OA19281A - Trispecific antibodies against II-17A, II-17F and another proinflammatory molecule. - Google Patents

Trispecific antibodies against II-17A, II-17F and another proinflammatory molecule. Download PDF

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Publication number
OA19281A
OA19281A OA1201800424 OA19281A OA 19281 A OA19281 A OA 19281A OA 1201800424 OA1201800424 OA 1201800424 OA 19281 A OA19281 A OA 19281A
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tri
binding
seq
antibody
amino acid
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OA1201800424
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Dmitry Valentinovich MOROZOV
Andrei Borisovich ULITIN
Ekaterina Sergeevna Zubareva.
Iakov Iurevich Ustiugov.
Aleksandr Vladimirovich Karabelskii.
Dmitry Yaleryevich Korzhavin
Tatiana Veniaminovna CHERNOVSKAYA
Aleksandr Valerevich GRACHEV
Yury Ivanovich BASOVSKIY
Ekaterina Aleksandrovna LOMKOVA
Veronika Sergeevna USATOVA
Aleksei Aleksandrovich ALEKSANDROV
Elena Leonidovna Morozova
Roman Alekseevich Ivanov.
Pavel Andreevich IAKOVLEV
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Joint Stock Company ‘‘Biocad’’
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Publication of OA19281A publication Critical patent/OA19281A/en

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Abstract

The present invention relates to the field of biotechnology and proposes monoclonal trispecific binding molecules which specifically bind to human IL-17A, human IL-17F and a human proinflammatory molecule (in particular, TNF-alpha) with a high degree of affinity. The invention also relates to DNA constructs which code for such binding molecules, corresponding expression vectors and production methods, and also treatment methods using said binding molecules.

Description

The main function of the immune system is to protect body against infections, such as bacteria or viruses, as well as from cancer lésion. The immune response includes inflammation, i.e., accumulation of immune cells systematically or in a particular part of the body. Cells of the immune system include several types of myeloid and lymphoid cells, such as monocytes, macrophages, dendritic cells, eosinophils, T cells, B cells and neutrophils. In response to an infection or foreign substance, immune cells secrete signaling proteins known as cytokines which, in turn, modulate the prolifération, development, différentiation, and/or migration of immune cells. In some cases, the immune response can resuit in pathological processes, such as autoimmune diseases (see e.g., Abbas et al. (eds.) (2000) Cellular and Molecular Immunology, W.B. Saunders Co., Philadelphia, Pa.; Oppenheim and Feldmann (eds.) (2001) Cytokine Reference, Academie Press, San Diego, Calif; von Andrian and Mackay, New Engl J Med 343:1020-1034 (2000); Davidson and Diamond, New Engl J Med 345:340-350 (2001)).
CD4+ T-cells play a central rôle in immune responses, aiding other cells of the adaptive or innate immune system. Early research identified two classes of CD4+ T-cells (Thl and Th2). Recently, a new subset of CD4+ T cells has been identified: Thl7 cells. Thl7 cells hâve emerged as an offshoot of the adaptive immune system, specializing in strengthening the protection of the host against extracellular bacteria and some fungi and bacteria from which Thl and Th2 cells do not provide protection.
Th 17 cells hâve been identified in the context of discovery of a new family of cytokines, the II,-17 family, which consists of six members at présent (IL-17A-F). IL-17 (previously called CTLA-8) is substantially expressed by Thl7-cells and has been designated as IL-17A, as the original molécule in the IL-17 cytokine family. The sequences of IL-17 family members do not hâve homology with other currently known mammalian proteins; thus, the IL-17 family constitutes a separate family of cytokines. The structural features of IL-17 family members, established on the basis of the crystal structure of IL-17F, are similar to many cytokines; each of the family members is likely produced as a homodimer, although structural analyses imply that heterodimers may be présent as well. More recently, it has been found that heterodimeric IL-17A and IL-17F, expressed by activated CD4+ T cells, transmits a signal through a complex of IL17R/IL-17RC (Wright J.F. et al., JImmunol 181:2799-2805 (2001)).
The gene encoding human IL-17F is located adjacent to the gene encoding human IL17A (Hymowitz et al., Embo J 20(19):5332-41 (2001)). IL-17A and IL-17F hâve 44% amino acid sequence identity, while other members of the IL-17 family hâve lower sequence identity (15-27%). This suggests that IL-17A and IL-17F form a spécial subgroup of the IL-17 family (Starnes et al., J Immunol 167(8):4137-40 (2001); Aggarwal and Gurney, J. Leukoc Biol 71(1):18 (2002)). It has been found that the biological effects of IL-17F are similar to those of IL-17A, e.g., the ability to stimulate the production of IL-6, IL-8 and G-CSF in a wide variety of cells. Like IL-17A, IL-17F is able to induce the sécrétion of cartilage matrix and inhibit the synthesis of new cartilage matrix (see US 2002/0177188). Thus, like IL-17A, IL-17F may contribute to the pathology of inflammatory diseases. Recently, the authors observed that both IL-17A and IL17F are induced in T-cells exposed to interleukin 23 (IL-23) (Aggarwal et al., J Biol Chem 278(3):1910-4 (2003)). The observation that IL-17A and IL-17F hâve similar chromosomal localization and significant sequence similarity, and the fact that IL-17A and IL-17F are induced in the same cell populations in response to spécifie stimuli, led to the identification of a new human cytokine that contains a covalent heterodimer of IL-17A and IL-17F.
Further, IL-17A acts synergistically with tumor necrosis factor alpha (TNFa) and interleukin-1 beta (IL-Ιβ) to achieve a significant pro-inflammatory State. To treat a variety of inflammatory, immune, and proliférative conditions, including rheumatoid arthritis (RA), osteoarthritis, rheumatoid arthritis osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis and cirrhosis), gingivitis, periodontosis or periodontal diseases, inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis and inflammatory bowel disease), asthma (including allergie asthma), allergies, chrome obstructive pulmonary disease (COPD), multiple sclerosis, psoriasis, cancer and others, it was proposed to reduce the activity of IL-17A with antibodies or antigen-binding antibody fragments that are antagonists of these molécules (see, e.g., US 2003/0166862, WO 2005/108616, WO 2005/051422, and WO 2006/013107).
TNFa antagonists are known to treat arthritis by reducing inflammation and inhibiting the progression of cartilage and bone destruction (van den Berg, Arthritis Res 3: 18-26 (2001)). However, a significant percentage of patients hâve an inadéquate response to TNFa antagonistcontaining drugs. Preclinical research shows that TNFa and IL-17A exhibit the same properties in the pathophysiology of arthritis. While singly TNFa and IL-17A hâve a negligible effect on the expression of inflammatory genes, their combination leads to a strong synergistic response. The interaction of TNFa and IL-17A increases the expression of cytokines (LeGrand et al., Arthritis Rheum 44:2078-2083 (2001)) and pro-inflammatory chemokines (Chabaud et ah, J. Immunol 167:6015-6020 (2001)), and also induces cartilage and bone destruction (Van Bczooijen et al., Ann Rheum Pis 61:870-876 (2002)).
Currently there is a number of IL-17A bispecific antibodies and other pro-inflammatory molécules, e.g., DVD (Abbott) and COVA322 (Covagen-Janssen).
International Patent Publication No. WO 2015/014979 describes IL-17A and TNFa tetravalent bispecific antibodies, which contain two binding sites for IL-17A and two binding sites for TNFa. These antibodies demonstrated a synergistic effect in inhibiting inflammation and tissue destruction in rheumatoid arthritis.
International Patent Application WO 2014/137961 describes IL-17A and TNFa bispecific antibodies that comprise two polypeptides. These bispecific antibodies are thermally and chemically stable, exhibit low aggregation ability, and can simultaneously bind to IL-17A and TNFa.
US Patent No. 8,496,936 describes IL-17A, IL-17F and IL-23pl9 tri-specific antibodies that exhibit different activities against various ligands in cellular assays and in vivo animal models, but did not show any physical-chemical characteristics necessary to create stable formulation candidates.
The production of antibodies that can effectively neutralize IL-17A, IL-17F, and TNFa activity in the context of inflammatory reactions and autoimmune disease, especially antibodies that remain stable in formulation, remains relevant.
Summary of the Invention
The présent invention provides tri-specific binding molécules directed against IL-17A, IL-17F and a pro-inflammatory molécule such as TNFa. Such antibodies may be used for treatment of autoimmune and/or inflammatory disorders such as rheumatoid arthritis, psoriasis, and psoriatic arthritis. Compared to currently available treatments for such disorders, including antibody treatments, it is contemplated that the tri-specific binding molécules of the invention may provide a superior clinical response either alone or in combination with other antiinflammatory and/or immunosuppressive thérapies.
In one aspect, the invention provides a tri-specific binding molécule comprising a first binding domain that binds to human IL-17A and human IL-17-F and a second binding domain that binds to a human pro-inflammatory molécule. In certain embodiments, the human proinflammatory molécule is human TNFa. In a particular embodiment, the binding molécule is an antibody or an antigen-binding portion thereof.
In some embodiments, the first binding domain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first binding domain comprises the amino acid sequences of SEQ ID NOs: 1-3. In some embodiments, the first binding domain competes for binding with or binds to the same epitope as a binding domain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first binding domain is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the first binding domain comprises the amino acid sequence of SEQ ID NO: 4. In a particular embodiment, the first binding domain consists of the amino acid sequence of SEQ ID NO: 4.
In some embodiments, the first binding domain is humanized. In certain embodiments, the first binding domain is a humanized VHH.
In some embodiments, the second binding domain competes for binding with or binds to the same epitope as a binding domain comprising:
o a heavy chain comprising the amino acid sequences of SEQ ID NOs: 10-12 and a light chain comprising the amino acid sequences of SEQ ID NOs: 13-15;
* a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 9; or ® a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.
In some embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequences of SEQ ID NOs: 10-12 and a light chain comprising the amino acid sequences of SEQ ID NOs: 13-15. In some embodiments, the second binding domain comprises a heavy chain variable domain at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 8, a variable domain at least 90% identical to the amino acid sequence of SEQ ID NO: 9, or both. In some embodiments, the second binding domain comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or both. In a particular embodiment, the second binding domain comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 9.
In some embodiments, the second binding domain is a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody, Fv, Fab, F(ab’)2, Fd, single chain Fv molécule (scFv), diabody, or single domain antibody (dAb). In some embodiments, the second binding domain is a humanized Fab.
In some embodiments, the second binding domain comprises a heavy chain at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 5, a light chain at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 6, or both. In some embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5, a light chain comprising the amino acid sequence of SEQ ID NO: 6, or both. In a particular embodiment, the second binding domain comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.
In one embodiment, the invention provides a tri-specific binding molécule comprising any of the first binding domains described herein in combination with any of the second binding domains described herein.
In one embodiment, the invention provides a tri-specific binding molécule comprising a first binding domain that binds to human IL-17A and human IL-17F and a second binding domain that binds to human TNFa, wherein said first binding domain comprises the amino acid sequence of SEQ ID NO: 4 and said second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence ofSEQ ID NO: 6.
In one embodiment, the invention provides a tri-specific binding molécule comprising a first binding domain that binds to human IL-17A and human IL-17F and a second binding domain that binds to human TNFa, wherein said first binding domain comprises the amino acid sequence of SEQ ID NO: 4 and said second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 8 and a light chain comprising the amino acid sequence ofSEQ ID NO: 9.
In one embodiment, the invention provides a tri-specific binding molécule that binds to human IL-17A, human IL-17F, and human TNFa, wherein said binding molécule comprises the amino acid sequences of SEQ ID NOs: 5 and 7.
In some embodiments, the first binding domain and the second binding domain are joined by a peptide linker of greater than five amino acids. In certain embodiments, the amino acid residues of the peptide linker are selected from G, A, S, P, E, T, D, and K. In a particular embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 16. The first binding domain may be joined to the N-terminus or the C-terminus of either the heavy or light chain of the second binding domain via the peptide linker.
In some embodiments, the binding molécule is an antibody of isotype IgG or an antigenbinding portion thereof. The antibody may be, e.g., of isotype subtype IgGl.
In some embodiments, the binding molécule comprises an Fc région with at least one mutation that reduces ADCC and/or CDC in comparison to the same binding molécule without the mutation.
In some embodiments, the binding molécule has at least one of the following properties:
• a Kd for IL-17 A of 10'11 M or less;
• a Kd for IL-17F of 10'7 M or less;
• a KD for TNFa of 10'11 M or less;
• inhibits the activity of IL-17A and/or IL-17A/TNFa by at least 50% (e.g., 60%, 70%, 80%, or 90%) at concentrations below 200 ng/mL in an IL-6 assay in HT1080 cells;
• inhibits IL-17A-dependent IL-6 release by HT080 cells;
® inhibits production of IL-6 in HT1080 cells;
• inhibits TNFa-dependent cytotoxicity by at least 50% (e.g., 60%, 70%, 80%, or 90%) at concentrations below 100 ng/mL in WEHI-13VAR cells;
• does not bind to rabbit, mouse, or guinea pig TNFa;
• simultaneously binds to IL-17A and TNFa;
• binds to cynomolgus IL-17A and TNFa;
• remains stable after at least three, four, five, six, or seven days of incubation (e.g., after seven days of incubation) at 37°C in human sérum;
• has ahalf-life of over 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days in vivo (e.g., 12 days);
e has an anti-inflammatory effect in an in vivo arthritis model; and • inhibits the activity of IL-17 and TNFa in vivo.
In one aspect, the invention provides a pharmaceutical composition comprising any of the tri-specific binding molécules described herein and a pharmaceutically acceptable excipient.
In one aspect, the invention provides an isolated nucleic acid molécule comprising a nucléotide sequence that encodes the first binding domain of any of the tri-specific binding molécules described herein.
The invention also provides an isolated nucleic acid molécule comprising a nucléotide sequence that encodes the heavy chain, the light chain, or both, of the second binding domain of any of the tri-specific binding molécules described herein.
In some aspects, the invention provides an isolated nucleic acid molécule comprising: « a nucléotide sequence that encodes the first binding domain of any of the tri-specific binding molécules described herein;
• a nucléotide sequence that encodes the heavy chain of the second binding domain of any of the tri-specific binding molécules described herein;
• a nucléotide sequence that encodes the first binding domain of any of the tri-specific binding molécules described herein; or • any combination of the above.
In some embodiments, the isolated nucleic acid molécule comprises a nucléotide sequence that encodes the first binding domain, and a nucléotide sequence that encodes the light chain of the second binding domain, of any of the tri-specific binding molécules described herein, optionally further comprising a nucléotide sequence encoding a peptide linker. The isolated nucleic acid molécule may comprise, for example, a nucléotide sequence encoding SEQ ID NO: 4 and a nucléotide sequence encoding SEQ ID NO: 6, optionally further comprising a nucléotide sequence encoding a peptide linker. In one embodiment, the amino acid sequence of the peptide linker is SEQ ID NO: 16. In one embodiment, the isolated nucleic acid molécule encodes SEQ ID NO: 7.
In some embodiments, the isolated nucleic acid molécule encodes the amino acid sequence of any one of SEQ ID NOs: 1-15.
The invention also provides a vector comprising any of the isolated nucleic acid molécules described above, wherein said vector further comprises an expression control sequence.
The invention also provides a host cell comprising:
o a nucléotide sequence that encodes the first binding domain of any of the tri-specific binding molécules described herein;
• a nucléotide sequence that encodes the heavy chain of the second binding domain of any of the tri-specific binding molécules described herein; and o a nucléotide sequence that encodes the first binding domain of any of the tri-specific binding molécules described herein.
In a particular embodiment, the host cell comprises a nucléotide sequence that encodes SEQ ID NO: 5 and a nucléotide sequence that encodes SEQ ID NO: 7.
In one aspect, the invention provides a method for producing any of the tri-specific binding molécules described herein, comprising providing a host cell as described above, cultivating said host cell under conditions suitable for expression of the binding molécule, and isolating the resulting binding molécule.
In one aspect, the invention provides a method for treating a patient in need thereof, comprising administering to the patient any of the tri-specific binding molécules or pharmaceutical compositions described herein. In some embodiments, the patient has a disorder mediated by IL-17A, IL-17F, or TNFa. In a particular embodiment, the invention provides a method for treating rheumatoid arthritis, psoriasis, or psoriatic arthritis in a patient, comprising administering to the patient any of the tri-specific binding molécules or pharmaceutical compositions described herein. In some embodiments, the method further comprises administering an anti-inflammatory or immunosuppressive agent. In any of the above methods of treatment, the patient may be a human.
Brief Description of the Figures
Fig. 1 shows a schematic of production of the tri-specific anti-IL-17A/anti-IL-17F/antiTNFa antibody.
Fig. 2 shows the protein sequence of IL-17A-His6-FLAG.
Fig.3 is a gel showing purified IL-17A-His6-FLAG protein.
Fig. 4 is a gel showing purified control anti-IL-17A antibodies.
Fig. 5 is a graph showing a titer of anti-human IL-17A antibodies produced by immunization of Lama glama.
Fig. 6 is a map showing the pH5 plasmid for phage display of antibody Fab fragments.
Fig. 7 is a map showing the pLL-Fab plasmid for secretory expression of antibody Fab fragments in E.coli.
Fig. 8 is a graph showing the binding of polyclonal phage with spécifie and nonspecific antigens after sélection of anti-IL17-A fragments from an immune phage library.
Fig. 9 shows the amino acid sequence of a high affinity VHH domain (VHH17; SEQ ID NO: 4) and its parent VHH domain (VHH; SEQ ID NO: 17) spécifie for human IL-17A and IL17F. Amino acids substituted during the humanization process are shown in bold. CDRs are highlighted in gray. Fig. 9 also shows the heavy and light chain variable domain amino acid sequences (SEQ ID NOs: 18 and 9, respectively) of an anti-human TNFa antibody (RU 2303604).
