month of screening or within 5 half-lives if known (whichever is longer); alanine transaminase (ALT) or aspartate transaminase (AST) above the upper limit of normal (ULN) more than twice; hemoglobin less than 11 g / 100 mL for men and less than 10 g / 100 mL for women; 3 Less than 1000 neutrophils / mm3 (excluding less than 100,000 / mm 3 abnormal laboratory tests at screening from either platelets less than 100 μmol / L or creatine greater than 150 μmol / L; a current history of drug and / or alcohol abuse; pregnant or breastfeeding women; and women of childbearing potential not protected by a highly effective method of birth control and / or women unwilling or unable to test for pregnancy.

[0182] Medication regimen After the screening period, on Day 1 (D1, Figure 2), each eligible subject was randomly assigned to receive one of the following two arms: (1) RKB 200 mg administered subcutaneously once weekly (qw); and (2) placebo administered subcutaneously once weekly (qw) (Figure 2). Treatment with RKB or placebo began on Day 1, with a treatment duration of 24 weeks. The study included eight on-site visits and five telephone calls. The first screening visit was between Days 28 and 1; the second baseline visit was Day 1, when the first dose was administered; visits 3 through 6 were at Weeks 2, 4, 8, and 12 of the treatment period; the seventh visit was during the last week of dosing (Week 24); and the eighth visit was the last study visit for the Week 35 follow-up. During the on-site visit, RKB or placebo was administered after clinical procedures and blood sampling. For safety considerations, telephone calls to subjects were conducted at weeks 6, 16, 18, and 20 during the treatment period and at week 30 during the follow-up period. The post-dosing follow-up period (Figure 2) was conducted to assess adverse events (AEs) and pharmacokinetic analysis.

[0183] C trough Pharmacokinetic parameters were estimated using population PK methods for

[0184] Formulation and route of administration A 100 mg / mL RKB solution for injection was prepared from RKB supplied as a sterile lyophilized powder in glass vials. Each vial was filled with 125 mg of RKB lyophilized powder, and the entire contents of the vial were reconstituted with 1.1 mL of sterile water for injection to obtain a final solution for injection equivalent to 125 mg of RKB drug substance in a total volume of 1.25 mL, equivalent to a 100 mg / mL RKB solution. One mL of this 100 mg / mL RKB solution was then withdrawn for dose administration. Thus, two drug product vials were required to reach a 200 mg dose and prepare a 2 mL syringe of RKB solution.

[0185] For the placebo preparation, the vial containing the excipient was reconstituted with 1.1 mL of sterile water to give a total volume of 1.25 mL. Two placebo product vials were required, and 1 mL was taken from each vial to prepare a 2 mL syringe of placebo solution.

[0186] The route of administration was subcutaneous in the abdomen. Subcutaneous injection sites alternated between quadrants of the abdomen (avoiding the navel and lumbar region), so that the same site was not injected for two consecutive weeks. The site was preferably free of SSc lesions.

[0187] RKB or placebo was administered every 7 ± 2 days from the first dose, a window allowed per protocol to accommodate various circumstances (e.g., pending test results, management of adverse events, visit scheduling difficulties).

[0188] Efficacy endpoints The primary efficacy endpoint for evaluating the efficacy of RKB on skin fibrosis in patients with dcSSC was by assessing the change in modified Rodnan skin score (mRSS) from baseline to week 24. Two secondary endpoints for evaluating the efficacy of RKB on other aspects of dcSSc were: 1) change in HAQ-DI, as assessed by the SHAQ, from baseline to week 24; and 2) change in respiratory function, as measured by observed FVC and observed DLco (corrected for hemoglobin), from baseline to week 24. Exploratory endpoints included: change from baseline to week 24 in the visual analog scale (VAS) for pain, respiratory function, vascular function (Raynaud's phenomenon), gastrointestinal function, digital ulcers, and global assessment from the SHAQ; change in respiratory function, as measured by % predicted FVC and % predicted DLco (corrected for hemoglobin), from baseline to week 24; and change in UCLA Scleroderma Clinical Trials Consortium Gastrointestinal 2.0 (UCLA SCTC GIT) from baseline to week 24. change in TJC28 from baseline to week 24; change in digital ulcer count from baseline to week 24; CRISS from baseline to week 24; change in EQ-5D-5L index from baseline to week 24; change in efficacy endpoints (mRSS, HAQ-DI, VAS from SHAQ, observed FVC, predicted FVC%, observed DLco [corrected for hemoglobin], predicted DLco% [corrected for hemoglobin], UCLA SCTC GIT2.0, TJC28, digital ulcer count, CRISS, and EQ-5D-5L) from baseline to week 35 (up to the end of follow-up period), and proportion of patients with at least 20%, 40%, and 60% improvement in mRSS from baseline to week 35; and proportion of patients with improvement in SHAQ (HAS-DI and VAS) and EQ-5D-5L (index value and VAS) based on MIC at week 24.

