Binding protein for human interleukin-6 receptor

Abstract

This disclosure provides an antibody (or antigen-binding protein) comprising at least one, preferably two, antigen-binding domains that bind to the human IL-6 receptor, wherein the antibody inhibits IL-6 transsignaling via the human soluble IL-6 receptor while maintaining IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antibody is capable of binding to both the human soluble IL-6 receptor and the human membrane-bound IL-6 receptor. Compositions comprising the antibody or antigen-binding protein are also provided, along with encoding nucleic acid molecules and expression vectors.

Description

Technical Field

[0001] As a whole, the present invention relates to antibodies and binding proteins that bind to the human interleukin-6 receptor (IL-6R), particularly anti-IL-6R antibodies that inhibit IL-6-mediated trans-signaling while maintaining (e.g., having no effect on) IL-6-mediated classical signaling. Such anti-IL-6R antibodies or binding proteins have therapeutic uses, such as the treatment of inflammatory diseases and cancer. Compositions, methods, and kits based on binding proteins and antibodies are also provided.

Background Art

[0002] Interleukin-6 (IL-6) is a pleiotropic cytokine that functions as both a pro-inflammatory mediator and an immunomodulatory or anti-inflammatory mediator depending on the signaling mode (Non-Patent Document 1). IL-6 plays an important role in both innate and acquired immunity, in the maintenance and regeneration of nerve cells, and as a myokine in the regulation of metabolism (Non-Patent Document 1, supra). IL-6 is also a well-known growth factor for tumor cells and plays a major role in inflammatory diseases and cancer (Non-Patent Document 1, supra).

[0003] The interleukin-6 receptor (IL-6R) functions as a component of the signaling complex for IL-6. IL-6R exists in two forms: a membrane-bound form (membrane-bound IL-6R) expressed on the cell membrane of a limited number of cells (e.g., neutrophils, naive T cells, macrophages, monocytes, and hepatocytes), and a soluble form (soluble IL-6R, sIL-6R) present in the extracellular space (e.g., in the systemic circulation) (Non-Patent Document 1, supra).

[0004] IL-6 signaling via IL-6R can occur through two distinct pathways: classical signaling and transsignaling. Both pathways are initiated by the interaction of circulating IL-6 cytokines with IL-6R and depend on subsequent interactions between the IL-6 / IL-6R complex and the ubiquitously expressed (e.g., expressed on virtually all cells) transmembrane protein gp130 (Non-Patent Literature 1, above). A soluble form of gp130 (sgp130) also exists in the extracellular space. When IL-6 binds to membrane-bound IL-6R or soluble IL-6R, dimerization of membrane-bound gp130 activates signaling via members of the JAK tyrosine kinase family, leading to phosphorylation and activation of the transcription factor STAT3 at Y705 (Non-Patent Literature 2). Activated STAT3, along with other activated transcription factors, can induce the expression of downstream target genes involved in differentiation, survival, apoptosis, and proliferation, and can activate pro-inflammatory or anti-inflammatory pathways depending on the signaling pathway used (i.e., classical or trans) (Non-Patent Literature 1, Non-Patent Literature 2, above).

[0005] In the case of IL-6 classical signaling, signaling via membrane-bound IL-6R is limited to cells that express IL-6R on their cell surface. IL-6 classical signaling is recognized as having anti-inflammatory properties and playing a crucial role in protective, homeostatic, and regenerative functions, including proliferation, inhibition of apoptosis, and defense against bacterial infection (Non-Patent Literature 1, above).

[0006] In IL-6 transsignaling, IL-6 binds to soluble IL-6R in the extracellular space, and subsequent binding to gp130 on the cell membrane stimulates IL-6 transsignaling. Because gp130 is ubiquitously expressed, IL-6 transsignaling can occur in all (or essentially all) cells of the body. Therefore, such IL-6 transsignaling can occur even in cells that do not express membrane-bound IL-6R. In other words, the expression of IL-6R on the cell surface is not required for transsignaling. IL-6 transsignaling is pro-inflammatory and is recognized to play an important role in pathological inflammation and cancer (Non-Patent Literature 1, above).

[0007] It is thought that molecules capable of interacting with the IL-6 / soluble IL-6R transsignaling complex to form an inactive complex, such as soluble gp130, lower free extracellular IL-6 levels, and that a natural buffering system that prevents indiscriminate IL-6 signaling regulates extracellular IL-6 levels (Non-Patent Literature 3).

[0008] The crucial role of IL-6 transsignaling in disease has led to the development of antibodies and other types of biologics that inhibit IL-6 signaling, such as Silvant (siltuximab), Actemra (tocilizumab), and Kevzara (sarilumab), which are monoclonal antibodies targeting human IL-6 or IL-6R (Non-Patent Literature 1, Non-Patent Literature 2, see above). These therapeutics interfere with both classical and transsignaling of IL-6. Thus, such antibodies can lead to adverse effects for patients resulting from the inhibition of classical IL-6 signaling, in addition to other functionally relevant cytokine signaling pathways (e.g., susceptibility to bacterial infections, neutropenia, malignancies, blood cell count changes, gastrointestinal perforation, hepatic signs, and cardiovascular risks).

[0009] While antibodies that selectively target mouse IL-6 transsignaling have been described previously, methods for developing antibodies against the corresponding epitopes on human IL-6R have failed to selectively inhibit human IL-6 transsignaling, highlighting the complex problem of developing antibodies that selectively inhibit IL-6 transsignaling in humans (Non-Patent Literature 4).

[0010] A potential alternative to inhibiting human IL-6 transsignaling targets the human soluble IL-6R / IL-6 complex. However, this approach may result in undesirable inhibition of IL-6 classical signaling due to IL-6 trapping in the IL-6 receptor complex, and therefore does not result in selective inhibition of IL-6 transsignaling. Furthermore, this approach may result in off-target inhibition of other signaling pathways that require gp130 for signaling, such as inhibition of IL-11 signaling due to IL-11 trapping in the IL-11 receptor complex, thus leading to undesirable and potentially harmful off-target effects.

[0011] Other biologics targeting the human soluble IL-6R / IL-6 complex have also been described. For example, VHH6, a single-domain antibody that binds to the soluble IL-6R / IL-6 complex, has been found to capture IL-6 in the complex, resulting in increased IL-6 transsignaling, which is predicted to lead to local or systemic pathological inflammation (Non-Patent Literature 3, above). Such capture of IL-6 may also result in the undesirable inhibition of IL-6 classical signaling discussed above. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Rose-John et al., Cytokine, Vol. 144, p. 155577 (2021) [Non-Patent Document 2] Johnson et al., Nature Reviews Clinical Oncology, Vol. 15, No. 4, pp. 234-248 (2018). [Non-Patent Document 3] Baran et al., Journal of Biological Chemistry, Vol. 293, No. 18, pp. 6762-6775 (2018). [Non-Patent Document 4] Lacroix et al., Journal of Biological Chemistry, Vol. 290, No. 45, pp. 26943-26953 (2015). [Overview of the project] [Means for solving the problem]

[0013] Therefore, there is a need for alternative and preferably improved therapeutic agents that have improved function and, ideally, an improved side effect profile in humans. Specifically, there is a need for therapeutic agents that selectively inhibit human IL-6 transsignaling (i.e., do not inhibit or significantly inhibit IL-6 classical signaling), preferably do not capture IL-6 in the soluble IL-6 receptor complex (or other inactive complex), and / or do not inhibit other gp130-dependent signaling pathways, such as IL-11 signaling. Surprisingly, we have identified such antibodies that bind to the human IL-6 receptor and have the ability to selectively inhibit or target IL-6 transsignaling in contrast to IL-6 classical signaling in humans, for example, the ability to selectively inhibit or target the IL-6 transsignaling ring without affecting IL-6 classical signaling in humans.

[0014] Accordingly, the present invention provides an alternative and improved therapeutic option in the form of an antibody and binding protein (antigen-binding protein) that binds to human IL-6R and inhibits IL-6 transsignaling while maintaining (e.g., not affecting) IL-6 classical signaling. Thus, the antibodies produced by the inventors have advantageous properties that make them ideal agents for the therapeutic applications described above and other applications described elsewhere in this specification.

[0015] As will be described elsewhere in this specification, the antibodies of the present invention have been shown to bind to human IL-6R and inhibit IL-6 transsignaling via human soluble IL-6R while maintaining (e.g., not significantly affecting or significantly reducing) IL-6 classical signaling via human membrane-bound IL-6R.

[0016] Therefore, in its broadest sense, the present invention provides antigen-binding proteins, such as antibodies, that bind to (or are capable of binding to) human IL-6R and inhibit IL-6 transsignaling via human soluble IL-6R while maintaining (e.g., not significantly affecting or significantly reducing) IL-6 classical signaling (sometimes also called "classic" signaling) via human membrane-bound IL-6R.

[0017] From a different perspective, the present invention provides an antigen-binding protein, such as an antibody, comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, the antigen-binding protein or antibody inhibiting IL-6 transsignaling via human soluble IL-6R while maintaining IL-6 classical signaling via human membrane-bound IL-6R.

[0018] The present invention further provides an antigen-binding protein, such as an antibody, comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antigen-binding protein or antibody inhibits IL-6 transsignaling via human soluble IL-6R while maintaining IL-6 classical signaling via human membrane-bound IL-6R, and the antigen-binding protein or antibody can bind to both human soluble IL-6R and human membrane-bound IL-6R.

[0019] Such antibodies of the present invention (or other binding proteins of the present invention comprising, for example, the human IL-6R antigen-binding domain described herein) can be conveniently and advantageously used for the treatment of diseases associated with IL-6 signaling (preferably IL-6 transsignaling), particularly for the treatment of inflammatory diseases, CNS symptoms, and cancer. By inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling (for example, by selectively or specifically targeting IL-6 transsignaling), it is possible to treat the aforementioned diseases while preventing (or minimizing or limiting) the side effects associated with the inhibition of IL-6 classical signaling.

[0020] To the best of our knowledge, no other antibody or binding protein that binds to human IL-6R has been disclosed to possess the advantageous property of inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling in humans (e.g., not affecting, not significantly affecting, or not significantly reducing IL-6).

[0021] In one embodiment, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antigen-binding protein or antibody inhibits IL-6 trans-signaling via the human soluble IL-6 receptor while maintaining (e.g., not significantly affecting or not significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antigen-binding domain comprises a heavy-chain variable region containing three complementarity-determining regions (CDRs) and a light-chain variable region containing three CDRs. a) The heavy-chain variable region comprises (i) a variable heavy (VH) CDR1 comprising the amino acid sequence GFTFSSYA or a sequence substantially homologous thereto, (ii) a VH CDR2 comprising the amino acid sequence INSNGGST or a sequence substantially homologous thereto, (iii) a VH CDR3 comprising the amino acid sequence AREGYYTMDY or a sequence substantially homologous thereto, and / or (preferably "and"), the light-chain variable region comprises (iv) a variable light (VL) CDR1 comprising the amino acid sequence ESVDSYGNSF or a sequence substantially homologous thereto, (v) a VL CDR2 comprising the amino acid sequence LAS or a sequence substantially homologous thereto, (vi) a VL CDR3 comprising the amino acid sequence QQNNEDPYT or a sequence substantially homologous thereto, or b) The heavy-chain variable region comprises (i) a variable heavy (VH) CDR1 comprising the amino acid sequence GFTFSNYG or a sequence substantially homologous thereto, (ii) a VH CDR2 comprising the amino acid sequence INSNGGST or a sequence substantially homologous thereto, (iii) a VH CDR3 comprising the amino acid sequence ARDGNYVSDY or a sequence substantially homologous thereto, and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPWT or a sequence substantially homologous thereto, including, or c) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of TRDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, including, or d) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of MNSKGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGYYTMDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of KSVDSFGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, including, or e) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 having the amino acid sequence of ESVDSYGNRF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes.

[0022] In one embodiment, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two antigen-binding domains that bind to human IL-6R, wherein the antigen-binding protein or antibody inhibits IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. a) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSSYA or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of AREGYYTMDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are sequences that contain one, two, or three amino acid substitutions compared to these predetermined CDR sequences, or b) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGNYVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPWT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are sequences that contain one, two, or three amino acid substitutions compared to these predetermined CDR sequences, or c) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of TRDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are sequences that contain one, two, or three amino acid substitutions compared to these predetermined CDR sequences, or d) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of MNSKGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGYYTMDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of KSVDSFGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are sequences that contain one, two, or three amino acid substitutions compared to these predetermined CDR sequences, or e) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 having the amino acid sequence of ESVDSYGNRF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are sequences that contain one, two, or three amino acid substitutions compared to these predetermined CDR sequences.

[0023] In one embodiment, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two antigen-binding domains that bind to human IL-6R, wherein the antigen-binding protein or antibody inhibits IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. a) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSSYA or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence AREGYYTMDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or b) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a substantially homologous sequence, wherein the substantially homologous sequence is a VH CDR1 comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGNYVSDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPWT or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or c) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence TRDGNFVSDY or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or d) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of MNSKGGST or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGYYTMDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of KSVDSFGNSF or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or e) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGNFVSDY or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNRF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, Includes.

[0024] In one embodiment, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. a) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSSYA or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of AREGYYTMDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are, for example, sequences containing one, two, or three amino acid substitutions compared to these given CDR sequences, or b) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGNYVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPWT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are, for example, sequences containing one, two, or three amino acid substitutions compared to these given CDR sequences, or c) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of TRDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are, for example, sequences containing one, two, or three amino acid substitutions compared to these given CDR sequences, or d) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of MNSKGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGYYTMDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of KSVDSFGNSF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are, for example, sequences containing one, two, or three amino acid substitutions compared to these given CDR sequences, or e) The heavy chain variable region is, (i) Variable weight (VH)CDR1 containing the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereto, (ii) VH CDR2 containing the amino acid sequence of INSNGGST or a sequence substantially homologous thereto, (iii) VH CDR3 containing the amino acid sequence of ARDGNFVSDY or a sequence substantially homologous thereto, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 having the amino acid sequence of ESVDSYGNRF or a sequence substantially homologous thereto, (v) VL CDR2 containing the amino acid sequence of LAS or a sequence substantially homologous thereto, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, Includes, The substantially homologous sequences are, for example, sequences containing one, two, or three amino acid substitutions compared to these given CDR sequences.

[0025] In one embodiment, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. a) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSSYA or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence AREGYYTMDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or b) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGNYVSDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPWT or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or c) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence TRDGNFVSDY or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNSF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or d) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of MNSKGGST or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGYYTMDY or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of KSVDSFGNSF or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, including, or e) The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (ii) A VH CDR2 comprising the amino acid sequence of INSNGGST or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, (iii) A VH CDR3 comprising the amino acid sequence of ARDGNFVSDY or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence, including and / or (preferably "and") The aforementioned light chain variable region is (iv) A variable light (VL) CDR1 comprising the amino acid sequence of ESVDSYGNRF or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions compared to a given CDR sequence, (v) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereof, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution compared to a given CDR sequence, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions compared to a given CDR sequence, Includes.

[0026] In a preferred embodiment, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antibody (or binding protein) inhibits IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. a) The heavy chain variable region is, (i) Variable weight (VH) CDR1 containing the amino acid sequence GFTFSSYA, (ii) VH CDR2 containing the amino acid sequence of INSNGGST, (iii) VH CDR3 containing the amino acid sequence AREGYYTMDY, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF, (v) VL CDR2 containing the amino acid sequence of LAS, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT, including, or b) The heavy chain variable region is, (i) Variable weight (VH) CDR1 containing the amino acid sequence of GFTFSNYG, (ii) VH CDR2 containing the amino acid sequence of INSNGGST, (iii) VH CDR3 containing the amino acid sequence ARDGNYVSDY, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF, (v) VL CDR2 containing the amino acid sequence of LAS, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPWT, Includes, or, c) The heavy chain variable region is, (i) Variable weight (VH) CDR1 containing the amino acid sequence of GFTFSNYG, (ii) VH CDR2 containing the amino acid sequence of INSNGGST, (iii) VH CDR3 containing the amino acid sequence TRDGNFVSDY, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNSF, (v) VL CDR2 containing the amino acid sequence of LAS, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT, including, or d) The heavy chain variable region is, (i) Variable weight (VH) CDR1 containing the amino acid sequence of GFTFSNYG, (ii) VH CDR2 containing the amino acid sequence of MNSKGGST, (iii) VH CDR3 containing the amino acid sequence of ARDGYYTMDY, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of KSVDSFGNSF, (v) VL CDR2 containing the amino acid sequence of LAS, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT, including, or e) The heavy chain variable region is, (i) Variable weight (VH) CDR1 containing the amino acid sequence of GFTFSNYG, (ii) VH CDR2 containing the amino acid sequence of INSNGGST, (iii) VH CDR3 containing the amino acid sequence ARDGNFVSDY, including and / or (preferably "and") The aforementioned light chain variable region is (iv) Variable light (VL)CDR1 containing the amino acid sequence of ESVDSYGNRF, (v) VL CDR2 containing the amino acid sequence of LAS, (vi) VL CDR3 containing the amino acid sequence of QQNNEDPYT, Includes.

[0027] In another aspect, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, the antibody (or binding protein) inhibiting IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, the antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. The aforementioned heavy chain region is a) A variable weight (VH) CDR1 comprising the amino acid sequence GFTFSX6YX8, wherein X6 and X8 are any amino acids, where X6 is preferably N or S, and / or X8 is preferably G or A. b) A VH CDR2 comprising the amino acid sequence X1NSX4GGST, wherein X1 and X4 are any amino acids, where X1 is preferably I or M, and / or X4 is preferably N or K. c) A VH CDR3 comprising the amino acid sequence X1RX3GX5X6X7X8DY, where X1, X3, X5, X6, X7, and X8 are any amino acids, where X1 is preferably A or T, X3 is preferably D or E, X5 is preferably N or Y, X6 is preferably Y or F, X7 is preferably V or T, and / or X8 is preferably S or M. Preferably, the VH CDR3 amino acid sequence comprises ARX3GYYTMDY, where X3 is any amino acid, preferably E or D, or the VH CDR3 amino acid sequence comprises X1RDGNX6VSDY, where X1 and X6 are any amino acids, where X1 is preferably A or T, and / or X6 is preferably F or Y. VH CDR3 and, including and / or (preferably "and") The aforementioned light chain variable region is d) A variable light (VL) CDR1 comprising the amino acid sequence X1SVDSX6GNX9F, wherein X1, X6, and X9 are any amino acids, X1 is preferably E or K, X6 is preferably Y or F, and / or X9 is preferably S or R. e) A VL CDR2 comprising the amino acid sequence of LAS or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, or three amino acid substitutions, preferably one amino acid substitution. f) A VL CDR3 comprising the amino acid sequence QQNNEDPX8T, wherein X8 is any amino acid, preferably Y or W, Includes.

[0028] In some embodiments, VH CDR1 has the amino acid sequence GFTFSX6YX8 or includes . In these embodiments, X6 and X8 may be any amino acid. Preferably, one or both of these X residues are selected from the following group, where X6 is N or S and X8 is G or A. Thus, preferred VH CDR1 has the amino acid sequence GFTFS[N / S]Y[G / A] or includes . For example, preferred VH CDR1 sequences in this embodiment have the sequence GFTFSNYG or GFTFSSYA or include .

[0029] In some embodiments, VH CDR2 has the amino acid sequence X1NSX4GGST or includes . In these embodiments, X1 and X4 may be any amino acid. Preferably, one or both of these X residues are selected from the following group, where X1 is I or M and X4 is N or K. Thus, preferred VH CDR2 has the amino acid sequence [I / M]NS[N / K]GGST or includes . For example, preferred VH CDR2 sequences in this embodiment have the sequence INSNGGST or MNSKGGST or include .

[0030] In some embodiments, VH CDR3 has the amino acid sequence X1RX3GX5X6X7X8DY or includes . In these embodiments, X1, X3, X5, X6, X7, and X8 may be any amino acid. Preferably, one or more of these X residues, for example one, two, three, four, five, or six, most preferably all, are selected from the following groups: X1 is A or T, X3 is D or E, X5 is N or Y, X6 is Y or F, X7 is V or T, and X8 is S or M. Thus, a preferred VH CDR3 has the amino acid sequence [A / T]R[D / E]G[N / Y][Y / F][V / T][S / M]DY or includes . For example, a preferred VH CDR3 sequence in this embodiment has or includes the sequence AREGYYTMDY, ARDGNYVSDY, TRDGNFVSDY, ARDGYYTMDY, or ARDGNFVSDY.

[0031] In a preferred embodiment, VH CDR3 has the amino acid sequence ARX3GYYTMDY or comprises . In this embodiment, X3 may be any amino acid. Preferably, the X residue is selected from the following group, where X3 is E or D. Therefore, a preferred VH CDR3 has the amino acid sequence AR[E / D]GYYTMDY or comprises . For example, a preferred VH CDR3 sequence in this embodiment has the sequence AREGYYTMDY or ARDGYYTMDY or comprises .

[0032] In another preferred embodiment, the VH CDR3 has the amino acid sequence X1RDGNX6VSDY or comprises . In this embodiment, X1 and X6 may be any amino acid. Preferably, one or both of these X residues are selected from the following group, where X1 is A or T and X6 is F or Y. Thus, an alternative preferred VH CDR3 has the amino acid sequence [A / T]RDGN[F / Y]VSDY or comprises . For example, preferred VH CDR3 sequences in this embodiment have the sequence ARDGNYVSDY, TRDGNFVSDY or ARDGNFVSDY or comprises .

[0033] In some embodiments, VL CDR1 has the amino acid sequence X1SVDSX6GNX9F or includes . In these embodiments, X1, X6, and X9 may be any amino acids. Preferably, one or more of these X residues, for example one, two, or three, most preferably all, are selected from the following group: X1 is E or K, X6 is Y or F, and X9 is S or R. Thus, preferred VL CDR1 has the amino acid sequence [E / K]SVDS[Y / F]GN[S / R]F or includes . For example, preferred VL CDR1 sequences in this embodiment have the sequence ESVDSYGNSF, KSVDSFGNSF, or ESVDSYGNRF or include .

[0034] In some embodiments, VL CDR2 has or includes the amino acid sequence of LAS or a sequence substantially homologous thereto. A preferred substantially homologous sequence is one that includes one, two, or three amino acid substitutions, more preferably one or two amino acid substitutions, and most preferably one amino acid substitution.

[0035] In some embodiments, VL CDR3 has the amino acid sequence QQNNEDPX8T or includes . In these embodiments, X8 may be any amino acid. Preferably, the X residue is selected from the following group: X8 is Y or W. Therefore, preferred VL CDR3 has the amino acid sequence QQNNEDP[Y / W]T or includes . For example, preferred VL CDR3 sequences in this embodiment have the sequence QQNNEDPYT or QQNNEDPWT or include .

[0036] The present invention further provides an antigen-binding protein (e.g., an antibody) described in any of the above embodiments or aspects, which can bind to human soluble IL-6R and human membrane-bound IL-6R.

[0037] In some embodiments, the antibody (or antigen-binding protein) of the present invention comprises one or more of the above CDRs, for example, one, two, three, four, five, or all six of the above CDR sequences. A preferred antibody (antigen-binding protein) of the present invention comprises all three VH CDRs and / or (preferably "and") all three VL CDRs. Therefore, a particularly preferred antibody (or antigen-binding protein or antigen-binding domain) of the present invention comprises six CDRs.

[0038] In some embodiments, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antibody (or antigen-binding protein) inhibits IL-6 transsignaling via a soluble human IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via a human membrane-bound IL-6 receptor, and the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs) and a light chain variable region comprising three CDRs. (a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3, or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), and / or (preferably "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:4, or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity), or (b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:21 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), and / or (preferably "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:22 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity (for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity)), or (c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity thereto (for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto) and / or (preferably "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:40 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity), or (d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:57 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), and / or (preferably, "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:58 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity), or, (e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:75 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), and / or (preferably "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:76 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity).

[0039] In another embodiment, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, the antibody (or antigen-binding protein) inhibiting IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, wherein the antigen-binding domains (a) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), wherein the heavy chain variable region comprises three CDRs, preferably the amino acid sequences of GFTFSSYA, INSNGGST, and AREGYYTMDY or sequences substantially homologous thereto, as defined elsewhere herein, and / or (preferably "and") A light chain variable region comprising the amino acid sequence of SEQ ID NO:4 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably the amino acid sequences of ESVDSYGNSF, LAS, and QQNNEDPYT, or sequences substantially homologous thereto as defined elsewhere herein, or (b) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), wherein the heavy chain variable region comprises three CDRs, preferably the amino acid sequences of GFTFSNYG, INSNGGST, and ARDGNYVSDY or sequences substantially homologous thereto, as defined elsewhere herein, and / or (preferably "and") A light chain variable region comprising the amino acid sequence of SEQ ID NO:22 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably the amino acid sequences of ESVDSYGNSF, LAS, and QQNNEDPWT or sequences substantially homologous thereto as defined elsewhere herein, or (c) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:39 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), wherein the heavy chain variable region comprises three CDRs, preferably the amino acid sequences of GFTFSNYG, INSNGGST, and TRDGNFVSDY or sequences substantially homologous thereto, as defined elsewhere herein, and / or (preferably "and") A light chain variable region comprising the amino acid sequence of SEQ ID NO:40 or a substantially homologous sequence thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably comprising the amino acid sequences of ESVDSYGNSF, LAS, and QQNNEDPYT or substantially homologous sequences thereto as defined elsewhere herein, or (d) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:57 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), wherein the heavy chain variable region comprises three CDRs, preferably the amino acid sequences of GFTFSNYG, MNSKGGST, and ARDGYYTMDY or sequences substantially homologous thereto, as defined elsewhere herein, and / or (preferably "and") A light chain variable region comprising the amino acid sequence of SEQ ID NO:58 or a substantially homologous sequence thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably the amino acid sequences of KSVDSFGNSF, LAS, and QQNNEDPYT or sequences substantially homologous thereto as defined elsewhere herein, or (e) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:75 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably comprising the amino acid sequences of GFTFSNYG, INSNGGST, and ARDGNFVSDY or sequences substantially homologous thereto as defined elsewhere herein, and / or (preferably "and") A light chain variable region comprising the amino acid sequence of SEQ ID NO:76 or a sequence substantially homologous thereto (for example, a sequence having at least 80% sequence identity, for example, a sequence having at least 85%, 90%, 95%, or 98% sequence identity thereto), further comprising three CDRs, preferably comprising the amino acid sequences of ESVDSYGNRF, LAS, and QQNNEDPYT or sequences substantially homologous thereto as defined elsewhere herein. Includes.

[0040] In a preferred embodiment, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, the antibody (or binding protein) inhibiting IL-6 transsignaling via the human soluble IL-6 receptor while maintaining (e.g., without significantly affecting or significantly reducing) IL-6 classical signaling via the human membrane-bound IL-6 receptor, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs) and at least one light chain variable region comprising three CDRs. (a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3 and / or (preferably "and") The light chain variable region contains the amino acid sequence of SEQ ID NO:4, or (b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:21 and / or (preferably "and") The light chain variable region contains the amino acid sequence of SEQ ID NO:22, or (c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 and / or (preferably "and") The light chain variable region contains the amino acid sequence of SEQ ID NO:40, or (d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:57 and / or (preferably "and") The light chain variable region contains the amino acid sequence of SEQ ID NO:58, or (e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:75 and / or (preferably "and") The light chain variable region includes the amino acid sequence of SEQ ID NO:76.