Fig. 10 shows a table of mutations in the CDRs of anti-TNFa antibody variants used as part of anti-IL-17A/anti-IL-17F/anti-TNFa tri-specific antibodies (from top to bottom, SEQ ID NOs: 19-32).
Fig. 11 shows the results of transient development of different anti-IL-17A/anti-IL17F/anti-TNFa tri-specific antibody variants.
Fig. 12 shows a comparative analysis of the anti-TNFa activity of different anti-IL17A/anti-IL-17F/anti-TNFa tri-specific antibody variants in a cytotoxic TNFa-dependent assay using WEHI-13VAR cells. Variants with maximum inhibitory activity are marked in grey.
Fig. 13 shows a graph of the anti-TNFa activity of the three anti-IL-17A/anti-IL17F/anti-TNFa tri-specific antibody variants (Mabl2, Mab31, and Mab32) in a cytotoxic TNFadependent assay using WEHI-13VAR cells.
Fig. 14 shows a graph of anti-TNFa activity for anti-IL-17A/anti-IL-17F/anti-TNFa trispecific antibody BCD-121 and the two commercial monospecific anti-TNFa antibodies adalimumab and infliximab in a TNFa-dependent cytotoxicity assay using WEHI-13VAR cells.
Fig. 15 is a graph showing the ability of BCD-121 to block function of IL-17A-dependent IL-6 production in HT 1080 cells as compared to a positive control (monospecific anti-IL-17A antibody BCD-085) and a négative control (anti-TNFa antibody adalimumab).
Fig. 16 is a graph showing the ability of BCDJ121 to block dual IL-17A/TNFa-dependent IL-6 production in HT1080 cells as compared to a positive control (monospecific anti-IL-17A antibody BCD-085) and a négative control (reproduced anti-TNFa antibody adalimumab).
Fig. 17 is a graph showing antibody-dependent cellular cytotoxicity (ADCC) from antibody BCD-121 as compared to three commercial monospecific anti-TNFa antibodies (adalimumab, infliximab, and cimzia) based on Jurkat-tm TNFa c 1.8 cell culture.
Fig. 18 is a graph showing complement-dependent cytotoxicity (CDC) from antibody BCD-121 as compared to three commercial monospecific anti-TNFa antibodies (adalimumab, infliximab, and cimzia) based on Jurkat-tm TNFa c 1.8 cell culture.
Fig. 19 is a sensogram of simultaneous “sandwich” binding of BCD-121 to both IL-17A and human TNFa using Octet RED96.
Fig. 20 is a graph showing ELISA results for BCD-121 antibody binding to different ligands in the human IL-17 family.
Fig. 21 is a graph showing ELISA results for BCD-121 antibody binding to different orthologous TNFa proteins.
Fig. 22 is a graph showing the thermal stability of BCD-121 protein aggregation using dynamic light scattering (DLS). The graph shows the average particle size (Z-average) versus température for four buffers.
Fig. 23 is a graph showing the thermal stability of BCD-121 during long-term exposure using Thermostress 50°C in four different buffer solutions.
Fig. 24 shows a purity analysis of BCD-121 obtained from the culture medium of a stable cell line producer according to an optimized purification scheme. Panel A: reducing conditions (+ β-ΜΕ). Panel B: non-reducing conditions (- β-ΜΕ). Lanes 2 and 3:10 and 40 pg applied, respectively.
Fig. 25 is a graph demonstrating BCD-121 stability in pure human sérum by ELISA.
Fig. 26 is a graph showing the pharmacodynamie properties of BCD-121 in a M. fascicularis collagen-induced arthritis (CIA) model.
Fig.27 is a graph showing the pharmacokinetic profile by ELISA of BCD-121 in monkeys following a single SC administration at a dose of 10, 50 or 100 mg/kg.
Fig. 28 is a graph showing that BCD-121 can dual block the ability of IL-17A and TNFa to induce production of IL-6 in HT1080 cells. BCD-121 more potently blocks IL-6 release than a monospecifîc anti-TNF Abl, an anti-TNF Fab-PEG, or an anti-IL17 Ab (monoclonal antibody against IL-17), with an IC50 of 20 ng/ml.
Fig. 29 is a graph showing a shaking stress aggregation assay (20 at 500 rpm) by Zetasizer nano ZSP. The data shows that BCD-121 is a highly soluble tri-specific molécule that can be used in high-concentration formulations for subcutaneous injection (above 100 mg/ml).
Detailed Description of the Invention
Définitions and General Techniques
Unless otherwise defîned herein, scientific and technical terms used in connection with the présent invention shall hâve the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or équivalent to those described herein can also be used in the practice or testing of the présent, invention. AU publications and other référencés mentioned herein are incorporated by reference in their entirety. In case of conflict, the présent spécification, including définitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, médicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s spécifications, as commonly accomplished in the art or as described herein.
Throughout this spécification and embodiments, the words “hâve” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Antibody-related définitions
Unless otherwise stated, as used herein, “IL-17A,” “IL-17F,” and “TNFa” refer to human IL-17A, IL-17F, and TNFa, respectively.
Interleukin-17 (“IL-17”) is a 20-30 kD glycosylated homodimeric protein. The IL-17A human gene encodes a 155 amino acid protein, which has a 19 amino acid signal sequence and a 136 amino acid mature segment. The IL-17F human gene encodes a 163 amino acid protein, which has a 30 amino acid signal sequence and a 133 amino acid mature segment. A human IL17A polypeptide sequence is available under Uniprot Accession No. Q16552. A human IL-17F polypeptide sequence is available under Uniprot Accession No. Q96PD4.
The term Tumor Necrosis Factor alpha” or “TNFa, as used herein, refers to a human TNFa molécule having the polypeptide sequence described in Pennica et al., Nature 312:721 (1984) or in Aggarwal et al., JBC 260:2345 (1985).
The terms “immune response,” “autoimmune reaction,” and “autoimmune inflammation” refer to, for example, the action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complément that resuit in sélective damage, destruction, or élimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).
The term “binding molécule” encompasses antibodies and immunoglobulins. The term “antibody” (Ab) or “immunoglobulin” (Ig), as used herein, refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant région (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant région (CL). The VH and VL domains can be subdivided further into régions of hypervariability, termed “complementarity determining régions” (CDRs), interspersed with régions that are more conserved, termed “framework régions” (FRs). Each VH and VL is composed of three CDRs (H-CDR herein désignâtes a CDR from the heavy chain; and L-CDR herein désignâtes a CDR from the light chain) and four FRs, arranged from aminoterminus to carboxyl-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each région may be in accordance with IMGT définitions (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003); or the définitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989).
In some embodiments, an antibody or immunoglobulin as described herein may comprise in addition to the heavy and light chains a VHH domain, or “nanobody.” These single domains are the antigen-binding parts of heavy chain antibodies (hcAb), which are devoid of light chains and are typically found in camelid species such as Hamas and alpacas.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human IL-17A, human IL-17F, TNFa, or portions thereof). It has been shown that certain fragments of a full-length antibody can perform the antigenbinding function of the antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” include (i) a Fab fragment: a monovalent fragment consisting of the Vl, Vh, Cl and ChI domains; (ii) a F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge région; (iii) an Fd fragment consisting of the VH and ChI domains; (iv) a Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody, (v) a single domain antibody (dAb) fragment, which consists of a Vh domain; and (vi) an isolated complementarity determining région (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, Vl and Vh, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vl and Vh régions pair to form monovalent molécules (known as single chain Fv (scFv)). Also, within the invention are antigen-binding molécules comprising a Vh and/or a Vl. In the case of a Vh, the molécule may also comprise one or more of a CHI, hinge, CH2, or CH3 région. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites.
Antibody portions, such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molécules can be obtained using standard recombinant DNA techniques, e.g., as described herein.
The term “recombinant antibody” refers to an antibody that is expressed from a cell or cell line comprising the nucléotide sequence(s) that encode the antibody, wherein said nucléotide sequence(s) are not naturally associated with the cell.
The term “variant” antibody as used herein refers to an antibody having an amino acid sequence which differs from the amino acid sequence of its “parent” antibody by addition, délétion and/or substitution of one or more amino acid residues relative to the parent antibody sequence. In a preferred embodiment, the variant antibody comprises at least one or more (for example, from one to about twelve, e.g., two, three, four, five, six, seven, eight, or nine, ten, eleven, or twelve; and in some embodiments, from one to about ten) amino acid additions, délétions, and/or substitutions relative to the parent antibody. In some embodiments, the amino acid additions, délétions, and/or substitutions are in the CDRs of the variant antibody. Identity or homology with respect to the variant antibody sequence is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to the parent antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The variant antibody retains the ability to bind to the same antigen, and preferably epitope, as that to which the parent antibody binds, and in some embodiments has at least one property or bioactivity that is superior to similar properties of the parent antibody. For example, a variant antibody may hâve, e.g., stronger binding affînity, longer half-life, lower IC 50, or enhanced ability to inhibit the biological activity of the antigen as compared to the parent antibody. A variant antibody of particular interest herein is an antibody exhibiting at least about a 2-fold (preferably at least about a 5-fold, 10-fold or 20-fold) increase in biological activity as compared to the parent antibody.
The term “chimeric antibody” refers in its broadest sense to an antibody that contains one or more régions from one antibody and one or more régions from one or more other antibodies, typically an antibody that is partially of human origin and partially of non-human origin, i.e., derived in part from a non-human animal, for example a mouse, rat or other rodent, or a camelid such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of a human anti-antibody response, e.g., a human anti-mouse antibody response in the case of a murine antibody. An example of a typical chimeric antibody is one in which the variable région sequences are murine while the constant région sequences are human. In the case of a chimeric antibody, the non-human parts may be subjected to further alteration in order to humanize the antibody.
The term “humanize” refers to the fact that where an antibody is wholly or partially of non-human origin, for example a murine or llama antibody obtained from immunization of mice or Hamas, respectively, with an antigen of interest or a chimeric antibody based on such a murine or llama antibody, it is possible to replace certain amino acids, in particular in the framework régions and constant domains of the heavy and light chains, in order to avoid or minimize an immune response in humans. The specificity of an antibody’s interaction with a target antigen résides primarily in the amino acid residues located in the six CDRs of the heavy and light chain. The amino acid sequences within CDRs are therefore much more variable between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a spécifie naturally occurring antibody, or more generally any spécifie antibody with a given amino acid sequence, e.g., by constructing expression vectors that express CDR sequences from the spécifie antibody grafted into framework sequences from a different antibody. As a resuit, it is possible to “humanize” a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody. Although it is not possible to precisely predict the immunogenicity and thereby the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic than human antibodies. Chimeric antibodies, where the foreign (e.g. rodent or camelid) constant régions hâve been replaced with sequences of human origin, hâve been shown to be generally less immunogenic than antibodies of fully foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. Chimeric antibodies or other antibodies of non-human origin thus can be humanized to reduce the risk of a human anti-antibody response.
For chimeric antibodies, humanization typically involves modification of the framework régions of the variable région sequences. Amino acid residues that are part of complementarity determining régions (CDRs) most often will not be altered in connection with humanization, although in certain cases it may be désirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site, an aspartate isomerization site or an undesired cysteine or méthionine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-XThr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser/Thr residue to a different residue, preferably by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when présent in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is présent in a CDR sequence, it may therefore be désirable to remove the site, typically by conservative substitution to remove one of the implicated residues.
Numerous methods for humanization of an antibody sequence are known in the art; see, e.g., the review by Almagro & Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is CDR grafting, which for, e.g., a murine-derived chimeric antibody involves identification of human germline gene counterparts to the murine variable région genes and grafting of the murine CDR sequences into this framework. CDR grafting may be based on the Kabat CDR définitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) has suggested that the IMGT® définition (the international ImMunoGeneTics information System®, www.imgt.org) may improve the resuit of the humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)). In some cases, CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR-grafted non-human antibody as compared to the parent antibody from which the CDRs are obtained. Back mutations (sometimes referred to as “framework repair”) may be introduced at selected positions of the CDR-grafted antibody, typically in the framework régions, in order to reestablish the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back mutations are typically those that are located at the surface of an antibody molécule, while residues that are buried or that hâve a low degree of surface exposure will not normally be altered. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues.
In certain cases, it may also be désirable to alter one or more CDR amino acid residues in order to improve binding affinity to the target epitope. This is known as “affinity maturation” and may optionally be performed in connection with humanization, for example in situations where humanization of an antibody leads to reduced binding specificity or affinity and it is not possible to sufficiently improve the binding specificity or affinity by back mutations alone. Varions affinity maturation methods are known in the art, for example the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method of Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).
The term “isolated protein”, “isolated polypeptide” or “isolated antibody” refers to a protein, polypeptide or antibody that by virtue of its origin or source of dérivation (1) is not associated with naturally associated components that accompany it in its native State, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular System different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
As used herein, the term “germline” refers to the nucléotide and amino acid sequences of antibody genes and gene segments as they are passed from parents to offspring via germ cells. Germline sequences are distinguished from the nucléotide sequences encoding antibodies in mature B cells, which hâve been altered by recombination and hypermutation events during the course of B cell maturation. An antibody that “utilizes” a particular germline sequence has a nucléotide or amino acid sequence that aligns with that germline nucléotide sequence or with the amino acid sequence that it spécifiés more closely than with any other germline nucléotide or amino acid sequence.
The term “affmity” refers to a measure of the attraction between an antigen and a binding molécule, e.g., an antibody. The intrinsic attractiveness of the binding molécule for the antigen is typically expressed as the binding affmity equilibrium constant (Kd) of a particular binding molecule-antigen interaction. A binding molécule is said to specifically bind to an antigen when the Kd is < 1 mM, preferably <100 nM. A Kd binding affmity constant can be measured, e.g., by surface plasmon résonance (BIAcore™) or Bio-Layer Interferometry, for example using the ProteOn™ XPR36 SPR system from Bio-Rad or the Octet™ system.
The term “kOff” refers to the dissociation rate constant of a particular binding molecule-antigen interaction. A kon- dissociation rate constant can be measured by Bio-Layer Interferometry, for example using the Octet™ System.
The term “epitope” as used herein refers to a portion (déterminant) of an antigen that specifically binds to a binding molécule (e.g., and antibody or a related molécule such as a bispecific binding molécule). Epitopic déterminants generally consist of chemically active surface groupings of molécules such as amino acids or carbohydrate or sugar side chains and generally hâve spécifie three-dimensional structural characteristics, as well as spécifie charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, ail of the points of interaction between a protein (e.g., an antigen) and an interacting molécule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope using techniques well known in the art. Further, the génération and characterization of antibodies or other binding molécules may elucidate information about désirable epitopes. From this information, it is then possible to competitively screen binding molécules for binding to the same or similar epitopes, e.g., by conducting compétition studies to find binding molécules that compete for binding to the antigen.
One can détermine whether an antibody or other binding molécule binds to the same epitope of or cross competes for binding to IL-17 A, IL-17F, or TNFa with a tri-specific binding molécule of the invention by using methods known in the art. In one embodiment, one allows the tri-specific binding molécule of the invention to bind to IL-17A, IL-17F, or TNFa under saturating conditions and then measures the ability of the test antibody to bind to said target antigen. If the test antibody is able to bind to the target antigen at the same time as the reference tri-specific binding molécule, then the test antibody binds to a different epitope than the reference tri-specific binding molécule. However, if the test antibody is not able to bind to the target antigen at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the tri-specific binding molécule. This experiment can be performed using ELISA, RIA, BIACORE™, Bio-Layer Interferometry or flow cytometry. To test whether a tri-specific binding molécule of the invention cross-competes with another binding molécule, one may use the compétition method described above in two directions, i.e., determining if the known binding molécule blocks the test binding molécule and vice versa. Such cross-competition experiments may be performed e.g. using an IBIS MX96 SPR instrument or the Octet™ System.
In one embodiment, the binding molécule of the invention is a monoclonal antibody. As used herein, the acronym “mAb” refers to a monoclonal antibody, i.e., an antibody synthesized and secreted by an individual clonal population of cells. The clonal population can be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells - hybridomas - typically produced by the fusion of individual B lymphocytes from an immunized animal with individual cells from a lymphocytic tumour. Hybridomas are an engineered cell type and do not occur in nature.
The class (isotype) and subclass of antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are spécifie for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA, Western Blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing ail or a portion of the constant domains of the heavy and/or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.
The term “pro-inflammatory molécule” includes, but is not limited to, lymphokines, monokines, traditional polypeptide hormones, growth hormones such as human growth hormone, N-methionyl human growth hormone and cattle growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyrotrophic hormone (TSH) and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, factor and tumor necrosis factor, Mullerian inhibiting substance, gonadotropin-associated mouse peptide, inhibin, activin, vascular endothélial growth factor, integrin, thrombopoietin (TPO), nerve growth factor and platelet-derived growth factor, transforming growth factors (TGF), insulin-like growth factors I and II, erythropoietin (EPO), osteoinductive factors, interferons, colony-stimulating factors (CSF) such as CSF macrophages (M-CSF), CSF granulocyte-macrophage (GM-CSF) and CSF granulocyte (G-CSF), interleukins (IL) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL8, IL-9, IL-IO, IL-11, IL15, IL-12, IL-17A, IL-22, and IL-23, tumor necrosis factors, and other polypeptide factors inducing leukemia inhibitory factor (LIF) and kit ligand (KL). As used herein, the term “pro-inflammatory molécule” includes proteins isolated from natural sources or from recombinant cell culture and biologically active équivalents to said proteins.
Tri-Specific Anti-TNFa/Anti-IL-I7A/Anti-IL-17F Binding Molécules
The présent invention provides tri-specific binding molécules directed against IL-17A, IL-17F, and a pro-inflammatory molécule that is not IL-17A or IL-17F (e.g., TNFa). In a particular embodiment, the tri-specific binding molécules are tri-specific antibodies or antigenbinding portions thereof. The binding molécules comprise domains that bind to IL-17A, IL-17F, and a pro-inflammatory molécule (e.g., TNFa). In some embodiments, the binding molécules comprise a first binding domain that binds to IL-17A and IL-17F and a second binding domain that binds to TNFa. In some embodiments, the binding domains for IL-17A and IL-17F may be separate.