[0189] Modified Rodnan skin score Skin fibrosis was assessed using the mRSS, which is performed by palpation of the skin in 17 areas of the body (fingers, hands, forearms, arms, feet, legs, and thighs, face, chest, and abdomen) using a scale of 0 to 3 (0 = normal, 1 = mild thickness, 2 = moderate thickness, and 3 = severe thickness). The total skin score could range from 0 (no thickening) to 51 (severe thickening in all 17 areas). Only physicians or licensed healthcare professionals with standardized training were allowed to assess skin thickening. To minimize inter-rater variability, an attempt was made to have the same healthcare professional assess a given patient from baseline through EOS participation. The baseline and Week 24 / 7 visit mRSS assessments must be performed by the same healthcare professional.

[0190] respiratory function Pulmonary function testing is a secondary endpoint assessing change in respiratory function as measured by observed FVC and observed DLco (corrected for hemoglobin) from baseline to week 24. Absolute changes in observed and predicted % change in FVC and DLco from baseline to week 24 and / or week 35 were assessed as exploratory endpoints. Manual correction of DLco for hemoglobin, unless automatically corrected between measurements, was based on the following equation: 1) For male patients: DLco 観察 / (coefficient), where coefficient = (1.7 × Hb) / (10.22 + Hb); 2) For female patients: DLco 観察 / (coefficient), where coefficient = (1.7 × Hb) / (9.38 + Hb). Hb refers to hemoglobin, and values ​​were obtained from the same visit when DLCO was performed. Spirometry was performed according to the 2005 ATS / ERS guidelines (Miller MR et al., Eur. Respir. J. 2005, 26:319-38), while DLCO was performed according to standard guidelines (MacIntyre N. et al., Eur. Respir. J. 2005, 26:720-35).

[0191] Gastrointestinal symptoms The UCLA SCTC GIT2.0 instrument is a validated self-report questionnaire used to assess quality of life (QOL) related to gastrointestinal function in patients with SSc (Khanna D. et al., Arthritis Rheum. 2009, 61:1257-63). It utilizes seven multi-item scales in the domains of reflux, distension / bloating, diarrhea, fecal incontinence, constipation, emotional well-being, and social functioning. It was obtained at all visits except visits 1 and 3.

[0192] renal function Renal function was assessed at all visits except V3 by measuring blood urea nitrogen and creatinine and by dipstick urinalysis. Urinalysis (dipstick) included specific gravity, pH, glucose, ketones, blood, protein, nitrates, leukocyte esterase, urobilinogen, and bilirubin. If any parameter was abnormal on dipstick, a urine sample was sent to a central laboratory for testing. If the dipstick was positive for protein and / or red blood cells, microscopic analysis was performed by the central laboratory.

[0193] Heart symptoms Systemic sclerosis-related cardiac symptoms were assessed by physical examination and ECG, an established method for monitoring cardiac conduction and underlying coronary artery disease and cardiomyopathy. Electrocardiograms were obtained at all scheduled visits except visit 3. Cardiovascular events were reported as adverse events.

[0194] Assessment of joint pain The TJC28 is a global joint pain assessment based on examination of 28 major joints. It is a reliable and validated method of assessing general joint pain and was obtained at all visits except visits 1 and 3. The 28 joints that were part of the assessment included: shoulder (2 joints), elbow (2 joints), wrist (2 joints), metacarpophalangeal joints (10 joints), proximal interphalangeal joints (10 joints), and knee (2 joints).

[0195] Counting finger ulcers The digital ulcer count captures the number of active open wounds (or digital ulcers) on the fingertips secondary to SSc (and not secondary to local trauma or injury). Fissures, cracks, or further skin defects associated with calcification were not included. Digital ulcer counts were performed at all visits except visits 1 and 3.

[0196] Scleroderma Health Assessment Questionnaire The standard HAQ-DI to measure functional disability and the SHAQ, which includes five SSc-specific VAS assessments, were completed by patients at baseline and throughout the study except for visits 1 and 3 (Steen VD and Medsger TA, Arthritis Rheum. 1997, 40:1984-91). The SHAQ is a standard, validated, and accepted health assessment questionnaire for patients with SSc to assess physical and functional disability associated with cutaneous and systemic fibrosis.