[0041] The present invention further provides an antibody (or antigen-binding protein) described in any of the above embodiments or aspects, the antigen-binding protein or antibody being capable of binding to human soluble IL-6R and human membrane-bound IL-6R.

[0042] In another embodiment, the present invention provides an antibody (or antigen-binding protein), for example, an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs) and at least one light-chain variable region comprising three CDRs, the heavy-chain variable region and / or (preferably "and") the light-chain variable region as described elsewhere herein.

[0043] In alternative embodiments of the present invention, a sequence shown to have sequence identity with a predetermined sequence, for example, a sequence having a predetermined SEQ ID NO, may have at least 55%, 60%, 65%, 70%, or 75% identity with that sequence, or at least 96%, 97%, or 99% identity with that sequence. Other appropriate levels of sequence identity are provided elsewhere in this specification.

[0044] The CDR sequences of the specific antibodies of the present invention are specified in Tables A, B, C, D, E, and F herein. In some other embodiments, the CDR sequence of the antibody of the present invention is identified using any suitable method (or tool), for example, using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, Vol. 7, pp. 132-136 (1999), www.imgt.org), for example, Tables A, B, C, D, and E, or as shown in Chothia (e.g., Chothia, C, et al., Nature, Vol. 342, pp. 877-883 (1989), or Al-Lazikani et al., Journal of Molecular Biology, Vol. 273, pp. 927-948 (1997)), or the well-known method of Kabat (e.g., Kabat et al., "Sequences of Immunologically Important Proteins"). This may refer to the CDR sequences in the VH and VL domains of the antibody of the present invention, as identified by "Proteins of Immunological Interest," 5th edition, edited by the Public Health Service, National Institutes of Health, Bethesda, Maryland (MD), pp. 647–669 (1991)) or by AbM numbering (e.g., Abhinandan and Martin, Molecular Immunology, Vol. 45, pp. 3832–3839 (2008)).

[0045] Anti-IL-6R antibodies (and antigen-binding proteins) based on the C07, C09, C12, C19, and C20 antibody sequences specified in Tables A, B, C, D, and E, respectively, are preferred. The CDR domain, FR domain, VH, and VL domains are shown in Tables A, B, C, D, and E herein. Antibodies (or antigen-binding proteins) containing the CDR domain or these pairs of VH and VL domains, or IgG-containing formats containing such domains (or substantially homologous sequences), such as IgG1, or full-length antibody formats, are preferred embodiments of the present invention.

[0046] Accordingly, preferred antigen-binding proteins of the present invention are or include the C07, C09, C12, C19, or C20 antibodies (or their six CDRs or their VH and VL domains) as specified in Tables A, B, C, D, and E, respectively. Antigen-binding proteins having sequences substantially homologous thereto, as defined elsewhere herein, are also preferred.

[0047] In some embodiments, the preferred antigen-binding protein of the present invention is a C20 antibody (or its six CDRs or their VH and VL domains) or a sequence substantially homologous thereto, or comprises the above.

[0048] In some embodiments, the preferred antigen-binding protein of the present invention is a C20, C07, C12, or C19 antibody (or its six CDRs, or their VH and VL domains) or a sequence substantially homologous thereto.

[0049] An antigen-binding protein according to any aspect of the present invention and disclosure may be defined as a binding protein comprising at least one antigen-binding domain, which includes an antigen-binding domain, for example, obtained from or derived from an antibody, or based on or corresponding to the antigen-binding domain of an antibody. Thus, for example, the light chain and heavy chain variable domains (i.e., light chain and heavy chain variable regions) described herein are obtained from or derived from an antibody, or based on or corresponding to the antigen-binding domain of an antibody. In other words, the antigen-binding domains referred to herein generally include a heavy chain variable region containing three CDRs and a light chain variable region containing three CDRs.

[0050] As described above, the present invention provides antigen-binding proteins, such as antibodies, or antigen-binding proteins comprising an antibody or an antigen-binding domain of an antibody, that bind to (or specifically recognize or specifically bind to) human IL-6R. Preferred antigen-binding proteins of the present invention are antibodies or their antigen-binding fragments (also known as antibody fragments, e.g., Fab fragments). The term "antibody" is used as an abbreviation for "antibody or antigen-binding fragment of an antibody" unless otherwise specified.

[0051] However, the embodiments described herein relating to antibodies are equally applicable to other types of antigen-binding proteins with necessary modifications, and vice versa. Thus, other antigen-binding proteins may include the antibodies of the present invention, or may include the antigen-binding domains of the antibodies of the present invention, for example, three VL CDR regions and three VH CDR regions of the antibody of the present invention, or one VL domain and one VH domain of the antibody of the present invention (the domains typically include, as appropriate, three VH or three VL CDR regions (CDR1, CDR2, and CDR3) and four VH or four VL framework ("FR") regions (FR1, FR2, FR3, and FR4)).

[0052] A preferred antigen-binding protein is any polypeptide chain capable of binding to (e.g., specifically binding to) human IL-6R. Suitable types of antigen-binding proteins that can be used in the present invention are known in the art. For example, in some embodiments, an immunoglobulin-based polypeptide is used, generally containing a CDR region (and optionally an FR region or immunoglobulin-based scaffold), so that the CDR region (and optionally an FR region) of the antibody of the present invention can be grafted onto a suitable scaffold or framework, such as an immunoglobulin scaffold. Alternatively, the antigen-binding domain or antibody of the present invention can be incorporated into any suitable antigen-binding fragment or antibody-containing format.

[0053] Other embodiments include immunoglobulin (Ig) forms, such as IgG, IgA, IgD, IgE, or IgM forms, or forms comprising all or part of the immunoglobulin constant region, such as all or part of the IgG, IgA, IgD, IgE, or IgM constant regions of various antibodies as defined herein, such as full-length (or whole) Ig or IgG, IgM, or IgA forms. The IgG form of the antibodies of the present invention described herein is preferred, preferably the full-length (or whole) IgG form (e.g., IgG1, IgG2, IgG3, or IgG4 forms), with IgG1 being the most preferred. A full-length IgG antibody typically comprises two substantially identical heavy chains (having appropriate variable and constant regions) and two substantially identical light chains (having appropriate variable and constant regions). In a preferred embodiment, the antibody, such as the IgG antibody, is monoclonal.

[0054] The IgG (or other Ig) form comprises a heavy chain variable region (VH) and a light chain variable region (VL) as described herein, and further comprises suitable IgG (or other Ig) heavy chain and light chain constant regions, for example, an IgG (or other Ig) Fc region. Sequences of such constant regions are well known and described in the art, and any of them may be used. Thus, these regions may be derived from any suitable source or species, such as mouse or human. Preferably, such IgG (or other Ig) sequence is a human IgG (or other) sequence, most preferably a human IgG1 sequence.

[0055] Other embodiments are antigen-binding fragments of the antibodies of the present invention as described herein (e.g., antibody fragments), such as fragments of C07, C09, C12, C19, and C20 antibodies (or antibodies based thereon, e.g., substantially homologous antibodies). Any type of antigen-binding fragment is assumed to include one or more antigen-binding domains of the present invention, i.e., one or more antigen-binding domains that bind to human IL-6R. Exemplary types of antigen-binding fragments are described elsewhere in this specification. In some embodiments, Fab fragments are preferred. A Fab fragment typically includes one heavy chain variable region (with a suitable heavy chain constant region or CH1 region) and one light chain variable region (with a suitable light chain constant region), and includes one antigen-binding domain. F(ab′)2 fragments may also be used. Thus, in some embodiments, the antibody is a Fab fragment or F(ab′)2 fragment, preferably a Fab fragment, containing a CDR sequence and / or heavy chain variable domain and / or light chain variable domain (or a sequence substantially homologous thereto), as described in Tables A, B, C, D, and E. In a preferred embodiment, the Fab fragment is monoclonal.

[0056] Accordingly, in some embodiments, the present invention provides a Fab fragment that binds to (or is capable of binding to) human IL-6R, inhibiting IL-6 transsignaling via human soluble IL-6R while maintaining IL-6 classical signaling via human membrane-bound IL-6R. Preferably, this Fab fragment binds to (or is capable of binding to) both human soluble IL-6R and human membrane-bound IL-6R. Other aspects of the present invention are applied to this aspect of the present invention with necessary modifications.

[0057] Particularly preferred Fab fragments include six CDR sequences (SEQ ID NO77-82) and / or heavy chain variable domain and / or light chain variable domain sequences (SEQ ID NO75 and 76) of the C20 antibody, or sequences substantially homologous thereto. These sequences are also shown in Table E.

[0058] Other preferred Fab fragments include six CDR sequences and / or heavy chain variable domain sequences and / or light chain variable domain sequences of the C07, C12, C19, or C09 antibodies shown in Tables A, C, D, or B, or sequences substantially homologous thereto.

[0059] In some embodiments, the antigen-binding protein or antibody of the present invention is covalently or noncovalently bound to a molecule that provides advantageous properties, such as reduced immunoantigenicity and / or increased circulating half-life. Such modifications, such as PEGylation, are known to those skilled in the art.

[0060] In some embodiments, the antigen-binding protein or antibody of the present invention is PEGylated.

[0061] The term “PEGylation” is used herein to describe the process of covalent and noncovalent attachment or association of polyethylene glycol (PEG) polymer chains to antibodies or binding proteins, and the antibody or binding protein is described as “PEGylated.” As is known to those skilled in the art, association of PEG with antibodies or binding proteins is known to maintain antigen binding while creating a stable hydrate layer that can reduce immunogenicity by limiting excretion by the renal system and extend the circulating half-life.

[0062] The antigen-binding protein or antibody CDR of the present invention is preferably separated by appropriate framework regions, such as those found in natural antibodies and / or effective designer antibodies. Therefore, the VH, VL, and individual CDR sequences of the present invention are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen (here, human IL-6R) binding. Such framework sequences or regions may appropriately correspond to natural framework regions FR1, FR2, FR3, and / or FR4 to form an appropriate scaffold, or they may correspond to a consensus framework region identified, for example, by comparing various natural framework regions. Alternatively, a non-antibody scaffold or framework, such as a T-cell receptor framework, may be used.

[0063] Suitable sequences that can be used for framework regions are well known in the art and described in the literature, and any of these may be used. Exemplary sequences for framework regions are one or more framework regions that make up the VH and / or VL domains of the antibody (or antigen-binding protein) of the present invention, for example, the framework regions of the C07, C09, C12, C19, or C20 antibodies disclosed in Table A or B or C or D or E, respectively, or framework regions substantially homologous thereto, and one or more framework regions that enable the maintenance of antigen specificity, for example, one or more framework regions that result in substantially the same or identical 3D structure of the antibody.

[0064] In certain embodiments, all four variable heavy chain and / or variable light chain framework regions (FRs) (disclosed, for example, in Tables A, B, C, D, and E) are appropriately, or substantially homologous, to FR regions found in the antibody (or binding protein) of the present invention.

[0065] In some embodiments, the antibodies of the present invention may be humanized antibodies. A “humanized” antibody based substantially on non-human variable region domains is an antibody in which certain amino acids have been modified to better correspond to amino acids typically present in human antibodies. Methods for generating humanized antibodies are well known in the art. For example, a humanized antibody can be achieved by inserting an appropriate CDR (e.g., a mouse / rat CDR, such as those present in the antibody of the present invention) into a human antibody “scaffold.” In some cases, one or more CDR residues may be modified to better correspond to amino acids typically present in human antibodies. Thus, in some embodiments, a set of six CDRs from the antibody of the present invention, for example, from the exemplary C07, C09, C12, C19, or C20 antibodies of the present invention or sequences substantially homologous thereto, may be present within the human antibody framework (or combined with, or inserted into, or grafted into) using an appropriate FR region found, for example, in a human antibody.

[0066] Exemplary C07, C09, C12, C19, and C20 antibodies of the present invention contain mouse / mouse VH and VL domains. Therefore, in certain embodiments, chimeric antibodies are preferred. Such antibodies typically contain mouse / mouse VH and VL domains (and related, substantially homologous sequences, e.g., described herein) with a constant region from another (non-mouse / rat) species, preferably a human constant region including, for example, a human Fc region, more preferably a human Ig region such as IgG, e.g., an IgG1 constant region. Therefore, chimeric human antibodies are preferred.

[0067] Exemplary antibodies of the present invention (C07, C09, C12, C19, and C20) that bind to human IL-6R and inhibit IL-6 transsignaling while maintaining IL-6 classical signaling were generated using a subtractive immunization protocol followed by phage presentation. The subtractive immunization technique is described, for example, in Sandrock, Journal of Immunological Methods, Vol. 100, pp. 73-82 (1987), and a specific protocol including an IP injection tolerance cycle using cells expressing human membrane-bound IL-6R (e.g., JV1 cells) followed by treatment with a cytotoxic agent (e.g., cyclophosphamide treatment) and IP immunization injection using recombinant human soluble IL-6R alpha is provided in the examples section of this specification. Other methods may be used, but such subtractive immunization protocols have been found to be particularly suitable for generating antibodies with the described functional properties. While phage presentation is a preferred technique for use in combination with such subtractive immunization protocols, other techniques may be used for antibody screening after subtractive immunization.

[0068] Therefore, the antibodies (or antigen-binding proteins) of the present invention bind to (or specifically recognize or specifically bind to) human IL-6R. Such antibodies (or antigen-binding proteins) may also bind to IL-6R from other species, for example, other mammalian species. However, as described elsewhere in this specification, in some embodiments, the antibodies (or antigen-binding proteins) of the present invention do not bind to (or do not significantly bind to) mouse IL-6R.

[0069] Antibodies (or antigen-binding proteins) can also bind to any suitable form of human IL-6R, such as human soluble IL-6R and / or (preferably "and") human membrane-bound IL-6R, specific examples of which are described elsewhere in this specification. Such binding is preferably dose-dependent, such that, for example, an increase in the concentration of the antibody (or binding protein) results in an increase in the level of binding to human IL-6R.

[0070] In preferred embodiments, the antibody (or antigen-binding protein) of the present invention binds to (or specifically recognizes or specifically binds to) human soluble IL-6R. Soluble IL-6R (sometimes also called the IL-6R alpha subunit) is generated by limited protein hydrolysis of membrane-bound IL-6R by the protease ADAM17 or by alternating splicing, and soluble IL-6R in the extracellular space can stimulate transsignaling in all cell types (when IL-6 is bound) because gp130 is ubiquitously expressed. IL-6 transsignaling via soluble IL-6R is recognized as fundamental in pathological inflammation and cancer and is a target for therapeutic purposes.

[0071] Therefore, in certain embodiments, the antibody (or antigen-binding protein) of the present invention can bind to human soluble IL-6R. Such antibodies (or antigen-binding proteins) may also bind to sIL-6R from other species, for example, other mammalian species. However, as described elsewhere in this specification, in some embodiments, the antibody (or antigen-binding protein) of the present invention does not bind (or does not significantly bind) to mouse soluble IL-6R.

[0072] This antibody (or antigen-binding protein) can bind to any suitable form of human soluble IL-6R. Preferred forms of human soluble IL-6R to which the antibody (or antigen-binding protein) of the present invention can bind include recombinant human soluble IL-6R, or native or natural human soluble IL-6R, such as human soluble IL-6R as it is present in the circulatory system. Such binding is preferably dose-dependent, for example, an increase in the concentration of the antibody (or binding protein) results in an increase in the level of binding to human soluble IL-6R.

[0073] The sequence of human soluble IL-6R is well known and described in the art. An exemplary sequence is provided herein (SEQ ID NO: 113). Recombinant human soluble IL-6R is also commercially available (e.g., PeproTech, catalog no. 200-06RC).

[0074] In some embodiments, the antibody (or antigen-binding protein) of the present invention binds to (or specifically recognizes or specifically binds to) human membrane-bound IL-6R. Membrane-bound IL-6R (typically composed of the IL-6R / IL-6R alpha complex and gp130 / IL-6R beta) is expressed on the cell surface of a limited number of cells (e.g., neutrophils, naive T cells, macrophages, monocytes, and hepatocytes). IL-6 classical signaling via membrane-bound IL-6R is limited to cells expressing IL-6R on their cell surface and is recognized to play a crucial role in protective, homeostatic, and regenerative functions, including proliferation, inhibition of apoptosis, and defense against bacterial infections.

[0075] Therefore, in certain embodiments, the antibody (or antigen-binding protein) of the present invention can bind to human membrane-bound IL-6R. Such antibodies (or antigen-binding proteins) may also bind to membrane-bound IL-6R from other species, for example, other mammalian species. However, as described elsewhere in this specification, in some embodiments, the antibody (or antigen-binding protein) of the present invention does not bind (or does not significantly bind) to mouse membrane-bound IL-6R.

[0076] The antibody (or antigen-binding protein) can bind to any suitable form of human membrane-bound IL-6R. Preferred forms of human membrane-bound IL-6R to which the antibody (or antigen-binding protein) of the present invention can bind include native or natural human membrane-bound IL-6R, for example, human IL-6R present on the cell surface (e.g., endogenously expressed or naturally / natively expressed human IL-6R on the cell surface), or human membrane-bound IL-6R expressed (or overexpressed) on the cell surface by recombination or genetic engineering of cells that do not naturally express membrane-bound IL-6R. Such binding is preferably dose-dependent, such that an increase in the concentration of the antibody (or binding protein) indicates an increase in the level of binding to human membrane-bound IL-6R.

[0077] The sequences of human membrane-bound IL-6R are well-known and documented in this field and can be obtained from various sequence databases. For example, Uniprot entry P08887 provides the sequence of human IL-6R subunit alpha (SEQ ID NO: 112), and Uniprot entry P40189 provides the sequence of human IL-6R subunit beta (gp130) (SEQ ID NO: 114).

[0078] Therefore, the antibody (or binding protein) of the present invention has the ability to bind to human soluble IL-6R and / or (preferably "and") human membrane-bound IL-6R, inhibiting IL-6 transsignaling via the human soluble IL-6 receptor while maintaining IL-6 classical signaling via the human membrane-bound IL-6 receptor.

[0079] Methods for evaluating antibodies (or antigen-binding proteins) (or their ability to bind) to human soluble or membrane-bound IL-6R may be any appropriate method that would be well known to those with common knowledge in this field, such as flow cytometry to determine binding to membrane-bound IL-6R and ELISA assays to determine binding to soluble IL-6R.

[0080] Therefore, in some embodiments, the binding (or ability to bind) of an antibody (or antigen-binding protein) to human soluble IL-6R may be evaluated using an ELISA assay. Those skilled in the art will be familiar with ELISA assays and will readily be able to establish appropriate conditions for evaluating the ability of an antibody (or antigen-binding protein) to bind to soluble IL-6R in such assays. For example, in a preferred assay, first, an ELISA plate is coated with biotinylated recombinant IL-6 presented in streptavidin (to allow proper folding of soluble IL-6R), and then incubated with recombinant human soluble IL-6R. Next, the plate is incubated with antibodies (or antigen-binding proteins) in any suitable format of various concentrations, for example, full-length (whole) antibodies (or antigen-binding proteins), and then the bound antibodies are detected and quantified. Typically, the antibodies (or antigen-binding proteins) of the present invention can bind to recombinant human soluble IL-6R in an ELISA assay, for example, the ELISA assay described above. In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention exhibits dose-dependent binding to recombinant human soluble IL-6R in an ELISA assay, such as the ELISA assay described above. In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention can bind to recombinant human soluble IL-6R (or be detected as significantly binding to or binding to) in an ELISA assay, such as the ELISA assay described above, when present at 1 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, or 500 ng / ml, or at least 1 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, or 500 ng / ml. Such values ​​may be appropriate, for example, when the antibody is in full-length (whole) antibody format, such as a full-length chimeric antibody. Such binding can be easily measured compared to a suitable control, such as no antibody or an unrelated control antibody (e.g., a non-IL-6R binding antibody).

[0081] In some embodiments, flow cytometry may be used to evaluate the binding (or ability to bind) of an antibody (or antigen-binding protein) to human membrane-bound IL-6R. Those skilled in the art will be familiar with flow cytometry assays and will readily be able to establish suitable conditions for evaluating the ability of an antibody (or antigen-binding protein) to bind to human IL-6R (membrane-bound IL-6R) on the cell surface in such assays. For example, in a preferred assay, cells expressing membrane-bound IL-6R are cultured in or without recombinant IL-6, incubated with an antibody (or antigen-binding protein) directly or indirectly conjugated with a fluorescent label, and subsequently analyzed (or detected or quantified or fluorescence-quantified) by flow cytometry. In a preferred embodiment, the cells used to evaluate the binding (or ability to bind) of an antibody (or antigen-binding protein) to human membrane-bound IL-6R are human THP1 cells (a human monocyte cell lineage derived from leukemia patients) or Ba / F3_gp130_IL-6R cells (a pre-mouse B cell lineage genetically designed to express human gp130 and human membrane-bound IL-6R), both of which express human IL-6R on their cell surface. Typically, the antibody (or antigen-binding protein) of the present invention can bind (or significantly bind, or be detected as binding) to human membrane-bound IL-6R in the presence or absence of IL-6, for example, in a flow cytometry assay such as the assay described above. Such binding can be conveniently measured by the mean increase in fluorescence of cells stained with the antibody of the present invention and cells stained with a secondary antibody compared to cells stained with a suitable control, for example, cells stained with a secondary antibody alone.

[0082] In some embodiments, the antibodies (or antigen-binding proteins) of the present invention do not bind (or do not significantly bind) to IL-6R in non-human species, such as mice (or rats) (e.g., they do not bind (or do not significantly bind) to soluble and / or (preferably "and") membrane-bound IL-6R, and / or (preferably "and") affect or inhibit (or do not significantly affect or inhibit) IL-6 classical signaling or IL-6 transsignaling in non-human species, such as mice (or rats). In other words, the preferred antibodies (or antigen-binding proteins) of the present invention do not cross-react with IL-6R in non-human species, such as mouse IL-6R (or rat IL-6R) (e.g., they do not cross-react (or do not significantly cross-react) with soluble and / or (preferably "and") membrane-bound IL-6R). The antibodies of the present invention, as illustrated, were shown not to bind (or not significantly bind) to mouse soluble IL-6R and mouse membrane-bound IL-6R. Furthermore, the antibodies of the present invention, as illustrated, were shown not to affect (or not significantly affect) IL-6 classical signaling or IL-6 transsignaling via mouse IL-6R.

[0083] The definition of “maintain (or not inhibit or significantly inhibit) IL-6 classical signaling” as used elsewhere in this Specification shall apply, after any necessary modifications, to “not affect or inhibit (or significantly affect or inhibit) IL-6 classical signaling or IL-6 transsignaling in a non-human species, e.g., a mouse (or rat).”

[0084] The sequences of the mouse membrane-bound IL-6R are well-known and documented in this field and can be obtained from various sequence databases. For example, Uniplot entry P22272 provides the sequence of the mouse IL-6R subunit alpha (SEQ ID NO: 115).

[0085] The sequences of mouse-soluble IL-6R are well known and described in the art. An exemplary sequence is provided herein (SEQ ID NO: 116). Recombinant mouse-soluble IL-6R is also commercially available (e.g., Bio-techne, catalog no. 1830-SR).

[0086] The antibodies (or antigen-binding proteins) of the present invention typically exhibit the ability to bind to human IL-6R, such as soluble and / or (preferably "and") membrane-bound human IL-6R, in or without the presence of IL-6; that is, they can bind to human IL-6R that is complexed with IL-6 or not complexed with IL-6, such as soluble and / or (preferably "and") membrane-bound human IL-6R. For example, the antibodies (or antigen-binding proteins) of the present invention typically exhibit the ability to bind to human membrane IL-6R in or without the presence of IL-6. In other words, the presence or absence of IL-6 does not affect the ability of the antibodies (or antigen-binding proteins) to bind to human IL-6R, such as human membrane-bound IL-6R and / or human soluble IL-6R.

[0087] The antibody (or antigen-binding protein) of the present invention has the ability to bind to human IL-6R (and was selected for this ability). Therefore, the binding site (or epitope) of the antibody (or antigen-binding protein) of the present invention is located only on human IL-6R (e.g., soluble and / or membrane human IL-6R) or human IL-6R (e.g., soluble and / or membrane human IL-6R). In other words, the binding site (or epitope) does not contain any human IL-6 residues (or contains only human IL-6R residues). Therefore, the antibody (or antigen-binding protein) of the present invention has the ability to bind only to human IL-6R, and for example, exhibits binding only to measurable and / or significant human IL-6R (e.g., soluble and / or membrane human IL-6R) (e.g., binding at a functionally, biologically, or clinically meaningful level of affinity).

[0088] Therefore, the dominant epitope (or primary epitope or preferred epitope) to which the antibody (or antigen-binding protein) of the present invention is bound is considered to be human IL-6R (e.g., human soluble and / or membrane-bound IL-6R). In other words, the dominant epitope (or primary epitope or preferred epitope) does not contain any human IL-6 residues (or only contains human IL-6R residues).

[0089] More specifically, the antibody (or antigen-binding protein) of the present invention is thought to bind or specifically bind to an epitope (or dominant epitope or primary epitope or preferred epitope) that is located only in human IL-6R (e.g., soluble and / or membrane-bound human IL-6R) (or is located globally or overall). In other words, the epitope does not contain any human IL-6 residues (or contains only human IL-6R residues).

[0090] Therefore, in some embodiments, the antibody (or antigen-binding protein) of the present invention does not preferentially bind to the human IL-6 / IL-6R complex (e.g., soluble and / or membrane-bound human IL-6 / IL-6R complex) compared to human IL-6R (e.g., soluble and / or membrane-bound human IL-6R) alone. In other words, the preferred antibody (or antigen-binding protein) of the present invention does not bind to human IL-6 / IL-6R complexes (e.g., soluble and / or membrane-bound human IL-6 / IL-6R complexes) to a greater extent or with greater affinity than the antibody (or antigen-binding protein) of the present invention binds to human IL-6R (e.g., soluble and / or membrane-bound human IL-6R) alone, preferably to a significantly greater extent or with significantly greater affinity, when measured by a suitable assay as described elsewhere in this specification (e.g., flow cytometry for membrane-bound IL-6R or membrane-bound IL-6 / IL-6R complexes, or ELISA assay for soluble IL-6R or soluble IL-6 / IL-6R complexes). In other words, IL-6 (the presence of IL-6) does not affect or propagate the binding of the preferred antibody (or antigen-binding protein) of the present invention to human IL-6R (soluble and / or membrane-bound human IL-6R), preferably not significantly affecting or propagating it.

[0091] As used herein, the term “human IL-6R only,” for example, “soluble and / or membrane-bound human IL-6R only,” refers to human IL-6R when it is not complexed with another molecule, for example, when it is not complexed with IL-6 / human IL-6. This term may also refer to empty or unloaded human IL-6R.

[0092] Therefore, generally speaking, the antibodies (or antigen-binding proteins) of the present invention do not bind (or do not significantly bind) to IL-6 or human IL-6 alone. As used herein, the terms "IL-6 alone" or "human IL-6 alone" refer to IL-6 or human IL-6 when not complexed with another molecule, for example, when not complexed with IL-6R / human IL-6R. This term may also refer to free IL-6.

[0093] In this specification, the terms “dominant epitope,” “primary epitope,” “preferred epitope,” “first epitope,” etc., or equivalent terms, may refer to the epitope that is involved in the best or highest affinity binding between an antibody (or antigen-binding protein) and human IL-6R, such as human soluble IL-6R and / or human membrane-bound IL-6R.