In one embodiment, a tri-specific anti-TNFa/anti-IL-17A/anti-IL-17F binding molécule of the invention competes for binding to human IL-17A and/or IL-17F with, or binds to the same epitope(s) of said antigen(s) as, a VHH comprising:
• SEQIDNOs: 1-3; or • SEQ ID NO: 4.
In one embodiment, a tri-specific anti-TNFa/IL-17A/IL-17F binding molécule of the invention competes for binding to human TNFa with, or binds to the same epitope(s) of said antigen(s) as, an antibody comprising:
• SEQIDNOs: 10-15;
• SEQ ID NOs: 8 and 9; or • SEQ ID NOs: 5 and 6.
In certain embodiments, a tri-specific anti-TNFa/IL-17A/IL-17F binding molécule of the invention competes for binding to human IL-17A and/or IL-17F with, or binds to the same epitope(s) of said antigen(s) as, a VHH comprising:
• SEQIDNOs: 1-3; or • SEQ ID NO: 4;
and competes for binding to human TNFa with, or binds to the same epitope(s) of said antigen(s) as, an antibody comprising:
• SEQIDNOs: 10-15;
• SEQ ID NOs: 8 and 9; or • SEQ ID NOs: 5 and 6.
In one embodiment, the tri-specific binding molécule comprises an anti-IL-17A/anti-IL17F VHH domain. In certain embodiments, the anti-IL-17A/anti-IL-17F VHH domain is humanized. In some embodiments, the anti-IL-17A/anti-IL-17F VHH domain comprises the heavy chain CDR1 (H-CDR1) amino acid sequence of SEQ ID NO: 1, the heavy chain CDR2 (H-CDR2) amino acid sequence of SEQ ID NO: 2, the heavy chain CDR3 (H-CDR3) amino acid sequence of SEQ ID NO: 3, or any combination thereof. In one embodiment, the VHH domain comprises the H-CDR1, H-CDR2, and H-CDR3 amino acid sequences of SEQ ID NOs: 1-3. In some embodiments, the VHH domain is at least 60% identical in sequence to SEQ ID NO: 4, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 4. In some embodiments, the VHH domain is at least 90% identical in sequence to SEQ ID NO: 4, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In a particular embodiment, the VHH domain comprises or consists of the amino acid sequence of SEQ ID NO: 4.
In one embodiment, the tri-specific binding molécule comprises an anti-human TNFa antibody or an antigen-binding portion thereof. In certain embodiments, the anti-human TNFalpha antibody is a humanized antibody or an antigen-binding portion thereof (e.g., a humanized Fab).
In some embodiments, the heavy chain of the anti-human TNFa antibody comprises the H-CDR1 amino acid sequence of SEQ ID NO: 10, the H-CDR2 amino acid sequence of SEQ ID NO: 11, the H-CDR3 amino acid sequence of SEQ ID NO: 12, or any combination thereof. In one embodiment, the heavy chain of the anti-human TNFa antibody comprises the H-CDR1, HCDR2, and H-CDR3 amino acid sequences of SEQ ID NOs: 10-12. In some embodiments, the heavy chain of the anti-human TNFa antibody has a heavy chain variable domain (VH) that is at least 60% identical in sequence to SEQ ID NO: 8, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 8. In some embodiments, the heavy chain of the anti-human TNFa antibody has a heavy chain variable domain (VH) that is at least 90% identical in sequence to SEQ ID NO: 8, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In a particular embodiment, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 8. The VH may be joined to a heavy chain constant domain (CH) of isotype IgG, an IgM, an IgE, an IgA, or an IgD molécule. In certain embodiments, the CH is of isotype IgG, e.g., of isotype subtype IgGl, IgG2a or b, IgG3 or IgG4. In a particular embodiment, the CH is of isotype subtype IgGl.
In some embodiments, the heavy chain of the anti-human TNFa antibody (HC) is at least 60% identical in sequence to SEQ ID NO: 5, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 5. In some embodiments, the heavy chain of the anti-human TNFa antibody (HC) is at least 90% identical in sequence to SEQ ID NO: 5, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to SEQ ID NO: 5. In a particular embodiment, the HC comprises or consists of the amino acid sequence of SEQ ID NO: 5.
In some embodiments, the light chain of the anti-human TNFa antibody comprises the light chain CDR1 (L-CDR1) amino acid sequence of SEQ ID NO: 13, the light chain CDR2 (LCDR2) amino acid sequence of SEQ ID NO: 14, the light chain CDR3 (L-CDR3) amino acid sequence of SEQ ID NO: 15, or any combination thereof. In one embodiment, the light chain of the anti-human TNFa antibody comprises the L-CDR1, L-CDR2, and L-CDR3 amino acid sequences of SEQ ID NOs: 13-15. In some embodiments, the light chain of the anti-human TNFa antibody has a light chain variable domain (VL) that is at least 60% identical in sequence to SEQ ID NO: 9, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 9. In some embodiments, the light chain of the anti-human TNFa antibody has a light chain variable domain (VL) that is at least 90% identical in sequence to SEQ ID NO: 9, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. In a particular embodiment, the VL comprises or consists of the amino acid sequence of SEQ ID NO: 9.
In some embodiments, the light chain of the anti-human TNFa antibody (LC) is at least 60% identical in sequence to SEQ ID NO: 6, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 6. In some embodiments, the light chain of the anti-human TNFa antibody (LC) is at least 90?/o identical in sequence to SEQ ID NO: 6, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. In a particular embodiment, the VL comprises or consists of the amino acid sequence of SEQ ID NO: 6.
In some embodiments, a tri-specific binding molécule of the invention comprises aa antiIL-17A/anti-IL-17F binding domain (e.g., a VHH domain) joined directly or via a linker to the heavy or light chain of the anti-TNFa binding domain (e.g., an antibody or an antigen-binding portion thereof). In one embodiment, the binding molécule comprises an anti-IL-17A/anti-IL17F VHH domain joined via a linker to the light chain of an anti-TNFa antibody to form a fusion molécule. In some embodiments, the linker is at least 60% identical in sequence to SEQ ID NO: 16, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 16. In some embodiments, the linker is at least 90% identical in sequence to SEQ ID NO: 16, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In a particular embodiment, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the fusion molécule is at least 60% identical in sequence to SEQ ID NO: 7, e.g., at least 60%, 70%, or 80% identical to SEQ ID NO: 7. In some embodiments, the fusion molécule is at least 90% identical in sequence to SEQ ID NO: 7, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In a particular embodiment, the fusion molécule comprises or consists of the amino acid sequence of SEQ ID NO: 7.
In some embodiments, the tri-specific binding molécule comprises an anti-TNFa binding domain comprising any one of the above-described heavy chains and any one of the abovedescribed light chains, or antigen-binding portions thereof. In certain embodiments, the trispecific binding molécule further comprises any one of the above-described anti-IL-17A/anti-IL17F binding domains. In a particular embodiment, the tri-specific binding molécule comprises any one of the above-described fusion molécules in combination with any of the above-described anti-TNFa binding domain heavy chains.
The class of a tri-specific binding molécule obtained by the methods described herein may be switchcd with another class or subclass. In one aspect of the invention, a nucleic acid molécule encoding VL or VH is isolated using methods well-known in the art such that it does not include nucleic acid sequences encoding CL or CH. The nucleic acid molécules encoding VL or VH then are operatively linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class of immunoglobulin molécule. This may be achieved using a vector or nucleic acid molécule that comprises a CL or CH chain, as described above. For example, a binding molécule that was originally IgM may be class switched to IgG. Further, the class switching may be used to couvert one IgG subclass to another, e.g., from IgGl to IgG2. An exemplary method for producing a binding molécule of the invention with a desired isotype comprises the steps of isolating a nucleic acid molécule encoding the heavy chain of a binding molécule and a nucleic acid molécule encoding the light chain of a binding molécule, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain with the constant domain of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and collecting the binding molécule with the desired isotype.
The tri-specific binding molécule of the invention can be an IgG, an IgM, an IgE, an IgA, or an IgD molécule, but is typically of the IgG isotype, e.g. of IgG subclass IgGl, IgG2a or b, IgG3 or IgG4. In one embodiment, the binding molécule is an antibody of the IgG subclass IgGl.
In one embodiment, the tri-specific binding molécule comprises an Fc région with at least one mutation. A number of different Fc mutations are known, where these mutations provide altered effector function. For example, in many cases it will be désirable to reduce or eliminate effector function, e.g. where ligand/receptor interactions are undesired or in the case of antibodydrug conjugates. Fc région amino acid positions that may be advantageous to mutate in order to reduce effector function include one or more of positions 228, 233, 234 and 235, where amino acid positions are numbered according to the Kabat numbering scheme. In some embodiments, the tri-specific binding molécule comprises an Fc région with one or more mutations that reduce ADCC or CDC in comparison to the same binding molécule without the mutation.
In certain embodiments, a tri-specific binding molécule of the invention may be part of a larger immunoadhesion molécule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molécules include use of the streptavidin core région to make a tetrameric scFv molécule (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molécules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include where one or more CDRs from an antibody are incorporated into a molécule either covalently or noncovalently to make it an immunoadhesin that specifically binds to an antigen of interest. In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incoiporated noncovalently.
In another embodiment, a fusion antibody or immunoadhesin may be made that comprises ail or a portion of a tri-specific binding molécule of the invention linked to another polypeptide. In certain embodiments, only the variable domains of the tri-specific binding molécule are linked to the polypeptide. In certain embodiments, a VH domain of a tri-specific binding molécule is linked to a first polypeptide, while a VL domain of the tri-specific binding molécule is linked to a second polypeptide that associâtes with the first polypeptide in a manner such that the VH and VL domains can interact with one another to form an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker such that the VH and VL domains can interact with one another (e.g., single-chain antibodies). The VH-linker-VL antibody is then linked to the polypeptide of interest. In addition, fusion antibodies can be created in which two (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a multi-specific antibody.
To create a single chain antibody (scFv), the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker. See, e.g., Bird étal., Science 242:423-426 (1988); Huston étal., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used; bivalent, if two VH and VL are used; or polyvalent, if more than two VH and VL are used.
In other embodiments, other modified antibodies may be prepared using tri-specific binding molecule-encoding nucleic acid molécules. For instance, “kappa bodies” (111 et al.,
Protein Eng. 10:949-57 (1997)), “minibodies” (Martin étal., EMBO J. 13:5303-9 (1994)), “diabodies” (Holliger étal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or “Janusins” (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standard molecular biological techniques following the teachings of the spécification.
A tri-specific binding molécule of the invention can be derivatized or linked to another molécule (e.g., another peptide or protein). In general, binding molécules (e.g., antibodies or antigen-binding portions thereof) are derivatized such that IL-17A, IL-17F, and/or TNFa binding is not affected adversely by the derivatization or labeling. Accordingly, the binding molécules of the invention may include both intact and modified forms of the tri-specific binding molécules described herein. For example, a binding molécule of the invention can be functionally linked (by Chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody, a détection agent, a pharmaceutical agent, and/or a protein or peptide that can médiate association of the binding molécule with another molécule (such as a streptavidin core région or a polyhistidine tag).
One type of derivatized binding molécule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.
A tri-specific binding molécule of the invention can also be derivatized with a Chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the binding molécule, e.g., to increase sérum half-life.
A tri-specific binding molécule according to the présent invention may also be labeled. As used herein, the ternis “label” or “labeled” refers to incorporation of another molécule in the binding molécule. In one embodiment, the label is a détectable marker, e.g., incorporation of a radiolabeled amino acid or attachaient to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimétrie methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, lllln, 1251, 1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, métal binding domains, epitope tags), magnetic agents, such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of varions lengths to reduce potential steric hindrance.
In certain embodiments, the tri-specific binding molécules of the invention may be présent in a neutral form (including zwitter ionic forms) or as a positively or negatively-charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable sait.
The term “pharmaceutically acceptable sait” refers to a complex comprising one or more tri-specific binding molécules and one or more counterions, wherein the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.
Pharmaceutically acceptable inorganic bases include metallic ions including, but are not limited to, appropriate alkali métal salts, alkaline earth métal salts and other physiological acceptable métal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium, manganèse, chromium, sélénium, tin, copper, ferrie, ferrous, lithium, magnésium, manganic or manganous salts, potassium, rubidium, sodium, and zinc, e.g., in their usual valences.
Pharmaceutically acceptable acid addition salts of the tri-specific binding molécules of the présent invention can be prepared from the following acids, including, without limitation, formic, acetic, acetamidobenzoic, adipic, ascorbic, boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic, malonic, edetic, ethylsulfuric, fendizoic, metaphosphoric, succinic, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric, ascorbic, glucuronic, maleic, folie, fumaric, propionic, pyruvic, aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic, lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic, mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic, aminosalicylic, silicate, phydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic, ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic, pantothenic, naphthalenesulfonic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuric acid monomethyl ester, cyclohexylaminosulfonic, β-hydroxybutyric, glycine, glycylglycine, glutamic, cacodylate, diaminohexanoic, camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal 5-phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-Laspartic, galactaric and galacturonic acids.
Pharmaceutically acceptable organic bases include trimethylamine, diethylamine, N, N'5 dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diéthanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, cyclic amines, quatemary ammonium cations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine, butylamine, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine, 10 glucosamine, histidine, hydrabamine, imidazole, isopropylamine, methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine, triethanolamine, tromethamine, methylamine, taurine, mholate,' 6-ammo-2-inethyl-2-heptanol, 2-amino-2-methyl-l,3-propanediol, 2-amino-2ί methyl-Upropanol, aliphatic mono- and dicarboxylic acids, phenyi-substituted alkanoic acids, 15 hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine, phenylcyclohexylamine, 2-(N-morpholino)ethanesulfonic acid, bis(2hydroxyethyl)amino-tris(hydiOxymethyl)methane, N-(2-acetamido)-2-aminoethanesulfonic acid, 1,4-piperazinediethanssulfonic acid, 3-morphoIino-2-hydroxypropanesulfonic acid, 1,3bis[tris(hydroxymethyl)methylamino]propane, 4-morpholinepropanesulfonic acid, 4-(220 hydroxyethyl)piperazine-1 -ethanesulfonic acid, 2- [(2-hydroxy-1,1 -bis(hydroxymethyl)ethyl) amino]ethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 4-(Nmorpholino)butanesulfonic acid, 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid,/ 2hydroxy-3-[tris(hydroxymethyl)methylamino]-l-propanesulfbnic acid, 4-(2hydroxyethyl)piperazine-l-(2-liydroxypropanesulfbnic acid), piperazine- l,4-bis(225 hydroxypropanesulfonic acid) dihydrate, 4-(2-hydroxyethyl)-l-piperazinepropanesulfonic acid,
N,N-bis(2-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N[tris(hydroxymethyl)m ethyl] -3 -aminopropanesulfonic acid, N-tri s(Hydroxymethyl)methyl-4aminobutanesulfonic acid, N-( 1,1 -dimethyl-2-hydroxyethyl)-3 -amino-2-hydroxypropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-l30 propanesulfonic acid, 3 -(cyclohexylamino)-l -propanesulfonic acid, N-(2-acetamido) iminodiacetic acid, 4-(cyclohexylamino)-l-butanesulfonic acid, N-[tris(hydroxymethyl)methyl] glycine, 2-amino-2-(hydroxymethyl)-l,3-propanediol, and trometamol.
In sonie embodiments, bispecific antibody molécules of the présent disclosure comprise an anti-TNFa antibody comprising two heavy chains and two light chains, or an antigen-binding 35 portion thereof, and an anti-IL-17A/anti-IL-17F VHH connected (directly or through a linker) to the antibody. The anti-IL-l7A/anti-IL-17F VHH may be connected by its N-terminus or Cterminus to the N-terminus or C-terminus of the heavy or light chain of the anti-TNFa antibody. In certain embodiments, the anti-IL-17A/anti-IL-17F VHH is connected to the N-terminus of the light chain ofthe anti-TNFa antibody. Any such fusion described herein may be made according to methods known in the art. See, e.g., Ahmad et al., Clinical and Developmental Immunology 2012, Article ID 980250 (2012). In certain embodiments, the anti-TNFa antibody is joined to the anti-IL-17A/anti-IL-17F VHH by a linker, more preferably by a peptide linker, and most preferably by a peptide linker that lacks a proteolytic cleavage site. In some embodiments, the amino acid residues of the linker are selected from G, A, S, P, E, T, D, and K. In some
10. embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 16. Nucleic Acid Molécules and Vectors
The présent invention also provides nucleic acid molécules and sequences encoding tri- • spécifie binding molécules ofthe invention described herein. In some embôdifnents; different r nucleic acid molécules encode the fïrst domain and second domain amino acid sequences ofthe tri-specific binding molécule. Where the first domain and/or the second domain comprises a heavy chain and a light chain, in some embodiments, different nucleic acids encode the heavy chain and the light chain amino acid sequences. In other embodiments, the same nucleic· acid moiecule encodes the heavy chain and light chain amino acid sequences. In certain embodiments, a nucleic acid moiecule may encode any combination of the amino acid sequences (e.g., heavy and light chain sequences) of the first and second domains. In a particular embodiment, a nucleic acid moiecule may encode the amino acid sequence of the first binding • domain and the light chain amino acid sequence of the second binding domain, optionally . inchiding any peptide linker sequence joining the two.
A reference to a nucléotide sequence encompasses its complément unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term “polynucleotide” as referred to herein means a polymeric form of nucléotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucléotide. The term includes single and double stranded forms.