[0197] The HAQ-DI includes eight activity domains (dressing, standing, eating, walking, hygiene, stretching, grip strength, and general daily activities), each with at least two questions, for a total of 20 items. For each item, patients report the degree of difficulty they experienced performing the activity. There are four possible responses for each item, ranging from 0 (no difficulty) to 3 (unable to do). For each of the eight domains included in the HAQ-DI, the score is a single response within the domain with the highest score. If an aid or device is used and the highest score is 0 or 1, the score is increased to 2; if the highest score is 2 or 3, the score remains the same. The HAQ-DI composite score is then calculated as the average of the scores for the eight domains. If one or two domains are missing, the HAQ-DI composite score is obtained by dividing the sum of the domains by the number of domains answered. If three or more domains are missing, the HAQ-DI composite score is missing. The composite score is reported on an ordinal scale ranging from 0 to 3. Scores are interpreted as 0 (no impairment) to 3 (maximum impairment).

[0198] The HAQ-DI also includes a VAS that patients use to report the degree of pain they have experienced over the past week. The VAS is a 10 cm line that is converted to a continuous scale from 0 to 3, with 1 cm corresponding to 0.3 points. The VAS anchors range from 0 (no pain) to 100 (very severe pain). To obtain the patient's score, a metric ruler was used to measure the distance in centimeters from the left anchor to the patient's mark and then multiplied by 0.3. The VAS pain score is not incorporated into the HAQ-DI composite score.

[0199] For the other five VAS, patients were assessed for respiratory, vascular (Raynaud's phenomenon), gastrointestinal function, digital ulcers, and global assessment. Patients were asked to mark a 10 cm line to indicate severity from 0 to 100, with 0 indicating no severity and 100 indicating the worst severity.

[0200] Euro-QOL-5D-5L The EQ-5D-5L questionnaire is a standardized measure of health status developed by the EuroQol group to provide a simple, general measure of health for clinical and economic evaluation. The EQ-5D-5L is designed for self-administration by patients.

[0201] The EQ-5D includes two separate scales: the EQ-5D Descriptive System and the EQ Visual Analogue Scale (EQ VAS). The EQ-5D Descriptive System has five items, each measuring one aspect of health: mobility, self-care, daily activities, pain / discomfort, and anxiety / depression. Each aspect / item has a five-level Likert-type response scale: no problems, mild problems, moderate problems, severe problems, and extreme problems. Responses to the five aspects can be combined into a single five-digit number describing the respondent's health profile and converted into a single index value for calculating quality-adjusted life years (QALYs) and informing economic evaluations of health interventions. The EQ VAS provides a quantitative measure of health as judged by individual respondents on a vertical visual analogue scale. The EQ VAS "thermometer" has endpoints ranging from 100 ("the best health you can imagine") to 0 ("the worst health you can imagine").

[0202] In this analysis, index values ​​were considered as continuous variables.

[0203] Composite response index in diffuse cutaneous systemic sclerosis The CRISS tool summarizes changes in clinical and patient-reported outcomes using a single composite score that reflects the probability that patients with dcSSc will improve (Khanna D. et al., Arthritis Care Res. (Hoboken) 2016, 68(2):167-78). For effective treatments for dcSSc, CRISS can summarize a higher probability of improvement in subjects treated with RKB versus ineffective agents (e.g., placebo). CRISS is a two-step process, as described below.

[0204] Step 1: Patients who develop new or worsening cardiopulmonary and / or renal impairment due to SSc are considered unimproved (regardless of improvement in other primary outcomes) and are assigned a probability of improvement equal to 0.0. Specifically, subjects experience any of the following: a new scleroderma renal crisis; a 15% or greater (relative) decline in predicted FVC% and a predicted FVC% of less than 80% predicted, confirmed by another FVC% within 1 month on high-resolution computed tomography (HRCT) to confirm ILD (if a previous chest HRCT did not show ILD); new onset of left ventricular failure (defined as a left ventricular ejection fraction of 45% or less) requiring treatment; or new onset of PAH (attributable to SSc) on right heart catheterization requiring treatment (PAH is defined as a mean pulmonary artery pressure of 25 mmHg or greater at rest, an end-expiratory pulmonary artery wedge pressure of 15 mmHg or less, and a pulmonary vascular resistance of more than 3 Wood units).

[0205] Step 2: For the remaining patients, step 2 involves computing the predicted probability of improvement for each subject using the following equation (the equation derived the predicted probability from a logistic regression model):

number

[0206] Patient and physician global assessments of overall health were used in calculating CRISS Step 2. These two assessments were based on a Likert scale ranging from 0 (very good) to 10 (very bad) (Khanna D. et al., Arthritis Care Res. 2016, 68(2):167-78).