[0094] The exemplified antibodies of the present invention exhibit similar functional properties (for example, binding to soluble and membrane-bound human IL-6R as defined elsewhere herein, inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling) and have relevant sequences, such as relevant CDR sequences; therefore, the exemplified antibodies of the present invention may bind to the same epitope on IL-6R (e.g., a common epitope). If the functional properties of the exemplified antibodies had not been previously described, the epitope to which the antibody (or antigen-binding protein) of the present invention is bound would be considered unique and previously undisclosed.

[0095] Without adhering strictly to theory, it is believed that the antibody (or antigen-binding protein) of the present invention binds to or specifically binds to epitopes on IL-6R that are not involved in, not significantly involved in, or unnecessary for, the IL-6 / IL-6R complex (e.g., soluble and / or membrane IL-6 / IL-6R complex). In other words, it is believed that the antibody (or antigen-binding protein) of the present invention binds to or specifically binds to epitopes on IL-6R that are not involved in, not significantly involved in, or unnecessary for IL-6 binding.

[0096] Therefore, the antibody (or antigen-binding protein) of the present invention does not capture or significantly capture IL-6 in complex with soluble IL-6R or other inactive complexes, and thus, the antibody (or antigen-binding protein) is considered to have no effect (or no significant effect) on the extracellular IL-6 buffer system. In other words, the antibody (or antigen-binding protein) does not affect (or has no significant effect) the equilibrium of extracellular IL-6, and / or does not reduce (or significantly reduce) the level or amount of free IL-6 in the extracellular space.

[0097] As used herein, the term "capture" refers to IL-6 complexing with other molecules (e.g., soluble IL-6R and soluble gp130), thereby rendering the complexed IL-6 unable to interact with other signaling molecules / transducers or induce other signaling pathways.

[0098] As used herein, the terms "IL-6 buffer system" and "equilibrium" refer to the ratio or relative concentration of free IL-6 to IL-6 complexed with other molecules, which determines the amount or level of free IL-6 in the extracellular space.

[0099] Accordingly, in some embodiments, the antibodies (or antigen-binding proteins) of the present invention have the ability to bind to human soluble IL-6R and / or (preferably "and") membrane-bound human IL-6R, inhibit IL-6 transsignaling while maintaining IL-6 classical signaling, and / or (preferably "and") not affect (or significantly affect) the IL-6 buffer system (or not reduce (or significantly reduce) the level or amount of free IL-6 in the extracellular space).

[0100] Undesirable off-target inhibition of IL-6 classical signaling, resulting from IL-6 being captured in complexes with other molecules (e.g., soluble IL-6R and soluble gp130), is evident under certain physiological conditions (e.g., when the extracellular concentration of soluble IL-6R exceeds the extracellular concentration of IL-6). The exemplified antibodies of the present invention have been shown to maintain IL-6 classical signaling (i.e., not inhibit or significantly inhibit IL-6 classical signaling) under these experimental conditions (i.e., when the concentration of soluble IL-6R exceeds the concentration of IL-6). Therefore, the exemplified antibodies of the present invention are not considered to exhibit the property of inhibiting IL-6 classical signaling due to IL-6 capture, as defined elsewhere in this specification.

[0101] Therefore, in some embodiments, the antibody (or antigen-binding protein) of the present invention inhibits IL-6 transsignaling while maintaining IL-6 classical signaling under conditions (e.g., experimental conditions, e.g., cell-based IL-6 proliferation assays described elsewhere herein) in which soluble IL-6R is used at an excess concentration compared to IL-6 (i.e., a higher concentration of soluble IL-6R than IL-6).

[0102] In some embodiments, the antibody (or antigen-binding protein) of the present invention has the ability to inhibit IL-6 transsignaling (for example, inhibiting IL-6 transsignaling mediated by (or induced by, or mediated by, or associated with) human soluble IL-6R. In some embodiments, such inhibition is dose-dependent, and for example, increasing the concentration of the antibody (or binding protein) results in an increasing level of inhibition of IL-6 transsignaling.

[0103] As described elsewhere in this specification, IL-6 transsignaling via soluble IL-6R is recognized as pro-inflammatory and contributing to pathological inflammation and cancer. Therefore, inhibition of IL-6 transsignaling by the antibodies (or antigen-binding proteins) of the present invention may favorably inhibit (or reduce or limit) pro-inflammatory signaling and inhibit (or reduce or limit) pathological inflammation associated with IL-6 signaling.

[0104] Therefore, in some embodiments, the antibodies (or antigen-binding proteins) of the present invention that have the ability to inhibit IL-6 transsignaling have the ability to inhibit (or reduce or limit) pro-inflammatory signaling and / or inhibit (or reduce or limit) pathological inflammation.

[0105] In some embodiments, the antibody (or antigen-binding protein) of the present invention has the ability to maintain IL-6 classical signaling (for example, maintaining IL-6 classical signaling via (or induced by, mediated by, or associated with) human membrane-bound IL-6R.

[0106] As described elsewhere in this specification, IL-6 classical signaling via membrane-bound IL-6R is recognized as anti-inflammatory and beneficial to protective, homeostatic, and regenerative functions (e.g., defense against bacterial infections). Therefore, the maintenance of IL-6 classical signaling by the antibody (or antigen-binding protein) of the present invention is highly advantageous. For example, such an antibody (or antigen-binding protein) should maintain or not affect (e.g., not reduce or inhibit, or significantly reduce or inhibit) anti-inflammatory signaling, and should maintain or not affect (e.g., not reduce or inhibit, or significantly reduce or inhibit) immune function (e.g., defense against bacterial infections).

[0107] Therefore, in some embodiments, the antibodies (or antigen-binding proteins) of the present invention that have the ability to maintain IL-6 classical signaling do not affect, reduce, inhibit, or interfere with IL-6 classical signaling (or do not significantly affect, reduce, inhibit, or interfere with IL-6 classical signaling). For example, such antibodies (or antigen-binding proteins) do not affect, reduce, inhibit, or interfere with anti-inflammatory signaling (or do not significantly affect, reduce, inhibit, or interfere with IL-6 classical signaling), and / or do not affect, reduce, inhibit, or interfere with immune function (such as beneficial immune function as protection against bacterial infection).

[0108] Therefore, in a preferred embodiment, the antibody (or antigen-binding protein) of the present invention has the advantageous property of inhibiting (or reducing or limiting) the effects of IL-6 transsignaling, or IL-6 transsignaling such as pro-inflammatory signaling and / or pathological inflammation, while maintaining (e.g., not significantly affecting, reducing, inhibiting, or interfering with) the effects of IL-6 classical signaling, or IL-6 classical signaling such as anti-inflammatory signaling and / or immune function (e.g., beneficial immune function such as protection against bacterial infection).

[0109] Appropriate methods and assays for measuring IL-6 classical signaling and IL-6 transsignaling are described elsewhere in this specification.

[0110] As discussed elsewhere in this specification, some embodiments of the present invention describe antibodies (or antigen-binding proteins) that bind to human IL-6R, for example, to human membrane-bound and soluble IL-6R, and these antibodies have the ability to inhibit transsignaling via soluble IL-6R while maintaining classical signaling via membrane-bound IL-6R.

[0111] It is remarkable that the antibody of the present invention binds to human soluble IL-6R and human membrane-bound IL-6R, yet possesses the ability to selectively inhibit IL-6 transsignaling. Without adhering to theory, it is thought that the antibody of the present invention selectively inhibits IL-6 transsignaling (for example, inhibiting transsignaling while maintaining classical signaling) through steric hindrance and / or stereoconversion of human soluble IL-6R. In this mechanism, binding of the antibody (or antigen-binding protein) of the present invention to a site on human IL-6R, for example, a site adjacent to the ligand (IL-6) binding site, may act to sterically hinder (or inhibit) the subsequent formation of the IL-6 / soluble IL-6R / gp130 signaling complex (hexameric signaling complex) in the cell membrane, while enabling (for example, not inhibiting, not significantly inhibiting, not significantly affecting, or maintaining) the formation of the IL-6 / membrane-bound IL-6R / gp130 signaling complex (hexameric signaling complex). Alternatively, or further, binding of the antibody (or antigen-binding protein) of the present invention may result in the stereoconversion of soluble IL-6R, thereby preventing refolding from forming the active IL-6 transsignaling receptor complex, while the fixation of membrane-bound IL-6R in the cell membrane may stabilize receptor folding and suppress the stereoconversion of membrane-bound IL-6R. Therefore, the formation of soluble IL-6R signaling complexes (hexameric signaling complexes thought to contain sIL-6R, 2IL-6:sIL-6 receptor, and gp130 dimers) is hindered, inhibited, or reduced by the antibody (or antigen-binding protein) of the present invention, while the formation of membrane-bound IL-6 signaling complexes (hexameric signaling complexes thought to contain membrane-bound IL-6R, 2IL-6:membrane-bound IL-6R dimers, and gp130 dimers) is not affected (or is not significantly affected, inhibited, or maintained) by the antibody (or antigen-binding protein) of the present invention.

[0112] Furthermore, the ability of the antibody (or antigen-binding protein) of the present invention to specifically bind to human IL-6R and selectively inhibit IL-6 transsignaling is advantageous in preventing undesirable off-target effects (e.g., inhibition of other signaling pathways or functions, including gp130). Without being bound by theory, the ability of the antibody (or antigen-binding protein) of the present invention to bind to (or specifically recognize or specifically bind to) IL-6R prevents off-target effects (e.g., inhibition of other signaling pathways, such as those containing gp130) that may occur when this antibody (or antigen-binding protein) or other biologics target (e.g., interfere with, inhibit, or include) a signaling component common to multiple signaling pathways (e.g., receptor coactivators or signal transducer components), such as gp130. For example, signaling via other IL-6 family cytokines such as IL-11 also requires the recruitment of gp130 for downstream signaling downstream of cytokine / receptor complex formation. The antibodies of the present invention, as illustrated, maintain (or do not inhibit or significantly inhibit) IL-11 signaling. Therefore, the antibodies (or antigen-binding proteins) of the present invention have the beneficial property of maintaining other signaling pathways that require gp130 for signaling (i.e., other than IL-6), such as IL-11.

[0113] Therefore, in some embodiments, the antibody (or antigen-binding protein) of the present invention inhibits IL-6 transsignaling while maintaining IL-6 classical signaling and maintaining (or not inhibiting or significantly inhibiting) other gp130-dependent signaling pathways.

[0114] The definition of “maintain (or not inhibit or significantly inhibit) IL-6 classical signaling” as used elsewhere in this specification applies to “maintain (or not inhibit or significantly inhibit) other gp130-dependent signaling pathways (e.g., IL-11 signaling)” with necessary modifications.

[0115] As used herein, “other gp130-dependent pathways” refers to, but is not limited to, one or more signaling pathways, such as cytokines, including IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin 1 (CT1), cardiotrophin-like cytokine (CLC), and IL-27.

[0116] Therefore, in some embodiments, the antibody (or antigen-binding protein) of the present invention inhibits IL-6 transsignaling while maintaining IL-6 classical signaling and maintaining IL-11 signaling.

[0117] In some embodiments, the antibody (or binding protein) of the present invention binds to (or specifically binds to) human IL-6R, inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling, and possesses the following properties: i. Soluble and / or (preferably "and") binds to membrane-bound human IL-6R, ii. In some non-human species, e.g., rats (or mice), IL-6R (soluble and / or (preferably "and") membrane-bound IL-6R) does not bind, and / or (preferably "and") in some non-human species, e.g., suitable non-human IL-6R, e.g., rat (or mouse) IL-6R, maintain (or do not inhibit or significantly inhibit) or affect IL-6 classical signaling or IL-6 transsignaling. iii. Maintain (or not inhibit or significantly inhibit) other gp130-dependent signaling pathways, for example, maintain (or not inhibit or significantly inhibit) IL-11 signaling. It has one or more of these, for example, two or three.

[0118] In some embodiments, the antibody (or binding protein) of the present invention has the following characteristics: iv. It does not preferentially bind to human IL-6 / IL-6R complexes (soluble and / or membrane-bound human IL-6 / IL-6R complexes) compared to human IL-6R (soluble and / or membrane-bound human IL-6R) alone (or does not bind with greater affinity or significantly greater affinity), v. Does not affect (or does not significantly affect) the IL-6 buffer system (or does not reduce (or significantly reduce) the level or amount of free IL-6 in the extracellular space), It further has one or both of the above.

[0119] Methods for evaluating IL-6 classical signaling and / or IL-6 transsignaling and / or IL-11 signaling (including IL-11 classical and transsignaling) may be any appropriate method and will be well known to those of ordinary skill in the art. Preferred methods or assays for evaluating the signaling pathways described herein are discussed.

[0120] In some embodiments, human IL-6 classical signaling may be evaluated using a cell model for cell proliferation (also referred to herein as a cell proliferation assay) in which cells express both membrane-bound IL-6R and gp130 (in this specification, human membrane-bound IL-6R and gp130) (endogenously and / or by genetic engineering) and are cultured in the presence of human IL-6 cytokines. This system can be used to measure (or evaluate or quantify) cell proliferation induced solely by IL-6 classical signaling, in which there is a lack of additional growth factors to IL-6 and a lack of soluble IL-6R.

[0121] In a preferred embodiment, to measure (or evaluate or quantify) human IL-6 classical signaling using a cell model for cell proliferation, the cell model is a cell line expressing human gp130 and human membrane-bound IL-6R, for example, a pre-mouse B cell line genetically engineered to express human gp130 and human membrane-bound IL-6R (Ba / F3_gp130_IL-6R). In a preferred embodiment, cells are cultured with human IL-6 cytokines (preferably 10 ng / ml) and treated with the antibodies (or antigen-binding proteins) of the present invention, for example C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at concentrations of 1 to 10 μg / ml, more preferably 1 or 10 μg / ml), or treated with an antibody-free control (i.e., a cytokine-only control). After an appropriate period, for example 48 hours, the number of metabolically active cells is measured (or quantified) by (for example, Cell Titer Blue). The number of viable cells (and therefore cell proliferation) is determined using the Blue reagent. In a preferred embodiment, the number of viable cells in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the number of viable cells in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided by and then multiplied by 100) relative to the number of viable cells in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of the antibody (or antigen-binding protein) to affect IL-6 classical signaling.

[0122] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using cell proliferation assays such as those described above, exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) human IL-6 classical signaling via human membrane-bound IL-6R. Therefore, when evaluated in such assays, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) the level of cell proliferation induced by IL-6 classical signaling (IL-6 classical signaling via human membrane-bound IL-6R) compared to a suitable control level or assay, e.g., the level of cell proliferation observed in the absence of the antibody (or antigen-binding protein) in the same assay except in the absence of the antibody (or antigen-binding protein). Thus, proliferation observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control. Exemplary maintenance of IL-6 classical signaling levels in terms of percentage levels of inhibition (here, low percentage levels of inhibition) is described elsewhere in this specification.

[0123] In some embodiments, human IL-6 transsignaling may be evaluated using a cell model for cell proliferation (also referred to herein as a cell proliferation assay) in which cells express gp130 (here human gp130) (endogenously or by genetic engineering) and are cultured in the presence of human soluble IL-6R and IL-6 cytokines. This system can be used to measure (or evaluate or quantify) only cell proliferation induced by IL-6 transsignaling, as it contains soluble IL-6R but lacks membrane-bound IL-6R and additional growth factors to IL-6.

[0124] In a preferred embodiment, to measure (or evaluate or quantify) human IL-6 transsignaling using a cell model for cell proliferation, the cell model used is a cell line expressing human gp130, for example, a mouse pre-B cell line (Ba / F3_gp130) genetically engineered to express human gp130. In a preferred embodiment, cells are cultured with human IL-6 cytokine (preferably at a concentration of 10 ng / ml) and human soluble IL-6R (preferably at a concentration of 100 ng / ml or 200 ng / ml) or Hyper-IL-6 (Hy-IL-6, a recombinant fusion protein consisting of IL-6 and soluble IL-6R (Peters et al., Journal of Immunology) The cells are cultured with the antibody (or antigen-binding protein) of the present invention, for example, C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at a concentration of 1 to 10 μg / ml, more preferably at a concentration of 1 or 10 μg / ml), or treated with an antibody-free control (i.e., a control with only cytokines and soluble IL-6R). After an appropriate period, for example 48 hours, the number of viable cells is determined by measuring (or quantifying) the number of metabolically active cells (for example, using cell titer blue reagent). (Thus cell proliferation) is determined. In a preferred embodiment, the number of viable cells in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the number of viable cells in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided and then multiplied by 100) to the number of viable cells in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of the antibody (or antigen-binding protein) to affect IL-6 transsignaling.

[0125] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using a cell proliferation assay such as the assay described above, exhibit the ability to inhibit (e.g., reduce or limit, or, for example, significantly reduce or limit) human IL-6 transsignaling via human soluble IL-6R. Therefore, when evaluated in such an assay, for example, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to inhibit (e.g., reduce or limit, or, for example, significantly reduce or limit) the level of cell proliferation induced by IL-6 transsignaling (transsignaling via human soluble IL-6R) compared to a suitable control level or assay, e.g., the level of cell proliferation observed in the absence of the antibody (or antigen-binding protein) in the same assay except in the absence of the antibody (or antigen-binding protein). Therefore, the proliferation observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control level. Exemplary levels of inhibition of IL-6 transsignaling (here, high percentage levels of inhibition) in terms of percentage levels of inhibition are described elsewhere in this specification.

[0126] In some embodiments, combined human IL-6 classical and transsignaling (also referred to herein as "IL-6 mixed signaling") may be evaluated using a cell model for cell proliferation (also referred to herein as a cell proliferation assay) in which cells express both membrane-bound IL-6R and gp130 (here, human membrane-bound IL-6R and gp130) (endogenously and / or by genetic engineering) and are cultured in the presence of both human IL-6 cytokine and soluble IL-6R. In the body, cells confront both IL-6 cytokine and soluble IL-6R, enabling combined IL-6 classical and transsignaling (mixed signaling) in cells expressing membrane-bound IL-6R. However, high expression of membrane-bound IL-6R is favorable for classical signaling (Reeh et al., Cell Communication and Signaling, Vol. 17, No. 1, p. 46 (2019)). Therefore, this system allows for the evaluation of classical signaling (and its maintenance) under more physiological conditions.

[0127] In a preferred embodiment, to measure (or evaluate or quantify) human IL-6 mixed signaling using a cell model for cell proliferation, the cell model is a cell line expressing human gp130 and membrane-bound IL-6R, for example, a mouse pre-B cell line genetically engineered to express human gp130 and human membrane-bound IL-6R (Ba / F3_gp130_IL-6R). In a preferred embodiment, the cells are cultured with human IL-6 cytokine (preferably at a concentration of 10 ng / ml) and human soluble IL-6R (preferably at a concentration of 100 ng / ml or 200 ng / ml) and treated with the antibody (or antigen-binding protein) of the present invention, for example, C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at a concentration of 1 to 10 μg / ml, more preferably at a concentration of 1 or 10 μg / ml), or treated with an antibody-free control (i.e., a control with only cytokine and soluble IL-6R). After an appropriate period, for example 48 hours, the number of viable cells (and therefore cell proliferation) is determined by measuring (or quantifying) the number of metabolically active cells (e.g., using cell titer blue reagent). In a preferred embodiment, the number of viable cells in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the number of viable cells in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided by 100) relative to the number of viable cells in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of the antibody (or antigen-binding protein) to affect IL-6 classical signaling.

[0128] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using a mixed signaling cell proliferation assay such as the assay described above, exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) human IL-6 classical signaling via human membrane-bound IL-6R. Therefore, when evaluated in such an assay, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) the level of cell proliferation induced by IL-6 classical signaling (IL-6 classical signaling via human membrane-bound IL-6R) compared to a suitable control level or the level of cell proliferation observed in the same assay except in the absence of the antibody (or antigen-binding protein). Thus, the proliferation observed in the absence of this antibody (or antigen-binding protein) is a suitable and exemplary control level. Exemplary maintenance of IL-6 classical signaling levels (here, low inhibition percentage levels) in terms of percentage levels of inhibition is described elsewhere in this specification.

[0129] In a preferred embodiment, human IL-11 classical, trans, and mixed signaling can be measured (or evaluated or quantified) using the above cell assay for IL-6 classical, trans, and mixed signaling, but by replacing Ba / F3_gp130_IL-6R cells with Ba / F3_gp130_IL-11R cells (expressing human gp130 and human membrane-bound IL-11R), replacing human IL-6 cytokine with human IL-11 cytokine (preferably 10 ng / ml), replacing human soluble IL-6R with human soluble IL-11R (preferably at a concentration of 200 ng / ml), and treating with the antibody (or antigen-binding protein) of the present invention at a preferred concentration of 10 μg / ml.

[0130] Exemplary antibodies (or antigen-binding proteins) of the present invention demonstrate the ability to maintain human IL-11 classical, trans, and mixed signaling (e.g., not significantly affecting, significantly reducing, inhibiting, or significantly inhibiting it), and evaluations of such assays described above for "maintaining IL-6 classical signaling" are applicable to this aspect of the present invention with necessary modifications.

[0131] In a preferred embodiment, the above cell assays for IL-6 classical, trans, and mixed signaling can be used, but Ba / F3_gp130_IL-6R cells are replaced with Ba / F3_gp130_muIL-6R cells (expressing human gp130 and murine membrane-bound IL-6R), human IL-6 cytokines are replaced with murine IL-6 cytokines (preferably 10 ng / ml), human soluble IL-6R is replaced with murine soluble IL-6R (preferably at a concentration of 200 ng / ml), and the cells are treated with the antibody (or antigen-binding protein) of the present invention at a preferred concentration of 10 μg / ml to measure (or evaluate or quantify) murine IL-6 classical, trans, and mixed signaling. In particular, murine IL-6R can form an active receptor complex with human gp130.

[0132] Exemplary antibodies (or antigen-binding proteins) of the present invention demonstrate the ability to maintain mouse IL-6 classical, trans, and mixed signaling (e.g., without significantly affecting, significantly reducing, inhibiting, or significantly inhibiting it), and evaluations of such assays described above for “maintaining IL-6 classical signaling” are applicable to this aspect of the present invention with necessary modifications.

[0133] In some embodiments, IL-3 signaling may be evaluated using a cell model for cell proliferation (also referred to herein as a cell proliferation assay), in which cells express membrane-bound IL-3R (here, mouse membrane-bound IL-3R) (endogenously and / or by genetic engineering) and are cultured in the presence of mouse IL-3 cytokines. This system lacks additional growth factors for IL-3 and can therefore be used to measure (or quantify) only the cell proliferation induced by IL-3 signaling, in which case the IL-6-independent effect of the antibody (or binding protein) of the present invention on cell proliferation can be evaluated.

[0134] In a preferred embodiment, to measure (or evaluate or quantify) IL-3 signaling using a cell model for cell proliferation, the cell model is a cell lineage expressing moulineal membrane-bound IL-3R, for example, a moulineal pre-B cell lineage (Ba / F3) that expresses moulineal membrane-bound IL-3R endogenously. In a preferred embodiment, cells are cultured with recombinant moulineal IL-3 cytokine (preferably 10 ng / ml) and treated with the antibody (or antigen-binding protein) of the present invention, for example C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at a concentration of 1 to 10 μg / ml, more preferably 1 or 10 μg / ml) or with an antibody-free control (i.e., a control of cytokine (IL-3) only). After a suitable period, for example 48 hours, the number of viable cells (and thus proliferating cells) is determined by measuring (or quantifying) the number of metabolically active cells (for example, using cell titer blue reagent). In a preferred embodiment, the number of viable cells in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the number of viable cells in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided and then multiplied by 100) to the number of viable cells in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of the antibody (or antigen-binding protein) to affect IL-3 signaling, or non-IL-6 signaling, or IL-6 independent signaling. In other words, they can be used to obtain an indicator of the specificity of the antibody (or antigen-binding protein) for IL-6R.

[0135] Preferred exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using a cell proliferation assay such as the assay described above, show no or only slight effect on IL-3 signaling via moulin membrane-bound IL-3R. Therefore, when evaluated in such an assay, such antibodies (or antigen-binding proteins) of the present invention show no or only slight effect on the level of cell proliferation induced by IL-3 signaling (IL-3 signaling via moulin membrane-bound IL-3R) compared to a suitable control level or the level of cell proliferation observed in the same assay except in the absence of the antibody (or antigen-binding protein). Thus, the proliferation observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control level.

[0136] In some embodiments, IL-6 classical signaling may be evaluated using a cell model for intracellular IL-6 signaling (also referred to herein as an intracellular IL-6 signaling assay or intracellular signaling assay), in which cells express both membrane-bound IL-6R and gp130 (here, human membrane-bound IL-6R and gp130) (endogenously and / or genetically engineered) and are cultured in the presence of human IL-6 cytokines. This system can be used to measure (or evaluate or quantify) only intracellular signaling induced by IL-6 classical signaling, lacking additional growth factors to IL-6 and soluble IL-6R.

[0137] In a preferred embodiment, to measure (or evaluate or quantify) IL-6 classical signaling using a cell model for intracellular IL-6 signaling, the cell model used is a cell lineage expressing human gp130 and membrane-bound IL-6R, for example, HEK293 cells (HEK293_IL-6R) that endogenously express human gp130 and are genetically engineered to express human membrane-bound IL-6R. In a preferred embodiment, the cells are cultured with human IL-6 cytokines (preferably 10 ng / ml) and treated with the antibodies (or antigen-binding proteins) of the present invention, for example, C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at concentrations of 1 to 10 μg / ml, more preferably 1 μg / ml), or treated with an antibody-free control (i.e., a cytokine-only control). After a suitable period, for example 48 hours, intracellular IL-6 signaling is measured (or quantified) by measuring the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705, for example using flow cytometry (for example, using a fluorophore-conjugated antibody that binds to (or specifically binds to or detects) STAT3 phosphorylated at Y705). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided and then multiplied by 100) to the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of an antibody (or antigen-binding protein) to affect IL-6 classical signaling.

[0138] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using an intracellular IL-6 signaling assay such as the assay described above, exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) IL-6 classical signaling via human membrane-bound IL-6R. Therefore, when evaluated in such an assay, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) the level of intracellular IL-6 signaling induced by IL-6 classical signaling (IL-6 classical signaling via human membrane-bound IL-6R) compared to a suitable control level or the level of intracellular IL-6 signaling observed in the same assay except in the absence of the antibody (or antigen-binding protein). Thus, intracellular IL-6 signaling observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control level. Exemplary maintenance of IL-6 classical signaling levels in terms of percentage levels of inhibition (in this case, percentage levels of low inhibition) is described elsewhere in this specification.

[0139] In some embodiments, IL-6 transsignaling may be evaluated using a cell model for intracellular IL-6 signaling (also referred to herein as an intracellular IL-6 signaling assay or intracellular signaling assay), in which cells express gp130 (here human gp130) (endogenously or genetically) and are cultured in the presence of human soluble IL-6R and IL-6 cytokines. This system can be used to measure (or evaluate or quantify) only intracellular signaling induced by IL-6 transsignaling, as it contains soluble IL-6R but lacks membrane-bound IL-6R and additional growth factors for IL-6.