· The invention also provides nucléotide sequences that are at least 70%, 75%, 80%, 85%,
90%, 95%, 97%, 98% or 99% identical to one or more of the above-recited nucléotide sequences or to a nucléotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-16. In certain embodiments, the nucléotide sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucléotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-9. The term “percent η 19281 sequence identity” in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucléotides, usually at least about 18 nucléotides, more usually at least about 24 nucléotides, typically at least about 28 5 nucléotides, more typically at least about 32 nucléotides, and preferably at least about 36, 48 or more nucléotides. There are a number of different algorithms known in the art which can be used to measure nucléotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, e.g., the 10 programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the régions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol· Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227'.-258 (1996); Pearson, J. Mol. Biol. .276:71-84 (1998); incorporated herein by référencé). ‘Uniess otherwise specified, default parameters for a particular program or algorithm are used.
For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, incorporated herein by référencé.
In one aspect, the invention provides a nucleic acid molécule comprising a nucléotide 20 sequence that encodes an amino acid sequence selected from SEQ ID NOs: 1-16. The nucleic acid molécule may also comprise any combination of said nucléotide sequences. In one embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes SEQ ID NO: 4. In another embodiment, the nucleic acid molécule comprises a nucléotide sequence thatencodcs SEQ ID NOs: 4 and 6. In another embodiment, the nucleic acid molécule comprises a 25 nucléotide sequence that encodes SEQ ID NOs: 4, 6, and 16. In another embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes SEQ ID NO: 7. In another embodiment, the nucleic acid molécule comprises a nucléotide sequence that encodes SEQ ID NO: 5.
In any of the above embodiments, the nucleic acid molécules may be isolated.
In a further aspect, the présent invention provides a vector suitable for expressing any of the nucléotide sequences described herein. The term “vector”, as used herein, means a nucleic acid molécule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circulai' double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, 35 wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous réplication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of réplication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along 5 with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
The invention provides vectors comprising nucleic acid molécules that encode any of the amino acid sequences of tri-specific binding molécules or portions thereof (e.g., heavy and/or 10 light chain sequences of the first and/or second binding domains) as described herein. The invention further provides vectors comprising nucleic acid molécules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.
... .. A.nucleic acid molécule of the invention can . be isolated from any source that produces a tri-specific binding molécule or a portion thereof. In certain embodiments, a nucleic acid 15 moiccuie of the invention can be synthesized rather than isolated.
In some embodiments, a nucleic acid molécule of the. invention can comprise a nucléotide sequence encoding a VH domain from the first or second domain of a tri-specific binding molécule of the invention joined in-frame to a nucieotide sequence encoding a heavy chain : constant domain from any source. Similarly, a nucleic acid molécule of the invention can 20 comprise a nucieotide sequence encoding a VL domain from the first or second domain of a trispecific binding molécule of the invention joined in-frame to a nucieotide sequence encoding a light chain constant domain from any source.
In a further aspect of the invention, nucleic acid molécules encoding the variable domain of the heavy (VH) and/or light (VL) chains of the first or second binding domain may be 25 “converted” to full-length antibody genes. In one embodiment, nucleic acid molécules encoding the VH or VL domains are converted to full-length antibody genes by insertion into an expression vector already encoding heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operatively linked to the CH segment(s) whhin the vector, and/or the VL segment is operatively linked to the CL segment within the 30 vector. In another embodiment, nucleic acid molécules encoding the VH and/or VL domains are converted into full-length antibody genes by linking, e.g., ligating, a nucleic acid molécule encoding a VH and/or VL domains to a nucleic acid molécule encoding a CH and/or CL domain using standard molecular biological techniques. Nucleic acid molécules encoding the full-length heavy and/or light chains may then be expressed from a cell into which they hâve been 35 introduced.
The nucleic acid molécules may be used to récombinantly express large quantities of trispecific binding molécules. The nucleic acid molécules also may be-used to produce human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody dérivatives, as 5 described herein.
In another embodiment, a nucleic acid molécule of the invention is used as a probe or PCR primer for a spécifie amino acid sequence. For instance, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify régions of DNA that could be used, e.g., to isolate additional nucleic acid molécules encoding portions (e.g., variable domains) of tri10 spécifie binding molécules. In some embodiments, the nucleic acid molécules are oligonucleotides. In some embodiments, the oligonucleotides are from highly variable domains . of the tri-specific binding molécule. In some embodiments, the oligonucleotides encode ail or a . > partpf one or· .more of the · CDRs of the tri-specific binding molécule of the invention as described herein.
In another embodiment, the nucleic acid molécules and vectors may be used· to make : mutated tri-specific binding molécules. The antibodies may be mutated in thé variable domains of the heavy and/or light chains of the first and/or second binding domain, e.g., to alter a binding property of the tri-specific binding molécule. For example, a mutation may be made in one or more of the CDR régions to increase or decrease the KD of the tri-specific binding molécule, to 20 increase or decrease kog·, or to alter the binding specificity of the antibody, with regard to L-17A, IL-17F, and/or TNFa. In another embodiment, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in an antibody corresponding to the first or second binding domain of the tri-specific binding molécule of the invention. The mutations may be made in a CDR région or framework région of a variable domain, or in a constant domain. In a preferred embodiment, the mutations are made in a variable domain. In some embodiments, one or more mutations are made at an amino acid residue that is known to be changed compared to the germline in a CDR région or framework région of a variable domain of a tri-specific binding molécule of the invention.
In another embodiment, the framework region(s) are mutated so that the resulting 30 framework region(s) hâve the amino acid sequence of the corresponding germline gene. A mutation may be made in a framework région or constant domain to increase the half-life of the tri-specific binding molécule. See, e.g., WO 00/09560. A mutation in a framework région or constant domain also can be made to alter the immunogenicity of the tri-specific· binding molécule, and/or to provide a site for covalent or non-covalent binding to another molécule.
According to the invention, a tri-specific binding molécule may hâve mutations in any one or more ofthe CDRs or framework régions of a variable! domain or in a constant domain.
In some embodiments, the tri-specific binding molécules of the invention are expressed by inserting DNAs encoding partial or full-length sequences of the first and second binding 5 domains (e.g., heavy and light chain sequences where the binding domain comprises a heavy and light chain sequence), obtained as described above, into expression vectors such that the genes are operatively linked to necessary expression control sequences such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic 10 viras, cosmids, YACs, EBV derived episomes, and the like. The DNAs may be ligated into a vector such that transcriptional and translational control sequences. within the vector serve their intended function of regulating the transcription and translation of the DNAs. The expression ..... vector and.expression controlsequences may be chosen to be compatible with the expression host cell used. DNAs encoding partial or full-length sequences of the first and second binding 15 domains (e.g., heavy and light chain sequences where the binding domain comprises a heavy and light chain sequence) can be inserted into separate vectors can be inserted into separate vectors. In one embodiment, any combination of the above DNAs is inserted into the same expression vector. The DNAs may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end 20 ligation if no restriction sites are présent).
A convenient vector is one that encodes a functionally complété human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can easily be inserted and expressed, as described above. The HC- and LC-encoding genes in such vectors may contain intron sequences that will resuit in enhanced overall antibody 25 protein yields by stabilizing the related mRNA. The intron sequences are flanked by splice donor and splice acceptor sites, which détermine where RNA splicing will occur. Location of intron sequences can be either in variable or constant régions of the antibody chains, or in both variable and constant régions when multiple introns are used. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding régions. The 30 recombinant expression vector also can encode a signal peptide that facilitâtes sécrétion of an antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal pèptide (i.e., a signal peptide from a non-immunoglobulin protein).
3i 19281
In addition to the antibody chain genes, the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the ait that the design of the expression vector, including the sélection of regulatory sequences, may dépend on such factors as the choice 5 of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral éléments that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived Irom retroviral LTRs, cytomégalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major 10 late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters. For further description of viral regulatory éléments, and sequences .thereof, see e.g., US Patents 5,168,062, 4,510,245 and 4,968,615. Methods for • - e^pressing binding molécules such as antibodies in plants, including a description of promoters and vectors, as well as transformation of plants, are known in the art. See, e.g., US Patent 15 ' 6,517,529. Methods of expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells, are also well known in the art.
In addition to the antibody chain genes and regulatory sequences, the recombinant expression .vectors of the invention may carry additional sequences, such as sequences that regulate réplication of the vector in host cells (e.g., origins of réplication) and selectable marker 20 genes. The selectable marker gene facilitâtes sélection of host cells into which the vector has been introduced (see e.g., US Patents 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers résistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with 25 methotrexate selection/amplification), the neo gene (for G418 sélection), and the glutamate synthetase gene.
The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, 30 promoter and enhancer sequences; efficient RNA processing signais such as splicing and polyadenylation signais; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein sécrétion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences 35 generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, ail components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner 5 sequences.
Host Cells and Methods of Tri-Specific Binding Molécule Production
An additional aspect of the invention relates to methods for producing the tri-specific binding molécules of the invention. One embodiment of this aspect of the invention relates to a method for producing a tri-specific binding molécule as defmed herein,· comprising providing a 10 recombinant host cell capable of expressing the tri-specific binding molécule, cultivating said host cell under conditions suitable for expression of the tri-specific binding molécule, and isolating the resulting tri-specific binding molécule. Tri-specific binding molécules produced by .
•. such expression in such recombinant host cells are referrcd to herein as “recômbinant tri-specific > binding molécules.” Where the tri-specific binding molécules are antibodies, they are referred to 15 as “recômbinant antibodies.” The invention also provides progeny cells of such host cells, and tri-specific binding molécules produced by same.
The term “recômbinant host cell” (or simply “host cell”), as used herein, means a cell into which a recombinant expression vector has been introduced. The invention provides host cells that may comprise, e.g., a vector according to the invention described above. The invention also 20 provides host cells that comprise, e.g., a nucléotide sequence encoding the heavy chain or an antigen-binding portion thereof, a nucléotide sequence encoding the light chain or an antigenbinding portion thereof, or both, of the first binding domain and/or the second binding domain of a tri-specific binding molécule of the invention. It should be understood that “recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell.
Because certain modifications may occur in succeeding générations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
Nucleic acid molécules encoding tri-specific binding molécules of the invention and vectors comprising these nucleic acid molécules can be used for transfection of a suitable 30 mammalian, plant, bacterial or yeast host cell. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate précipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of 35 the DNA into nuclei. In addition, nucleic acid molécules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., US Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods of 5 transforming bacterial and yeast cells are also well known in the art.
Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell Unes available from the American Type Culture Collection (ATCC). These include, inter alla, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster 10 kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines hâve high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells.’ When recombinant expression vectors encoding tri-specific binding molécules are introduced into mammalian host 15 cells, the binding molécules are produced by culturing the host cells for a period of time sufficicnt to allow for expression of the binding molécule in the host cells or, more preferably, sécrétion of the binding molécule into the culture medium in which the host cells are grown. Tri-specific binding molécules can be recovered from the culture medium using standard protein purification methods. Plant host cells include, e.g., Nicotiana, Arabidopsis, duckweed, corn, 20 wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.
Further, expression of tri-specific binding molécules of the invention from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression System (the GS System) is a common approach for enhancing 25 expression under certain conditions. The GS System is discussed in whole or part in connection with EP patents 0 216 846, 0 256 055, 0 323 997 and 0 338 841.
It is likely that tri-specific binding molécules expressed by different cell lines or in transgenic animais will hâve different glycosylation patterns from each other. However, ail trispecific binding molécules encoded by the nucleic acid molécules provided herein, or 30 comprising rhe amino acid sequences provided herein, are part of the instant invention, regardless of the glycosylation State of the binding molécules, and more generally, regardless of the presence or absence of post-translational modification(s).
The tri-specific binding molécules can be prepared using a variety of methods. For example, the domains binding to IL-17A, IL-17F, and/or TNFa, or any combination thereof, can 35 be prepared separately (e.g. using Chemical protein synthesis, recombinant expression methods, hybridoma technology, etc.) and then chemically attached to each other, either directly or through a linker. Means of chemically conjugating molécules (e.g., antibodies or antigenbinding portions thereof) are well known to those of skill in the art. Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH) or free amine (— NH2) 5 groups, that are available for reaction with suitable functional groups on the corresponding polypeptide or on a linker. The antibodies can also be derivatized to expose or attach additional réactivé functional groups, and can involve attachment of one or more linker molécules such as those available from Pierce Chemical Company, Rockford 111. The linkers used in the trispecific binding molécules of the invention may be any of a variety of suitable linkers known in 10 the art.
hi certain embodiments of the invention, the domains binding to IL-17A, IL-17F, and/or TNFa, or any combination thereof, are produced by expression of recombinant antibodies or antigen-binding portions in host cells. The sequences encoding any combination of the binding domains can be connected (directly or through a linker). The resulting nucleic acid molécules 15 encoding the domains binding to IL-17A, IL- 17 F, and TNFa are inserted into expression vectors and introduced into host cells. The resulting tri-specific binding molécules are then expressed, isolated and purified from the expression system.
In certain embodiments of the invention, binding domains of the tri-specific binding molécule can be paired together with a novel linker molécule designed to protect against 20 proteolytic dégradation of the binding molécule. Such a linker typically lacks a proteolytic cleavage site.
Pharmaceutical Compositions
Another aspect of the invention is a pharmaceutical composition comprising as an active ingrédient (or as the sole active ingrédient) a tri-specific binding molécule of the invention. The 25 pharmaceutical composition may comprise any tri-specific binding molécule as described herein. In some embodiments, the compositions are intended for amelioration, prévention, and/or treatment of a disorder that can be affected by activity of IL-17A, IL-17F, TNFa, and/or an autoimmune or inflammatory disease.
Generally, the tri-specific binding molécules of the invention are suitable to be 30 administered as a formulation in association with one or more pharmaceutically acceptable excipient(s) e.g. as described below.
Pharmaceutical compositions of the invention may comprise at least one tri-specific binding molécule, and one or more additional binding molécules (e.g., antibodies) that target one or more relevant cell surface receptors, e.g., a cell surface receptor involved in an autoimmune or 35 inflammatory response.
The term “excipient” is used herein te describe any ingrédient other than the compound(s) of the invention. The choice of excipient will to a large extent dépend on factors . such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable 5 excipient” includes any and ail solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonie and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol,. éthanol and the like, as well as combinations thereof. In many cases, it ' will be préférable to include isotonie agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
· Pharmaceutical compositions of the présent invention and methods foi their préparation will be readily apparent to those skilled in the art. Such compositions and methods for their préparation may be found, for example, in Remingion’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably manufactured under GMP (good manufacturing practices) conditions.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in 20 bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrète amount of the pharmaceutical composition comprising a predetermined amount of the active ingrédient. The amount of the active ingrédient is generally equal to the dosage of the active ingrédient which would be administered to a subjcct or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for the tri-specific binding molécule of the invention.
The pharmaceutical compositions of the invention are typically suitable for parentéral administration. As used herein, “parentéral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject 30 and administration of the pharmaceutical composition through the breach in the tissue,. thus generally resulting in the direct administration into the blood stream, into muscle, or into an internai organ. Parentéral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non35 surgical wound, and the like. In particular, parentéral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. Intratumoral delivery, e.g; intratumoral injection, may also be advantageous. Régional perfusion is also contemplated. Preferred 5 embodiments include the intravenous and the subcutaneous routes. .
Formulations of a pharmaceutical composition suitable for parentéral administration typically comprise the active ingrédient combined with a pharmaceutically acceptable carrier, such as stérile water or stérile isotonie saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuons administration. Injectable 10 formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for patenterai administration include, but are not limited to, suspensions, solutions, émulsions in oily or aqüeous vehicles, .· pastes, and the like. Such formulations may further comprise one or more additional ingrédients - including, büt not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of 15 a formulation for parentéral administration, the active ingrédient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., stérile pyrogen-free water) prior to parentéral administration of the reconstituted composition. Parentéral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pFI of from 3 to 9), but, for some applications, they may be 20 more suitably formulated as a stérile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as stérile, pyrogen-free water. Exemplary parentéral administration forms include solutions or suspensions in stérile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which 25 comprise the active ingrédient in microcrystalline form, or in a liposomal préparation.
Formulations for parentéral administration may be formulated to be immédiate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
For example, in one aspect, stérile injectable solutions can be prepared by incorporating 30 the tri-specific binding molécule in the required amount in an appropriate solvent with one or a combination of ingrédients enumerated above, as required, foliowed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a stérile vehicle that contains a basic dispersion medium and the required other ingrédients from those enumerated above. In the case of stérile powders for the préparation of stérile injectable 35 solutions, the preferred methods of préparation are vacuum drying and freeze-drying that yields a powder of the active ingrédient plus any additional desired ingrédient from a previously sterilefïltered solution thereof. The proper fluidity of a solution can be maintained, for example, by the . use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be 5 brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin, and/or by using modified-release coatings (e.g., slow-release coatings).
The tri-specific binding molécules ofthe invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, or as a mixed 10 component particle, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aérosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamies to produce a fine mist), or • j nebuliser, with or v/ithout the use of a suitable propeliant, or as nasal drops.
The pressurised container, pump, spray, ammizer, or nebuliser generally contains a 15 solution or suspension of a binding molécule ofthe invention comprising, for example, a suitable agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent.
Prior to use in a dry powder or suspension formulation, the drug product is generally micronised to a size suitable for delivery by inhalation (typically less than-5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet 20 milling, supercritical fluid processing to form nanoparticles, high pressure homogénisation, or spray drying.
Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a: suitable powder ' base and a performance modifier.
A suitable solution formulation for use in an atomiser using electrohydrodynamies to produce a fine mist may contain a suitable dose of the tri-specific binding molécule of the invention per actuation and the actuation volume may for example vary from 1 pL to 100 pL.
Suitable fiavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for 30 inhaled/intranasal administration.
Formulations for inhaled/intranasal administration may be formulated to be immédiate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
In the case of dry powder inhalers and aérosols, the dosage unit is determined by means 35 of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or “puff” of a binding molécule of the invention. The overall daily dose will typically be administered in a single dose or, more usually, as divided doses throughout the day.
The tri-specific binding molécules of the invention may also be formulated for an oral 5 route administration. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.