[0207] statistics Primary Efficacy Analysis Changes in mRSS from baseline to week 24 were analyzed in the intention-to-treat population using a MMRM approach. All available post-baseline data for the analysis window from week 4 to week 24 were included in the analysis, regardless of treatment adherence. The model included fixed categorical effects of treatment group (placebo, RKB), randomization stratum (by IRT, SSc-ILD: yes / no), time point (week 4, week 8, week 12, week 24), randomization stratum-time point interaction and treatment-time point interaction, as well as continuous fixed covariates for baseline mRSS value and baseline value-time point interaction.

[0208] result research patients A total of 143 patients were screened, and 97 patients were randomized: 49 patients in the placebo group and 48 patients in the SAR15697 group. All randomized patients were exposed to the investigational product (IMP), and therefore all 97 patients were included in the safety population. All 97 patients were included in the intention-to-treat population (Table 1).

[0209] [Table 1]

[0210] Patient breakdown [Table 2]

[0211] Demographic and baseline characteristics Overall demographic and patient characteristics at baseline were similar in the two treatment groups, although patients in the RKB group were slightly older (Table 3).

[0212] [Table 3]

[0213] Although overall disease characteristics and medical history at baseline were similar between the two treatment groups, patients in the RKB group had a slightly shorter mean disease duration from the time of first non-Raynaud's phenomenon and slightly higher FVC and DLco at baseline compared with the placebo group (Table 4).

[0214] [Table 4]

[0215] In the placebo group, 29 patients (59.2%) received background therapy within the 3 months before baseline and during the course of the study, compared with 25 patients (52.1%) in the RKB group (Table 5).

[0216] [Table 5]

[0217] Ten (10.3%) patients were incorrectly stratified in the IVRS based on examination of the patient's medical history (Table 6). More specifically, six patients who were stratified as having SSc-ILD in the IVRS did not have a documented medical history in the clinical database, whereas four patients who were not stratified as having SSc-ILD in the IVRS did have a documented medical history in the clinical database.

[0218] [Table 6]

[0219] Historical antibody profiles (Table 7) were relatively evenly distributed between the two treatment groups, which was indirectly confirmed by examining the ANA staining patterns obtained at baseline (Table 8), particularly for the centromere-positive group.

[0220] [Table 7]

[0221] [Table 8]

[0222] [Table 9]

[0223] efficacy Primary Efficacy Endpoint Key Analysis There was a significant difference between the RKB group compared to placebo, as shown in Table 10 and Figure 3. The mean change in mRSS from baseline to week 24 was -2.45 (0.85) and -4.76 (0.86) for the placebo and RKB groups, respectively, resulting in a decrease of 2.31 (1.21) with an associated one-sided p-value = 0.0291.

[0224] [Table 10]

[0225] Subgroup analysis Subgroup analyses were performed based on randomization strata and background therapy.

[0226] When examining data stratified by history of SSc-ILD via IVRS (Table 11), the mean change in mRSS from baseline to week 24 was -1.48 (1.09) and -4.09 (1.08) for the placebo and RKB groups, respectively, for patients without a history of SSc-ILD. For patients with a history of SSc-ILD, the mean change in mRSS from baseline to week 24 was -4.08 (1.41) and -5.82 (1.42), respectively. The mean differences between strata were not significant.

[0227] [Table 11]

[0228] In the subgroup analysis based on background therapy (Table 12), the mean change in mRSS from baseline to week 24 was -0.95 (1.34) and -3.64 (1.24) for the placebo and RKB groups, respectively, for patients without background therapy. For patients with background therapy, the mean change in mRSS from baseline to week 24 was -3.48 (1.08) and -5.81 (1.17), respectively. Similarly, the mean differences between subgroups were not significant.

[0229] [Table 12]

[0230] Key secondary efficacy endpoints HAQ-DI No significant differences were observed between treatment groups for HAQ-DI. As shown in Table 13 and Figure 4, the mean change in HAQ-DI from baseline to week 24 was -0.12 (0.08) and -0.09 (0.08) for the placebo and RKB groups, respectively, yielding a difference of -0.03 (0.11) with an associated one-sided p-value of 0.3975.

[0231] [Table 13]

[0232] FVC No significant differences were observed between treatment groups for FVC (Table 14 and Figure 5). The mean change in absolute FVC(L) from baseline to week 24 was -0.08 (0.04) and -0.01 (0.04) for the placebo and RKB groups, respectively, yielding a difference of -0.07 (0.06) with an associated one-sided p-value = 0.0964.

[0233] [Table 14]

[0234] Observed DLco (corrected for hemoglobin) No significant differences were observed between treatment groups for DLco (Table 15 and Figure 6). The mean change in absolute DLco (mmol / min / kPa) from baseline to week 24 was -0.27 (0.10) and -0.12 (0.10) for the placebo and RKB groups, respectively, yielding a difference of -0.15 (0.14) with an associated one-sided p-value = 0.1352.