[0140] In a preferred embodiment, to measure (or evaluate or quantify) IL-6 transsignaling using a cell model for intracellular IL-6 signaling, the cell model used is a cell line expressing human gp130, for example, endogenously expressing human gp130 HEK293 cells (HEK293). In a preferred embodiment, the cells are cultured with human IL-6 cytokine (preferably 10 ng / ml) and human soluble IL-6R (preferably at a concentration of 100 ng / ml or 200 ng / ml) and treated with the antibody (or antigen-binding protein) of the present invention, for example, C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at a concentration of 1 to 10 μg / ml, more preferably 1 μg / ml), or treated with an antibody-free control (i.e., a control with only cytokine and soluble IL-6R). After an appropriate time, for example 48 hours, intracellular IL-6 signaling is measured (or quantified) by measuring the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705, for example using flow cytometry (for example, using a fluorophore-conjugated antibody that binds to (or specifically binds to or detects) STAT3 phosphorylated at Y705). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided and then multiplied by 100) to the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of an antibody (or antigen-binding protein) to affect IL-6 transsignaling.

[0141] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using an intracellular IL-6 signaling assay such as the assay described above, exhibit the ability to inhibit (e.g., not significantly affect, e.g., not significantly reduce or limit) IL-6 transsignaling via human soluble IL-6R. Therefore, when evaluated in such an assay, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to inhibit (e.g., reduce or limit, e.g., significantly inhibit or limit) the level of intracellular IL-6 signaling induced by IL-6 transsignaling (transsignaling via human soluble IL-6R) compared to a suitable control level or assay, e.g., the level of intracellular IL-6 signaling observed in the absence of the antibody (or antigen-binding protein) in the same assay except for the absence of the antibody (or antigen-binding protein). Thus, intracellular IL-6 signaling observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control level. Exemplary inhibition levels of IL-6 transsignaling are described elsewhere in this specification in terms of percentage levels of inhibition (in this case, percentage levels of high inhibition).

[0142] In some embodiments, combined IL-6 classical and transsignaling (IL-6 mixed signaling) may be evaluated using a cell model for intracellular IL-6 signaling (also referred to herein as an intracellular IL-6 signaling assay or intracellular signaling assay), in which cells express both membrane-bound IL-6R and gp130 (here, human membrane-bound IL-6R and gp130) (endogenously and / or genetically engineered) and are cultured in the presence of both human IL-6 cytokines and soluble IL-6R. As previously described, this system allows for the observation of classical signaling under physiological conditions.

[0143] In a preferred embodiment, to measure (or evaluate or quantify) mixed IL-6 signaling using a cell model for intracellular IL-6 signaling, the cell model is a cell line expressing human gp130 and membrane-bound IL-6R, for example, HEK293 cells (HEK293_IL-6R) that endogenously express human gp130 and are genetically engineered to express membrane-bound IL-6R. In a preferred embodiment, the cells are cultured with human IL-6 cytokine (preferably 10 ng / ml) and human soluble IL-6R (preferably at a concentration of 100 ng / ml or 200 ng / ml) and treated with the antibody (or antigen-binding protein) of the present invention, for example, C07 and / or C09 and / or C12 and / or C19 and / or C20 (preferably at a concentration of 1 to 10 μg / ml, more preferably 1 μg / ml), or treated with an antibody-free control (i.e., a control with only cytokine and soluble IL-6R). After an appropriate period, for example 48 hours, intracellular IL-6 signaling is measured (or quantified) by measuring the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705, for example, using flow cytometry (for example, using a fluorophore-conjugated antibody that binds to (or specifically binds to or detects) STAT3 phosphorylated at Y705). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control represents (or is set as) the baseline signaling level (or 100% signaling level or value). In a preferred embodiment, the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with the antibody (or antigen-binding protein) of the present invention is normalized (i.e., divided and then multiplied by 100) to the level (or amount or fluorescence intensity) of STAT3 phosphorylated at Y705 in a sample treated with an antibody-free control. These values ​​can then be used to measure (or evaluate or quantify) the ability of an antibody (or antigen-binding protein) to affect IL-6 classical signaling.

[0144] Exemplary antibodies (or antigen-binding proteins) of the present invention, when measured (or evaluated or quantified) using a mixed signaling intracellular IL-6 signaling assay such as the assay described above, exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) IL-6 classical signaling via human membrane-bound IL-6R. Therefore, for example, when evaluated in such an assay, such antibodies (or antigen-binding proteins) of the present invention exhibit the ability to maintain (e.g., not significantly affect or significantly reduce) the level of intracellular IL-6 signaling induced by IL-6 classical signaling (IL-6 classical signaling via human membrane-bound IL-6R) compared to a suitable control level or the level of intracellular IL-6 signaling observed in the same assay except in the absence of the antibody (or antigen-binding protein). Therefore, intracellular IL-6 signaling observed in the absence of the antibody (or antigen-binding protein) is a suitable and exemplary control level. Exemplary maintenance of IL-6 classical signaling levels in terms of percentage levels of inhibition (in this case, percentage levels of low inhibition) is described elsewhere in this specification.

[0145] Therefore, in some embodiments, a suitable cell model expressing a specific combination of receptors (e.g., gp130 and / or membrane-bound IL-6R) can be used, and the cell model can be cultured with a suitable cytokine and / or soluble receptor (e.g., IL-6 and / or soluble IL-6R) to stimulate any of the IL-6 classical, trans, and / or mixed signaling pathways, so that one or more of the IL-6 classical signaling, IL-6 trans signaling, and IL-6 mixed signaling can be measured (or evaluated or quantified) separately. In some embodiments, the IL-6 classical, trans, and mixed signaling can be measured (or evaluated or quantified) using downstream IL-6 signaling events (e.g., measuring STAT3 phosphorylation at Y705, e.g., intracellular signaling assays) and / or downstream cellular responses to IL-6 signaling (e.g., cell proliferation) that are common to both the IL-6 classical, trans, and mixed signaling pathways.

[0146] As described above, in one aspect of the present invention, the antibody (or antigen-binding protein) of the present invention binds to human IL-6R and inhibits IL-6 transsignaling via human soluble IL-6R (IL-6-mediated transsignaling), while maintaining IL-6 classical signaling via human membrane-bound IL-6R (IL-6-mediated classical signaling).

[0147] In some embodiments, the ability of the antibody or antigen-binding protein of the present invention to inhibit IL-6 transsignaling while maintaining IL-6 classical signaling can be determined by the ratio of classical signaling to transsignaling (also referred to herein as the "classical:transsignaling ratio").

[0148] In some embodiments, to determine the classical:transsignaling ratio, the levels (or amounts or measurable amounts) of active (or residual) IL-6 classical signaling and IL-6 transsignaling are determined separately after treatment with the antibody (or antigen-binding protein) of the present invention, compared to (or normalized to, or relative to), a suitable control, e.g., a control without the antibody, or compared to a control having an antibody that does not bind to IL-6R, and the levels (or amounts or measurable amounts) of both IL-6 classical signaling and IL-6 transsignaling observed using the control represent (or are set as) the baseline signaling level (or 100% signaling level or value) for each pathway (i.e., the levels of IL-6 classical signaling and IL-6 transsignaling are individually set as 100% signaling after treatment with the control). Next, the classical:transsignaling ratio can be calculated or determined by dividing the residual level (or amount or measurable amount) of IL-6 classical signaling (normalized to the control) by the residual level (or amount or measurable amount) of IL-6 transsignaling (normalized to the control) for each antibody (or antigen-binding protein) after treatment with the antibody (or binding protein). Thus, in some embodiments, the classical:transsignaling ratios discussed elsewhere in this specification are normalized to (or relative to, or in comparison to) the baseline signaling level observed using a suitable control, e.g., an antibody-free control or a control having an antibody that does not bind to IL-6R, preferably to (or relative to, or in comparison to) the baseline signaling level observed using an antibody-free control.

[0149] Therefore, after treatment with the antibody (or antigen-binding protein) of the present invention, a classical:transsignaling ratio of 1.0 indicates similar or residual levels of IL-6 classical signaling and IL-6 transsignaling, and similar levels of inhibition (or lack thereof) of both pathways by the antibody (or binding protein). A ratio >1.0 indicates a higher residual level of IL-6 classical signaling compared to IL-6 transsignaling, and therefore indicates greater or increased inhibition of IL-6 transsignaling compared to IL-6 classical signaling by the antibody (or binding protein). A ratio <1.0 indicates a higher residual level of IL-6 transsignaling compared to IL-6 classical signaling, and therefore indicates greater or increased inhibition of IL-6 classical signaling compared to IL-6 transsignaling by the antibody (or binding protein).

[0150] Therefore, in some embodiments, the antibodies (or antigen-binding proteins) of the present invention that inhibit IL-6 transsignaling while maintaining IL-6 classical signaling are defined by a classical:transsignaling ratio greater than 1.0.

[0151] In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention that inhibits IL-6 transsignaling while maintaining IL-6 classical signaling has at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, Defined by classical:transsignaling ratios of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, 12.0, 14.0, 15.0, 16.0, 18.0, 20.0, 25.0, 30.0, 40.0, 50.0, 75.0, 100, 150, 200, 250, 300, 350, 400, 450, or 500.

[0152] In some embodiments, antibodies (or antigen-binding proteins) that inhibit IL-6 transsignaling while maintaining IL-6 classical signaling can be determined by performing appropriate tests (or assays) to determine (or quantify) the level (or amount or residual amount) of IL-6 transsignaling via soluble IL-6R and appropriate tests (or assays) to determine (or quantify) the level (or amount or residual amount) of IL-6 classical signaling via membrane-bound IL-6R. Appropriate assays are described elsewhere in this Spectrum, for example, assays for measuring IL-6 classical signaling, IL-6 transsignaling, and IL-6 mixed (complex) signaling are described herein and can be used, for example, in some embodiments, assays for measuring IL-6 classical signaling and IL-6 transsignaling described herein (e.g., cell proliferation or, for example, the intracellular IL-6 signaling assay described herein) can be used. However, while both assays for measuring IL-6 transsignaling and IL-6 classical signaling are suitable (and can be used) for determining the classical:transsignaling ratio as defined herein, cell-based assays are preferred.

[0153] In a preferred embodiment, values ​​for IL-6 classical signaling and IL-6 transsignaling are determined using the same assay (e.g., the cell proliferation or intracellular IL-6 signaling assay described herein) to determine the classical:transsignaling ratio.

[0154] Furthermore, typically, the same concentration of antibody (or binding protein) is used in both of such assays (i.e., the same concentration of antibody or binding protein is used in the tests that determine the level of inhibition of IL-6 transsignaling and the level of inhibition of IL-6 classical signaling). Therefore, in some embodiments, the antibody (or binding protein) that inhibits IL-6 transsignaling while maintaining IL-6 classical signaling is determined when the antibody (or binding protein) is used at a concentration of 0.01, 0.1, 0.3, 0.5, 0.7, 1, 5, or 10 μg / ml, preferably 1 to 10 μg / ml). Therefore, in a preferred embodiment, the above classical:transsignaling ratio is applied when the antibody (or binding protein) is used at a concentration of 1 or 10 μg / ml, for example, 10 μg / ml.

[0155] Alternatively, the classical:transsignaling ratio of the antibody (or antigen-binding protein) of the present invention can be determined by calculating the percentage inhibition of IL-6 classical signaling and IL-6 transsignaling (i.e., the concentration of antibody (or antigen-binding protein) required to inhibit a biological process, e.g., cell proliferation, by half (50%)) using the percentage inhibition of IL-6 classical signaling and IL-6 transsignaling as described elsewhere in this specification, within a range of antibody concentrations (e.g., 0.01, 0.1, 0.3, 0.5, 0.7, 1, 5, and 10 μg / ml). In this aspect, the classical:transsignaling IC50 ratio (as referred to elsewhere in this specification) is calculated by dividing the IC50 of IL-6 classical signaling by the IC50 of IL-6 transsignaling. Those skilled in the art will be familiar with calculating the IC50 value based on the percentage inhibition of IL-6 classical signaling and IL-6 transsignaling, as defined elsewhere in this specification.

[0156] In some embodiments, the antibodies (or antigen-binding proteins) of the present invention that inhibit IL-6 transsignaling while maintaining IL-6 classical signaling are defined by a classical:transsignaling IC50 ratio of at least 1.0, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, at least 11.0, at least 12.0, at least 13.0, at least 14.0, or at least 15.0, when the IC50 is calculated using a cell-utilized assay, preferably a cell-utilized proliferation assay.

[0157] In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention that inhibits IL-6 transsignaling while maintaining IL-6 classical signaling is defined by a classical:transsignaling IC50 ratio of at least 1.5, at least 3.0, at least 6.0, at least 9.0, at least 10.0, or at least 15.0, when the IC50 is calculated using a cell utilization assay, preferably a cell utilization growth assay.

[0158] In some embodiments, the above classical:transsignaling ratio applies when the antibody is a monoclonal antibody (for example, as opposed to a polyclonal antibody). In preferred embodiments, the above ratio applies when the antibody is a monoclonal full-length antibody, for example, a chimeric full-length antibody, or a Fab fragment, most preferably a monoclonal full-length antibody, for example, a chimeric full-length antibody.

[0159] For example, exemplary antibodies of the present invention have been shown to have a classical:transsignaling ratio of at least 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, or 12.0 when evaluated as chimeric full-length (whole) antibodies using a cell proliferation assay at a concentration of 10 μg / ml.

[0160] For example, exemplary antibodies of the present invention have classical:transsignaling IC50 ratios of at least 1.5, at least 8.0, at least 10.0, and at least 15.0 when used in cell-based proliferation assays.

[0161] As described above, in one aspect of the present invention, the antibody (or antigen-binding protein) of the present invention binds to human IL-6R and inhibits IL-6 transsignaling via human soluble IL-6R while maintaining IL-6 classical signaling via human membrane-bound IL-6R.

[0162] The inhibition (or equivalent term) of IL-6 transsignaling as referred to herein by the antibody (or antigen-binding protein) of the present invention includes any measurable or significant inhibition, reduction, limitation, prevention or interference of IL-6 transsignaling (e.g., compared to a suitable control, e.g., a control without the antibody, or a control having an antibody that does not bind to IL-6R), more preferably a statistically significant inhibition, reduction, limitation, prevention or interference.

[0163] In some embodiments, the level (or amount) of inhibition of IL-6 transsignaling observed (or caused by or derived by) using a control, e.g., an antibody-free control or a control antibody that does not bind to IL-6R, represents (or is set as) a zero inhibition level (or zero inhibition value or 0% inhibition level or value). Thus, in some embodiments, the % inhibition of IL-6 transsignaling discussed elsewhere in this specification is normalized (or relative to or compared to) the inhibition observed (or caused by or derived by) using a control, e.g., an antibody-free control or a control antibody that does not bind to IL-6R, preferably normalized (or relative to or compared to) the inhibition observed using an antibody-free control.

[0164] In some embodiments, the inhibition of IL-6 transsignaling by the antibody (or antigen-binding protein) of the present invention is, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% inhibition compared to a suitable control considered herein.

[0165] In preferred embodiments, the inhibition of IL-6 transsignaling by the antibody (or antigen-binding protein) of the present invention is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or 100% inhibition compared to, for example, suitable controls considered herein.

[0166] In some embodiments, an antibody (or antigen-binding protein) that inhibits IL-6 transsignaling can be determined by performing a suitable test (or assay) to determine (or quantify) the level (or amount or residue) of IL-6 transsignaling via soluble IL-6R. Suitable assays for measuring IL-6 transsignaling are described elsewhere herein and can be used. For example, in a preferred embodiment, a level or value for IL-6 transsignaling (or inhibition of IL-6 transsignaling) is determined using a cell proliferation or intracellular IL-6 signaling assay, for example, described herein. However, any assay for measuring IL-6 transsignaling to determine the level (or amount) of inhibition of IL-6 transsignaling as defined herein is suitable (can be used), but cell utilization assays are preferred.

[0167] Furthermore, in such assays, an appropriate concentration of antibody (or binding protein) is used. Therefore, in some embodiments, the antibody (or binding protein) that inhibits IL-6 transsignaling is determined when the antibody (or binding protein) is used at a concentration of 0.01, 0.1, 0.3, 0.5, 0.7, 1, 5, or 10 μg / ml, preferably 1 to 10 μg / ml). Therefore, in a preferred embodiment, the above levels or values ​​for inhibition of IL-6 transsignaling are applied when the antibody (or binding protein) is used at a concentration of 1 or 10 μg / ml, for example, 10 μg / ml.

[0168] Alternatively, the inhibition of IL-6 transsignaling (or equivalent term) by the antibody (or antigen-binding protein) of the present invention can be determined by calculating the IC50 value of IL-6 transsignaling (i.e., the concentration of antibody (or antigen-binding protein) required to inhibit a biological process, such as cell proliferation, by half (50%), as defined elsewhere in this specification, using the percentage inhibition of IL-6 transsignaling for a range of increasing antibody concentrations (e.g., antibodies at 0.01, 0.1, 0.3, 0.5, 0.7, 1, 5, and 10 μg / ml). Those skilled in the art will be familiar with calculating the IC50 value based on the percentage inhibition of IL-6 transsignaling as defined elsewhere in this specification.

[0169] In some embodiments, inhibition of IL-6 transsignaling by the antibody (or antigen-binding protein) of the present invention is defined by an IC50 of IL-6 transsignaling of 50 nM or less (or less), 45 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less, when the IC50 is calculated using a cell utilization assay, preferably, for example, the cell utilization proliferation assay described herein.

[0170] In a preferred embodiment, inhibition of IL-6 transsignaling by the antibody (or antigen-binding protein) of the present invention is defined by an IC50 of IL-6 transsignaling of 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 8 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3.5 nM or less, 3 nM or less, 2 nM or less, 1.5 nM or less, 1 nM or less, or 0.5 nM or less, when the IC50 is calculated using a cell utilization assay, preferably, for example, the cell utilization proliferation assay described herein.

[0171] Alternatively, in some embodiments, inhibition of IL-6 trans-signaling by an antibody (or antigen-binding protein) of the invention is defined by an IC50 of IL-6 trans-signaling of 5500 ng / ml or less (or less than), 5000 ng / ml or less, 4500 ng / ml or less, 4000 ng / ml or less, 3500 ng / ml or less, 3000 ng / ml or less, 2500 ng / ml or less, 2000 ng / ml or less, 1500 ng / ml or less, 1000 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less, 300 ng / ml or less, 200 ng / ml or less, 150 ng / ml or less, 100 ng / ml or less, or 50 ng / ml or less, preferably when calculated using a cell-based assay, preferably a cell-based proliferation assay such as described herein.

[0172] In preferred embodiments, inhibition of IL-6 trans-signaling by an antibody (or antigen-binding protein) of the invention is an IC50 of IL-6 trans-signaling of 1300 ng / ml or less (or less than), 900 ng / ml or less, 800 ng / ml or less, 700 ng / ml or less, 600 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less, 350 ng / ml or less, or 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, or 100 ng / ml or less, preferably when calculated using a cell-based assay, preferably a cell-based proliferation assay such as described herein.

[0173] In some embodiments, the above levels or values for inhibition of IL-6 trans-signaling apply when the antibody is a monoclonal antibody (e.g., as contrasted with a polyclonal antibody). In preferred embodiments, the above levels or values apply when the antibody is a monoclonal full-length (whole) antibody, e.g., a chimeric full-length (whole) antibody, or a Fab fragment, most preferably a monoclonal full-length (whole) antibody, e.g., a chimeric full-length (whole) antibody.

[0174] For example, exemplary antibodies of the present invention, when evaluated as chimeric full-length (whole) antibodies using a cell proliferation assay at a concentration of 10 μg / ml, have been shown to have the ability to inhibit IL-6 transsignaling by at least 70%, 75%, 80%, 85%, 88%, or 90%.

[0175] For example, the exemplified anti-IL-6R IgG antibodies of the present invention have an IC50 of 2 nM (304 ng / ml), 3.5 nM (531 ng / ml), 4.3 nM (644 ng / ml), 5 nM (757 ng / ml), 5.8 nM (863 ng / ml), 8 nM (1214 ng / ml), 28 nM (4130 ng / ml), or less than 35 nM (5260 ng / ml) for IL-6 transsignaling, preferably determined from a cell-utilized assay as described herein.

[0176] For example, the anti-IL-6R Fab fragments of the present invention, as illustrated, preferably have an IC50 determined from, for example, a cell utilization assay described herein, of IL-6 transsignaling of 1.9 nM (98 ng / ml) or less (or less) 3.2 nM (159 ng / ml).

[0177] Conversely, as readouts for inhibiting IL-6 transsignaling, the anti-IL-6R IgG antibodies of the present invention, as exemplified, preferably reduce (or inhibit) cell proliferation by 50% at concentrations of 2 nM (304 ng / ml), 3.5 nM (531 ng / ml), 4.3 nM (644 ng / ml), 5 nM (757 ng / ml), 5.8 nM (863 ng / ml), 8 nM (1214 ng / ml), 28 nM (4130 ng / ml), or 35 nM (5260 ng / ml) or less in the cell utilization assay described herein.

[0178] Conversely, as a readout for inhibiting IL-6 transsignaling, the anti-IL-6R Fab fragments of the present invention, as exemplified, preferably reduce (or inhibit) cell proliferation by 50% at concentrations of 1.9 nM (98 ng / ml) or 3.2 nM (159 ng / ml) or less in the cell utilization assay described herein.

[0179] Accordingly, in some embodiments, the antibody (or antigen-binding protein) of the present invention has the ability to inhibit IL-6 transsignaling as defined elsewhere herein at concentrations of 0.3 μg / ml, 0.5 μg / ml, 0.7 μg / ml, 1 μg / ml, or 10 μg / ml, preferably at a concentration of 10 μg / ml.

[0180] As described above, in this aspect of the present invention, the antibody (or antigen-binding protein) of the present invention binds to human IL-6R and inhibits IL-6 transsignaling via human soluble IL-6R while maintaining IL-6 classical signaling via human membrane-bound IL-6R.

[0181] Maintenance of IL-6 classical signaling (or equivalent terms) by the antibody (or antigen-binding protein) of the present invention includes no effect or change (or no measurable or significant, e.g., statistically significant effect or change) on IL-6 classical signaling (e.g., compared to a suitable control, e.g., a control without antibody, or a control having an antibody that does not bind to IL-6R), e.g., no decrease, inhibition or prevention of IL-6 classical signaling, e.g., no measurable or significant (e.g., statistically significant) decrease, inhibition or prevention. Conversely, the maintenance (or equivalent term) of IL-6 classical signaling as referred herein by the antibody (or antigen-binding protein) of the present invention is substantially the same (or not significantly different, similar, or equivalent) inhibition of IL-6 classical signaling as observed (or caused by, or elicited by) using a suitable control, e.g., an antibody-free control or a control having an antibody that does not bind to IL-6R, preferably an antibody-free control.

[0182] Therefore, the preferred antibodies or antigen-binding proteins of the present invention do not affect, inhibit, reduce, prevent, or interfere with IL-6 classical signaling, for example, not significantly (e.g., statistically significantly) affecting, inhibiting, reducing, preventing, or interfering with it. Thus, conversely, the present invention provides antibodies (or antigen-binding proteins) that bind to human IL-6R and inhibit IL-6 transsignaling via human soluble IL-6R without significantly inhibiting IL-6 classical signaling via human membrane-bound IL-6R. Considerations of various antibody features and preferred embodiments of other aspects of the present invention are applied to this aspect of the present invention with necessary modifications.

[0183] In some embodiments, the level (or amount) of inhibition of IL-6 classical signaling observed (or caused by or elicited by) using a suitable control, e.g., an antibody-free control, represents (or is set as) a zero inhibition level (or zero inhibition value or 0% inhibition level or value). Thus, in some embodiments, the % inhibition of IL-6 classical signaling discussed elsewhere in this specification is normalized (or relative to or compared to) the inhibition observed (or caused by or elicited by) using a suitable control, e.g., an antibody-free control or a control antibody that does not bind to IL-6R, preferably normalized (or relative to or compared to) the inhibition observed using an antibody-free control.

[0184] In some embodiments, maintenance of IL-6 classical signaling is defined as inhibition of 30% or less, 25% or less, 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.

[0185] In some embodiments, maintenance of IL-6 classical signaling is defined as inhibition of less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0%.

[0186] In some preferred embodiments, the antibody (or antigen-binding protein) of the present invention does not produce (or elicit) measurable inhibition of IL-6 classical signaling, or more preferably does not produce (or elicit) significant inhibition (preferably statistically significant inhibition) of IL-6 classical signaling.

[0187] In some embodiments, the antibody (or antigen-binding protein) that maintains IL-6 classical signaling can be determined by performing an appropriate test (or assay) to determine (or quantify) the level (or amount or persistence) of IL-6 classical signaling via membrane-bound IL-6R. Appropriate assays for measuring IL-6 classical signaling are described and can be used elsewhere in this specification. For example, in a preferred embodiment, a cell proliferation assay or intracellular IL-6 signaling assay, for example, as described herein, is used to determine the level or value for IL-6 classical signaling (or inhibition of IL-6 classical signaling). However, any assay for measuring IL-6 classical signaling is suitable and can be used to determine the maintenance of IL-6 classical signaling as defined herein, but cell utilization assays are preferred.

[0188] Furthermore, in such assays, an appropriate concentration of antibody (or binding protein) is used. Therefore, in some embodiments, the antibody (or binding protein) that maintains IL-6 classical signaling is determined when the antibody (or binding protein) is used at a concentration of 1 or 10 μg / ml. Thus, in preferred embodiments, the above level or value of IL-6 classical signaling is applied when the antibody (or binding protein) is used at a concentration of 1 or 10 μg / ml, for example, 10 μg / ml.

[0189] In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention, preferably when anti-IL-6R IgG is used, has the ability to inhibit IL-6 transsignaling at a concentration of 10 μg / ml (or 53 nM) and maintain IL-6 classical signaling at a concentration of 10 μg / ml (or 53 nM).

[0190] In a preferred embodiment, the antibody (or antigen-binding protein) of the present invention, preferably when an anti-IL-6R Fab fragment is used, has the ability to inhibit IL-6 transsignaling at a concentration of 3.3 μg / ml (or 53 nM) and maintain IL-6 classical signaling at a concentration of 3.3 μg / ml (or 53 nM).

[0191] Alternatively, the maintenance (or equivalent term) of IL-6 classical signaling by the antibodies (or antigen-binding proteins) of the present invention, as referenced herein, can be determined by calculating the IC50 value (or minimum IC50 value) of IL-6 classical signaling (i.e., the concentration of the antibody (or antigen-binding protein) required to inhibit a biological process, such as cell proliferation, by half (50%), using the percentage inhibition of IL-6 classical signaling by the antibodies (or antigen-binding proteins) of the present invention, as referenced herein, over an increasing range of concentrations as defined elsewhere herein (e.g., anti-IL-6R IgG antibodies at 0.01, 0.1, 0.3, 0.5, 0.7, 1, 5, and 10 μg / ml, or anti-IL-6R Fab at 3.3, 33.3, 100, 166.7, 233.3, 333.3, or 3300 ng / ml). Those skilled in the art will be familiar with the calculation of the IC50 value based on the percentage inhibition of IL-6 classical signaling as defined elsewhere herein. The minimum IC50 can be calculated if an antibody (or antigen-binding protein) does not inhibit a biological process, such as cell proliferation, by half (50%) at any concentration. For example, if the antibody (or antigen-binding protein) does not inhibit cell proliferation by half (50%) at the maximum concentration tested of 10 μg / ml, the IC50 value is greater than 10 μg / ml (>10 μg / ml).