. Formulations suitable for oral administration include solid, semi-solid and liquid Systems such as tablets; soft or hard capsules containing muiti- or nano-particulates, liquids, or powders; 10 lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
Liquid formulations include suspensions, solutions, syrups and élixirs. Such - formulations may be employed as fillers in soft or hard capsules (made, for· example, from vgelatin or hydroxypropylmethylceilulose) and typically comprise a carrier, for example, water, 15 éthanol, polyethyiene glycol, propylene glycol, methylceilulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
In one embodiment, the tri-specific binding molécules of the invention are formulated in a composition comprising histidine and acetate at a concentration of 1 to 100 mM, in a buffer 20 with pH 5.0 to 6.5.
Therapeutic uses of tri-specific binding molécules of the invention
In one aspect, the tri-specific binding molécules of the invention are used in the treatment of a disorder that can be affected by activity of IL-17A, IL-17F, and/or TNFa. For example, a physician can treat an autoimmune or inflammatory disorder by administering a tri-specific 25 binding molécule of the présent invention, alone or in combination with other therapeutic agents (sequentially or concurrently). The tri-specific binding molécule modulâtes the activity of IL17A, IL-17F, and/or TNFa, resulting in réduction of an autoimmune or inflammatory response. Disorders that can be treated by the tri-specific binding molécules and methods of the présent invention may include any of the following: rheumatoid arthritis, osteoarthritis, juvénile chrome 30 arthritis, septic arthritis, Lyme osteoarthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dépendent diabètes mellitus, thyroiditis, asthma, allergie diseases, psoriasis, plaque psoriasis, atopie dermatitis, scleroderma, reaction graft versus host organ transplant rejection, acute or chronic immune disease associated with organ 35 transplantation, sarcoidosis, Kawasaki disease, Graves disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopie rénal vasculitis, chronic active hepatitis, uvenita, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, 5 hemolytic anémia, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, séronégative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy associated with ulcerative colitis arthropathy, atopie allergy, autoimmune bullous diseases, pemphigus vulgaris, sheet-like pemphigus, pemphigoid disease, linear IgA, an autoimmune hemolytic anémia, Coombs-positive hemolytic anémia, acquired pemicious anémia, juvénile pemicious anémia, 10 arthritis, primary sclerosing hepatitis A, cryptogenic autoimmune hepatitis, fîbrosis lung disease, cryptogenic fîbrosis alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, chronic éosinophilie pneumonia, post-infections interstitial lung disease, gouty arthritis, autoimmune hepatitis, autoimmune hepatitis type I (classical autoimmune hepatitis or lupôid), autoimmune hepatitis type II Osteoarthritis, primary sclerosing cholangitis, psoriasis type 15 1, type II psoriasis, idiopathic leucopenia, autoimmune neutropenia, rénal NOS-disease, glomerulonephritis, microscopie rénal vasculitis, discoid lupus erythematosus, idiopathic or NOS-male infertility, autoimmunity to sperm, multiple sclerosis (ail subtypes), sympathetic ophthahnia, pulmonary hypertension secondary to connective tissue disease, syndrome Gôodpasture, pulmonary manifestations of polyarthritis nodosa, acute rheumatic fever, 20 rheumatoid spondylitis, ankylosing spondylitis, Still's disease, systemic sclerosis, syndrome
Shengrena, Sjôgren's syndrome, Takayasu's arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopcnia, autoimmune thyroid disease, hyperthyroidism, goiter autoimmune hypothyroidism (Hashimoto's disease), autoimmune atrophie hypothyroidism, primary myxedema, phacogenic uveitis, primary vasculitis, vitiligo, acute liver disease, chronic liver 25 disease, allergies, asthma, psychiatrie disorders (including dépréssion and schizophrenia), mediated type Th2 and Thl type diseases, conjunctivitis, allergie contact dermatitis, allergie rhinitis, a deficiency of alpha- 1-antitrypsin, amyotrophie latéral sclerosis, anémia, cystic fîbrosis, associated with cytokine therapy disorders, demyelinating disease, dermatitis, iridocyclitis / uveitis / optic neuritis, damage in ischemia-reperfusion injury, ischémie stroke, juvénile 30 rheumatoid arthritis, autoimmune enteropathy, autoimmune hearing loss, autoimmune lymphoproliférative syndrome, autoimmune myocarditis, autoimmune prématuré ovarian failure, and blepharitis. The tri-specific binding moiecule may also treat any combination of the above disorders.
In certain embodiments, a tri-specific binding moiecule of the invention is used to treat a 35 disorder selected from (but not limited to) rheumatoid arthritis (RA), osteoarthritis, rheumatoid arthritis osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis and cirrhosis), gingivitis, periodontosis or periodontal diseases, inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis and inflammatory bowel disease), asthma (including allergie asthma), allergies, chrome obstructive pulmonary disease (COPD), multiple sclerosis, psoriasis, 5 and cancer.
In a particular embodiment, a tri-specific binding molécule of the invention is used to treat rheumatoid arthritis, psoriasis, or psoriatic arthritis.
“Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a 10 disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment.
- ’·' In one aspect, the subject of· treatment, or patient, is a mammal, preferably· a human .
subject. Said subject may be either maie or female, of any âge.
“Therapeuticaily effective amount” refers to that amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
The tri-specific binding molécules of the invention may be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). The 20 pharmaceutical compositions, methods and uses of the invention thus also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.
As used herein, the terrns “co-administration”, “co-administered” and “in combination with,” referring to the tri-specific binding molécules with one or more other therapeutic agents, is intended to mean, and does refer to and include the following:
e simultaneous administration of such combination of tri-specific binding molécule of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are fonnulated together into a single dosage form which releases said components at substantially the same time to said patient, • substantially simultaneous administration of such combination of tri-specific 30 binding molécule of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient, ® sequential administration of such combination of tri-specific binding molécule of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time 5 interval between each administration, whereupon said components are released at substantially different times to said patient; and « sequential administration of such combination of tri-specific binding molécule of the invention and therapeutic agent(s) to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said 10 components in a controlled manner whereupon they are concurrently, consecutively, and/or overlappingly released at the same and/or different times to said patient, where each part may be administered by either the same or · a different route.
' The tri-specific binding molécules of the invention may be administered without 15 additional therapeutic treatments, i.e., as a stand-alone therapy. Alternatively, treatment with the tri-specific binding molécules of the invention may include at least one additional therapeutic * treatment (combination therapy). In some embodiments, the tri-specific binding molécule may be co-administered or formulated with another medication/drug for the treatment of an autoimmune or inflammatory disorder.
In some embodiments, a tri-specific binding molécule of the invention is administered in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the tri-specific binding molécules of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, méthylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti25 inflammatory drugs (e.g., balsalazide, celecoxib, aiclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin. ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, olsalazine, oxaprozin, phenylbutazone, piroxicam, salsalate. sulindac, tenoxicam, tiaprofenic acid, and tolmetin), as well as acetaminophen, antihistamines, aminoarylcarboxyhc acid dérivatives, arylacetic acid dérivatives, arylbutyric acid 30 dérivatives, arylcarboxylic acids, arylpropionic acid dérivatives, pyrazoles, pyrazolones, salicylic acid dérivatives (e.g. sulfasalazone and mesalamine), thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-ammo-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and 35 tenidap.
4? 19281
In some embodiments, a tri-specific binding molécule of the invention is administered in combination with an immunosuppressive agent such as, but not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, natalizumab, and other immunosuppressive agents that act by suppressing 5 the function of responding T cells.
In some embodiments, a tri-specific binding molécule of the invention is administered in combination with an immunosuppressant such as, but not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ .(tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, AVONEX™ 10 (interferon-beta IA), and RAPAMUNE™ (sirolimus). In one embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In some embodiments, a tri-specific binding molécule of the invention is administered in combination with a TNF antagonist. TNF antagonists that may be administered with the binding molécules of the invention include, but are net limited to, infliximab (REMICADE™), 15 adalimumab (HUMIRA™), certolizumab pegol (CIMZIA™), golimumab (SIMPONI™), etanercept (ENBREL™), xanthine dérivatives (e.g. pentoxifyline) and bupropion (WELLBURTIN™, ZYBAN™). In certain embodiments, the TNF antagonist is a TNFa antagonist.
In some embodiments, a tri-specific binding molécule of the invention is administered in 20 combination with an antagonist from the list including, but not limited to, IL-6R antagonists (e.g., tocilizumab and serulumab), IL-6 antagonists, IL-la or IL-lb antagonists, IL-2o antagonists, IL-22 antagonists, and GM-CSF antagonists.
In some embodiments, a tri-specific binding molécule of the invention is administered in combination with an IL-17A and/or IL-17F antagonist. In certain embodiments, said antagonist 25 is an anti-IL-17A and/or anti-IL-17F antibody or an antigen-binding portion thereof.
Pharmaceutical articles comprising a tri-specific binding molécule of the invention and at least one other agent (e.g., an immunosuppressive or anti-inflammatory agent) may be used as a combination treatment for simultaneous, separate or successive administration in treatment of an autoimmune or inflammatory disorder.
It is understood that the tri-specific binding molécules of the invention may be used in a method of treatment as described above, may be for use in a treatment as described above, and/or may be for use in the manufacture of a médicament for a treatment as described above.
Dose and Route of Administration
The tri-specific binding molécules of the invention will be administered in an effective 35 amount for treatment of the condition in question, Le., at dosages and for periods of time necessary to achieve a desired resuit. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the âge, sex and weight of the patient, and wbether the tri-specific binding molécules are being administered as a stand-alone treatment or in combination with one or more additional anti-autoimmune or anti-inflammatory treatments.
Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or.the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parentéral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrète units suited as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The spécification for the dosage unit fomis of the invention are generally dictated by and directly dépendent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inhérent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a détectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a détectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the présent invention.
It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, spécifie dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration ofthe compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the embodied composition. Further, the dosage regimen with the compositions of this invention may be based on a variety of factors, including the type of disease, the âge, weight, sex, medical condition of the patient, the severity ofthe condition, the route of administration, and the particular tri-specific binding molécule employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamie parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the présent invention encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
Examples of suitable routes of administration are provided above.
It is contemplated that a suitable dose of a tri-specific binding molécule of the invention will be in the range of 0.1-100 mg/kg, such as about 0.5-50 mg/kg, e.g., about 1-20 mg/kg. The tri-specific binding molécule may for example be administered in a dosage of at least 0.25 mg/kg, e.g., at least 0.5 mg/kg, such as at least 1 mg/kg, e.g., at least 1.5 mg/kg, such as at least 2 mg/kg, e.g., at least 3 mg/kg, such as at least 4 mg/kg, e.g., at least 5 mg/kg; and e.g., up to at most 50 mg/kg, such as up to at the most 30 mg/kg, e.g., up to at the most 20 mg/kg, such as up to at the most 15 mg/kg. Administration will normally be repeated at suitable intervals, e.g., once every week, once every two weeks, once every three weeks, or once every four weeks, and for as long as deemed appropriate by the responsable doctor, who may optionally increase or decrease the dosage as necessary.
An effective amount for autoimmune or inflammatory disorder therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression. The ability of a tri-specific binding molécule of the invention to inhibit an autoimmune or inflammatory disorder may be evaluated by in vitro assays, e.g., as described in the examples, as well as in suitable animal models that are prédictive of the efficacy in such disorders. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a • single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.
Diagnostic Uses and Compositions
The tri-specific binding molécules of the présent invention also are useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the tri-specific binding molécules can be used to detect and/or measure the level of IL-17A, IL-17F, and/or TNFa m a sample from a patient (e.g., a tissue sample, or a body fluid sample such as an inflammatory exudate, blood, sérum, bowel fluid, saliva, or urine). Suitable détection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassay, and immunohistology. The invention further encompasses kits (e.g., diagnostic kits) comprising the tri-specific binding molécules described herein.
In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
Ail publications, patents, and patent applications cited in this spécification are 5 incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.
Examples , v Example 1; Création and optimizatiôn-of tri-specific anti-IL-17A/anti-IL-17F/anti-TNF« antibodies
Trispecific anti-IL-UAfrmti-IL-WF/anti-TNFa antibodies were created and optimized as shown in Fig. 1.
Example 2: Production of recombinant antigens and antibodies in suspension culture of mammalian cells
Antibodies and antigens were produced in CHO-K1 cells constitutively expressing
EBNA 1 (Epstein-Barr virus nuclear antigen 1), according to published protocols (Longo et al., Methods Enzymol 529:227-240 (2013)). The cells were cultivated in suspension on orbital shakers using a sérum-free media produced by Life Technologies Corporation, according to the manufacturer's instructions. For transient expression, cells were transfected at a concentration of 25 2 x 10E6 / ml with a linear polyethyleneimine (PEI MAX, Polysciences). The DNA/PEI ratio was 1:3. 5-7 Days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a 0.22 pm filter. The target proteins were extracted from the cultuie fluid by affinity chromatography.
Recombinant protein IL-17A containing six amino acids at the C-terminus ofthe protein 30 and FLAG-peptide (Fig. 2) was extracted and purified from the culture fluid using Profinity IMAC Ni-charged resin (Bio-Pvad Company). Prior to purification, NiCl2 was added to the culture fluid to a concentration of 1 mM. 5 ml of Profinity IMAC Ni-charged resin were then added to the culture fluid and stirred on a shaker for 1 h at room température. The sorbent (5 ml) was transferred to a Thermo Scientific polypropylene column and washed with 5 column 35 volumes of PBS to remove non-specific binding components. Bound antigen was eluted using
0.3 M imidazole, pH 8, 150 mm NaCl. The protein then was transferred to PBS (pH 7.4) by dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 pm), transferred to tubes, and stored at -70°C. Purity of the resulting protein solution was assessed by SDS-gel electrophoresis (Fig. 3).
Test and control IgGl antibodies were purified on a HiTrap Protein FF 1 ml column (GE
Healthcare) according to the procedure described above for the IL-17A-Fc antigen. Purity of the resulting protein solution was assessed by SDS-gel electrophoresis (Fig. 4).
According to electrophoresis purity data under denaturing conditions, the recombinant antigen products and IL-17A-H6F and IL-17A-Fc are suitable for immunization and further 10 research. Furthermore, control préparation AIN457 (Novartis; reproduced (US7807155 B2)) of satisfactory purity can be used in studies.
: v, > .\ Example 3: Immunization of Hamas with human Il-17a and création of llama Fab phage antibody librarv ' .
Llamas (Lama Glama) were immunized 5 fîmes successively by subcutaneous injection of the anti génie material mixed with an equal volume of complété (during the first injection) or incomplète (for the remaining injections) Freund’s adjuvant. A mixture of the recombinant protein was used as the antigen (0.2 mg of each protein per 1 injection), one of which was human IL-17A-H6F (Example 2). A second injection (the immunization step) was performed 3 weeks 20 after the first, and then at intervals of two weeks, the immunization was carried out three more fîmes. Blood samples (50 ml) were taken 5 days after each injection, starting with the third.
The collected llama blood after immunization was diluted 2 times with PBS solution containing 1 mM EDTA. 35 ml of diluted blood was layered on the medium solution Histopaque® -1077 (Sigma) at a density of 1.077 g/ml, volume of 15 ml, and then centrifuged 25 for 20 min at 800 g. Mononuclear cells (lymphocytes and monocytes) were selected from the interphase zone of the plasma/Histopaque medium and then washed with PBS solution containing 1 mM EDTA.
The resulting sérum immunoglobulin titer against IL-17A, certified according to the standard protocol, was not less than 1/10000, which is satisfactory for the construction of 30 antibody libraries (5).
Mononuclear cell total RNA was extracted using the llama RNeasy Mini Kit according to the suggested protocol (QIAGEN). RNA concentration was determined by Nanovue (GE Healthcare) and the quality of extracted RNA was checked by electrophoresis on a 1.5% agarose gel.
4? 19281
The reverse transcription reaction was performed using a MMLV RT kit (Evrogen) according to the recommended protocol using reverse transcriptase and random-MMuLV hexameric oligonucleotides as primers.
Reverse transcription products were used as a template in a two-stage PCR to produce variable domain genes flanked by restriction sites using a set of oligonucleotides and published protocols (de Haard et al., J Biol Chem 274(26):18218-30 (1999); De Genst et al., Dev Comp Immunol 30(1-2):187-98 (2006)). Then, the compound of the genes of the variable light and heavy chain domains of one fragment was carried out by sequential restriction, ligation, and amplification. The genes of heavy chains were combined with kappa genes and separately with lambda genes of light chains. Thus, the estimated number of molécules of the matrix in ail reactions was not less than 1011. The resulting DNA product VL-CK-VH was treated with restriction enzymes Nhel / Eco91I and ligated into the original phagemid pH5. Fig. 6 shows the resulting structure .of the phasmids. The ligaticm products . were transformed into elecüOcompetent SS320 cells obtained according to known protocols (Bond et al., J Mol Biol 332(3):643-55 (2003)). The répertoire of kappa dérivatives of the Fab library was 5.1 * 10E8 and of lambda dérivatives of the Fab library was - 3.7 * 10E8, respectively.
Example 4: Sélection of Fab libraries of phage antibodies
Spécifie anti-IL-17A phage Fabs were isolated from the Fab-display library on recombinant human IL-17A (R&D Systems) by performing a sériés of sélection cycles, as previously described (Marks et al., J Mol Biol 222(3):581-597 (1991); de Haard et al., J Biol Chem 274(26):18218-30 (1999)). To carry out the sélection by panning, human IL-17A in 50 mM carbonate buffet (pH 9.5) was adsorbed overnight at 4°C on the surface of sorbent tubes High Sorb (Nunc). The tubes were washed with PBS (pH 7.4), then blocked with PB S solution (pH 7.4) - skim milk (0.5% wt./Vol.) for 1 h. Next, 2.4 ml of phage solution in PBS (pH 7.4) skim milk (0.5% wt./Vol.) at a concentration of 1012 phage particles in 1 ml were added to a test tube with antigen and incubated for 1 hour with stirring. Nonbinding phage was removed during a sériés of wash cycles using PBS solution (pH 7.4) -Tween 20 (0.1% vol./vol.). The number of washes was increased from the first round to the third round to 20-30-40 times, respectively. The remàining bound phage particles were eluted with 100 ml of a Gly-HCl (pH 2.5) solution for 15 min with stirring, and then neutralized with 1 M Tris-HCl (pH 7.6). Bacteria strain E. coii TGI was infected with obtained phages, then phages were isolated and used in the next cycle of sélection.