[0235] [Table 15]

[0236] The results of the exploratory efficacy endpoints are shown in Table 16. The exploratory endpoints suggested a possible effect of RKB on overall pain, Raynaud's and digital ulcers.

[0237] Romilkimab produced statistically significant improvements in the EQ-5D-5L index compared with placebo; the LS mean (SE) change from baseline to week 24 was 0.00 (0.03) for placebo versus 0.07 (0.03) for romilkimab, yielding a difference of 0.07 [95% CI: -0.01, 0.15; p = 0.04] (Table 2). There were numerical improvements (i.e., decreases) across the SHAQ VAS scales for overall disease severity, pain severity, digital ulcer impact on vascular function, and activity, and less deterioration for GI function and respiratory function from baseline to week 24 for romilkimab versus placebo, but these did not reach statistical significance (Table 2).

[0238] Further exploratory efficacy endpoints are summarized in Supplement 4. At week 24, there was a numerical improvement (i.e., greater reduction) with romilkimab versus placebo in the UCLA SCTC GIT2.0 total score, resulting in a LS mean difference of -0.02 [95% CI: -0.14, 0.10; p = 0.39], a difference of -1.08 [95% CI: -2.74, 0.58; p = 0.10] in the number of tender joints, but no difference in the number of digital ulcers (LS mean difference: 0.10 [95% CI: -0.37, 0.57; p = 0.33]). The mean (SD) predicted probability of improvement in CRISS was slightly higher with romilkimab than placebo: 0.4245 (0.4266) vs. 0.3811 (0.4372), respectively.

[0239] [Table 16]

[0240] CRISS probability No significant differences were observed between treatment groups for the Step 1 analysis of CRISS events (Table 17) and CRISS predicted probability of improvement (Table 18). There were a total of two events that met the CRISS criteria, with both events (decline in FVC) occurring in the placebo group. One additional CRISS event (scleroderma renal crisis) was reported by the RKB group at Week 35 and is reflected in the CRISS analysis for CSR.

[0241] [Table 17]

[0242] [Table 18]

[0243] Pre-specified subgroup analyses The LS mean difference in mRSS was statistically significantly beneficial for romilkimab versus placebo in patients with more severely affected skin (i.e., baseline mRSS ≥ 15 (-3.42 [95% CI: -6.21, -0.64; p = 0.01]). Responder proportion analysis showed that 20%, 40%, and 60% improvements in mRSS from baseline to week 24 were higher with romilkimab than with placebo; the between-group difference for a 40% improvement in mRSS was statistically significant (p = 0.02). The LS mean difference in mRSS was numerically beneficial for romilkimab versus placebo at week 24, regardless of baseline disease duration (< 20 months and ≥ 20 months), use of background therapy, or history of SSc-ILD (Table 19).

[0244] [Table 19]

[0245] Post-mortem analysis The time to the first event was longer with romilkimab versus placebo (Figure 7). The time to an event reflecting disease worsening tended to favor romilkimab compared with placebo: 9 (18.8%) vs. 15 (30.6%), respectively [hazard ratio: 0.47 [95% CI: 0.20, 1.11; p=0.09, two-sided] (Table 20). This was driven by pulmonary and cutaneous events for romilkimab and pulmonary, cutaneous, and other CRISS events for placebo.

[0246] Pharmacokinetics, immunogenicity and biomarker endpoints Pharmacokinetic analysis showed that steady state was reached for romilkimab by week 4. Arithmetic mean (SD) C troughThe TARC values ​​were 38.23 (17.96) μg / mL at week 4 and 47.45 (30.23) μg / mL at week 24, respectively. Immunogenicity testing demonstrated that no patients in either treatment group had pre-existing positive ADAs at baseline. Three patients in the romilkimab group and none in the placebo group developed positive ADAs by week 24; all were considered low titers. None of the ADA positivity cases were associated with TEAEs. Romilkimab was associated with a statistically significant reduction in TARC compared with placebo; the LS mean difference at week 24 was -115.56 ng / L [95% CI; -216.87, -14.26; p = 0.03] (Figure 8A). Periostin showed a strong trend toward greater reduction with romilkimab versus placebo; the LS mean difference at week 24 was -16.92 μg / L [-35.19, 1.35; p=0.07] (Figure 8B). No additional biomarkers were significantly different between romilkimab and placebo (Table 21).