[0192] In some embodiments, the maintenance of IL-6 classical signaling by the antibody (or antigen-binding protein) of the present invention is preferably defined by an IC50 of at least 100 nM, at least 95 nM, at least 90 nM, at least 85 nM, at least 80 nM, at least 75 nM, at least 70 nM, at least 65 nM, at least 60 nM, at least 55 nM, at least 50 nM, at least 45 nM, at least 40 nM, at least 35 nM, at least 30 nM, at least 25 nM or at least 20 nM of IL-6 classical signaling when calculated using a cell utilization assay, preferably a cell utilization proliferation assay such as described herein.

[0193] In a preferred embodiment, the maintenance of IL-6 classical signaling by the antibody (or antigen-binding protein) of the present invention is preferably defined by an IC50 of at least 20 nM, or at least 25 nM, or at least 30 nM, or at least 35 nM, or at least 40 nM, or at least 45 nM, or at least 50 nM, or at least 53 nM of IL-6 classical signaling when calculated using a cell utilization assay, preferably a cell utilization proliferation assay such as described herein.

[0194] Alternatively, in some embodiments, the maintenance of IL-6 classical signaling by the antibody (or antigen-binding protein) of the present invention is preferably defined by an IC50 of at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 30 μg / ml, at least 35 μg / ml, at least 40 μg / ml, at least 45 μg / ml or at least 50 μg / ml of IL-6 classical signaling when calculated using a cell utilization assay, preferably a cell utilization proliferation assay such as described herein, preferably when an anti-IL-6R IgG antibody is used.

[0195] In preferred embodiments, maintenance of IL-6 classical signaling by the antibody (or antigen-binding protein) of the present invention is defined by an IC50 of at least 5 μg / ml, at least 6 μg / ml, at least 7 μg / ml, at least 8 μg / ml, at least 9 μg / ml, or at least 10 μg / ml of IL-6 classical signaling, preferably when an anti-IL-6R IgG antibody is used and the IC50 is calculated using a cell utilization assay, preferably, for example, the cell utilization proliferation assay described herein.

[0196] In some embodiments, the above levels or values ​​for IL-6 classical signaling apply when the antibody is a monoclonal antibody (e.g., in contrast to a polyclonal antibody). In preferred embodiments, the above levels or values ​​apply when the antibody is a monoclonal full-length antibody, e.g., a chimeric full-length antibody, or a Fab fragment, most preferably a monoclonal full-length antibody, e.g., a chimeric full-length antibody.

[0197] For example, the exemplary antibodies of the present invention, when evaluated as full-length chimeric antibodies using a cell proliferation assay at a concentration of 10 μg / ml, were shown to inhibit IL-6 classical signaling by 12%, 9%, 8%, 7%, or 6% or less.

[0198] For example, the anti-IL-6R IgG antibody of the present invention, as illustrated, has an IC50 of at least 53 nM (or 10 μg / ml) of IL-6 classical signaling when used, for example, with the cell-based proliferation assay described herein.

[0199] Accordingly, in some embodiments, the anti-IL-6R IgG antibody (or antigen-binding domain) of the present invention maintains IL-6 classical signaling, preferably, the maintenance of IL-6 classical signaling is defined by an IC50 of at least 53 nM (or 10 μg / ml) using, for example, the cell-utilized cell proliferation assay described herein.

[0200] The exemplified anti-IL-6R IgG antibody of the present invention does not inhibit (or does not significantly inhibit) (i.e., maintains) IL-6 classical signaling at the highest concentration tested, 10 μg / ml (or 53 nM).

[0201] Thus, in some embodiments, the anti-IL-6R IgG antibody (or antigen-binding protein) of the present invention has the ability to maintain IL-6 classical signaling at a concentration of 1 μg / ml or 10 μg / ml, preferably at a concentration of 10 μg / ml (or 53 nM), as defined elsewhere herein. As shown above, in this aspect of the present invention, the antibody (or antigen-binding protein) inhibits IL-6 trans-signaling via soluble IL-6R while maintaining IL-6 classical signaling via membrane-bound IL-6R. This means that a given antibody (or binding protein) inhibits IL-6 trans-signaling (or has the ability to inhibit IL-6 trans-signaling) to a greater extent than it inhibits (or has the ability to inhibit) IL-6 classical signaling. Thus, if a given antibody (or binding protein) inhibits IL-6 trans-signaling by X%, the same antibody (or binding protein) inhibits IL-6 classical signaling by <X%. This property may also be referred to herein as preferential inhibition or selective inhibition.

[0202] In some embodiments, the % inhibition (or % inhibition value) of IL-6 trans-signaling is at least 5% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, at least 70% higher, at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher or 100% higher than the % inhibition (or % inhibition value) of IL-6 classical signaling.

[0203] In a preferred embodiment, to compare the % inhibition (or % inhibition value) of IL-6 transsignaling with (or relative to) IL-6 classical signaling, the % inhibition (% inhibition value) is typically taken from the same assay (e.g., measuring cell proliferation or measuring intracellular IL-6 signaling) and the same antibody (or binding protein) concentration (e.g., 1 μg / ml or 10 μg / ml) in the assay. In such embodiments, the % inhibition (or % inhibition value) of IL-6 transsignaling is at least 15% higher than the % inhibition (or % inhibition value) of IL-6 classical signaling, but typically at least 50% higher, at least 60% higher, preferably at least 75% higher, at least 80% higher, at least 85% higher, at least 90% higher, at least 95% higher, or 100% higher.

[0204] For example, the exemplary antibodies of the present invention, when evaluated as full-length chimeric antibodies using a cell proliferation assay at a concentration of 10 μg / ml, were shown to have the ability to inhibit IL-6 transsignaling by at least 68%, 80%, 81%, or 82% compared to IL-6 classical signaling.

[0205] In some embodiments, the % inhibition (or % inhibition value) of IL-6 signaling is determined by performing a test (or assay) to determine (or quantify) the level (or amount) of inhibition of IL-6 transsignaling via soluble IL-6R and / or a test (or assay) to determine (or quantify) the level (or amount) of inhibition of IL-6 classical signaling via membrane-bound IL-6R. Appropriate assays are described elsewhere in this Specification, for example assays for measuring IL-6 classical signaling, IL-6 transsignaling and IL-6 mixed (complex) signaling are described herein and can be used, for example assays for measuring IL-6 classical signaling and IL-6 transsignaling described herein can be used in some embodiments. However, any assay for measuring IL-6 transsignaling and IL-6 classical signaling is suitable and can be used to determine the percentage inhibition (or percentage inhibition value) of IL-6 transsignaling relative to IL-6 classical signaling as defined herein.

[0206] In a preferred embodiment, to determine the % inhibition (or % inhibition value) of IL-6 transsignaling relative to IL-6 classical signaling, values ​​for IL-6 classical signaling and IL-6 transsignaling are determined using, for example, the same assay described herein (e.g., cell proliferation or intracellular IL-6 signaling).

[0207] Furthermore, typically, the percentage inhibition (or percentage inhibition value) of IL-6 transsignaling and the percentage inhibition (or percentage inhibition value) of IL-6 classical signaling are determined when the antibody (or binding protein) is used at the same concentration in both such assays (i.e., the same concentration of antibody is used in the tests determining the percentage inhibition of IL-6 transsignaling and the percentage inhibition of IL-6 classical signaling).

[0208] In some embodiments, the above % inhibition (or % inhibition value) is determined when the antibody (or binding protein) is used at a concentration of 0.01, 0.1, 0.3, 0.5, 0.7, 1, or 10 μg / ml, preferably 1 to 10 μg / ml. Therefore, in a preferred embodiment, the above % inhibition (or % inhibition value) is applied when the antibody (or binding protein) is used at a concentration of 1 or 10 μg / ml, for example, 10 μg / ml.

[0209] In some embodiments, the above % inhibition (or % inhibition value) applies when the antibody is a monoclonal antibody (for example, in contrast to a polyclonal antibody). In preferred embodiments, the above % inhibition (or % inhibition value) applies when the antibody is a monoclonal full-length antibody, for example, a chimeric full-length antibody or a Fab fragment, most preferably a monoclonal full-length antibody, for example, a chimeric full-length antibody.

[0210] Conversely, the present invention provides an antigen-binding protein, such as an antibody, such as an isolated antibody (or antigen-binding protein), comprising at least one, preferably two, antigen-binding domains that bind to human IL-6R, and the antigen-binding protein or antibody preferentially or selectively inhibits IL-6 transsignaling via human soluble IL-6R, for example, in comparison to (or compared to) IL-6 classical signaling via human membrane-bound IL-6R. This aspect of the present invention is applied to the consideration of various features and preferred embodiments of the antibody (or antigen-binding protein) of the other aspects of the present invention with necessary modifications.

[0211] Nucleic acid molecules (e.g., one or more nucleic acid molecules) containing nucleotide sequences encoding antigen-binding proteins (e.g., antibodies) or portions or fragments thereof as defined herein, or nucleic acid molecules substantially homologous thereto (e.g., having at least 80% sequence identity with a specific nucleic acid sequence as defined herein), form yet another aspect of the present invention.

[0212] Preferred nucleic acid molecules are those encoding antibodies (or antigen-binding proteins) of the present invention as described elsewhere herein, which can bind to human IL-6R, for example, antibodies (or antigen-binding proteins) of the present invention having the CDR and optionally the FR and other regions as defined in any one of Tables A, B, C, D, E, or F, or antibodies having substantially homologous sequences thereto.

[0213] Preferred nucleic acid molecules are those that encode the antibody of the present invention that can bind to human IL-6R (for example, including nucleic acid sequences encoding SEQ ID NO:3 and / or SEQ ID NO:4, SEQ ID NO:21 and / or SEQ ID NO:22, SEQ ID NO:39 and / or SEQ ID NO:40, SEQ ID NO:57 and / or SEQ ID NO:58, or SEQ ID NO:75 and / or SEQ ID NO:76, for example, SEQ ID NO:1 and / or SEQ ID NO:2, SEQ ID NO:19 and / or SEQ ID NO:20, SEQ ID NO:37 and / or SEQ ID NO:38, SEQ ID NO:55 and / or SEQ ID NO:56, or SEQ ID NO:73 and / or SEQ ID NO:74, respectively).

[0214] Preferred nucleic acid molecules are those that encode the VH region of the antibody (or antigen-binding protein) of the present invention (for example, those encoding SEQ ID NO: 3 or 21 or 39 or 57 or 75, respectively; for example, SEQ ID NO: 1 or 19 or 37 or 55 or 73). Other preferred nucleic acid molecules are those that encode the VL region of the antibody (or antigen-binding protein) of the present invention (for example, those encoding SEQ ID NO: 4 or 22 or 40 or 58 or 76, respectively; for example, SEQ ID NO: 2 or 20 or 38 or 56 or 74). Other preferred nucleic acid molecules are those that encode the VH region of the antigen-binding protein (e.g., antibody) of the present invention and the VL region of the antigen-binding protein (e.g., antibody) of the present invention.

[0215] Therefore, a preferred nucleic acid molecule includes a sequence encoding a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO: 3 or 21 or 39 or 57 or 75 (preferably encoded by SEQ ID NO: 1 or 19 or 37 or 55 or 73, respectively), and / or a sequence encoding a light chain variable region (VL) having an amino acid sequence of SEQ ID NO: 4 or 22 or 40 or 58 or 76 (preferably encoded by SEQ ID NO: 2 or 20 or 38 or 56 or 74, respectively).

[0216] Nucleic acids encoding the following combinations are also preferred: SEQ ID NO:3 and 4, or SEQ ID NO:21 and 22, or SEQ ID NO:39 and 40, or SEQ ID NO:57 and 58, or SEQ ID NO:75 and 76.

[0217] Nucleic acid molecules containing the following combinations, such as SEQ ID NO:1 and 2, or SEQ ID NO:19 and 20, or SEQ ID NO:37 and 38, or SEQ ID NO:55 and 56, or SEQ ID NO:73 and 74, are also preferred.

[0218] Other preferred nucleic acid molecules include sequences encoding the human IgG (preferably IgG1) form of the antibody of the present invention, for example, sequences encoding the VH and VL chains of the antibody of the present invention that are chimeric (or conjugated) with human IgG (preferably IgG1) (see, for example, Tables A, B, C, D, and E).

[0219] Other preferred nucleic acid molecules include sequences encoding the Fab format of the antibody of the present invention or other fragments (preferably the Fab format of antibody C20 or the Fab format of antibodies C07, C12, C19, or C09).

[0220] In another aspect, the present invention provides a set (or collection) of nucleic acid molecules, each containing a nucleotide sequence (for example, a nucleotide sequence encoding a heavy chain variable region or a nucleotide sequence encoding a light chain variable region), wherein the set of nucleic acid molecules together (or collectively) encodes an antibody (or antigen-binding protein) according to the present invention. Such a set of nucleic acid molecules may be characterized in that, when the set is expressed (i.e., expressed together) in, for example, a host cell, the entire antibody (or binding protein) of the present invention is expressed, and preferably assembled.

[0221] In some embodiments, this set of nucleic acid molecules comprises at least one nucleic acid molecule containing a nucleotide sequence encoding a heavy chain variable region and at least one nucleic acid molecule containing a nucleotide sequence encoding a light chain variable region as described herein, preferably the nucleic acid molecule containing the nucleotide sequence encoding the heavy chain variable region and the nucleic acid molecule containing the nucleotide sequence encoding the light chain variable region are the preferred nucleic acid molecules described above.

[0222] Nucleic acid sequences that are substantially homologous to the sequences described above are also sometimes used.

[0223] As used herein, the terms “nucleic acid sequence” or “nucleic acid molecule” refer to a sequence of nucleosides or nucleotide monomers composed of native bases, sugars, and intersugar (backbone) bonds. This term also includes modified or substituted sequences containing non-natural monomers or parts thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and may contain native bases including adenine, guanine, cytosine, thymidine, and uracil. Sequences may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. Nucleic acid molecules may be double-stranded or single-stranded. Nucleic acid molecules may be synthesized in whole or in part, or recombinant.

[0224] An antigen-binding protein (e.g., antibody) of the present invention comprising one or more (e.g., one, two, three, four, five, or six) CDR sequences that are "substantially homologous" to one or more CDRs of an exemplary antibody of the present invention, and an antigen-binding protein (e.g., antibody) of the present invention comprising a heavy chain variable domain and / or light chain variable domain comprising an amino acid sequence substantially homologous to the VH and / or VL sequence of an exemplary antibody of the present invention, is referred to herein as a "substantially homologous antigen-binding protein" (e.g., a "substantially homologous antibody"). Accordingly, a "substantially homologous antigen-binding protein" (e.g., a "substantially homologous antibody") is any such antigen-binding protein / antibody comprising one or more CDR or variable domain sequences that are substantially homologous to a particular CDR sequence or variable domain sequence having a predetermined SEQ ID NO as described herein, for example. Substantially homologous antigen-binding proteins (e.g., antibodies) referenced herein are expressly limited to those having, for example, a predetermined SEQ ID NO as specified herein, and possessing the characteristics of antigen-binding proteins (e.g., antibodies) described herein, in particular the ability to bind to human IL-6R and, preferably, the ability to inhibit IL-6 transsignaling via human soluble IL-6 receptor while maintaining (e.g., not significantly affecting or significantly reducing) IL-6 classical signaling via human membrane-bound IL-6 receptor, and preferably the ability to bind to human soluble IL-6R and human membrane-bound IL-6R.

[0225] In this specification, the term “substantially homologous” as used in relation to an amino acid or nucleic acid sequence includes sequences having at least 55%, 60%, 65%, 70%, or 75%, preferably at least 80%, and more preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the disclosed amino acid or nucleic acid sequence. A substantially homologous sequence of the present invention therefore includes one or more base or amino acid changes (additions, substitutions, insertions, or deletions) to the sequence of the present invention.

[0226] A substantially homologous sequence at the amino acid level is one or more of the framework regions and / or one or more of the CDRs forming the sequence of the present invention, comprising up to six, for example, one, two, three, four, five, or six, for example, one, two, three, four, or five, preferably one, two, three, or four, preferably one, two, or three, more preferably one or two, more preferably one modified amino acid.

[0227] Other preferred examples of “substantially homologous” sequences are sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with one or more CDR regions or one or more FR regions disclosed in Tables A, B, C, D, or E. Thus, in some embodiments, a “substantially homologous” CDR sequence may be a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a given CDR sequence described herein.

[0228] In some embodiments, in an antibody (or antigen-binding protein) having a sequence that is "substantially homologous" to a given sequence, or having a certain degree of sequence identity to a given sequence, the modified amino acid residue is not in the CDR region. For example, in some embodiments, in an antibody (or antigen-binding protein) having a VH domain that has a certain degree of sequence identity to a given VH domain sequence of a particular antibody (or binding protein) of the present invention, the modified (or mutated) residue is not in the CDR region. Therefore, in some embodiments, in an antibody (or antigen-binding protein) having a sequence that is "substantially homologous" to a given sequence, or having a certain degree of sequence identity to a given sequence, the modified amino acid residue is in one or more framework regions.

[0229] As will be apparent from other parts of this specification, in other embodiments, in an antibody (or antigen-binding protein) having a sequence that is "substantially homologous" to a given sequence or has a certain degree of sequence identity to a given sequence, the modified amino acid residue may be located in the CDR region.

[0230] In some embodiments, in an antibody (or antigen-binding protein) having a sequence that is "substantially homologous" to a given sequence or has a certain degree of sequence identity to a given sequence, three VH CDR amino acid sequences (i.e., all three VH CDR sequences together) and three VL CDR amino acid sequences (i.e., all three VL CDR sequences together) that make up a total of six CDRs are considered together to be the whole CDR complement (or entire CDR complement) of the antibody (or antigen-binding protein), and the amino acid sequence of the whole CDR complement of the antibody (or antigen-binding protein) is at least 70%, preferably at least 80%, or at least 85%, or at least 90%, or at least 95% identical to the corresponding whole CDR complement (or entire CDR complement) of a given proto-(or reference) antibody (or antigen-binding protein). The progenitor (or reference) antibody (or antigen-binding protein) may have the CDR sequence of the C07, C09, C12, C19, or C20 antibody of the present invention shown in Tables A, B, C, D, and E, respectively.

[0231] Furthermore, at the amino acid level, preferred substantially homologous sequences include up to six, for example, just one, two, three, four, five, or six, for example, one, two, three, four, or five, preferably one, two, three, or four, preferably one, two, or three, more preferably one or two (preferably one) modified amino acids in the composite framework regions (e.g., four framework regions) and / or composite CDR regions (e.g., three CDR regions) that make up the VL or VH domain of the antibody (or antigen-binding protein) of the present invention. Furthermore, at the amino acid level, preferred substantially homologous sequences include up to six, for example, one, two, three, four, five, or six, preferably one, two, three, four, or five, preferably one, two, three, or four, more preferably one, two, or three, more preferably one or two (preferably one) modified amino acids in the VH domain and / or VL domain of the antibody (or antigen-binding protein) of the present invention.

[0232] In certain embodiments, if a given source sequence is relatively short (e.g., 3 or 8 amino acids in length), then a substantially homologous sequence may contain fewer amino acid substitutions compared to the number of amino acid substitutions that may be performed as optional substitutions in a substantially homologous sequence with a longer source sequence. For example, in certain embodiments, a VH CDR1 source sequence according to the present invention, for example, a VH CDR1 source sequence with a length of 8 amino acid residues, and a substantially homologous sequence to it, preferably has one, two, or three (more preferably one or two, e.g., one) modified amino acids compared to the source sequence. A VL CDR2 source sequence according to the present invention, for example, a VL CDR2 source sequence with a length of 3 amino acid residues, and a substantially homologous sequence to it, preferably has one or two, more preferably one, modified amino acid compared to the source sequence. Therefore, in some embodiments, the number of modified amino acids in a substantially homologous sequence (e.g., in a substantially homologous CDR sequence) may be adjusted to match the length of a given CDR source sequence. For example, depending on the length of a given CDR source sequence, various numbers of modified amino acids may exist to achieve a specific percentage of sequence identity in the CDR, such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0233] In all embodiments, the modified residue may be a conserved amino acid substitution residue, a non-conserved substitution residue, or a mixture thereof.

[0234] In such embodiments, preferred modifications are conservative amino acid substitutions.

[0235] To determine which amino acid residues of the CDR do not contribute to antigen binding or do not contribute significantly, and are therefore good candidates for modification or substitution in embodiments of the present invention that include substantially homologous sequences, routine methods in the art, such as alanine scanning mutagenesis and / or deep mutational scanning (aimed at performing all possible singular substitutions at all selected residues within a given protein sequence) and / or analysis of the crystalline structure of the antigen-antibody complex, can be used.

[0236] Once identified, the addition, deletion, substitution, or insertion of one or more amino acids in the amino acid sequence of a parent antibody (or antigen-binding protein), which is one of the antibodies (or antigen-binding proteins) of the present invention as defined elsewhere herein, to form a new antibody (or antigen-binding protein), and testing the resulting new antibody (or antigen-binding protein) to identify the antibody (or antigen-binding protein) that binds to human IL-6R according to the present invention, can be carried out using techniques that are commonplace in the art as described elsewhere herein. Such methods can be used to form several new antibodies (or antigen-binding proteins) that can all be tested for their ability to bind to human IL-6R. Preferably, the addition, deletion, substitution, or insertion of one or more amino acids is carried out in one or more CDR domains.

[0237] For example, the above operation could be conveniently carried out at the nucleic acid level by genetic engineering. In that case, the nucleic acid molecule encoding the appropriate antibody (or antigen-binding protein) and its domain would be modified, and the amino acid sequence of the resulting expressed protein would then be modified in an appropriate manner. The ability of one or more of the modified antibodies (or antigen-binding proteins) to bind to human IL-6R could be tested by any appropriate method known and described in the art. Appropriate methods are also described elsewhere in this specification and in the examples section.

[0238] The novel antibodies (or antigen-binding proteins) produced, obtained, or obtainable by these methods form yet another aspect of the present invention.

[0239] The term “substantially homologous” also includes modified amino acid and nucleotide sequence variants or chemical equivalents of the antibody (or binding protein) of the present invention that exert substantially the same function and manner as the protein or nucleic acid molecule of the antibody (or binding protein) of the present invention. For example, any substantially homologous antibody (or binding protein) should retain the ability to bind to human IL-6R as described above. Preferably, any substantially homologous antibody (or binding protein) should retain one or more (or all) of the functional capabilities of the proto-antibody (or binding protein), including the ability to inhibit IL-6 transsignaling while maintaining IL-6 classical signaling (e.g., not inhibiting or not significantly inhibiting it), and preferably the ability to bind to human soluble IL-6R and human membrane-bound IL-6R.

[0240] Substantially homologous sequences of the proteins of the present invention include, but are not limited to, conserved amino acid substitutions, or modifications that do not affect the VH, VL, or CDR domains of an antibody (or antigen-binding protein), such as an antibody (or antigen-binding protein) to which a tag sequence, label, or other component that does not contribute to binding to the IL-6R antigen has been added, or a modification that converts one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g., conversion from Fab to scFv or all antibodies or vice versa), or a conversion from an antibody molecule to a specific class or subclass of antibody molecule (e.g., conversion from an antibody molecule to IgG or its subclass, such as IgG1).

[0241] As used herein, “conservative amino acid substitution” refers to the replacement of an amino acid residue with another amino acid residue having a similar side chain. In this art, a family of amino acid residues having similar side chains is defined, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other examples, families of amino acid residues can be classified based on whether they have hydrophobic or hydrophilic side chains.

[0242] Homology or sequence identity can be evaluated by any simple method. However, to measure the degree of homology or identity between sequences, a computer program that performs multiple alignment of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Research, Vol. 22, pp. 4673-4680 (1994)), is useful. If desired, the Clustal W algorithm can be used with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proceedings of the National Academy of Sciences of the United States of America). The Smith and Waterman method (Advanceds in Applied Mathematics, Vol. 2, p. 482 (1981)) is used with a gap-opening penalty of 10 and a gap-extension penalty of 0.1, resulting in the highest degree of match between two sequences where at least 50% of the full length of one of the sequences is included in the alignment. Another method that may be used for sequence alignment is the Needleman and Wunsch alignment method (Needleman and Wunsch, Journal of Molecular Biology, Vol. 48, p. 443 (1970)), which was revised to determine the number of identical amino acids between two sequences and to obtain the highest degree of match between two sequences by Smith and Waterman (Advanceds in Applied Mathematics, Vol. 2, p. 482 (1981)).Other methods for calculating percentage identity between two amino acid sequences are generally recognized in this field, including, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM Journal of Applied Mathematics, Vol. 48, p. 1073 (1988)) and those described in Computational Molecular Biology, edited by Lesk, Oxford University Press, New York (1988), Biocomputing: Informatics and Genomics Projects.

[0243] Generally, computer programs are used for such calculations. (ALIGN (Myers and Miller, CABIOS (Computer Applications in the Biosciences), Vol. 4, pp. 11-17 (1988)), FASTA (Pearson and Lipman, Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, pp. 2444-2448 (1988)), Pearson, Methods in Enzymology) Programs that compare and sort pairs of sequences such as Enzymology (Vol. 183, pp. 63-98, 1990) and gap-type BLAST (Altschul et al., Newcraic Acids Research, Vol. 25, pp. 3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Newcraic Acids Research, Vol. 12, No. 387, 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics Institute provides sequences based on the structure of protein sequences (Holm, Trends in Biochemical Sciences, Vol. 20, pp. 478-480 (1995); Holm, Journal of Molecular Biology, Vol. 233, pp. 123-38 (1993); Holm, Newcraic Acids Research, Vol. 26, pp. 316-39 (1998)).

[0244] To provide a reference point, sequences according to the present invention having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc., may be determined using the ALIGN program, which has default parameters (for example, available on the GENESTREAM network server, IGH, Montpellier, France).

[0245] In all embodiments, the antibodies (or antigen-binding proteins) of the present invention containing substantially homologous sequences retain the ability to bind to human IL-6R. Preferably, the antibodies (or antigen-binding proteins) containing substantially homologous sequences retain one or more (preferably all) of the other properties described herein compared to the antibodies of the present invention, e.g., C07 and / or C09 and / or C12 and / or C19 and / or C20 antibodies. Thus, preferred antibodies (or antigen-binding proteins) containing substantially homologous sequences retain the property of binding to human IL-6R and inhibiting IL-6 transsignaling via human soluble IL-6R while maintaining (e.g., not significantly affecting or significantly reducing) IL-6 classical signaling via human membrane-bound IL-6R.

[0246] In some embodiments, the antibody (or antigen-binding protein) of the present invention, containing substantially homologous sequences, binds (or specifically binds) to human IL-6R, inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling, and exhibits the following properties: i. Soluble and / or (preferably "and") binds to membrane-bound human IL-6R, ii. In some non-human species, e.g., rats (or mice), IL-6R (soluble and / or (preferably "and") membrane-bound IL-6R) does not bind, and / or (preferably "and") in some non-human species, e.g., suitable non-human IL-6R, e.g., rat (or mouse) IL-6R, maintains (or does not inhibit or significantly inhibit) or affects IL-6 classical signaling or IL-6 transsignaling via appropriate non-human IL-6R, e.g., rat (or mouse) IL-6R. iii. Maintain (or not inhibit or significantly inhibit) other gp130-dependent signaling pathways, for example, maintain (or not inhibit or significantly inhibit) IL-11 signaling. It has one or more of these, for example, two or three.