After the second and third round of sélection on IL-17A, EIA analysis of the polyclonal phage product showed a significant enrichment (Fig. 8). The resulting pool of clones enriched by human Fab spécifie to anti-IL-J7A was recloned to the expression plasmid pLL (Fig. 7), containing myctag and His6 tag at the C-terminus of the heavy chain CH1 gene.
Example 5: Analysis of the spécifie binding of Fab to human IL-17a
EIA (ELISA) was used to measure binding of selected Fab fragments with human IL17A. As a positive control, a Fab antibody AIN457 with a published sequence (Novartis) was used. For the spécifie binding assay, wells on ELISA plates (Nunc Immuno Maxisorp) were coated with 50 μΐ IL-17A (0.5 pg/ml in IX coating carbonate buffer), then sealed and incubated ovemight at 4°C. Ail subséquent steps were performed by standard EIA protocol using an automated high-performance platform based on GenetixQ-pix2xt (Mole'cular Device) robotic Systems and Tecan Freedom EVO 200 (Tecan). To block non-specific binding, blocking buffer (BB) was added (200 μΐ 0.5% skim milk in PBS). Plates were incubated on a shaker for one hour at room température. After washes with PBS-Tween, 50 μΐ per well of the test cell supematant containing the analyzed Fab was added, mixed with an equal volume of blocking buffer. Plates were again incubated with shaking for one hour at room température, then each well of the plates was washed five times with PBS-Tween buffer. After washing, 50 μΐ/well of human anti-Fab HRP-conjugated secondary antibody (Pierce-Thermo Scientific) was added in a ratio of 1: 5000 in PBS-Tween. Plates were shaken on a rotary shaker (50 min, room température) and washed five times with PBS-Tween buffer, as described above. The colorimétrie signal was developed by adding TMB (100 μΐ/well) until saturation (on average 3-5 minutes), then further development was stopped by adding the terminating solution (100 μΐ/well, 10% sulfuric acid). The color signal was measured at a wavelength of 450 nm, using a suitable plate reader (Tecan-Sunrise; Tecan). The extern of antibody binding was proportional to color signal production. Clones in which the signal exceeded background color more than 5 times were tested in a compétitive EIA analysis to detect antagonistic Fabs that block the interaction between IL-17A ligand and receptor.
Example 6: Compétitive EIA analysis of blocking of the interaction between IL-17A ligand and IL-17R receptor
Compétitive ELISA was used to analyze the antagonistic ability of previously selected anti-human IL-17A Fabs. As a positive control, we used antagonist Fab antibodies AIN457 (Novartis). 50 μΐ of receptor IL-17ARA-Fc (R&D Systems) was immobilized to wells of ELISA plates (Nunc Immuno Maxisorp), at a concentration of 1 mg/ml in IX coating carbonate buffer, and incubated ovemight at 4°C. AU subséquent steps were performed by standard EIA protocol using an automated high-performance platform based on GenetixQ-pix2xt (Molecular Device) robotic Systems and Tecan Freedom EVO 200 (Tecan). To block non-spec'ific binding, blocking buffer (BB) was added (200 pl 0.5% of skimmed milk in PBS). Plates were incubated on a shaker for one hour at room température.
Simultaneously, 50 pl of the test cell supernatant containing the analyzed Fab was mixed 5 in a 96-well plate with 50 pl of IL-17A-His6-Flag at a concentration of 0.4 pg/ml in 1% milk PBS-Tween. It was then incubated for 1 hour at 37°C with stirring on a shaker at 500 rot/min.
Aller washing blocking buffer from the plates containing IL-17ARA-Fc receptor, the reaction mixture described above was transferred at 90 pl per well. The plates were incubated again with shaking for 45 minutes at room température, then each well of the plates was washed 10 five times with PBS-Tween buffer. 50 pl/well of mouse anti-FLAG M2 antibody (Sigma) was added at a concentration of 1 pg/ml. The plates were incubated for 45 min at room température, then each well of the plates was washed 5 times with PBS-Tween buffer. 50 pl/well of anti; mouse-IgG HRP-conjugated secondary antibody (Pierce-Thermo Scientific) diluted at the rate of 1:5000 in. PBS-Tween was added. Plates were shaken on a rotary shaker for 45 minutes at room 15 température and washed 5 times with PBS-Tween buffer, as described above. The colorimétrie signal was developed by adding TMB (100 pl/well) until saturation (on average 3-5 minutes), then further development was stopped by adding a terminating solution (10% sulfuric acid, 100 pl/well). The color signal was measured at a wavelength of 450 nm, using a suitable plate reader (Tecan-Sunrise; Tecan). The extent of antibody binding was proportional to the color 20 signal production.
Clones that showed blockage at the level of the control Fab antibody AIN457 were noted as positive and used for further analysis. Genes of the variable domains of positive clones were sequenced according to standard protocols using the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and analyzed. Clones containing VHH variable domains were selected for 25 further research. It was also found that one clone, 3\zHHFab, appeared in combination with twenty-three different light chain domain sequences, which may indicate its relative structural tolérance and may suggest that that VHH domain, but not the light chain, contributes to interaction with IL-17A.
Example 7: Comparative screening of anti-IL-Î7A VHHFab candidates by kinetic dissociation constant knff (kdis)
Comparative screening of koff for anti-IL-17A Fab candidates was performed using the device Octet Red 96 (Pall-ForteBio). Anti FABCH1 biosensors were rehydrated for 30 minutes in running buffer containing 10 mM PBS, pH 7.2-7.4, 0.1% Tween-20, and 0.1% BSA. E. coli 35 ΙΟχ-work buffer was added to the test samples to a final concentration of Ix. Anti FABCH1 biosensors were then immersed in tire samples of candidate Fab fragments and E. coli for 4 hours at 12°C. Sensors with immobilized Fab fragments on the surface were transferred into wells with a running buffer, where baseline was specified (60 sec.). Then sensors were transferred into wells with a solution of analyte (IL-17A, 30 pg/ml) for association of the antigen-antibody complex (300 sec.). Sensors were then retumed to wells containing running buffer for a subséquent dissociation step (300 sec.). After each experiment, used sensors were regenerated for use in the following experiment by placing them in a three-fold buffer for régénération (GlyHC1, pH 1.7). Analysis of the curves was performed using the Octet Data Analysis software (version 7.0) according to standard procedure using the interaction model 1:1.
The results of koS-screening of anti-IL-17A Fab candidates are presented in Table 1. Ail of the unique VHHFabs demonstrated spécifie high affmity binding with human IL-17A, while 3 VHHFab demonstrated very fast kon and very slow kdis(l/sec.), extending beyond the sensitivity ofthe instrument. · > .
Table 1. VHHFab Affmity for Human IL-17A and Specificity for Human IL- 17F.
•1 Kd(M) kon(l/Msec) kdjS(l/sec) Binding to human IL-17F *
1VHH 2.485E-08 2.498E04 6.206E-04 -
2 VHH 1.352E-08 1.628E05 2.201E-03 -
3VHHVK4B11 - - <2.272E-06 +++
* - no signal in the 31A with excess of not more than 50% above the ?ackground,
+++ strong signal exceeding more than 10 times over the background.
Thus, based on the analysis data, the VHH monodomain of 3VHHVK4B11 showed maximum binding affmity in comparison to the other two candidates.
Example 8: Spécifie binding of VHHFab to human IL-17F
ELIS A assays were performed on the three aforementioned VHHFabs according to the standard ELISA protocol described in Example 5, but for human IL-17F (R&D Systems). Fab antibody AIN457 (published sequence; Novartis) was used as a positive control. The results of the qualitative analysis ofthe interaction ofthe three VHHFab candidates are presented in Table 1. The VHH monodornain hereinafter designated as VHH 17 (SEQ ID NO: 1; Fig. 9,) with the mutations described below, was selected for further work on the basis of affmity and cross reactivity tests for 3VHHVK4B11 Fab.
Example 9: Scanning mutagenesis of CDRs of the anti-IL-17A VHH17 domain
Mutations to individual positions of the VHH 17 domain CDRs were inserted by means of . the NNK randomization technique using Q5® Site-Directed Mutagenesis Kit (NEB) in accordance with the manufacturer’s protocol. Plasmid pET22b-VHH17 was used as a matrix. PCR products were fractionated on low-melting agarose gels and purified on appropriate 5 columns. After ligation, DNA was transformed into E.coli expression strain BL21(DE3). The individual clones obtained by expression in 96-well plates, as described above. Supematants containing mutant VHH 17 were analyzed by ELIS A under the conditions described above and using the high-performance Genetix Q-pix2xt and Tecan Freedom EVO200 Systems. The concentration of immobilized IL-17A was 0.2 pg/ml. Bound VHH 17 monodomains were 10 stained with 1:5000 diluted mouse anti-myc-tag 9E10 and anti-mouse IgG (HRP) conjugate (Pierce) and TMB+H2O2/ H2SO4 dye; absorption was measured at a wavelength of 450 nm.
The results obtained by scanning mutagenesis are presented in Table >2. The table shows CDR substitutions that correspond.to <30% réduction of mutant VHH17/human 11-17A binding -signal when compared to the wild type sequence. In one aspect, the invention provides tri15 spécifie binding molécules comprising these mutations and any combinations thereof.
Table 2. VHH17 scanning mutagenesis
Mutation position Positive amino acids in mutants
HCDR3
V94 S, T, A, K, D, G
R95 K
R96 Y, H, W, K, D, G
R97 A, L, M, S, H, V
F98 -
D99 E, G, A, R, V, K, Q
G100 N,S
TlOOa G, P, V, R. S, N, K
S 100b V, Μ, T, L, T, A, H, G, I, C
YlOOc W, S
YlOOd R, L, W, K, A, G, Q, I, V
TlOOe A, L, S
GlOOf A, L, T, P, N, Q, F, I, D
DI 07 -
HCDR2
A50 G,L
S52 -
P52a A
S53 -
G54 -
G55 S, R, P, D, I, T, E, K , A, L
D56 -
R57 -
158 -
HCDR1
S32 N, K, R, E, W, M, Q, D, F, V, L, A
P33 S
M34 I
G35 L, A, I, S, R, V, N, Q, M
This screening identifîed amino acid positions in the CDRs of VHH17 that are tolérant of amino acid substitutions. It has been demonstrated that the présent panel of amino acid substitutions does not significantly change the binding affinity of VHH17 affinity for human IL17A. Thus, this substitution panel can be used to improve various properties of the candidate.
Example 10: Engineering and generating anti-IL-17A/anti-IL-17F and anti-TNFa tri-specific antibodies: VHH-IgGl (LALA)
The genes for the heavy and light chain variable domains of an antibody against human TNFa (RU 2303604) were synthesized in the original codon composition and cloned into pEEHc (IgGl) and pEE-Lc plasmids for the heavy and light chain, respectively. This allows joint transient expression in CHO-EBNA cells to produce IgGl isotype monoclonal antibodies. These constructions were validated by sequencing and were named pEE-aTNF-IgGlHc for the heavy chain and pEE-aTNFLc for the light chain (Fig. 9 shows the amino acid sequences of the variable domains).
To obtain a “no effect” LALA variant of anti-TNFa IgGl (Woodle et al., Transplantation 68(5):608-16 (1999)) with the variable domains shown in Fig. 9, amino acid substitutions L234A/L235A (Kabat nomenclature) were introduced using oligonucleotide-directed mutagenesis in the pEE-aTNF-IgGlHc construct via PfuUltraHS polymerase (Stratagene) according to the published protocol (Q5® Site-Directed Mutagenesis Kit (NEB)). PCR products were fractionated on a low melting point agarose gel and purified on columns. After the ligation reaction, the DNA was transformed into E. coli. The target sequence was validated by sequencing and designated hereinafter as pEE-aTNF-IgGlHcLALA.
In addition, using as a base the high affinity VHH sequences against IL-17A and IL-17F detected in Example 8, the VHH17 gene was synthesized (SEQ ID NO: 4; Fig. 9). An original 5 set of mutations spécifie to human VH germline was introduced (Q5V, SI IL, D61A, Y77N, R93V) as well as the combination of mutations E44G/R45T at the site of inter-subunit contact between the heavy and light chain variable domains (VH and VL). As shown previously (RU 2014138740), this provides stability of the VHH dérivative in combination with an antibody light chain.
Further, according to standard genetic engineering procedures, the VHH 17 gene (SEQ ID
NO: 1) was cloned at the N-terminus of the light chain pEE-aTNFLc construct, separated from the light chain by a spécifie 13-amino acid flexible linker necessary for correct independent folding of both polypeptides within fusion molécules and independent binding with different antigens. The resulting construct was named pEE-VHH17-77N-aTNFLc.
Different amino acid substitutions were introduced through oligonucleotide-directed mutagenesis in calculated positions in the CDRs of heavy chain pEE-aTNF-IgGlHcLALA and light chain pEE-VHH17-77N-aTNFLc (see the Table in Fig. 10) (nomenclature Kabat) using PfuUltraHS polymerase (Stratagene) according to the published prptocol (Q5® Site-Directed Mutagenesis Kit (NEB)) following the procedure described above. The mutations in the clones 20 selected for further work are indicated in Fig. 10.
Constructs containing the heavy and light chain mutations were combined pairwise to obtain varions paired combinations and to hold transient accumulation as described in Example 2. This resulted in génération of 1 variant (MabC) with no mutations in the CDRs of the antiTNFa domain, and 32 variants of the trispecific antibody (Mabl-32; Fig, 11). The productivity 25 of ail the variants was close to or above 100 mg/1, which is satisfactory for further research of their properties.
Example 11: Comparative analysis of neutralization of tumor-alpha necrosis factor by tri-specific anti-IL-17A/anti-IL-17F/anti-TNFa binding molécules
Test solutions of antibodies were titrated in a 96-well plate from a concentration of 500 ng/ml in incréments of 2, 100 μΐ of solution per well. A solution of recombinant human TNFa at a concentration of 500 pg/ml was prepared and then 50 μΐ added to each well with the test samples. Plates were incubated for 1 hour at 37°C in a CO2 incubator.
WEHI-13VAR cells were suspended at a concentration of l*106 cells/ml and introduced 35 into each well at 50 μΐ with the test solutions. The plates were incubated at 37°C in a CO2 incubator for 20-24 hours. The number of living cells was measured using the vital stain Alamar Blue. After completion of incubation, 20 μΐ of Alamar Blue reagent was added to each well of analyte plate, then plates were shaken for 5 minutes at room température on an orbital shaker. The plates were incubated at 37°C for 16-20 hours. Fluorescence readings were evaluated in 5 relative fluorescence units at excitation/emission wavelengths of 544/590 nm using a Fluoroskan
Ascent FL device. For data analysis, Microsoft Excel was used to plot dependency diagrams for the fluorescence signal and the concentration of the test and standard samples.
The table in Fig. 12 shows the results of comparative analysis of the antagonistic activity of the 32 mutants tri-specific antibodies compared to the non-mutated variant MabC and the 10 monospecific anti-TNFa antibody adalimumab. On the basis of this analysis, the six best variants were selected and tested to identify the best three: Mabl2 (ED50 = 4 ng/ml), Mab 31 (ED50 = 20 ng/ml), and Mab32 (ED50 = 4 ng/ml). Figure 13 shows a diagram of the comparative analysis of these variants relative to Controls. On the basis of this analysis, Mab 12 • variant has been selected for follow-up, and was named BCD-121.
Further, using the same method, trispecific candidate BCD-121 was compared to the two anti-TNFa monospecific antibodies infliximab and adalimumab. The results confirmed that BCD-121 demonstrates greater antagonistic activity than adalimumab (5-fold excess) and infliximab (10-fold excess), with an EC50 of 6 ng/ml (Fig. 14).
Example 12: Comparative analysis of the inhibition of ÏL-17A-dependent IL-6 production by trispecific anti-IL-17A/anti-IL-17F/anti-TNFa binding molécules
The neutralizing activity of trispecific candidate BCD-121 (see Example 10) was analyzed using the ability of IL-17A to induce the production of IL-6 by human HT1080 cell Unes (ATCC: CCL-121) in the presence of human recombinant IL-17A and TNFa. Cells were 25 grown in DMEM medium supplemented with 10% inactivated fêtai sérum, glutamine, and gentamycin. The cells were added to 96-well fiat bottom cell culture plates at 5 x 104 per well and were left for 5 hours to attach. A mixture of recombinant IL-17A at 40 ng/ml was incubated with dilutions of antibodies for one hour at 37°C. The cytokine/antibody mixture was then added to the cells and left overnight. The production of IL-6 in the HT1080 cells culture was 30 proportional to the added quantity of IL-17A. The amount of released IL-6 in cell supernatants was determined by ELISA using “DuoSet ELISA Development System Human IL6” (R&D System, Cat. No. DY206). The results are shown in Fig. 15. Reproduced anti-TNFa monospecific antibody adalimumab was used as a négative control and anti-IL-17A monospecific antibody BCD-085 was used as a positive control (BIOCAD). As can be seen 35 from the graph, while adalimumab did not inhibit IL-6 production, and BCD-085 inhibited IL-6 production with an IC50 of about 9.2 ng/mL, BCD-121 antibody advantageously inhibited IL-6 production with an IC50 of 3.5 ng/mL. Thus, candidate antibody BCD-121 is the most potent antagonist of IL-6 production in this assay.