[0247] [Table 20]

[0248] [Table 21]

[0249] safety Adverse events occurring during treatment Infections were the most frequently reported TEAE, occurring more frequently in the RKB group (54.2%) compared with the placebo group (46.9%). The most common infections were in the upper respiratory tract. More events of oral herpes occurred in the RKB group (10.4%) compared with the placebo group (2.0%).

[0250] Skin and subcutaneous tissue disorders were reported slightly more frequently in the placebo group (36.7%) compared with the RKB group (31.3%). The most commonly reported event was skin ulcers (or digital ulcers) in 30.6% and 16.7% of patients in the placebo and RKB groups, respectively.

[0251] Gastrointestinal disorders were reported more frequently in the RKB group (25.0%) compared with the placebo group (14.3%). The most commonly reported event was diarrhea, occurring in 8.2% and 14.6% of patients in the placebo and SAR156507 groups, respectively.

[0252] Musculoskeletal and connective tissue disorders were reported more frequently in the RKB group (22.9%) compared with the placebo group (14.3%). Respiratory, thoracic, and mediastinal disorders were reported slightly more frequently in the placebo group (16.3%) compared with the RKB group (12.5%). Nervous system disorders were reported more frequently in the RKB group (18.8%) compared with the placebo group (6.1%). From these latter three SOCs, some notable imbalances in PT levels included headache (2.0% vs. 8.3%), cough (0% vs. 6.3%), and arthralgia (2.0% vs. 8.3%) events in the placebo and RKB groups, respectively.

[0253] [Table 22]

[0254] [Table 23]

[0255] Serious treatment-emergent adverse events Nine patients (9.2%) experienced at least one TESAE, five (10.2%) and four (8.3%) patients in the placebo and RKB groups, respectively (Table 24). The most frequently reported TESAEs were under the SOCs for infectious and infestational disorders and cardiac disorders. TESAEs related to infections were reported slightly more frequently in the RKB group (4.2%) compared with the placebo group (2.0%). TESAEs related to cardiac events were reported more frequently in the placebo group (4.1%) compared with the RKB group (0.0%). There were no differences in the remaining TESAEs by SOC between the two treatment groups.

[0256] [Table 24]

[0257] Treatment-emergent adverse events leading to death Two patients experienced fatal TEAEs in this study, one in each of the two treatment groups (Table 25). Regarding the fatal TEAE in the RKB group, approximately 3 months after initiation of treatment, a 78-year-old female patient diagnosed with SSc (baseline mRSS of 35) since August 2016 and SSc-ILD and numerous other systemic medical conditions since December 2016, just prior to screening (January 26, 2017), experienced worsening renal dysfunction and was ultimately diagnosed with scleroderma renal crisis, leading to treatment discontinuation (Table 26). Of note, this patient had a baseline history of chronic renal failure, and renal function had already declined by the time of randomization (creatinine = 94.1 μmol / L in December 2016, screening creatinine = 103 μmol / L, and baseline creatinine = 122 μmol / L), which, according to the guidance of the renal consultation, was attributed to age-related processes. She was diagnosed with acute renal failure at 6 weeks, with a creatinine of 172.6 μmol / L, and was treated with furosemide and prednisolone. Several weeks after discontinuing IMP, the patient was hospitalized with bilateral pneumonia. This hospitalization led to rapid progression of respiratory failure, hypertension, and renal failure, leading to hemodialysis and death.

[0258] Regarding fatal TEAEs in the placebo group, a 31-year-old male patient who was receiving background therapy of methotrexate and low-dose prednisone before randomization into the study developed cardiomyopathy (primary SSc cardiomyopathy) and was treated with high-dose corticosteroids, leading to discontinuation of study treatment (Table 27). Ultimately, the patient died from this event approximately 9 months after discontinuation of study treatment.

[0259] [Table 25]

[0260] Adverse events leading to permanent discontinuation

[0261] [Table 26]

[0262] Other serious adverse events (AESI, including laboratory tests) A total of two patients experienced TEAEs considered AESIs per protocol, as seen in Table 27. No differences were observed in vital signs (Table 28) or ECG characteristics (Table 29) between the two treatment groups. No cases of vasculitis, tuberculosis, or anaphylaxis were reported.

[0263] Adverse events of special interest

[0264] [Table 27]

[0265] Vital signs and ECG monitoring

[0266] [Table 28]

[0267] [Table 29] [Table 30]

[0268] summary The primary efficacy endpoint, measured by absolute change in mRSS from baseline at week 24, showed a statistically significant difference between RKB and placebo: the absolute change in mRSS from baseline at week 24 was -2.45 (0.85) and -4.76 (0.86) for the placebo and RKB groups, respectively, resulting in a decrease of 2.31 (1.21) with an associated one-sided p-value = 0.0291.