[0247] In some embodiments, the antibody (or binding protein) of the present invention, which contains substantially homologous sequences, has the following characteristics: iv. Compared to human IL-6R (soluble and / or membrane-bound human IL-6R) alone, it does not preferentially bind to human IL-6 / IL-6R complexes (soluble and / or membrane-bound human IL-6 / IL-6R complexes) (or does not bind with greater affinity or significantly greater affinity), v. Does not affect (or significantly affects) the IL-6 buffer system (or does not reduce the level or amount of free IL-6 in the extracellular space (or does not significantly reduce the level or amount of IL-6)), It further has one or both of the above.

[0248] Methods used to determine the binding of substantially homologous antibody (or antigen-binding protein) sequences to human IL-6R, such as human soluble and / or membrane-bound IL-6R, are well known in the art and are described elsewhere in this specification.

[0249] Other parts of this specification describe methods used to determine whether substantially homologous antibody (or antigen-binding protein) sequences inhibit IL-6 transsignaling while maintaining IL-6 classical signaling. Therefore, substantially homologous antibody (or binding protein) sequences are expected to induce similar (or not significantly different) classical:transsignaling ratios and / or similar (or not significantly different) IL-6 transsignaling % inhibition (or % inhibition value) and / or IL-6 classical signaling for the antibodies of the present invention as described elsewhere in this specification for, for example, the C07 and / or C09 and / or C12 and / or C19 and / or C20 antibodies.

[0250] Further examples of substantially homologous amino acid sequences according to the present invention are described elsewhere in this specification.

[0251] Preferably, any substantially homologous antibody (or antigen-binding protein) of the present invention should retain the ability to bind to or specifically bind to the same human IL-6R epitope recognized by the proto-antibody (or binding protein) in question, for example, the same epitope recognized by one or more CDR domains of the antibody (or binding protein) of the present invention or one or more VH and VL domains of the antibody (or binding protein) of the present invention as described herein, for example, one or more of the various antibodies of the present invention (for example, one or more anti-IL-6R antibodies shown in Table A, B, C, D, or E).

[0252] Binding to the same epitope can be readily tested using known protein sequences of the antibodies of the present invention (e.g., the VH and / or VL domain sequences of C07 and / or C09 and / or C12 and / or C19 and / or C20 disclosed in various sequence listings herein) (or several known protein sequences) using methods well known and described in the art, such as binding assays and competition assays. Appropriate binding assays are considered elsewhere in this specification. Therefore, preferably, any substantially homologous antigen-binding protein (e.g., antibody) should retain the ability to compete for binding to one or more of the antigen-binding proteins of the present invention with the relevant antigen. For example, binding to the same epitope can be tested using epitope mapping assays, for example, by analyzing the crystal structure of the antigen-antibody complex, or by measuring the mutability of individual residues (e.g., using alanine scanning and / or deep mutational scanning DMS, or yeast presentation combined with DMS, see, for example, Sierocki et al., PLoS Neglected Tropical Diseases, Vol. 15, No. 3 (2021), e0009231, and Van Blarcom et al., Journal of Molecular Biology). See also Biology, Vol. 427, No. 6(B), pp. 1513–1534 (2015) and Medina-Cucurella and Whitehead, Methods in Molecular Biology, Vol. 1764, pp. 101–121 (2018). Any of the above analyses can be used to determine the epitope.

[0253] Therefore, antibodies (or binding proteins) that bind to the same epitope as one or more of the various antibodies of the present invention, which are evaluated or determined by one or more of the methods outlined above, for example, form yet another aspect of the present invention.

[0254] Therefore, a person with ordinary skill in the art will understand that such a method can be used to test whether any antibody or binding protein, for example, a "substantially homologous" antibody, has the same binding specificity as the antibody of the present invention, or for example, binds to the same epitope. A person skilled in the art will be aware of other suitable methods and variations.

[0255] As outlined below, competitive binding assays can be used to test whether an antibody (or binding protein), for example, a "substantially homologous" antibody (or binding protein), retains the ability to bind to or specifically bind to the same (or substantially identical) human IL-6R epitope recognized by one or more of the antibodies of the present invention shown in the various sequence listings herein, or whether it has the ability to compete with one of the various antibodies of the present invention shown in the various sequence listings herein. The method described below is merely one example of a suitable competitive assay. Those skilled in the art will be aware of other suitable methods and variations.

[0256] An exemplary competitive assay involves evaluating the binding of various effective concentrations of the antibody (or antigen-binding protein) of the present invention to human IL-6R in the presence of varying concentrations of a test antibody (or binding protein) (e.g., a substantially homologous antibody). The amount of binding inhibition induced by the test antibody / binding protein can then be evaluated. A test antibody / binding protein that shows increased competition with the antibody of the present invention with increasing concentration (i.e., the amount of the antibody of the present invention binding to IL-6R decreases correspondingly as the concentration of the test antibody / binding protein increases) is evidence of binding to the same or substantially the same epitope. Preferably, the test antibody / binding protein significantly reduces the amount of the antibody of the present invention binding to IL-6R. Preferably, the test antibody / binding protein reduces the amount of the antibody of the present invention binding to IL-6R by at least about 95%. In such competitive assays, ELISA or flow cytometry assays may be used to evaluate binding inhibition, but other suitable techniques are well known to those of ordinary skill in the art.

[0257] Therefore, yet another aspect of the present invention provides an antibody (or binding protein) having the ability to bind (or specifically bind) to human IL-6R and to the same (or substantially the same) epitopes as the C07 (Table A) and / or C09 (Table B) and / or C12 (Table C) and / or C19 (Table D) and / or C20 (Table E) antibodies, i.e., an antibody (or binding protein) having the ability to bind to the same (or substantially the same) epitopes as antibodies (or binding proteins) containing the same CDR, or the same VH and VL domains as the C07 (Table A) and / or C09 (Table B) and / or C12 (Table C) and / or C19 (Table D) and / or C20 (Table E) antibodies. In yet another aspect, an antibody (or binding protein) having the ability to compete for binding to IL-6R with one or more of the various antibodies of the present invention (for example, competing with one or more of the antibodies shown in Tables A, B, C, D, or E as defined above) is a further embodiment of the present invention. This aspect of the present invention is applied to other features and properties of other aspects of the present invention with necessary modifications.

[0258] As used herein, the term "competitive antibody (or antigen-binding protein)" refers to an antibody that binds to an epitope (or antigen-binding protein) that is similar, substantially, or essentially the same as, or even identical to, the "reference antibody." Therefore, a competitive antibody has the ability to effectively compete with the reference antibody for binding to human IL-6R. Preferably, the competitive antibody can bind to the same epitope as the reference antibody. Conversely, the competitive antibody preferably has the same epitope specificity as the reference antibody.

[0259] The “reference antibody” as used herein is preferably an antibody having VH and VL domains as defined in Tables A, B, C, D, or E herein, and capable of binding to human IL-6R according to the present invention.

[0260] Identifying one or more antibodies (or binding proteins) that bind to the same epitope, or competing antibodies (or binding proteins), is a straightforward technical problem now that reference antibodies, such as those outlined in the sequence listing herein, are provided. Since the identification of competing antigen-binding proteins (e.g., antibodies) is determined by comparison with a reference antigen-binding protein (e.g., antibody), it will be understood that actually determining the epitopes to which one or both antigen-binding proteins bind is not necessary in any way to identify competing antigen-binding proteins. However, epitope mapping can also be performed using standard techniques if desired. Such epitope mapping can also be supplemented by competitive assays that can be performed using standard techniques, for example, as an initial or supplemental screening step. For example, in this regard, anti-IL-6R antibodies can be generated by immunization protocols using human soluble and / or membrane-bound IL-6R antigens as described in the Examples section, for example, and then anti-IL-6R antibodies can be easily selected using the methods described herein, for example, to identify those that bind to the same epitope as the reference antibody of the present invention and therefore preferably exhibit the same functional properties, such as the ability to inhibit IL-6 transsignaling while maintaining IL-6 classical signaling as described herein. Alternatively, substantially homologous sequences derived from antibodies having sequences shown in Tables A, B, C, D, or E can be selected in this manner.

[0261] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunological conjugate containing an antigen-binding domain, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred.

[0262] As used herein, the term “antibody” refers to full-length antibodies and antibody fragments (e.g., Fab fragments) that form additional aspects of the present invention.

[0263] However, the antibodies and antigen-binding proteins of the present invention may have a structure or format that binds to human IL-6R, and may include, for example, an antibody or antibody fragment that binds to human IL-6R, or an antigen-binding domain (for example, of an antibody or antibody fragment).

[0264] Depending on the type of constant domain in the heavy chain, all antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM, and the antibodies of the present invention may be any one of these classes. Some of these can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. The constant heavy chain domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0265] Generally, when all antibodies are used in the present invention, IgG, and particularly IgG1, are preferred.

[0266] The "light chain" of a mammalian antibody is assigned to one of two distinct types, kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain and several amino acids in the framework region of the variable domain. In the antibodies of this invention, kappa (κ) light chains are sometimes preferred.

[0267] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to a hypervariable region within the heavy chain variable region (VH domain) of an antibody molecule. The heavy chain variable region has three CDRs called heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, arranged from the amino terminus to the carboxyl terminus. The heavy chain variable region also has four framework regions (FR1, FR2, FR3, and FR4, arranged from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs from each other.

[0268] As used herein, the term "heavy chain variable region" (VH domain) refers to the variable region of the heavy chain of an antibody molecule.

[0269] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to a hypervariable region within the light chain variable region (VL domain) of an antibody molecule. The light chain variable region has three CDRs, called light chain CDR1, light chain CDR2, and light chain CDR3, from the amino terminus to the carboxyl terminus. The light chain variable region also has four framework regions (FR1, FR2, FR3, and FR4, from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs from each other.

[0270] As used herein, the term "light chain variable region" (VL domain) refers to the variable region of the light chain of an antibody molecule.

[0271] As described elsewhere in this specification, the antibodies, antibody fragments (e.g., Fab fragments), and antigen-binding proteins of the present invention have a structure or format such that they are capable of binding to human IL-6R. Any suitable format, for example, any format of an antibody or antibody fragment containing at least one antigen-binding domain capable of binding to human IL-6R, may be used. Exemplary and preferred formats or fragments include full-length (whole) antibodies such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD antibodies, chimeric antibodies; dimeric, trimer, and multimeric antibodies; bispecific antibodies; triplicate antibodies; multispecific antibodies; recombinant and engineered antibodies; and antibody fragments (e.g., Fab fragments).

[0272] As used herein, the term “antibody fragment” refers to a biologically relevant fragment, such as one containing the antigen-binding domain described above, i.e., a fragment that contributes to antigen binding, for example, a fragment that forms part of the antigen-binding domain. Certain preferred fragments are the heavy chain variable region (V) of the antibody of the present invention. H Domain) and light chain variable region (V L Includes domains.

[0273] Antibodies can be fragmented using conventional techniques. For example, the F(ab')2 fragment can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can then be processed to reduce disulfide crosslinks to produce the Fab' fragment. Papain digestion can lead to the formation of the Fab fragment. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically. Techniques for generating antibody fragments are well known and described in this art.

[0274] Therefore, the term "antibody" is used to refer to any antibody-like molecule that has an immunoglobulin (Ig) antigen-binding domain, and this term is used for Fab, Fab', F(ab')2, single-domain antibodies (DAB), such as camelid VHH nanoantibodies or V NAR Alternatively, VH domain or VL domain, TandAbs dimer, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, scFv / Fc KIH Linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, scFv / Fab-Fc and scFv / Fab-Fc KIHThis includes, but is not limited to, bispecific or trispecific antibodies containing the following: sc-diabodies; single-chain bispecific diabodies (scDb); kappa (lambda) bodies (scFv-CL fusions); BiTEs (bispecific T-cell engagers, T-cell attracting scFv-scFv tandems) containing antigen-binding domains and antibody fragments and formats; DVD-Ig (bivariable domain antibody, bispecific format); SIP (small immunoprotein, a type of minibody); SMIP ("small modular immunotherapy" scFv-Fc dimer); DART (ds-stabilized diabodies "biaffinity retargeting"); DART-Fc; duabodies, crossumabs, duetmabs, DNL, ​​and small antibody mimetics.

[0275] In certain embodiments, the antibody (or antigen-binding protein) of the present invention comprises all or part of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region or part thereof. IgG1 is a preferred format for the antibody of the present invention. Preferably, the antibody of the present invention comprises or accommodates a human heavy chain constant region.

[0276] Furthermore, the antibody or antigen-binding protein of the present invention may contain all or part of the kappa light chain constant region or the lambda light chain constant region, or part thereof. Preferably, the antibody of the present invention contains or accommodates the human light chain constant region.

[0277] All or part of such constant regions may be generated spontaneously or all or part of them may be synthesized. Suitable sequences for such constant regions are well known in the art and described in the literature. When the antibody (or antigen-binding protein) of the present invention contains the complete complement of the constant regions from these heavy and light chains, such an antibody is typically referred to herein as a “full-length” antibody or “whole” antibody. In some embodiments, such full-length or whole antibodies are preferred. Therefore, in some embodiments, an antibody or antibody fragment that is divalent (or bivalent) to human IL-6R, i.e., an antibody or fragment having two antigen-binding domains that bind to human IL-6R, is preferred. In other embodiments, Fab format or fragment is preferred. In other embodiments, scFv format or fragment is preferred. Therefore, in some embodiments, an antibody or antibody fragment that is monovalent to human IL-6R, i.e., an antibody or fragment having one or a single antigen-binding domain that binds to human IL-6R, is preferred.

[0278] Antibodies or antigen-binding proteins can be produced spontaneously or entirely or partially synthetically.

[0279] The antigen-binding domain of the antibody or antigen-binding protein of the present invention generally comprises an antibody heavy chain variable region (VH) containing three CDR domains and an antibody light chain variable region (VL) containing three CDR domains.

[0280] However, the presence of three CDRs from the light chain variable domain and three CDRs from the heavy chain variable domain of the antibody is not always necessary for antigen binding, as has been well documented in the literature in this field. Therefore, it is well known that smaller constructs than the classical antigen-binding domain described above are effective.

[0281] For example, camelid VHH antibodies containing only the VH domain and other single-domain antibodies demonstrate that these domains can bind to antigens with an acceptablely high affinity. Therefore, three CDRs (or even a single CDR) can effectively bind to antigens and form antigen-binding domains.

[0282] Therefore, while a preferred antigen-binding domain in the antigen-binding protein (e.g., antibody) of the present invention may contain six CDR regions (three from the light chain and three from the heavy chain), antigen-binding proteins (e.g., antibodies) having an antigen-binding domain with fewer than six CDR regions (e.g., three CDR regions) are also included in the present invention. Antigen-binding proteins (e.g., antibodies) having an antigen-binding domain with CDRs from only the heavy chain or only the light chain, such as VH or VL domain antibodies, are also envisioned. Therefore, as used herein, the term “antibody” refers to any antibody-like molecule having an immunoglobulin (Ig) antigen-binding domain, and this term refers to antibody fragments and formats containing an antigen-binding domain with fewer than six CDR regions (preferably three CDR regions), such as single-chain antibodies (e.g., camelid or shark heavy-chain antibodies (HCAb)) and single-domain camel VHH antibodies (e.g., nanobodies) or V NAR or comprising a VH domain or a VL domain. Such single-domain antibodies may have the advantageous property of promoting entry into brain tissue, which makes their use beneficial in the treatment of central nervous system (CNS) disorders (Belanger et al., Antibodies (Basel), Vol. 8, No. 2, p. 27 (2019)). This aspect of the present invention is applied to the consideration of various antibody characteristics and preferred embodiments of other aspects of the present invention with necessary modifications.

[0283] Other parts of this specification describe preferred light chain CDR regions used to form an antigen-binding domain in conjunction with a specified heavy chain CDR region. However, other light chain variable regions, including three CDRs, that can be used in conjunction with the heavy chain variable region of the present invention are also conceivable. Suitable light chain variable regions that can be used in combination with the heavy chain variable region of the present invention and that produce antibodies that bind to human IL-6R by the present invention can be readily identified by those with ordinary skill in the art.

[0284] For example, the heavy chain variable region of the present invention can be combined with a single light chain variable region or a repertoire of light chain variable regions, and the binding of the resulting antibody to human IL-6R can be tested.

[0285] If desired, a similar method could be used to identify alternative heavy-chain variable regions to be used in combination with the preferred light-chain variable region of the present invention.

[0286] The antibodies, antigen-binding proteins, and nucleic acid molecules of the present invention are generally “isolated” or “purified” molecules insofar as they are distinct from any such components that may be present in situ in the body of a human or animal or in tissue samples derived from the body of a human or animal. However, the sequences may match or be substantially homologous to sequences found in the body of a human or animal. Therefore, the terms “isolated” or “purified” as used herein in reference to nucleic acid molecules or sequences and proteins or polypeptides, such as antigen-binding proteins or antibodies, refer to such molecules when they have been isolated, purified, or substantially released from the natural environment, for example, when they have been isolated or purified from the body of a human or animal (if they are actually natural products), or when they have been produced by a technical process, i.e., including recombinant molecules and synthetically produced molecules.

[0287] Therefore, when used in relation to protein or polypeptide molecules such as antibodies, antibody fragments, or antigen-binding proteins of the present invention, including light chain CDRs 1, 2, and 3, heavy chain CDRs 1, 2, and 3, light chain variable regions, heavy chain variable regions, and full-length antibodies, the terms “isolated” or “purified” typically refer to a protein that is substantially free of cellular material or other proteins from the derived source material. In some embodiments, particularly when the protein is administered to humans or animals, such isolated or purified proteins are substantially free of culture media when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

[0288] In embodiments, the antibodies (or antigen-binding proteins) of the present invention are artificial constructs in that they do not occur naturally and, in that sense, do not correspond to naturally occurring molecules. For example, preferred antibodies can be designed or recombinantly produced, or experimentally induced to be produced in animal species, for example, by immunization. In other words, in embodiments, the antibodies or binding proteins of the present invention are non-native and do not occur naturally.

[0289] Those with ordinary skill in the art will understand that the proteins and polypeptides of the present invention, such as heavy and light chain CDRs, heavy and light chain variable regions, antibodies, antibody fragments, and antigen-binding proteins, may be prepared by any of several methods well known and described in the art, but most preferably by recombinant methods.

[0290] The nucleic acid fragments encoding the heavy and / or light chain regions of the antibody or antigen-binding protein of the present invention can be appropriately induced or generated by any suitable method, for example, cloning or synthesis.

[0291] Once nucleic acid fragments encoding the heavy and / or light chain regions of the antibody or antigen-binding protein of the present invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert a variable region fragment into a full-length antigen-binding protein (e.g., antibody) molecule having a suitable constant region domain, or to convert antibody fragments of a specific format discussed elsewhere herein, such as Fab fragments or scFv fragments. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody (binding protein) molecule of the present invention are generally incorporated into one or more suitable expression vectors to facilitate the production or manipulation of the antibody (binding protein) of the present invention.

[0292] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), insofar as the vector is compatible with the host cell in which it is used. An expression vector is "suitable for host cell conversion," meaning that the expression vector comprises the nucleic acid molecule of the present invention and a control sequence selected based on the host cell used for expression and operably bound to the nucleic acid molecule. "Operatably bound" means that the nucleic acid is bound to the control sequence in a manner that enables nucleic acid expression.

[0293] Accordingly, the present invention envisions a recombinant expression vector containing or comprising an expression vector (or one or more expression vectors), for example, a nucleic acid molecule of the present invention (for example, one or more nucleic acid molecules) or a fragment thereof, and a control sequence necessary for the transcription and translation of a protein sequence encoded by the nucleic acid molecule of the present invention. In other words, an expression vector containing the nucleic acid molecule of the present invention or encoding an antibody or antigen-binding protein of the present invention is provided.

[0294] An expression vector can be introduced into host cells to generate transformed host cells. The terms “transformed using,” “transformed,” “transformed,” and “transformed” shall include the introduction of nucleic acids (e.g., vectors) into cells by one of the many possible techniques known in this art. Appropriate methods for transforming and transposing host cells can be found in Sambrook et al. 1989 (Sambrook, Fritsch, and Maniatis), “Molecular Cloning: A Laboratory Manual,” 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, (1989) and other laboratory textbooks.

[0295] Suitable host cells include a wide variety of eukaryotic and prokaryotic host cells, as is well known to those with common knowledge in this field. For example, the proteins of the present invention may be expressed in yeast cells or mammalian cells, such as HEK or CHO cells. Furthermore, if appropriate, the proteins of the present invention may be expressed in prokaryotic cells, such as bacterial cells like Escherichia coli (E. coli), when, for example, the Fab, scFv, or DAB format is selected. Cell-free expression systems may also be used.

[0296] The proteins of the present invention may also be prepared by chemical synthesis using well-known techniques in protein chemistry, such as solid-phase synthesis.

[0297] Another aspect provides an expression construct, expression vector, or expression system (e.g., for viruses, bacteria, yeast, or mammals, or other expression constructs, vectors, or systems) comprising one or more nucleic acid fragments, segments, or molecules of the present invention, for example, one or more expression constructs or expression vectors. Preferably, the expression construct, vector, or system is recombinant. Preferably, the construct, vector, or system further comprises a regulatory sequence necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0298] Another aspect provides a host cell (e.g., a mammalian, bacterial, or yeast host cell) or virus containing one or more expression constructs or expression vectors of the present invention, for example, one or more host cells or viruses. Also provided are a host cell (e.g., a mammalian, bacterial, or yeast host cell) or virus containing one or more nucleic acid molecules of the present invention, for example, one or more host cells or viruses. A host cell (e.g., a mammalian, bacterial, or yeast host cell) or virus expressing an antibody (or antigen-binding protein) of the present invention forms yet another aspect.

[0299] A further aspect of the present invention provides a method for generating (or producing) an antibody (or antigen-binding protein) of the present invention, comprising the step of culturing a host cell of the present invention. A preferred method comprises (i) culturing a host cell containing one or more nucleic acid molecules or one or more expression vectors of the present invention under conditions suitable for the expression of the encoded antibody or binding protein, and optionally (ii) isolating or obtaining the antibody or binding protein from the host cell or from the growth medium / supernatant. Such a generation (or production) method may also include the step of purifying the antibody or antigen-binding protein product and / or formulating the antibody or antigen-binding protein product into a composition, for example, a pharmaceutical composition comprising at least one additional component, for example, a pharmaceutically acceptable carrier or excipient or diluent.

[0300] In embodiments, when the antibody or antigen-binding protein of the present invention is constructed from more than one polypeptide chain (e.g., Fab format or whole antibody), all polypeptides are preferably expressed in host cells from either the same expression vector or different expression vectors, so that the complete protein, such as the antibody protein of the present invention, can be assembled, isolated, or purified in host cells.

[0301] A composition comprising at least one antibody (or antigen-binding protein) of the present invention, or at least one nucleic acid molecule or expression vector of the present invention, or at least one host cell of the present invention constitutes a further aspect of the present invention. A formulation (composition) comprising one or more antibodies, etc. of the present invention mixed with an optional suitable diluent, carrier, or excipient constitutes a preferred embodiment of the present invention. Such formulations may be for pharmaceutically acceptable use, and therefore compositions of the present invention may be pharmaceutically acceptable or otherwise acceptable for administration to humans or non-human animals, but are particularly intended for administration to humans. Suitable diluents, excipients, and carriers are known to those skilled in the art.

[0302] Any appropriate method of administration can be used. The compositions according to the present invention may be presented in a form suitable for oral, nasal, parenteral (e.g., intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration or mucosal delivery, and any of these methods of administration, or any other method that is actually appropriate, can be used. In a preferred embodiment, the compositions according to the present invention are presented in a form suitable for intravenous administration. In some embodiments, the compositions according to the present invention are presented in a form suitable for intraperitoneal (IP) administration. In some embodiments, the compositions according to the present invention are presented in a form suitable for direct (intratumoral) injection into a tumor.

[0303] The active compounds defined herein (for example, the antibodies or antigen-binding proteins of the present invention) may be presented in conventional pharmaceutical dosage forms such as tablets, sugar-coated tablets, nasal sprays, liquids, emulsions, liposomes, exosomes, powders, capsules, or sustained-release formulations. Conventional pharmaceutical excipients and common manufacturing methods may be used for the preparation of these forms. Furthermore, nucleic acids or nucleic acid-based vectors, such as mRNA-based vectors or virus-based vectors, may be used for the administration of the active compounds of the present invention, for example, by encoding the antibodies or antigen-binding proteins of the present invention.

[0304] For example, conventional methods may involve the addition of preservatives such as p-hydroxybenzoate or stabilizers such as EDTA to prepare the injectable solution. The solution may then be filled into injection vials or ampoules.

[0305] The pharmaceutical composition (formulation) of the present invention is preferably administered parenterally. Intravenous administration is preferred. In some embodiments, administration is intraperitoneal (IP) administration. In some embodiments, administration is by injection into a tumor. Parenteral administration may be carried out by subcutaneous, intramuscular, intraperitoneal or intravenous injection using a syringe. Alternatively, parenteral administration may be carried out using an infusion pump. A further option is a composition that may be a powder or liquid for administration of the antibody or binding protein in the form of a nasal or lung spray. Yet another option is that the antibody or binding protein of the present invention may be administered percutaneously, for example from a patch, optionally from an ionophoresis patch, or transmucosally, for example via the cheek.

[0306] Appropriate dosage units can be determined by someone with ordinary knowledge in this field.

[0307] A further aspect of the present invention provides antibodies (or antigen-binding proteins) or compositions of the present invention for use in therapy, particularly for use in the prevention or treatment of disease.

[0308] In some embodiments, the disease is characterized by or caused by undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced or excessive IL-6 signaling or gp130 signaling induced by IL-6R / sIL-6R, preferably undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced or excessive IL-6 transsignaling.

[0309] Conversely, the disease is characterized by, or is associated with, undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 signaling in cells that do not express homologous IL-6R (membrane-bound IL-6R) at the cell membrane.

[0310] In some embodiments, the disease is one or more of the following: cancer, arthritis, such as rheumatoid arthritis or systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis, and Covid-19 or Covid-19 complications.

[0311] In some embodiments, the disease is an inflammatory disease, including an autoimmune disease or cancer. In some embodiments, the disease includes pathological inflammation or pathogenic inflammation.

[0312] Therefore, the antibodies (or antigen-binding proteins) of the present invention can be used to suppress or reduce inflammation. The types of inflammatory diseases treated by the present invention include, but are not limited to, cancer, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), pancreatitis, acute lung injury (ALI), Alzheimer's disease, sepsis, and Covid-19 or Covid-19 complications.

[0313] The types of cancer to be treated by the present invention include, but are not limited to, colorectal cancer (i.e., colon cancer), liver cancer, lung cancer, colitis-related cancer, pancreatic cancer, multiple myeloma, lymphoma, leukemia, and Hodgkin's disease.

[0314] In some embodiments, the disease is a central nervous system (CNS) disorder or a neurodegenerative disorder. The types of central nervous system (CNS) disorders to be addressed by the present invention include, but are not limited to, multiple sclerosis, depression, and Alzheimer's disease.

[0315] In other embodiments, the compositions, nucleic acid molecules, expression vectors, host cells, or viruses of the present invention may also be used in the therapeutics described herein.