Example 13: Comparative analysis of inhibition of dual IL-17A/ TNFa -dépendent IL-6 production by tri-specific anti-IL-17A/anti-IL-17F/anti-TNFa binding molécule BCD121
The ability of IL-17A or dual IL-17A/TNFa to induce the production of IL-6 by human HT1080 cell lines (ATCC: CCL-121) was used to analyze the neutralizing activity of tri-specific binding molécule candidate BCD-121 (see Example 10) in the presence of human recombinant IL-17A and TNFa. Cells were grown in DMEM medium supplemented with 10% inactivated fêtai sérum, glutamine and gentamycin. 5 x 104 per well of cells were added to 96-well fiat bottom cell culture plates. The cells were left for five hours to attach. A mixture of recombinant IL-17A at 40 ng/ml and TNFa at 20 ng/ml was incubated with dilutions of antibodies for one hour at 37°C. Then, the mixtures of cytokines and antibodies were added to the cells and left ovemight. The production of IL-6 in HT1080 cell culture was proportional to the quantity of IL17A added. The amount of released IL-6 in cell supernatants was determined by ELISA using “DuoSet ELISA development System human IL6” (R&D System-, Cat. No. DY206). The results are shown in Fig. 16. Reproduced anti- TNFa monospecific antibody adalimumab and anti-IL17A monospecific antibody BCD-085 (BIOCAD) were used as Controls. As can be seen from the graph, the BCD-121 tri-specific antibody demonstrated an advantageous effect in comparison with the monospecific antibodies, showing an IC50 value of about 25 pM and an almost undetectable amount of IL-6 at the lower plateau. By contrast, the control antibody Aadalimumab had an IC50 value of about 100 pM. BCD-085 showed a similar IC50 value of about 25 pM, but had a lower threshold plateau 20 fîmes greater than BCD-121. Thus, BCD-121 is the most potent antagonist of IL-6 production in the assay used.
Example 14: Analysis of antibody-dependent cellular cytotoxicity (ADCC) of the trispecific anti-IL-17A/anti-IL-17F/anti-TNFa binding molécule BCD-121 in Jurkat-tmTNFa cells
For use in the quantitative assay below, Jurkat-tmTNFa cl.8 cell culture was collected from flasks and centrifuged for 5 minutes at 200 g. The supernatant was decanted, and washed once more in a medium for quantitative détermination.
The cell pellet was suspended in 5 ml of medium for quantitative détermination. Cell viability and number were determined. A cell suspension for seeding white 96-well culture plates was prepared at 3* 105cells/mL ’ '
The test sample was diluted into the wells of a 96-well plate. In wells with test samples, 5 the Jurkat-tmTNFa cl.8 cell suspension was added, and the plate was incubated in a CO2 incubator for 15-30 minutes.
A suspension of PBMC cells was prepared with a concentration of 7.5x106 cells/ml and added into the wells with test samples. The plate was incubated for four hours at 37°C in a CO2 incubator.
Assay Buffer was mixed with AAF-Glo™Substrate from the set “CytoTox-Glotm
Cytotoxicity Assay,” then added to each well with test samples. The plate was incubated for 15 minutes at room température. Luminescence was measured on a Fluoroskan Ascent FL device. By this method, the activity of intracellular proteases is determined; the luminescence signal is proportional to the number of lysed cells.
As shown in Fig. 17, BCD-121 demonstrated approximately 100-fold less ADCC activity compared to IgGl-derived antibodies adalimumab and infliximab, and is comparable in the level of this activity to cimzia (Fab-PEG).
Example 15: Analysis of complement-dcpendent cytotoxicity (CDC) of the tri20 spécifie anti-IL-17A/anti-IL-17F/anti-TNFa binding molécule BCD-121 in Jurkat-tmTNFa cells
Test solutions of antibodies were titrated from a concentration of 15 pg/ml in incréments of 2 in 96-well plates, 50 μΐ per well. Human complément was dissolved 1:3 in chilled medium for analysis (RPMI with 1% BSA) and 50 μΐ added into wells with test samples. 50 μΐ of Jurkat25 tmTNFa cell suspension was added to each well with samples at the concentration of 1χ106 cells/ml (5x104 cells per well). The plates were shaken for 3 min on an orbital shaker at room température and placed for 2-3 hours in a CO2 incubator at 37 C. The number of living cells was measured using the vital stain Alamar Blue. After completion of incubation, 15 .μΐ of Alamar Blue reagent were added to each well of analyte plate, then the plates were shaken for 5 minutes 30 at room température on an orbital shaker. The plates were incubated at 37°C for 18 to 24 hours.
Fluorescence readings were evaluated in relative fluorescence units at excitation/emission wavelengths of 544/590 nm using a Fluoroskan Ascent FL device.
As seen in Fig. 18, BCD-121 does not hâve CDC activity in Jurkat-tm TNFa cl.8 cells in comparison with IgGl-derived antibodies adalimumab and infliximab, and is comparable in the 35 level of this activity to cimzia (Fab-PEG).
Example 16: Analysis of simultaneous interactions of BCD-121 with human IL-17A and human TNFa using the OctetRED96 device
Analysis of simultaneous binding of BCD-121 to human IL-17A and human TNFa was carried out using the classic Sandwich analysis method by the Octet Red96 device (PallForteBio) (Fig. 19). Analysis was performed at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as running buffer. During the experiment, the first antigen (IL-17A-biotin) was immobilized on streptavidin-coated sensors at a concentration of 20 pg/ml and a volume of 200 μΐ/well. Further sensors were immersed in an antibody solution of 200 μΐ/well of antibody at a concentration of 20 μg/ml. Sensors subsequently were immersed in TNFa solution at 200 μΐ/well at a concentration of 1 mg/ml and into saline containing no protein.·
Binding curves minus baseline signal were analyzed using Octet Data Analysis (Version 7.0) according to the standard procedure.
The sensogram shown in Fig. 19 indicates that the tri-specific binding molécule BCD121 can simultaneously bind IL-17A and TNFa.
Example 17: Analysis of BCD-121 binding with orthologous IL-17A and TNFa ligands using the Biacore T200 device
The analysis was conducted using the Sandwich approach using IL-17A and TNFa préparations produced by Sino Biologics. After activation of the chips, individual ligand was nonspecifically (by NH2 groups) immobilized on the biosensor surface at a concentration of 20 pg/ml. Loading of the antibody was then performed at a concentration from 1 to 25 pg/ml. Thereafter, the sensors were immersed in the running buffer for the complex dissociation step. The flow rate was 10 μΐ/min.
Analysis of the obtained data was performed using BIA Evolution 3.1 software and the Heterogeneous Ligand model.
Table 3 shows data on the interaction of BCD-121 with orthologous IL-17A and TNFa ligands from human and primate. BCD-121 demonstrates very high affinity (below pM) for ail of the ligands.
Table 3. Affinity of BCD-121 to IL-17A, IL-17F and TNFa
IL-17A human KD, M IL-17A cyno KD, M IL-17F human KD, M (OctetRED96)
BCD-121 «l,0E-12* ~l,0E-12 ~4,0E-9
TNFa human KD, M TNFa cyno KD, M TNFa multta KD, M
BCD-121 «l,0E-12* «l,0E-12* ~l,0E-12
*- below the threshold sensitivity of the Biacore T200 device
Example 18: Enzyme immunoassay of the interaction between BCD-121 and various IL-17 family antigens (A, B, C, D, E, F and A/F)
ELISA was used to measure the comparative binding of BCD-121 to human IL17 family antigens. For the binding analysis, the wells of ELISA plates (medium binding from Greiner bio one) were individually coated with 50 μΐ of human IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, or IL-17A/F (R&D Systems) at a concentration of 1 pg/ml (in lx coating carbonate buffer), hermetically sealed, and incubated ovemight at 4 C. Ail subséquent steps were performed by standard EIA protocol. To block non-specific binding blocking, buffer BB was added (200 pl of 0.5% skim milk in PB S). Plates were incubated on a shaker for one hour at room température. After washes with PBS-Tween, 50 pl of the test antibodies were added to each well at a concentration of 5 pg/ml in PBS-Tween. Plates were again incubated with shaking for one hour at room température, then each well of the plates was washed three times with PBS-Tween buffer. After washing, human anti-Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific) was added (50 pl/well) at a ratio of 1: 5000 in PBS-Tween. Plates were shaken on a rotary shaker (50 min, room température) and washed three times with PBS-Tween buffer, as described above. The colorimétrie signal was developed by addition of TMB (50 pl/well) until saturation (on average 3-5 minutes), then further development was stopped by adding the terminating solution (30 1/well 10% sulfuric acid). The color signal was measured at 450 nm using a suitable plate-reader (Tecan-Sunrise; Tecan). The extent of antibody binding was proportional to color signal production.
The resuit of the interaction of BCD-121 with members of the human IL-17 family is presented in Fig. 20. As can be seen from the curve of optical absorption concentration for BCD-121, the tri-specific binding molécule specifically interacts with IL-17A and IL-17F with high affinity, and with their natural heterodimer IL-17A/F with low affinity. BCD-121 is not cross-reactive with other members of the family.
Example 19: Enzyme immunoassay of the interaction of BCD-121 with guinea pig, rabbit, and mouse TNFa
An ELISA assay was performed using a similar protocol to that described in Example 16. Human, guinea pig, rabbit, and mouse TNFa antigens are from R&D Systems.
The resuit of interaction of BCD-121 with human, guinea pig, rabbit, and mouse TNFa is shown in Fig. 21. As can be seen from the curve for optical absorption-concentration of BCD121, the tri-specific binding molécule reacts specifically and with high affinity only to human TNFa. BCD-121 is not cross-reactive with TNFa orthologs (except primate TNFa orthologs, as shown in Example 17).
Example 20: Détermination of thermal stability by assessing the protein aggregation point using the dynamic light scattering method (DLS).
Détermination of the aggregation point ofthe samples (1 mg/ml) was performed on the Zetasizer Nano ZSP device. 0.5 ml of solution was placed in a dust-free quartz cuvette, which was gradually heated in the device from 50 to 85°C in 1.5°C steps. Each température was maintained for 30 seconds with constant measuring of scattered light intensity. The intensity of the scattered light was detected at an angle 0 = 173°.
Data obtained using the dynamic light scattering method is shown in Fig. 22 and Table 4 for protein aggregation points in four different buffers.
Table 4. BCD-121 Aggregation Point Measured by Dynamic Light Scattering
Incurred samples Aggregation point
BCD-121 Acetate buffer, pH 5.0 69.5°C
Citrate buffer, pH 5.0 66.5°C
Histidine buffer, pH 6.0 69.5°C
Phosphate buffer, pH 6.0 65.0°C
Example 21: Détermination of thermal stability during long-term exposure by the thermostress 50°C method.
Samples of protein at a concentration of ~ 5 mg/ml were divided into 3 portions of 200 μΐ each and placed in separate tubes: 1 tube for each composition was stored in the refrigerator at + 4°C, and the other two were set in a test tube heatef and incubated at 50°C for 6 and 24 hours, respectively. After warming, the tubes were removed from the heater, allowed to stand at room température for 15 min, centrifuged at 8000 rpm for 5 minutes, and the supematants transferred for gel filtration with a UV detector.
The research standard for size exclusion chromatography was used (Bio-rad; cat. number 151 -1901 ). The data for BCD-121 stability in varions buffers under prolonged heating at 50°C is shown in the table in Fig. 23.
The results show that ail tested buffers provide satisfactory BCD-121 thermostress 5 stability.
Example 22: Création of stable cell lines for production of BCD-121
A stable producer cell line for BCD-121 was obtained by transfection with electroporation using the Néon Transfection System (Life Technologies) slurry parental cell line 10 CHO-S vector constructions, containing the light and heavy antibody chains in an optimized ratio. Clonal lines with high productivity (up to 1000 mg/L) were obtained using a ClonePix robot platform (Molecular Devices) and the preliminary stages of minipool sélection were performed using antibiotics in varions culture formats. Productivity analysis was performed using the analytical System Octet RED96 (Pall Life Sciences). DOE on the sélection of basic 15 medium and cultivation schemes was implemented using the autoinated System Biomek FX robotics (Beckman Coulter). For culturing the producer cells, a serum-free medium and fittings were used, which did not contain animal protein. Accumulation of BCD-121 for preclinical research was carried out in a HyCJone single-use bioreactor (Thermo Fisher Scientifïc) with a working volume of 50 1.
Example 23: Isolation of BCD-121 from the productive stable cell line environment
Clarification of the culture liquid containing BCD-121 was performed on a deep-bed filter Millistak C0HC (Merck-Millipore). Primary antibody purification was performed on affinity resin with protein A, using a spécifie target protein, and the antibody was eluted with 25 glycine buffer at pH3.3-3.8. The eluate was incubated at an acidic pH for 30-60 minutes for viral inactivation and then neutralized with a solution of IM Tris-OH. Final chromatographie purification was performed on the sorbent CHT™ Ceramic Hydroxyapatite (Bio-Rad) to remove residual DNA, protein producing cells, cleaved ligand affinity sorbent aggregates, and antibody fragments. For this method, protein solution was applied to the sorbent prepared at pH 7.5, 30 followed by a spécifie elution gradient with sodium chloride. The purified protein was filtered for viruses using a set of Viresolve PRO filters (Merck Millipore). The protein was concentrated in a tangential flow on the tapes with a cut-off limit of 50 kDa followed by diafiltration against a final buffer containing acetate buffer (pH5.0), sorbitol, and polysorbate-80. Protein concentration in the final solution was at least 70 mg/ml.
Purification results are presented in Figure 24. Vertical electrophoresis using a polyacrylamide gel with SDS under reducing and non-reducing conditions demonstrated the purity of the product, which is suffîcient for use in a pharmaceutical composition.
Example 24: Enzyme immunoassay showing BCD-121 stability in pure human sérum
To estimate the stability of BCD-121 in pure human sérum, the tri-specific antibody was added at concentrations of 2, 10, and 50 pg/ml to a pure human sérum mix obtained from seven healthy donors, preserved with 0.01 % of thimerosal, and stored for 7 days at 37°C. After 7 10 days, the concentration of BCD-121 was measured by ELIS A in two assay formats: immobilized TNFa and biotinilated IL 17 with Streptavidin-HRP conjugate détection and immobilized TNFa with Goat Anti-human IgG Fc - HRP conjugate détection. Sérum samples in which BCD-121 was added to concentrations of 2, 10, and 50 pg/ml immediately before ELIS A measurement were used as a control. Measurement was performed as follows: control and stored sérum 15 samples with 2, 10, or 50 pg/ml of BCD-121 were diluted to 100 ng/ml (20, 100, and 500-fold, respectively) and actual concentration was measured against a standard calibration curve (from 6.25 to 200 ng/ml) dissolved in PBS-Tween. Ail ELISA steps were performed by standard EIA protocol. 100 pl of diluted samples or calibration standards were added to microwells precoated by TNFa. The concentration of coated TNFa was 2 pg/ml in 20 mM carbonate buffer at pH 9.5.
A blocking buffer (BB: 200 pl of 0.5% skim milk in PBS) was then added to the plates. The plates were incubated on a shaker for one hour at room température. After washes with PBSTween, 100 pl of the biotinilated IL 17 (3 pg/ml) or Goat Anti-human IgG Fc - HRP (from Sigma, in a ratio 1:20000) were added per well with concentration in PBS-Tween. The plateswere again incubated with shaking for one hour at room température, then each well of the plates 25 was washed three times with PBS-Tween buffer. 100 pl of Streptavidin-HRP conjugate at a ratio of 1:20000 (Sigma) was additionally added into wells in which biotinilated IL17 was previously added. After subséquent incubation for one hour and washing, the colorimétrie signal was developed by addition of TMB (100 pl/well) until saturation (on average 10-15 minutes), then further development was stopped by adding the terminating solution (50 pl/well 10% sulfuric 30 acid). The color signal was measured at 450 nm using a suitable plate-reader (Tecan-Sunrise;
. Tecan). The amount of BCD-121 binding was proportional to color signal production. The calibration curve concentration was multiplied by the dilution factor and expressed as % of theoretically added in sérum BCD-121 concentration.
Example 25: Evaluating the effectiveness of the BCD-121 anti-IL-17A/anti-IL17F/anti-TNFa binding molécule in vivo
The research below was conducted on male cynomolgus monkeys (Macaca fascicularis) using a model of collagen-induced arthritis. The total number of animais in the experiment was 20, in five groups of four monkeys each. In the experiment, four doses of the product were used: 0.2 mg/kg; 1.0 mg/kg; 5.0 mg/kg; and 10.0 mg/kg. Control group animais received a placebo. Administration of the product and placebo started after preliminary sensitization with collagen. During this study, the animais of ail groups were measured to calculate the articular surface of the APW. At the end of the study, metacarpophalangeal and metatarsophalangeal-phalangeal joints were taken to assess the severity of destructive changes. The schematic for the experimental groups is shown in Table 5 below.
Table 5.
Group NumbeF Number of animais Product Route of administration Dose
1 40) BCD-121 subcutaneous 0.2 mg/kg
2 4 0) 1.0 mg/kg
5.0 mg/kg
3 4 0)
J- 4(d) 10.0 mg/kg
5 4(<î) Placebo -
For arthritis induction, an émulsion of bovine type II collagen (Sigma) was administered to the animais three times.
The first administration of collagen:
The total amount of administered collagen was 2 mg per experimental animal. For this purpose, 2 mg of collagen were dissolved in 0.7 ml of 0.1 M acetic acid. 0.7 ml of complété Freund's adjuvant was added to this solution.
Animais were kept for 28 days after the first administration of collagen.
The second administration of collagen:
The total amount of administered collagen was 3 mg per experimental animal. For this purpose, 3 mg of collagen were dissolved in 1 ml of 0.1 M acetic acid. 1 ml of complété Freund's adjuvant was added to this solution.
Animais were kept for 21 days after the second administration of collagen.
The third administration of collagen
The total amount of administered collagen was 3 mg per experimental animal. For this ' purpose, 3 mg of collagen were dissolved in 1 mi of 0.1 M acetic acid. 1 ml of complété 5 Freund's adjuvant was added to this solution.