[0269] Secondary efficacy endpoints measured by the HAQ-DI showed no differences between RKB and placebo. Secondary efficacy endpoints of FVC and DLco also showed no differences between the two groups, although the RKB group showed less decline in both parameters over 24 weeks compared with the placebo group.

[0270] The mean change in absolute FVC(L) from baseline at week 24 was -0.08 (0.04) and -0.01 (0.04) for the placebo and RKB groups, respectively, yielding a difference of -0.07 (0.06) with an associated one-sided p-value = 0.0964.

[0271] The mean change in absolute DLco (mmol / min / kPa) from baseline at week 24 was -0.27 (0.10) and -0.12 (0.10) for the placebo and RKB groups, respectively, yielding a difference of -0.15 (0.14) with an associated one-sided p-value = 0.1352.

[0272] There were similar incidences of treatment-emergent adverse events (TEAEs), treatment-emergent serious adverse events (TESAEs), TEAEs leading to death, and TEAEs leading to treatment discontinuation between the two treatment groups; for the RKB group, more TEAEs occurred within the system organ classes (SOCs) of infections and infestations and gastrointestinal disorders, while for the placebo group, more TEAEs occurred within the SOC of skin and subcutaneous tissue disorders.

Claims

1. 1. A pharmaceutical formulation comprising a dual V region bispecific antibody or antigen-binding fragment that specifically binds IL-4 and IL-13 for use in a method for treating systemic sclerosis (SSc) in a human subject with SSc, the method comprising subcutaneously administering about 200 mg of the dual V region bispecific antibody or antigen-binding fragment to the subject, wherein the treated human subject with SSc has an improved modified Rodnan Skin Score (mRSS) of at least about 20% compared to baseline about 24 weeks after the first administration of the bispecific antibody; A bispecific antibody or bispecific antibody fragment thereof comprises two identical light chain polypeptides and two identical heavy chain polypeptides, the light chain polypeptide comprises the structure N-VL hB-B13 -linker-VL hBD4-8 -CL-C and the heavy chain polypeptide comprises the structure N-VH hB-B13 -linker-VH hBD4-8 -CH1-C; VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2; The pharmaceutical preparation, wherein VH hBD4-8 comprises the amino acid sequence of SEQ ID NO:

4.

2. The pharmaceutical formulation described in claim 1, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6.

3. 3. The pharmaceutical formulation of claim 1 or 2, wherein 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days.

4. The pharmaceutical formulation of any one of claims 1 to 3, wherein the treatment is administered for at least about 24 weeks.

5. 5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and wherein the pH of the formulation is about 7.

0.

6. 6. The pharmaceutical formulation of claim 5, wherein the formulation is reconstituted from a lyophilized formulation.

7. The pharmaceutical formulation of any one of claims 1 to 6, wherein the bispecific antibody is administered in combination with another agent.

8. 8. The pharmaceutical formulation of claim 7, wherein the additional agent is administered prior to, simultaneously with, or after administration of the bispecific antibody.

9. The pharmaceutical preparation according to any one of claims 1 to 8, wherein the systemic sclerosis is diffuse cutaneous systemic sclerosis.

10. 10. The pharmaceutical formulation of claim 1, wherein the light chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO:22, and the heavy chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO:

23.

11. The pharmaceutical formulation of any one of claims 1 to 10, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO:22 and the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

23.

12. 1. A pharmaceutical formulation comprising an anti-IL-4 / anti-IL-13 bispecific antibody for use in a method for reducing sclerotic plaques in a human subject with SSc, the method comprising administering to the subject an effective amount of the anti-IL4 / anti-IL13 bispecific antibody, wherein sclerotic plaques are reduced by at least about 20%, 40%, 60%, 80%, or 100% about 24 weeks after a first administration of the bispecific antibody compared to baseline, wherein a portion of the treated human subjects with SSc have an improved modified Rodnan Skin Score (mRSS) of at least about 20% about 24 weeks after a first administration of the bispecific antibody compared to baseline, an anti-IL-4 / anti-IL-13 bispecific antibody comprising two identical light chain polypeptides and two identical heavy chain polypeptides; the light chain polypeptide comprises the structure N-VL hB-B13 -linker-VL hBD4-8 -CL-C and the heavy chain polypeptide comprises the structure N-VH hB-B13 -linker-VH hBD4-8 -CH1-C; VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2; The pharmaceutical preparation, wherein VH hBD4-8 comprises the amino acid sequence of SEQ ID NO:

4.

13. A pharmaceutical formulation as described in claim 12, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6.

14. 14. The pharmaceutical formulation of claim 12 or 13, wherein the portion of the treated human subjects with SSC have an improved modified Rodnan Skin Score (mRSS) of at least about 40%, or about 60%, compared to baseline about 24 weeks after the first administration of the bispecific antibody.