[0316] The administration of antibodies or antigen-binding proteins in the therapeutic methods and uses of the present invention is carried out in amounts that are pharmaceutically, therapeutically, or physiologically effective for the subject (e.g., animal, e.g., human or non-human mammal) requiring treatment. Therefore, the methods and uses may include an additional step of identifying the subject requiring treatment. The appropriate and effective concentration / dose to be administered can be readily determined by a person with ordinary skill in the art.

[0317] Treatment of a disease or symptom according to the present invention (for example, treatment of an existing disease) includes the cure of the disease or symptom, or any reduction or alleviation of the disease, such as a reduction in severity or symptoms of the disease.

[0318] The therapeutic methods and uses of the present invention are suitable for the prevention of disease and the active treatment of disease (e.g., treatment of existing disease). Therefore, preventive measures are also encompassed by the present invention. For this reason, in the methods and uses of the present invention, the treatment includes preventive methods or prevention as appropriate.

[0319] Such preventative (or protective) aspects can be readily implemented for healthy, normal, or at-risk subjects and may include both complete and significant prevention. Similarly, significant prevention may include scenarios in which the severity of the disease or symptoms of the disease is reduced (e.g., measurably or significantly) compared to what would be expected if no treatment were given.

[0320] Accordingly, subjects suitable for treatment according to the present invention include humans who have or are at risk of having any of the diseases referred above, including inflammatory diseases, central nervous system (CNS) disorders, or cancers associated with, or caused by, undesirable, inappropriate, abnormal, pathological, enhanced, or excessive IL-6 signaling, preferably undesirable, inappropriate, abnormal, pathological, enhanced, or excessive IL-6 transsignaling, or more specifically, any of the diseases referred above, including inflammatory diseases, central nervous system disorders, and cancers.

[0321] Therefore, in humans, the in vivo methods and uses described herein are commonly practiced.

[0322] Therefore, as used herein, the terms “patient” or “subject” preferably refer to a human being.

[0323] In another embodiment, the subject is a person who has, is thought to have (or develops), or potentially has (or develops) the disease or symptoms of the problem described above.

[0324] In alternative embodiments, the present invention provides a method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the antibody (or antigen-binding protein) of the present invention to a patient in need thereof. Suitable examples of diseases or disorders are defined above. Preferably, the diseases or disorders include cancer, arthritis, e.g., rheumatoid arthritis or systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), e.g., Crohn's disease or ulcerative colitis, pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis, and one or more of Covid-19 or Covid-19 complications. This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0325] In an alternative embodiment, the present invention provides a method for treating or preventing a disease or disorder characterized by, resulting from, or associated with, undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 signaling or gp130 signaling induced by IL-6R / sIL-6R, preferably undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 transsignaling, the method comprising administering a therapeutically effective amount of the antibody (or antigen-binding protein) of the present invention to a patient in need thereof. This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0326] In an alternative embodiment, the present invention provides a method for treating or preventing a disease or disorder characterized by, resulting from, or associated with, undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 signaling in cells that do not express homologous IL-6R (membrane-bound IL-6R) in the cell membrane, the method comprising administering a therapeutically effective amount of the antibody (or antigen-binding protein) of the present invention to a patient in need thereof. This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0327] A "therapeutally effective dose" means a dose sufficient to be effective for the subject's symptoms. Whether a dose is sufficient to be effective for a subject's symptoms may be determined by the subject themselves or by a physician, preferably by clinical evaluation, which can be easily monitored.

[0328] In alternative embodiments, the present invention provides the use of antibodies (or antigen-binding proteins) of the present invention in the manufacture of pharmaceuticals for therapeutic use, for example, for use in the treatment or prevention of disease. Appropriate examples of diseases or disorders are defined above. Preferably, the treatment is the treatment or prevention of one or more of the following: cancer, arthritis such as rheumatoid arthritis or systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis, pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis, and Covid-19 or complications of Covid-19.

[0329] In alternative embodiments, the present invention provides the use of an antibody (or antigen-binding protein) of the present invention in the manufacture of a pharmaceutical product for use in treating or preventing a disease or disorder characterized by, resulting from, or associated with, undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 signaling or gp130 signaling induced by IL-6R / sIL-6R, preferably undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 transsignaling. This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0330] In an alternative embodiment, the present invention provides the use of the antibody (or antigen-binding protein) of the present invention in the manufacture of pharmaceuticals for use in treating or preventing diseases or disorders characterized by, resulting from, or associated with, undesirable, inappropriate, abnormal, pathological, pathogenic, enhanced, or excessive IL-6 signaling in cells that do not express homologous IL-6R in the cell membrane (membrane-bound IL-6R). This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0331] From a different perspective, the present invention provides a use for the treatment or prevention of one or more diseases outlined above, based on the antigen-binding proteins (e.g., antibodies) of the present invention as defined herein. This aspect of the present invention is applied to the embodiments of therapeutic use of the present invention described herein with necessary modifications.

[0332] In some embodiments, the antibodies (or binding proteins) of the present invention can be used in monotherapy. In other embodiments, they can be used in combination with other standard cancer drugs and drugs used in the treatment of inflammatory diseases or central nervous system (CNS) disorders.

[0333] The present invention further comprises a kit comprising one or more antibodies (or antigen-binding proteins) or compositions of the present invention, one or more nucleic acid molecules encoding an antibody (or antigen-binding protein) of the present invention, one or more expression vectors comprising a nucleic acid sequence of the present invention, or one or more host cells or viruses comprising an expression vector or nucleic acid sequence of the present invention. Preferably, the kit is intended for use in the methods and uses described herein, for example, in the therapeutic methods or in vitro or in vivo methods / assays described herein. Preferably, the kit includes instructions for the use of the kit components. Preferably, the kit is intended to treat or prevent diseases or conditions described elsewhere herein, and optionally includes instructions for the use of kit components to treat or prevent such diseases or conditions.

[0334] The antibodies (or antigen-binding proteins) of the present invention as defined herein may also be used in vitro or in vivo for applications and assays, such as binding assays or diagnostic assays, as molecular tools. Since the antigen-binding proteins (e.g., antibodies) contain at least one antigen-binding domain that binds to IL-6R, for example, human IL-6R, they can be used in any assay where an IL-6R binding moiety is required.

[0335] Therefore, yet another aspect of the present invention provides reagents comprising the antibody (or antigen-binding protein) of the present invention as defined herein, and the use of such antibody (or antigen-binding protein) as a molecular tool in an in vitro or in vivo assay for the detection of, for example, IL-6R, for example, human IL-6R, in a sample of the present invention.

[0336] Throughout this application, the singular forms ("a" and "an") are used to mean "at least one," "at least first," "one or more," or "multiple" of the components or steps being referenced, except where an upper limit is subsequently specifically stated. Thus, as used herein, "an antibody" means "at least first antibody."

[0337] Furthermore, where the terms “comprise,” “comprises,” “has,” or “having,” or other equivalent terms are used herein, in some more specific embodiments, for example, in the definitions of CDR or FR sequences herein, these terms include the terms “consists of,” or “consists essentially of,” or other equivalent terms.

[0338] The terms “decrease,” “reduce,” “inhibit,” “limit,” “prevent,” or “block” (or equivalent terms) as used herein include any measurable reduction or decrease compared to a suitable control. A suitable control may be readily identifiable by a person of ordinary skill in the art and may include untreated or placebo-treated subjects or healthy subjects, or samples or assays in which the antibody is absent or a control antibody that does not bind to human IL-6R, such as an isotype control antibody. Preferably, the reduction, decrease, or inhibition is significant, for example, clinically or statistically significant.

[0339] As used herein, the term “increase” (or equivalent term) includes any measurable increase or rise compared to a suitable control. A suitable control may be readily identifiable by a person of ordinary skill in the art and may include untreated or placebo-treated subjects or healthy individuals, or samples or assays in which the antibody is absent or a control antibody that does not bind to human IL-6R, such as an isotype control antibody. Preferably, the increase is significant, for example, clinically or statistically significant.

[0340] Preferably, such increases (and actually other increases, improvements or positive effects as referred elsewhere herein) or decreases (and actually other decreases, reductions, inhibitions or negative effects as referred elsewhere herein) are measurable increases, decreases, etc., and (as appropriate) more preferably they are clinically significant or statistically significant increases, such as significant increases or decreases having, for example, <=0.05 or a probability value of <0.05 when compared to a suitable control level or value (e.g., compared to an untreated or placebo-treated subject, or compared to a healthy subject or a normal subject or the same subject before treatment, or compared to a sample or assay in which a control antibody is absent or does not bind to human IL-6R, e.g., an isotype control antibody is present).

[0341] Methods for determining the statistical significance of differences between test groups of subjects or differences at the level of a particular parameter are well known in the art and described in the literature. For example, in this specification, a decrease or increase at the level of a particular parameter, or a difference between test groups of subjects, is generally considered statistically significant if a statistical comparison using significance tests such as the Student's t-test, Mann-Whitney U rank-sum test, chi-squared test, or Fisher's exact test, one-way ANOVA, or two-way ANOVA test, as appropriate, shows a probability value of <= 0.05 or <0.05. In all embodiments, references to significant or insignificant differences are preferably references to statistically significant or insignificant differences.

[0342] [Table 1]

[0343] [Table 2]

[0344] [Table 3-1]

[0345] [Table 3-2]

[0346] [Table 4-1]

[0347] [Table 4-2]

[0348] [Table 5-1]

[0349]

Table 5-2

[0350]

Table 6

[0351] Amino acid sequence of human membrane-bound IL-6R subunit alpha (Uniplot entry P08887) (SEQ ID NO: 112) MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISP PVSPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFPR

[0352] Amino acid sequence of human soluble IL-6R (SEQ ID NO: 113) MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQ

[0353] Amino acid sequence of human membrane-bound IL-6R subunit beta (gp130) (Uniplot entry P40189) (SEQ ID NO: 114) MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ

[0354] Amino acid sequence of mouse membrane-bound IL-6R subunit alpha (Uniplot entry P22272) (SEQ ID NO: 115) MLTVGCTLLVALLAAPAVALVLGSCRALEVANGTVTSLPGATVTLICPGKEAAGNVTIHWVYSGSQNREWTTTGNTLVLRDVQLSDTGDYLCSLNDHLVGTVPLLVDVPPEEPKL SCFRKNPLVNAICEWRPSSTPSPTTKAVLFAKKINTTNGKSDFQVPCQYSQQLKSFSCQVEILEGDKVYHIVSLCVANSVGSKSSHNEAFHSLKMVQPDPPANLVVSAIPGRPRW LKVSWQHPETWDPSYYLLQFQLRYRPVWSKEFTVLLLPVAQYQCVIHDALRGVKHVVQVRGKEELDLGQWSEWSPEVTGTPWIAEPRTTPAGILWNPTQVSVEDSANHEDQYESS TEATSVLAPVQESSSMSLPTFLVAGGSLAFGLLLCVFIILRLKQKWKSEAEKESKTTSPPPPPYSLGPLKPTFLLVPLLTPHSSGSDNTVNHSCLGVRDAQSPYDNSNRDYLFPR

[0355] Amino acid sequence of mouse soluble IL-6R (SEQ ID NO: 116) LVLGSCRALEVANGTVTSLPGATVTLICPGKEAAGNVTIHWVYSGSQNREWTTTGNTLVLRDVQLSDTGDYLCSLNDHLVGTVPLLVDVPPEEPKLSCFRKNPLVNAICEWRPSSTPSPTTKAVLFAKKINTTNGKSDFQVPCQYSQQLKSFSCQVEILEGDKV YHIVSLCVANSVGSKSSHNEAFHSLKMVQPDPPANLVVSAIPGRPRWLKVSWQHPETWDPSYYLLQFQLRYRPVWSKEFTVLLLPVAQYQCVIHDALRGVKHVVQVRGKEELDLGQWSEWSPEVTGTPWIAEPRTTPAGILWNPTQVSVEDSANHEDQYESSTE

[0356] Next, the present invention is further described with reference to the following drawings in the following non-limiting embodiments. [Brief explanation of the drawing]

[0357] [Figure 1] An ELISA assay was performed to analyze the binding of increasing concentrations of anti-IL-6R IgG antibodies (clones 07, 09, 12, 19, and 20) to sIL-6R. Anti-mesothelin IgG antibody (clone 1H7) was used as a negative control. [Figure 2] Flow cytometry analysis of THP-1 cells stained with anti-IL-6R IgG antibodies (clones 07, 09, 12, 19, and 20) and APC-labeled secondary antibodies, as shown in the figure. Cells stained only with APC-conjugated secondary antibodies were used as a control for nonspecific binding of secondary antibodies. [Figure 3] Flow cytometry analysis of Ba / F3_gp130_IL-6R cells, membrane-stained with anti-IL-6R IgG C20 antibody and PE-labeled secondary antibody, and incubated with and without IL-6. The results are presented as histograms of the relative number of cells (rel.# cells) relative to PE fluorescence (IL-6R). Cells stained only with PE-conjugated secondary antibody were used as a control for nonspecific binding of the secondary antibody. [Figure 4] Analysis of IL-6 transsignaling-induced proliferation of Ba / F3_gp130 cells cultured with IL-6 (10 ng / ml), sIL-6R (200 ng / ml), and the indicated amounts of anti-IL-6R IgG antibody (clones 07, 09, 12, 19, and 20) or anti-mesothelin IgG (1H7). Proliferation responsive to IL-6 and sIL-6R was set to 100%. Mean ± standard deviation from independent n=3 experiments is shown. Data are also shown in Table 5. [Figure 5] Dose-dependent curves of inhibition of IL-6 transsignaling-induced cell proliferation in Ba / F3_gp130 cells cultured with IL-6 (10 ng / ml) and sIL-6R (200 ng / ml) by anti-IL-6R IgG antibodies (clones 07, 09, 12, 19, and 20). Data points from the mean values ​​in Tables 5 and 6 are plotted. IC50 values ​​for inhibition of IL-6 transsignaling-induced cell proliferation are also shown. [Figure 6]Analysis of IL-6 classical signaling-induced proliferation of Ba / F3_gp130_IL-6R cells cultured with IL-6 (10 ng / ml) and the indicated amounts of anti-IL-6R IgG antibody (clones 07, 09, 12, 19, and 20) or anti-mesothelin IgG (1H7). IL-6-responsive proliferation was set to 100%. Mean ± standard deviation from independent n=3 experiments is shown. Data are also shown in Table 9. [Figure 7] Analysis of combined IL-6 classical and transsignaling-induced proliferation of Ba / F3_gp130_IL-6R cells cultured with IL-6 (10 ng / ml), sIL-6R (200 ng / ml), and the indicated amounts of anti-IL-6R IgG antibody (clones 07, 09, 12, 19, and 20) or anti-mesothelin IgG (1H7). IL-6-responsive proliferation was set to 100%. Mean ± standard deviation from independent n=3 experiments is shown. Data are also shown in Table 11. [Figure 8] Dose-dependent curves of inhibition of IL-6 transsignaling-induced cell proliferation in Ba / F3_gp130 cells cultured with IL-6 (10 ng / ml) and sIL-6R (200 ng / ml) by anti-IL-6R Fab C20 (3.3 ng / ml, 33.3 ng / ml, 100 ng / ml, 167 ng / ml, 233 ng / ml, 333 ng / ml, and 3300 ng / ml). IC50 for inhibition of IL-6 transsignaling-induced cell proliferation is also shown. [Examples]

[0358] Example 1 - Fab generation and functional characterization material and method

[0359] Fab generation using a subtractive immunization protocol For example, antibodies were identified using a subtractive immunization protocol, a technique described in Sandrock, Journal of Immunological Methods, Vol. 100, pp. 73-82 (1987). Specifically, in this case, the protocol involved mice that were first treated with cyclophosphamide after four cycles of resistance-inducing IP injections using JV1 cells (Ba / F3 cells (ATCC) that stably express human membrane-bound IL-6R), and secondly, four IP immunization injections using recombinant human soluble IL-6R alpha. After this subtractive immunization protocol, the mice were sacrificed, and RNA from the splenocytes of each mouse was used to generate a Fab phage presentation library for screening. Bacteria were grown in 2TY medium + ampicillin + glucose, and Fab was generated from bacterial cultures supplemented with IPTG (isopropyl β-D-1-thiogalactopyranoside). The initial screening process yielded 27 candidate Fab for screening.

[0360] Cell proliferation assay to analyze Fab-mediated inhibition of the IL-6 signaling pathway Two mutants of the mouse premature B cell line (Ba / F3) were used as a cell model for cell proliferation. Ba / F3 wild-type cells do not express gp130 or IL-6R. 1) Ba / F3 cells stably express human gp130 (Ba / F3_gp130) (Gaering et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 3, pp. 1119-11123 (1994)). These cells lack membrane-bound IL-6R and therefore do not proliferate in response to IL-6 classical signaling. Proliferation of Ba / F3_gp130 cells is induced by a complex of IL-6 and soluble IL-6R. Therefore, Ba / F3_gp130 cells serve as a model for IL-6 transsignaling. 2) Ba / F3_gp130_IL-6R cells (Vollmer et al., European Journal of Biochemistry, Vol. 263, No. 2, pp. 438-446 (1999)) stably express both human membrane-bound gp130 and membrane-bound IL-6R. These cells proliferate in response to IL-6 stimulation and therefore serve as a model for IL-6 classical signaling. Upon stimulation with IL-6 and soluble IL-6R, both the classical and transsignaling pathways of IL-6 (IL-6 mixed signaling) are activated in Ba / F3_gp130_IL-6R cells.

[0361] To analyze cytokine-induced cell proliferation mediated by the IL-6 trans or combined IL-6 classical and trans (IL-6 mixed) pathways, 5,000 cells / well were inoculated into DMEM (10% FCS + penicillin / streptomycin) in 96-well plates. Cells were cultured in the presence of human IL-6 (Peprotech, 10 ng / ml) and human soluble IL-6R (200 ng / ml, SEQ ID NO: 113) to stimulate IL-6 transsignaling in Ba / F3_gp130 cells and combined IL-6 classical and transsignaling (mixed signaling) in Ba / F3_gp130_IL-6R cells. To determine the inhibitory potential of candidate Fabs, 1% periplasmic extract (PE) containing each of the 27 Fabs was added. As a control for nonspecific proliferation inhibition by PE, PE containing anti-mesothelin Fab (clone 1H7, MAB Designs) was used. After 48 hours, the number of viable cells was determined using Cell Titer Blue reagent (Promega) according to the manufacturer's instructions. Absorption was recorded at 570 nm and 600 nm. For normalization, the absorbance of cells grown without cytokines was subtracted from the absorbance of each sample. The absorbance of cells grown with cytokines was set to 100%.

[0362] Intracellular signaling assay to analyze Fab-mediated inhibition of the IL-6 signaling pathway Two variants of human embryonic kidney cells (HEK T-REx(trademark)-293 (ThermoFisher)) were used as cell models for intracellular signaling. 1) HEK293 cells endogenously express membrane-bound gp130 but do not express membrane-bound IL-6R. Because these cells lack membrane-bound IL-6R, they do not proliferate in response to IL-6 classical signaling. Proliferation of HEK293 cells is induced by a complex of IL-6 and soluble IL-6R. Therefore, they serve as a model for IL-6 transsignaling. 2) HEK293_IL-6R was induced from HEK293 cells by stable transduction of human IL-6R (Dittrich et al., Molecular Biosystems, Vol. 8, No. 8, pp. 2119-2134 (2012)). Stimulation with IL-6 activates IL-6 classical signaling. Stimulation with IL-6 and soluble IL-6R activates both the IL-6 classical signaling pathway and the IL-6 transsignaling pathway (IL-6 mixed signaling) in HEK293_IL-6R cells.

[0363] To analyze cytokine-induced intracellular signaling mediated by the IL-6 trans or complex IL-6 classical and trans (IL-6 mixed) pathways, IL-6-induced STAT3 Y705 phosphorylation was determined by intracellular flow cytometry. 2 × 10¹⁶ samples were collected in DMEM (10% FCS + penicillin / streptomycin) (HEK293) or DMEM (10% FCS + penicillin / streptomycin + 2 μg puromycin) (HEK293_IL-6R) in 12-well petri dishes. 5Cells were inoculated into wells. Cells were incubated at 37°C and 5% CO2 in a water-saturated atmosphere. After 48 hours, cells were starved for 2 hours in 900 μl of DMEM without FCS and antibiotics. Next, cells were treated with IL-6 (10 ng / ml) and soluble IL-6R (200 ng / ml) to stimulate IL-6 transsignaling in HEK293 cells and complex IL-6 classical and transsignaling (IL-6 mixed signaling) in HEK293_IL-6R cells. To determine the inhibitory potential of candidate Fabs, 1% PE containing each of the 27 Fabs was added for 30 minutes. After stimulation, the medium was removed and 150 μl of trypsin-EDTA was added per well. 100 μl of cell suspension was transferred to individual wells of a 96-well plate pre-filled with 100 μl of 4% paraformaldehyde. Cells were fixed at 37°C for 10 minutes. After fixation, the plate was centrifuged (5 minutes, 300g, 4°C). The supernatant was removed, and the cells were resuspended in 200 μl of 90% methanol, which was kept on ice. The plate was incubated at -20°C for 10 minutes, then centrifuged. The cells were washed twice with 200 μl of PBS with 1% BSA and 0.5 mM EDTA (FACS buffer). The cells were resuspended in 50 μl of antibody solution (anti-(p)Y STAT3, clone: ​​4 / P-STAT3 (BD ​​Biosciences), 1:200 in FACS buffer) and incubated overnight in the dark at 4°C. The cells were washed three times with FACS buffer. Finally, fluorescence was determined using a flow cytometer. For normalization, the fluorescence of unstimulated cells was reduced from each sample. The fluorescence of cytokine-stimulated cells was set to 100%.

[0364] result Selective inhibition of IL-6 transsignaling-induced cell proliferation by Fab

[0365] In the initial screening process, we identified 27 candidate Fabs that were most likely to be selective in inhibiting IL-6 transsignaling. To this end, we tested whether PEs containing one of the 27 Fabs selectively inhibited IL-6 transsignaling compared to IL-6 classical signaling. As indicators, we analyzed IL-6 transsignaling and classical signaling-induced cell proliferation.

[0366] First, we tested which of the 27 candidate Fabs inhibited IL-6 transsignaling-induced proliferation. To this end, we stimulated Ba / F3_gp130 cells with IL-6 and soluble IL-6R, and tested the inhibitory ability of Fab-containing PEs (Table 1). The control PE containing anti-mesothelin Fab did not reduce the proliferation of Ba / F3_gp130 cells, suggesting that PE alone does not inhibit the proliferation of these cells. In contrast, all 27 candidate Fabs inhibited IL-6 transsignaling-induced proliferation. The strength of inhibition varied by clone. For example, clones 01, 20, and 26 reduced IL-6 transsignaling-induced proliferation by more than 50%, while clones 08 and 23 only slightly reduced proliferation.

[0367] Table 1. Inhibition of IL-6 transsignaling-induced proliferation by PE containing each of the 27 candidate Fabs. The proliferation of Ba / F3_gp130 cells induced by 10 ng / ml IL-6 and 200 ng / ml soluble IL-6R (sIL-6R) was set to 100%. Results are presented as mean ± standard deviation from 3 independent experiments.

[0368] [Table 7]

[0369] To address whether the observed inhibition of IL-6-induced proliferation is selective for IL-6 transsignaling, we then tested whether IL-6 classical signaling-induced proliferation could also be inhibited by 27 candidate Fabs. In the body, cells confront both IL-6 and soluble IL-6R, and cells expressing membrane-bound IL-6R enable both classical and transsignaling (a complex classical and transsignaling, referred to as IL-6 mixed signaling). However, high expression of membrane-bound IL-6R is favorable to classical signaling (Reeh et al., Cell Communication and Signaling, Vol. 17, No. 1, p. 46 (2019)). Therefore, stimulating cells expressing a high number of IL-6Rs allows us to approximate the ability of Fabs to reduce IL-6 classical signaling under physiological conditions. Ba / F3_gp130_IL-6R can be stimulated by membrane-bound IL-6R and soluble IL-6R, and therefore this prerequisite (Lee et al. (2019), above) can be satisfied.

[0370] Ba / F3_gp130_IL-6R cells were stimulated with human IL-6 and soluble IL-6R to enable IL-6 classical and transsignaling-induced proliferation. PE containing anti-mesothelin Fab reduced Ba / F3_gp130_IL-6R cell proliferation by 85%, suggesting that PE alone only slightly inhibits the proliferation of these cells. Approximately half of the 27 Fabs tested (e.g., clones 01, 06, 16, and 26) inhibited Ba / F3_gp130_IL-6R cell proliferation more than anti-mesothelin Fabs, suggesting interference with IL-6 classical signaling-induced proliferation by these Fabs (Table 2). In comparison, all 27 candidate Fabs inhibited proliferation induced by IL-6 transsignaling. In summary, these data suggest that some of the 27 candidate Fabs selectively inhibit IL-6 transsignaling, while others inhibit both classical and transsignaling of IL-6.

[0371] Table 2. Inhibition of complex IL-6 classical and transsignaling-induced proliferation by PE containing each of the 27 candidate Fabs. The proliferation of Ba / F3_gp130_IL-6R cells induced by 10 ng / ml IL-6 and 200 ng / ml soluble IL-6R (sIL-6R) was set to 100%. Results are presented as mean ± standard deviation from three independent experiments.

[0372] [Table 8]

[0373] To quantify the selectivity of each Fab that inhibits IL-6 transsignaling, the normalized ratio of residual cytokine-induced proliferation was calculated between Ba / F3_gp130_IL-6R cells (measuring IL-6 classical signaling) (Table 2) and Ba / F3_gp130 cells (measuring IL-6 transsignaling) (Table 1) in the presence of each candidate Fab. This ratio is referred to as the classical:transsignaling ratio. Fabs that inhibit IL-6 transsignaling to a greater extent than IL-6 classical signaling (i.e., greater or more potent inhibition of IL-6 transsignaling) are characterized by a high classical:transsignaling ratio (e.g., a ratio >1.0). Fabs that inhibit IL-6 trans and classical signaling to the same degree have a classical:transsignaling ratio of approximately 1.0, while Fabs that inhibit IL-6 classical signaling to a greater extent than IL-6 transsignaling (i.e., greater or more potent inhibition of IL-6 classical signaling) are characterized by a low classical:transsignaling ratio (i.e., a ratio of <1.0). Therefore, Fabs were sorted in descending order of classical:transsignaling ratio (Table 3). In particular, Fabs 20, 07, 19, 09, and 12 have high classical:transsignaling ratios (2.2, 1.5, 1.4, 1.3, and 1.3, respectively) and are therefore promising candidates for selective inhibition (or greater inhibition) of IL-6 transsignaling.

[0374] Table 3 Ranking of candidate Fabs based on inhibition of proliferation Average residual cytokine-induced proliferation in the presence of indicated Fab cells, taken from Table 1 (Ba / F3_gp130, measuring IL-6 transsignaling) and Table 2 (Ba / F3_gp130_IL-6R, measuring IL-6 complex classical and transsignaling). Fab cells are sorted in descending order of classical:transsignaling ratio.