Measuring of the size of the joints was carried out by dividers at the following. time points:
• Before the first administration of collagen;
• At the time of the second administration of collagen; and « Weekly after the second administration immediately before the administration of the collagen for 7 weeks.
During the measurement process the longitudinal and transverse axis of the joint was estimated. This procedure has been carried out for ail the metacarpophalangeal and metatarsophalangeal joints except the thumb. Calculation of the area was carried out by the 15 formula:
JA = the value for the longitudinal axis x the value for the transverse axis x 3,14 x 0,25.
The data for 16 joints of each animal was used to calculate the percent of inflammation area (PIA). This calculation was carried out by the formula:
JA value on the day ofthe experiment x 100 the average value for JA before the induction of arthritis
The results, shown in Fig. 26, demonstrate the dose-dependent anti-inflammatory effect of BCD-121 in a monkey CIA model.
Example 26: Evaluation of toxicokinetics of the BCD-121 anti-IL-17A/anti-IL25 17F/anti-TNFa binding molécule
The toxicokinetics assay was performed on cynomolgus macaques (cynomolgus). The research used three dose levels of the BCD-121 product. The schematic of the experimental groups is shown in Table 6 below.
Table 6.
Group Number Number of animais Product Route of administration Dose
1 3(d) BCD-121 subcutaneous Minimal
2 3-0) BCD-121 Medium
3 3 (c?) BCD-121 Maximal
4 3«) Placebo -
In the course of the study, the following parameters were evaluated:
- Clinical examination results;
- Animal’s weight (before administration and on days 7, 14, 21, 28, 35 and 42 of the experiment);
- Body température (before administration and 1, 2, 4, 6, and 24 hours after administration on days 7, 14, 21, 28, 35, and 42 of the experiment);
- Urinalysis (before administration and on days 7, 14, 21, 28, 35, and 42 of the experiment);
- Clinical blood analysis on parameters: number of érythrocytes, number of leucocytes, hemogiobin concentration (before administration and on days 7, 14, 21, 28, 35, and 42 of the experiment);
- Biochemical analysis of sérum ratios: LDH, total bilirubin, total protein, glucose, aspartate aminotransferase, and alanine aminotransferase (before administration and on days 7, 14, 21, 28, 15 35, and 42 of the experiment); and
- Drug concentration in the blood sérum of primates (0.5, 1, 3, 6, 24, 30, 48, 72, 96, 120, 144, 168, 192, 264, 336, 408, 504, 624, 720, 816, 912 and 1008 hours after administration).
The level of the drug in blood sérum was assessed using ELISA and horseradish peroxidase as indicator enzyme.
Wells of 96-well microtiter plates (“Costar”, Cat. No. 3590) were sensitized with recombinant human TNFa (R&D Systems, Cat N 210-TA/CF) based on 250 ng/100 μΐ of 20 mM Tris HCI buffer solution, pH 9.0, per well and incubated for 16-18 hours at 4°C. Antigens which were not bound wrere removed from the wells. In order to block non-specific binding sites, 200 μΐ of 20 mM Tris HCI buffer solution, pH 7.2-7.4 (TB), containing 1.0% BSA, 0.14 M NaCI and
0.05% Tween-20 (blocking buffer - BB) were introduced into each well and then the wells were incubated for 30 minutes at 37°C. BB solution was removed, and 100 μΐ of test sérum diluted 1000-10 OOO-fold with BB were introduced into each well. Simultaneously 100 μΐ of BB containing different concentrations of BCD-121 in the range of 3.9 — 250 ng/ml were introduced into each well. The plates were incubated for 30 minutes at 37°C. The well contents were removed, the wells were washed 3 times each with 200 μΐ of TB containing 0.05 Tween-20, 100 μΐ of BB containing antibodies against human IgG Fc-fragment labelled with horseradish peroxidase («Sigma», USA, Cat. No. A0170 at working dilution) were introduced into each well and the wells were incubated for 30 minutes at 37°C. The well contents were removed, the wells were washed 4 times each with 200 μΐ of TB containing 0.05 Tween-20, and 100 μΐ of 0.1 M sodium acetate buffer solution, pH 5.0, containing 0.1 mg/ml tetramethylbenzidine and 0.003% H2O2 were introduced into each well. To quench the reaction, 50 μΐ of 2M H2SO4 were introduced into each well, then absorbance was measured in the wells using a plate spectrophotometer at 450 nm.
To détermine the concentration of BCD-121 in test samples, the calibration curve of the relationship between absorbance and concentration of BCD-121 in reference standards was plotted, and using the absorbance value obtained with the test sample, the concentration of BCD121 was determined.
The lower limit of reliable BCD-121 détection was 2 ng/ml. The concentration of BCD121 in test sera (X), in pg/ml, was calculated according to the following formula:
X = AxF where, A - concentration of BCD-121 table in test sample determined using calibration curve, ng/ml;
F - dilution of initial sérum;
Results of the PK profile demonstrated expected levels of BCD-121 (Fig. 27). The terminal disposition half-life was calculated above 12 days. The obtained PK profile of BCD121 in monkeys was similar to that of native human IgG antibody and had a typical dosedependent character.
Example 27: Evaluation of toxicity of repeated subcutaneous administration in cynomolgus monkeys for one month followed by a period free from administration for one month
A toxicity assay of repeated subcutaneous administration of BCD-121 for one month followed by a one month recovery period was carried out on Javanese macaques. Three dose levels were used in the experiment. The schematic of the experimental groups is shown in Table 7 below.
Table 7.
Group Number Number of animais Product Route of administration Dose
1 3 ca BCD-121 subeutaneous Minimal
3 (?)
2 3 (<?) BCD-121 Medium
3 (?) -
3 3 0)* BCD-121 Maximal
3 (?) *
3 (<n
3 (?)
4 3 (<?) Placebo -
3(Ç)
In the course of the study, the following parameters were evaluated:
- Clinical examination results;
- Animal weight (before administration and then weekly);
- Body température (before administration and then weekly until termination of the experiment);
- Influence on the cardiovascular System for which the bioelectric activity of the heart was evaluated using a Poly-Spectrum system; évaluation was made before administration and then on weeks 3, 5, and 7 of the experiment;
- Urinalysis (before administration and on weeks 3, 5, and 7 of the experiment);
- Clinical blood analysis on parameters: number of érythrocytes, number of leucocytes, hemoglobin concentration, number of lymphocytes, number of monocytes, neutrophils, eosinophils, and basophiles, and platelet count (before administration, then once a week, starting from the first week of the experiment);
- Assessment of the impact on the blood clotting system in terms of: activated partial thromboplastin time, fibrinogen concentration, and prothrombin time (before administration, then on weeks 3,5, and 7 of the experiment);
- Biochemical analysis of sérum ratios: sodium, potassium, créatinine, urea, alkaline phosphatase, LDH, total bilirubin, total protein, glucose, triglycérides, aspartate 20 aminotransferase, alanine aminotransferase, total cholestérol (before administration and on weeks 3,5, and 7 of the experiment);
- At the end of the administration period, satellite animais of group 3* were euthanized and subsequently pathologically examined; at the end of the study, animais of group 3 and the control group underwent the same; ’ . - As a part ofthe toxicity research, the influence of local-irritating drugs was also evaluated, for 5 which the soft tissue at the site of injection was selected and histological examination was performed.
Example 28: Investigation of immunotoxicity of the BCD-121 anti-IL-17A/anti-IL17F/anti-TNFa binding molécule
An immunotoxicity assay of repeated subcutaneous administration for 1 month followed by a one-month recovery period was carried ont on Javanese macaques. Three dose levels were used in the experiment. The schematic of the experimental groups is shown in Table 8 below.
Table 8.
Group Number Number of animais Product Route of administration Dose
1 3 0) BCD-121 subcutaneous Minimal
3 (?)
2 3 0) BCD-121 Medium
3 (?)
Maximal
3 3(Λ BCD-121
3 (?)
4 3(Λ Placebo -
3 (?)
In the course of the study, the following parameters were evaluated:
- Subpopulation composition of lymphocytes, which was assessed before administration and then at weeks 2, 4 and 6 of the experiment;
- Immunoglobulin classes ratio, which was evaluated before administration and at weeks 2, 4 and 6 of the experiment;
- Effect on phagocytosis was assessed before administration and then at weeks 2, 4 and 6 of the experiment.
Example 29: Evaluation of immunogenicity at repeated subcutaneous administration ofthe BCD-121 anti-IL-17A/anti-IL-17F/anti-TNFa binding molécule . An immunotoxicity assay of repeated subcutaneous administration for 1 month followed by a one-month recovery period was carried ont on Javanese macaques. Three dose levels were used in the experiment. The schematic of the experimental groups is shown in Table 9 below.
Table 9.
Group Number Number of animais Product Route of administration Dose
1 3«) BCD-121 subcutaneous Minimal
3 (?)
2 3 (d') BCD-121 Medium
3 (?)
3 3 (<?) BCD-121 Maximal
3 (?)
The immunogenichy assay is performed on the level of antibody binding, for which blood sérum is selected and isolated before administration and then at weeks 3, 5, and 7 of the experiment.
Example 30: Pharmacokinetics assay during repeated subcutaneous administration of the BCD-121 anti-IL!7A/anti-IL-l7F/anti-TNFa binding molécule
A pharmacokinetics assay of repeated subcutaneous administration for one month followed by a one-month recovery period was performed on Javanese macaques. Three dose levels were used. The schematic of the experimental groups is shown in Table 10 below.
Table 10.
Group Number Number of animais Product Route of administration Dose
1 3 (J BCD-121 subcutaneous Minimal
3 (?)
2 3(3) BCD-121 Medium
3 (?)
3 3(3) BCD-121 Maximal
3 (?)
To estimate the product level, blood sampling was performed on days 0, 1, 2, 8, 9, 15, 16,
22, 23, 29, 36, and 43 of the experiment.
Table 11: SEQ IDNO Chart
SEQ IDNO: Description
1. VHH17 CDR1 amino acid sequence
• 2· VHH17 CDR2 amino acid sequence
3 VHH17 CDR3 amino acid sequence
4 VHH17 amino acid sequence
5 anti-TNFa HC amino acid sequence
6 anti-TNFa LC amino acid sequence
7 VHH 17 + anti-TNFa LC amino acid sequence
8 anti-TNFa VH amino acid sequence
9 anti-TNFa VL amino acid sequence
10 anti-TNFa HC-CDR1 amino acid sequence
11 anti-TNFa HC-CDR2 amino acid sequence
12 anti-TNFa HC-CDR3 amino acid sequence
13 anti-TNFa LC-CDR1 amino acid sequence
14 anti-TNFa LC-CDR2 amino acid sequence
15 anti-TNFa LC-CDR3 amino acid sequence
16 peptide linker
17 parent VHH amino acid sequence
18 parent anti-TNFa VH amino acid sequence
19 VKY92F amino acid sequence
20 VKY94F amino acid sequence
21 VKS50A amino acid sequence
22 VKS50D amino acid sequence
23 VKS50G amino acid sequence
24 VKN33D amino acid sequence
25 VKN33A amino acid sequence
26 VHG33A amino acid sequence
27 VHG33S amino acid sequence
28 ΛΤΙΝ35Α amino acid sequence
29 VHN35H amino acid sequence
30 VHN35S amino acid sequence
31 VHN52T amino acid sequence
32 VHN52Q amino acid sequence

Claims (31)

1. A tri-specific antibody or antigen-binding fragment thereof comprising a first ' binding domain that binds to human IL-17A and human IL-17-F and a second binding domain that binds to a human TNFa, wherein said first binding domain comprises the amino acid sequence of SEQ ID NO: 3.
2. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said first binding domain comprises the amino acid sequences of SEQ ID NOs: 1-3.
3. The tri-specific antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein said first binding domain competes for binding with or binds to the same epitope as a binding domain comprising the amino acid sequence of SEQ ID NO: 4.
4. The tri-specific antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein said first binding domain is at least 90% identical to SEQ ID NO: 4.
5. The tri-specific antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein said first binding domain comprises the amino acid sequence of SEQ ID NO: 4.
6. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said first binding domain is humanized.
7. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain competes for binding with or binds to the same epitope as a binding domain comprising:
a) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 10-12 and a light chain comprising the amino acid sequences of SEQ ID NOs: 13-15;
b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 9; or
c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.
8. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ IDNO: 15.
9. The tri-specific antibody or antigen-binding fragment thereof according to claim 8, wherein said second binding domain comprises a heavy chain comprising the amino acid sequences of SEQ ID NOs: 10-12 and a light chain comprising the amino acid sequences ofSEQ IDNOs: 13-15.
10. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain comprises a heavy chain variable domain at least 90% identical to SEQ ID NO: 8, a variable domain at least 90% identical to SEQ ID NO: 9, or both.
11. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain comprises a heavy chain- variable domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 9, or both.
12. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein the second binding domain is a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody, Fv, Fab, F(ab’)2, Fd, single chain Fv molécule (scFv), diabody, or single domain antibody (dAb).
13. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain comprises a heavy chain at least 90% identical to SEQ ID NO: 5, a light chain at least 90% identical to SEQ ID NO: 6, or both.
14. The tri-specific antibody or antigen-binding fragment thereof according to claim 1, wherein said second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5, a light chain comprising the amino acid sequence of SEQ ID NO: 6, or both.
15. A tri-specific antibody or antigen-binding fragment thereof comprising a First binding domain that binds to human IL-17A and human IL-17F and a second binding domain that binds to human TNFa, wherein said first binding domain comprises the amino acid sequence of SEQ ID NO: 4 and said second binding domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain comprising the amino acid sequence of SEQ ID NO: 6. '
16. A tri-specific antibody or antigen-binding fragment thereof that binds to human IL-17A, human IL-17F, and human TNFa, wherein said antibody or antigen-binding fragment
5 thereof comprises the amino acid sequences of SEQ ID NOs: 5 and 7.
17. The tri-specific antibody or antigen-binding fragment thereof according to any one of claims 1,15 and 16, wherein said first binding domain and said second binding domain are joined by a peptide linker of greater than five amino acids.
18. The tri-specific antibody or antigen-binding fragment thereof according to claim 17, 10 wherein the amino acid residues of the peptide linker are selected from G, A, S, P, E, T,
D,and K.
19. The tri-specific antibody or antigen-binding fragment thereof according to claim 17, wherein the peptide linker comprises the amiilo acid sequence of SEQ ID NO: 16.
20. The tri-specific antibody according to any one of claims 1, 15 and 16, wherein said 15 - antibody is an antibody of isotype IgG.
21. The tri-specific antibody according to claim 20, wherein said antibody is of isotype subtype IgGl.
22. The tri-specific antibody or antigen-binding fragment thereof according to any one of claims 1,15 and 16, wherein the binding molécule comprises an Fc région with at least 20 one mutation that reduces the antibody-dependent cell-mediated cytotoxicity (ADCC) and/or the complément dépendent cytotoxicity (CDC) in comparison to the same binding molécule without the mutation.
23. The tri-specific antibody or antigen-binding fragment thereof according to any one of claims 1, 15 and 16, wherein said binding molécule has at least one of the following 25 properties:
a) a KD for IL-17A of 10-11 M or less;
b) a KD for IL-17F of 10-7 M or less;
c) a KD for TNFa of 10-11 M or less;
d) inhibits the activity of IL-17A arid/or IL-17A/TNFa by at least 50% at concentrations below 200 ng/mL in an IL-6 assay in HT1080 cells;
.e) inhibits IL-17A-dependent IL-6 release by HT080 cells;
f) inhibits production of IL-6 in HT 1080 cells;
g) inhibits TNFa-dependent cytotoxicity by at least 50% at concentrations below 100 ng/ml, in WEHI-13VAR cells;
h) does not bind to rabbit, mouse, or guinea pig TNFa;
i) simultaneously binds to IL-17A and FNFa;
j) binds to cynomolgus IL-17A and TNFa;
k) remains stable after seven days of incubation at 37°C in human sérum;
1) has a half-life of over 12 days in vivo;
m) has a pro-inflammatory effect in an in vivo arthritis model; and
1) inhibits the activity of IL-17 and TNFa in vivo.
24. A pharmaceutical composition comprising the tri-specific antibody or antigen-binding fragment thereof according to any one of claims 1 -23 and a pharmaceutically acceptable excipient.
25. An isolated nucleic acid molécule that encodes the tri-specific antibody or antigenbinding fragment thereof according to any one of claims 1-23.
26. N vector comprising an isolated nucleic acid molécule according to claim 25, wherein said vector further comprises an expression control sequence.
27. A host cell comprising an isolated nucleic acid molécule according to claim 25 or a vector according to claim 26.
28. A method for producing the tri-specific antibody or antigen-binding fragment thereof according to any one of claims 1-23, comprising providing a host cell according to claim 27, cultivating said host cell under conditions suitable for expression of the tri-specific antibody or antigen-binding fragment thereof, and isolating the resulting tri-specific antibody or antigen-binding fragment thereof.
29. A tri-specific antibody or antigen-binding fragment thereof according to any one of daims 1-23 or the pharmaceutical composition according to claim 24 for use in a method for treâting a disorder mediated by IL-17A, IL-17F, or TNFa, the method comprising administering the tri-specific antibody or antigen-binding fragment thereof or the 5 pharmaceutical composition to a patient in need thereof.
30. A tri-specific antibody or antigen-binding fragment thereof according to any one of daims 1-23 or the pharmaceutical composition according to claim 24 for use in a method for treâting rheumatoid arthritis, psoriasis, or psoriatic arthritis, the method comprising administering the tri-specific antibody or antigen-binding fragment thereof or the 10 pharmaceutical composition to a patient in need thereof.
31. The tri-specific antibody or antigen-binding fragment thereof or the pharmaceutical composition for use according to claim 30, wherein said method further comprises administering an anti-inflammatory or immunosuppressive agent.
OA1201800424 2016-04-29 2016-12-02 Trispecific antibodies against II-17A, II-17F and another proinflammatory molecule. OA19281A (en)

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