15. 15. The pharmaceutical formulation of claim 14, wherein the improved mRSS is measured as a least squares mean change from baseline.

16. 16. The pharmaceutical formulation of claim 14 or 15, wherein the least squares mean change from baseline is greater than about any of -3.00, -3.5, -4.0, -4.5, -5.0, -5.5, or -6.

0.

17. Any of claims 12 to 16, wherein the anti-IL4 / anti-IL13 antibody is romilukimab (RKB). The pharmaceutical formulation according to any one of claims 1 to 10.

18. The pharmaceutical formulation of any one of claims 12 to 17, wherein about 200 mg of the anti-IL4 / anti-IL13 antibody is administered subcutaneously to the subject.

19. 19. The pharmaceutical formulation of claim 18, wherein 200 mg of the bispecific antibody is administered to the subject about once a week or about every 5 to 9 days.

20. 20. The pharmaceutical preparation of claim 18 or 19, wherein the treatment is administered for at least about 24 weeks.

21. 21. The pharmaceutical formulation of any one of claims 12 to 20, wherein the pharmaceutical formulation comprises about 100 mg / ml of the bispecific antibody, about 6.3 mM monosodium phosphate, about 37 mM Tris, about 5% (w / v) sucrose, about 3% (w / v) proline, and about 0.2% (w / v) polysorbate 80, and wherein the pH of the formulation is about 7.

0.

22. The pharmaceutical formulation of any one of claims 12 to 21, wherein the formulation is reconstituted from a lyophilized formulation.

23. The pharmaceutical formulation of any one of claims 12 to 22, wherein the bispecific antibody is administered in combination with another agent.

24. 24. The pharmaceutical formulation of claim 23, wherein the additional agent is administered prior to, simultaneously with, or after administration of the bispecific antibody.

25. The pharmaceutical preparation according to any one of claims 12 to 24, wherein the systemic sclerosis is diffuse cutaneous systemic sclerosis.

26. 26. The pharmaceutical formulation of any one of claims 12 to 25, wherein the light chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO:22, and the heavy chain polypeptide comprises an amino acid sequence having at least about 90% identity to the amino acid sequence of SEQ ID NO:

23.

27. 27. The pharmaceutical formulation of any one of claims 12 to 26, wherein the light chain polypeptide comprises the amino acid sequence of SEQ ID NO:22 and the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

23.

28. 1. Use of a dual V region bispecific antibody or antigen-binding fragment in the manufacture of a medicament for treating systemic sclerosis (SSc) in a human subject with SSc, wherein the dual V region bispecific antibody or antigen-binding fragment is formulated for subcutaneous administration to the subject at a dose of about 200 mg and specifically binds IL-4 and IL-13, wherein a portion of the treated human subjects with SSc have an improved modified Rodnan Skin Score (mRSS) of at least about 20% about 24 weeks after the first administration of the bispecific antibody compared to baseline; A dual V region bispecific antibody or antigen-binding fragment comprises two identical light chain polypeptides and two identical heavy chain polypeptides; the light chain polypeptide comprises the structure N-VL hB-B13 -linker-VL hBD4-8 -CL-C and the heavy chain polypeptide comprises the structure N-VH hB-B13 -linker-VH hBD4-8 -CH1-C; VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2; The above use, wherein VH hBD4-8 comprises the amino acid sequence of SEQ ID NO:

4.

29. The use described in claim 28, wherein the linker comprises the amino acid sequence of SEQ ID NO:

6.

30. 1. Use of an anti-IL-4 / anti-IL-13 bispecific antibody in the manufacture of a medicament for reducing sclerotic plaques in a human subject with SSc, wherein the sclerotic plaques are reduced by at least about 20%, 40%, 60%, 80%, or 100% compared to baseline about 24 weeks after the first administration of the bispecific antibody; an anti-IL-4 / anti-IL-13 bispecific antibody comprising two identical light chain polypeptides and two identical heavy chain polypeptides; the light chain polypeptide comprises the structure N-VL hB-B13 -linker-VL hBD4-8 -CL-C and the heavy chain polypeptide comprises the structure N-VH hB-B13 -linker-VH hBD4-8 -CH1-C; VL hB-B13 comprises the amino acid sequence of SEQ ID NO: 1; VL hBD4-8 comprises the amino acid sequence of SEQ ID NO: 3; VH hB-B13 comprises the amino acid sequence of SEQ ID NO: 2; The above use, wherein VH hBD4-8 comprises the amino acid sequence of SEQ ID NO:

4.

31. The use described in claim 30, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.

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