[0375] [Table 9]

[0376] Fab-mediated IL-6 transsignaling induction and selective inhibition of intracellular signaling. Next, we tested the ability of 27 candidate Fabs to selectively inhibit intracellular IL-6 transsignaling. In contrast to cell proliferation, which takes hours to days, intracellular signaling occurs in seconds to minutes. Therefore, signaling is unaffected by indirect events and serves as a direct indicator for IL-6 signaling. Both classical and transsignaling of IL-6 lead to the activation of Janus kinases (JAKs) associated with gp130. Activated JAKs phosphorylate tyrosine (Y) residues within the intracellular portion of g130. These phosphorylated Y motifs recruit signaling and transcriptional activator 3 (STAT3). STAT3 is then phosphorylated by JAK at Y705 (see review by Schaper and Rose-John in Cytokine & Growth Factor Reviews, Vol. 26, No. 5, pp. 475-487 (October 2015)). Therefore, phosphorylation of STAT3 at Y705 serves as an indicator for IL-6-induced intracellular signaling.

[0377] To quantify the selectivity of each Fab that inhibits IL-6 transsignaling, the ratio of residual STAT3 activation (i.e., phosphorylation at Y705) induced by stimulation with IL-6 and soluble IL-6R (classical:transsignaling ratio) was calculated in HEK293_IL-6R cells (measuring combined classical and trans (mixed) signaling) and HEK293 cells (measuring IL-6 transsignaling) in the presence of individual candidate Fabs.

[0378] As described above, clones that selectively inhibit IL-6 transsignaling (i.e., greater inhibition of IL-6 transsignaling compared to IL-6 classical signaling) are characterized by a higher classical:transsignaling ratio (e.g., >1.0). Therefore, clones were sorted in descending order of classical:transsignaling ratio (Table 4). Notably, the same five Fabs (clones 20, 19, 07, 09, and 12) were ranked among the Fabs with the highest classical:transsignaling ratios in intracellular signaling.

[0379] Table 4. Ranking of candidate Fabs based on inhibition of intracellular signaling. Mean residual cytokine-induced STAT3 Y705 phosphorylation in the presence of Fab cells in HEK293_IL-6R cells (measuring IL-6 complex classical and trans (mixed) signaling) and HEK293 cells (measuring IL-6 trans signaling). Fab cells are sorted in descending order of classical:trans signaling ratio.

[0380] [Table 10]

[0381] Example 2 - Generation and functional characterization of anti-IL-6R IgG antibodies material and method

[0382] Generation of anti-IL-6R IgG antibodies by chimera formation of selected Fab fragments with human IgG1. Human IgG1 and a chimeric expression vector for Fab were transferred into CHO cells. The cell culture supernatant was collected, and the concentration of anti-IL-6R IgG antibody was determined.

[0383] ELISA assay to analyze the binding of anti-IL-6R IgG antibodies to soluble IL-6R. To enable proper convolution of soluble IL-6R, ELISA plates were first coated with neutraavidin (500 ng / well), followed by biotinylated IL-6 (500 ng / ml), and then incubated with recombinant human soluble IL-6R (500 ng / ml). Next, the plates were incubated with increasing concentrations of anti-IL-6R IgG antibody (1 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, and 500 ng / ml), and subsequently detected with HRP-conjugated anti-human Fc secondary antibody. Anti-mesothelin IgG antibody (clone 1H7) was used as a negative control.

[0384] Flow cytometry assay to analyze the binding of anti-IL-6R IgG antibodies to membrane-bound IL-6R. Human THP1 cells expressing membrane-bound IL-6R (a commercially available human monocyte cell lineage derived from leukemia patients) were incubated with anti-IL-6R IgG antibody. After washing, the cells were incubated with APC-conjugated secondary antibody and fluorescence quantification was performed using flow cytometry. As a control for nonspecific binding of the secondary antibody, cells stained only with APC-conjugated secondary antibody were used. The increase in mean fluorescence of cells stained with anti-IL-6R IgG antibody and cells stained with the secondary antibody compared to cells stained only with the secondary antibody indicates the binding of anti-IL-6R IgG antibody to membrane-bound IL-6R.

[0385] To analyze antibody binding to membrane-bound IL-6R in the presence of IL-6, Ba / F3_gp130_IL-6R cells were washed twice with 1% BSA-EDTA buffer (1% BSA in PBS, 0.5 mM EDTA). Next, cells were incubated on ice for 30 minutes with anti-IL-6R IgG C20 (1:100, 440 ng / ml in 1% BSA-EDTA buffer) in or without equimolar amounts of human IL-6 (Peprotec, 60 ng / ml). Subsequently, cells were stained for 30 minutes with a specific anti-human secondary antibody conjugated to PE (1:100, Biozole, in 1% BSA-EDTA buffer). Ba / F3_gp130_IL-6R cells incubated with the secondary antibody alone served as a control for nonspecific binding of the secondary antibody. Analysis using flow cytometry with FACS Canto II (BD Biosciences) revealed an increase in average fluorescence between cells stained with anti-IL-6R IgG C20 and cells stained with the secondary antibody, compared to cells stained with the secondary antibody alone. This indicates the binding of anti-IL-6R IgG C20 to membrane-bound IL-6R in the absence or presence of IL-6.

[0386] A cell proliferation assay analyzing the inhibition of human IL-6, human IL-11, mouse IL-6, and human IL-3 signaling pathways by anti-IL-6R IgG antibodies. Two variants of Ba / F3 cells were used as cell models for IL-6-mediated cell proliferation as described in Example 1.

[0387] To analyze cytokine-induced cell proliferation mediated by human IL-6 trans, IL-6 classical, or combined IL-6 classical and trans (IL-6 mixed) pathways, 5,000 cells / well were inoculated into DMEM (10% FCS + penicillin / streptomycin) in 96-well plates. Ba / F3_gp130_IL-6R cells were cultured in the presence of human IL-6 (Peprotec, 10 ng / ml) to stimulate IL-6 classical signaling, or in the presence of IL-6 (10 ng / ml) and soluble IL-6R (200 ng / ml) to stimulate combined IL-6 classical and trans (mixed) signaling. Ba / F3_gp130 cells were cultured in the presence of IL-6 (10 ng / ml) and soluble IL-6R (200 ng / ml) to stimulate IL-6 trans signaling. Furthermore, the cells were incubated with anti-IL-6R IgG antibody at concentrations that increase the certain amount of cells that can be collected (10 ng / ml, 100 ng / ml, 300 ng / ml, 500 ng / ml, 700 ng / ml, 1,000 ng / ml, and 10,000 ng / ml for Ba / F3_gp130 cells, or 1 μg / ml and 10 μg / ml for Ba / F3_gp130_IL-6R cells) or with anti-mesothelin IgG antibody (1H7) (1 μg / ml) used as a control for nonspecific inhibition of intracellular signaling by IgG antibody. After 48 hours, the number of viable cells was determined as described in Example 1.

[0388] To analyze cell proliferation induced by Hyper-IL-6 (Hy-IL-6) transsignaling, Ba / F3_gp130 cells were inoculated as described above, and Hy-IL-6 (28 ng / ml, equimolar to 10 ng / ml IL-6) was added to stimulate IL-6 transsignaling. Furthermore, the cells were incubated with anti-IL-6R IgG antibodies (10 ng / ml, 100 ng / ml, 300 ng / ml, 500 ng / ml, 700 ng / ml, 1,000 ng / ml, and 10,000 ng / ml) at concentrations that increased the constant yield of cells, or with anti-mesothelin IgG antibody (1H7) (1 μg / ml) used as a control for nonspecific inhibition of intracellular signaling by IgG antibodies. After 48 hours, the number of viable cells was determined as described in Example 1.

[0389] To analyze cytokine-induced cell proliferation mediated by the human IL-11 trans, IL-11 classical, or combined IL-11 classical and trans (IL-11 mixed) pathways, cells were inoculated, stimulated, and analyzed as described above, but with the addition of 10 μg / ml anti-IL-6R antibody. This was done by replacing Ba / F3_gp130_IL-6R cells with Ba / F3_gp130_IL-11R cells (expressing human gp130 and human IL-11R), replacing IL-6 and soluble IL-6R with IL-11 (biotechne, 10 ng / ml) and soluble IL-11R (biotechne, 200 ng / ml), respectively, and adding 10 μg / ml anti-IL-6R IgG antibody.

[0390] To analyze cytokine-induced cell proliferation mediated by the mouse IL-6 trans, IL-6 classical, combined IL-6 classical, and trans (IL-6 mixed) pathways, cells were inoculated, stimulated, and analyzed as described above, but Ba / F3_gp130_IL-6R cells were replaced with Ba / F3_gp130_muIL-6R cells (expressing human gp130 and mouse IL-6R), IL-6 and soluble IL-6R were replaced with mouse IL-6 (Bio-Techne, 10 ng / ml) and mouse soluble IL-6R (Bio-Techne, 200 ng / ml), respectively, and anti-IL-6R IgG antibody was added at a concentration of 10 μg / ml.

[0391] Ba / F3 wild-type cells were used to analyze cytokine-induced proliferation mediated by IL-3 signaling. Ba / F3 wild-type cells do not express membrane-bound gp130 or IL-6R and are therefore unresponsive to IL-6, but they proliferate in response to stimulation with IL-3. Therefore, Ba / F3 wild-type cells were inoculated as described above, stimulated with recombinant mouse IL-3 (10 ng / ml, Peprotec 213-13-2), and incubated with 1 or 10 μg / ml of anti-IL-6R IgG antibody. After 48 hours, the number of viable cells was determined as described in Example 1.

[0392] An intracellular signaling assay to analyze the inhibition of the human IL-6 signaling pathway by anti-IL-6R IgG antibodies. As described in Example 1, two variants of human embryonic kidney cells (HEK293) were used as cell models for intracellular signaling.

[0393] To analyze cytokine-induced intracellular signaling mediated by the IL-6 trans, IL-6 classical, or combined IL-6 classical and trans (IL-6 mixed) pathways, IL-6-induced STAT3 Y705 phosphorylation was determined by intracellular flow cytometry. HEK293 and HEK293_IL-6R cells were inoculated and cultured as described in Example 1. HEK293_IL-6R cells were treated with IL-6 (10 ng / ml) for 30 minutes to stimulate IL-6 classical signaling, or with IL-6 (10 ng / ml) and soluble IL-6R (200 ng / ml) for 30 minutes to stimulate combined IL-6 classical and trans (mixed) signaling. HEK293 cells were treated with IL-6 (10 ng / ml) and soluble IL-6R (200 ng / ml) for 30 minutes to stimulate IL-6 trans signaling. Furthermore, a fixed amount of cells was incubated with either anti-IL-6R IgG antibody (1 μg / ml) or anti-mesothelin IgG antibody (1H7) (1 μg / ml), which was used as a control for nonspecific inhibition of intracellular signaling by IgG antibodies. The cells were fixed, stained, and analyzed by flow cytometry as described in Example 1.

[0394] result Generation of five types of anti-IL-6R IgG antibodies through chimeration of Fab fragments with human IgG1. Initial Fab screening revealed five clones, namely clones 07, 09, 12, 19, and 20, as potential selective inhibitors of IL-6 transsignaling. To further characterize their ability to inhibit IL-6 transsignaling, these five Fabs were selected for chimeration with human IgG1.

[0395] Anti-IL-6R IgG antibodies bind to human soluble IL-6R and human membrane-bound IL-6R. First, the ability of five anti-IL-6R IgG antibodies to bind to soluble IL-6R was quantified by ELISA. All five anti-IL-6R IgG antibodies bound to soluble IL-6R in a dose-dependent manner, while no binding was detected with the anti-mesothelin IgG(1H7) control antibody (Figure 1).

[0396] Secondly, the binding ability of five anti-IL-6R IgG antibodies to membrane-bound IL-6R expressed on human THP1 cells was quantified by flow cytometry. Interestingly, all five anti-IL-6R IgG antibodies strongly bound to membrane-bound IL-6R, as indicated by the increase in average fluorescence of cells stained with anti-IL-6R IgG antibodies and secondary antibodies compared to cells stained with secondary antibodies alone (Figure 2).

[0397] This suggests that the inhibition of IL-6 transsignaling is not due to the specific binding of the anti-IL-6R IgG antibody to soluble IL-6R, but rather to interference with the formation of the active IL-6R complex.

[0398] Furthermore, the ability of anti-IL-6R IgG C20 to bind to membrane-bound IL-6R expressed on Ba / F3_gp130_IL-6R (expressing human IL-6R) was quantified by flow cytometry in the absence and presence of IL-6. Anti-IL-6R IgG C20 binds to membrane-bound IL-6R in both the absence and presence of IL-6, as indicated by the increase in mean fluorescence between cells stained with anti-IL-6R IgG C20 and cells stained with the secondary antibody compared to cells stained with the secondary antibody alone (Figure 3). Therefore, the presence or absence of IL-6 does not affect the binding of anti-IL-6R IgG C20 to membrane-bound IL-6R.

[0399] Selective inhibition of IL-6 transsignaling-induced cell proliferation by anti-IL-6R IgG antibodies. In the next step, we analyzed the ability of five anti-IL-6R IgG antibodies to selectively inhibit IL-6 transsignaling-induced cell proliferation using the Ba / F3 cell line.

[0400] As expected, Ba / F3_gp130 cells do not proliferate in response to stimulation with IL-6 alone or soluble IL-6R alone. Stimulation with both IL-6 and soluble IL-6R results in IL-6 transsignaling-induced proliferation. The anti-mesothelin IgG(1H7) control antibody did not affect IL-6 transsignaling-induced proliferation in Ba / F3_gp130 cells, suggesting that proliferation is not nonspecifically affected by IgG antibodies. All five anti-IL-6R IgG antibodies dose-dependently reduced IL-6 transsignaling-induced proliferation, with IgG O9 being the least potent inhibitor (Table 5 and Figure 4).

[0401] Table 5. Inhibition of IL-6 transsignaling-induced proliferation by anti-IL-6R IgG antibody. We set the proliferation of Ba / F3_gp130 cells induced by 10 ng / ml IL-6 and 200 ng / ml sIL-6R to 100%. Results are presented as mean ± standard deviation from 3 independent experiments.

[0402] [Table 11-1]

[0403] [Table 11-2]

[0404] In addition to one further experimental repeat (Table 6), dose-dependent curves (Figure 5) for the inhibition of IL-6 transsignaling-induced cell proliferation by anti-IL-6R IgG antibodies were plotted using the mean values ​​of IgG 07, 09, 12, and 19 for %IL-6 transsignaling-induced proliferation in Table 5, and the updated mean value for IgG 20, including the data used to calculate the mean in Table 5, and IC50 values ​​(Table 7) were calculated. Low nM IC50 values ​​for anti-IL-6R IgG antibodies indicate potent inhibition of IL-6 transsignaling by these antibodies.

[0405] Table 6. Inhibition of IL-6 transsignaling-induced proliferation by anti-IL-6R IgG C20. We set the proliferation of Ba / F3_gp130 cells induced by 10 ng / ml IL-6 and 200 ng / ml sIL-6R to 100%. Results are presented as mean ± standard deviation from 4 independent experiments.

[0406] [Table 12]

[0407] Table 7 IC50 of inhibition of IL-6 transsignaling-induced proliferation by anti-IL-6R IgG antibody. IC50 values ​​in ng / ml and nM for inhibition of Ba / F3_gp130 cell proliferation induced by 10 ng / ml IL-6 and 200 ng / ml sIL-6R for each of five anti-IL-6R IgG antibodies at increasing concentrations (10 ng / ml, 100 ng / ml, 300 ng / ml, 500 ng / ml, 700 ng / ml, 1,000 ng / ml and 10,000 ng / ml).

[0408] [Table 13]

[0409] To provide further evidence of inhibition of IL-6 transsignaling by five types of anti-IL-6R IgG antibodies, Hyper-IL-6 (Hy-IL-6) was used to induce IL-6 transsignaling. Hy-IL-6 is a recombinant fusion protein consisting of IL-6 and soluble IL-6R (Peters et al., Journal of Immunology, Vol. 161, No. 7, pp. 3575-81 (1998)), and is a frequently used tool to induce IL-6 transsignaling in situations where IL-6 classical and transsignaling are theoretically activated in parallel. Therefore, it is a useful tool for in vivo analysis of the biological consequences of IL-6 transsignaling. All five anti-IL-6R IgG antibodies inhibited Hy-IL-6 transsignaling-induced proliferation, and the low nM IC50 values ​​for inhibition of Hy-IL-6-induced cell proliferation by these antibodies further support the potent inhibition of IL-6 transsignaling by these antibodies (Table 8).

[0410] Table 8 IC50 of inhibition of Hy-IL-6 transsignaling-induced proliferation by anti-IL-6R IgG antibody IC50 values ​​in ng / ml and nM for inhibition of Hy-IL-6-induced Ba / F3_gp130 cell proliferation at 28 ng / ml for each of five types of anti-IL-6R IgG antibodies at increasing concentrations (10 ng / ml, 100 ng / ml, 300 ng / ml, 500 ng / ml, 700 ng / ml, 1,000 ng / ml, and 10,000 ng / ml).

[0411] [Table 14]

[0412] Next, we tested whether anti-IL-6R IgG antibodies reduced IL-6 classical signaling-induced proliferation in Ba / F3_gp130_IL-6R cells. As before, the anti-mesothelin IgG(1H7) control antibody did not affect the proliferation of Ba / F3_gp130_IL-6R cells, thus ruling out nonspecific inhibition of proliferation by IgG antibodies. It is noteworthy that none of the anti-IL-6R IgG antibodies examined significantly inhibited IL-6 classical signaling-induced proliferation, even at a concentration of 10 μg / ml (Table 5), which would cause significant inhibition of IL-6 transsignaling-induced proliferation (Table 9 and Figure 6). Since the inhibition is less than 50% required to calculate the IC50 value at the maximum concentration of 10 μg / ml (53 nM), all five anti-IL-6R IgG antibodies will have an IC50 greater than 10 μg / ml (53 nM) for inhibiting IL-6 classical signaling-induced proliferation. Therefore, all five anti-IL-6R IgG antibodies examined selectively inhibit IL-6 transsignaling-induced proliferation without affecting IL-6 classical signaling-induced proliferation (i.e., inhibiting IL-6 transsignaling while maintaining IL-6 classical signaling).

[0413] Table 9. Inhibition of IL-6 classical signaling-induced proliferation by anti-IL-6R IgG antibody. We set the proliferation of Ba / F3_gp130_IL-6R cells induced by 10 ng / ml of IL-6 to 100%. Results are presented as mean ± standard deviation from three independent experiments.

[0414] [Table 15]

[0415] As described in Example 1, antibodies that are thought to selectively inhibit IL-6 transsignaling (i.e., greater inhibition of IL-6 transsignaling compared to IL-6 classical signaling) are characterized by a larger classical:transsignaling ratio (e.g., >1.0), and a larger ratio indicates greater selectivity for inhibiting IL-6 transsignaling compared to IL-6 classical signaling. Therefore, the classical:transsignaling ratio was calculated for all five anti-IL-6R IgG antibodies (Table 10).

[0416] Table 10. Classical to transsignaling ratio of cell proliferation for anti-IL-6R IgG antibodies. Average residual cytokine-induced proliferation in the presence of the indicated IgG antibody, as taken from Table 5 (Ba / F3_gp130, IL-6 transsignaling measured) and Table 9 (Ba / F3_gp130_IL-6R, IL-6 classical signaling measured), respectively.

[0417] [Table 16]

[0418] To analyze the ability of five anti-IL-6R IgG antibodies to inhibit IL-6-induced proliferation under a wider range of physiological conditions, Ba / F3_gp130_IL-6R cells were stimulated with a mixture of IL-6 and soluble IL-6R. As described in Example 1, this condition allows both classical and trans (mixed) IL-6 signaling to occur. None of the anti-IL-6R IgG antibodies examined significantly inhibited combined classical and trans (mixed) IL-6 signaling-induced proliferation (Table 11 and Figure 7). Since the inhibition is less than 50% required to calculate the IC50 value at the maximum concentration of 10 μg / ml (53 nM), all five anti-IL-6R IgG antibodies would have an IC50 for inhibition of IL-6 mixed signaling-induced proliferation greater than 10 μg / ml (53 nM). Therefore, interference with IL-6 transsignaling by anti-IL-6R IgG antibodies does not interfere with IL-6 classical signaling under the analyzed conditions.

[0419] Table 11 Inhibition of combined IL-6 classical and transsignaling-induced proliferation by anti-IL-6R IgG antibodies. We set the proliferation of Ba / F3_gp130_IL-6R cells induced by 10 ng / ml IL-6 and 200 ng / ml sIL-6R to 100%. Results are presented as mean ± standard deviation from 3 independent experiments.

[0420] [Table 17]

[0421] As described in Example 1, combined IL-6 classical and trans (mixed) signaling represent IL-6 signaling under physiological conditions. The IL-6 signaling ratio (mixed:trans-signaling ratio) under physiological conditions was also calculated for all five types of anti-IL-6R IgG antibodies (Table 12).

[0422] Table 12 Cell proliferation mixture of anti-IL-6R IgG antibodies: transsignaling ratio Average residual cytokine-induced proliferation in the presence of indicated IgG antibodies, as obtained from Table 5 (Ba / F3_gp130, IL-6 transsignaling measured) and Table 11 (Ba / F3_gp130_IL-6R, combined IL-6 classical and trans (mixed) signaling measured).

[0423] [Table 18]

[0424] Using the IC50 values ​​for inhibition of IL-6 transsignaling-induced proliferation shown in Table 7, and the minimum IC50 values ​​of 10 μg / ml or 53 nM for inhibition of IL-6 classical signaling-induced proliferation, the classical:transsignaling IC50 ratios for five types of anti-IL-6R IgG antibodies were also calculated.

[0425] Table 13. Classical vs. Transsignaling IC50 ratio of anti-IL-6R IgG antibodies for cell proliferation. IC50 values ​​for inhibition of IL-6 transsignaling-induced proliferation by anti-IL-6R IgG antibodies (Table 7) and minimum IC50 values ​​for inhibition of IL-6 classical signaling-induced proliferation by anti-IL-6R IgG antibodies

[0426] [Table 19]

[0427] Next, to test whether the five anti-IL-6R IgG antibodies nonspecifically affected proliferation, Ba / F3 wild-type cells were stimulated with IL-3. These cells proliferated in response to IL-3 stimulation, and therefore, the proliferation of these cells should not be affected by the anti-IL-6R IgG antibodies. Regardless of the concentrations examined, the five anti-IL-6R IgG antibodies did not reduce IL-6-independent proliferation (Table 14).

[0428] Table 14 Inhibition of IL-3-induced proliferation by anti-IL-6R IgG antibody. Growth of Ba / F3 wild-type cells induced by 10 ng / ml IL-3 was set to 100%. Results are presented as mean ± standard deviation from three independent experiments.

[0429] [Table 20]

[0430] Next, to test whether the five types of anti-IL-6R IgG antibodies affect signaling via cytokines belonging to other human IL-6 family, we analyzed the effects of the five types of anti-IL-6R IgG antibodies on human IL-11 classical, trans, and mixed classical and trans (mixed) signaling.

[0431] To analyze IL-11 classical signaling, Ba / F3_gp130_IL-11R cells expressing human gp130 and human IL-11R were stimulated with IL-11. The anti-mesothelin IgG(1H7) control antibody did not affect the proliferation of Ba / F3_gp130_IL-11R cells, thus ruling out nonspecific inhibition of proliferation by IgG antibodies. None of the anti-IL-6R IgG antibodies examined significantly inhibited IL-11 classical signaling-induced proliferation, even at a concentration of 10 μg / ml (Table 15).

[0432] Table 15 Inhibition of IL-11 classical signaling-induced proliferation by anti-IL-6R IgG antibody. The proliferation of Ba / F3_gp130_IL-11R cells induced by 10 ng / ml IL-11 was set to 100%. Results are presented as mean ± standard deviation from three independent experiments.

[0433] [Table 21]

[0434] To analyze whether five types of anti-IL-6R IgG antibodies affect IL-11 transsignaling, Ba / F3_gp130 cells were stimulated with IL-11-soluble IL-11R. None of the anti-IL-6R IgG antibodies examined significantly inhibited IL-11 transsignaling-induced proliferation, even at a concentration of 10 μg / ml (Table 5), which was the concentration at which significant inhibition of IL-6 transsignaling was observed (Table 16).

[0435] Table 16 Inhibition of IL-11 transsignaling-induced proliferation by anti-IL-6R IgG antibody. Growth of Ba / F3_gp130 cells induced by 10 ng / ml IL-11 and 200 ng / ml soluble IL-11R (sIL-11R) was set to 100%. Results are presented as mean ± standard deviation from 3 independent experiments.

[0436] [Table 22]

[0437] To analyze whether five types of anti-IL-6R IgG antibodies affect combined IL-11 classical and trans (mixed) signaling, Ba / F3_gp130_IL-11R cells were stimulated with IL-11-soluble IL-11R. None of the anti-IL-6R IgG antibodies examined significantly inhibited IL-11 mixed signaling-ind...

Claims

1. An antibody comprising at least one, preferably two, antigen-binding domains that bind to the human IL-6 receptor, wherein the antibody inhibits IL-6 transsignaling via the human soluble IL-6 receptor while maintaining IL-6 classical signaling via the human membrane-bound IL-6 receptor, and the antibody is capable of binding to both the human soluble IL-6 receptor and the human membrane-bound IL-6 receptor.

2. The antigen-binding domain comprises a heavy chain variable region containing three complementarity-determining regions (CDRs) and a light chain variable region containing three CDRs. I. The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GTFFSNYG (SEQ ID NO: 77) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (ii) VH CDR2 comprising the amino acid sequence of INSNGGST (SEQ ID NO: 78) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (iii) VH CDR3 comprising the amino acid sequence of ARDGNFVSDY (SEQ ID NO: 79) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including and / or The aforementioned light chain variable region is A variable light (VL) CDR1 comprising the amino acid sequence of (iv)ESVDSYGNRF (SEQ ID NO: 80) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (v) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 81) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT (SEQ ID NO: 82) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including, or II. The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSSYA (SEQ ID NO: 5) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (ii) VH CDR2 comprising the amino acid sequence of INSNGGST (SEQ ID NO: 6) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (iii) VH CDR3 comprising the amino acid sequence of AREGYYTMDY (SEQ ID NO: 7) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including and / or The aforementioned light chain variable region is A variable light (VL) CDR1 comprising the amino acid sequence of (iv)ESVDSYGNSF (SEQ ID NO: 8) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (v) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 9) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including, or III. The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFFSNYG (SEQ ID NO: 41) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (ii) VH CDR2 comprising the amino acid sequence of INSNGGST (SEQ ID NO: 42) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (iii) VH CDR3 comprising the amino acid sequence of TRDGNFVSDY (SEQ ID NO: 43) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including and / or The aforementioned light chain variable region is A variable light (VL) CDR1 comprising the amino acid sequence of (iv)ESVDSYGNSF (SEQ ID NO: 44) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (v) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 45) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT (SEQ ID NO: 46) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including, or IV. The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFFSNYG (SEQ ID NO: 59) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (ii) VH CDR2 comprising the amino acid sequence of MNSKGGST (SEQ ID NO: 60) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (iii) VH CDR3 comprising the amino acid sequence of ARDGYYTMDY (SEQ ID NO: 61) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including and / or The aforementioned light chain variable region is A variable light (VL) CDR1 comprising the amino acid sequence of (iv) KSVDSFGNSF (SEQ ID NO: 62) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (v) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 63) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPYT (SEQ ID NO: 64) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including, or V. The heavy chain variable region is, (i) A variable weight (VH) CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 23) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (ii) VH CDR2 comprising the amino acid sequence of INSNGGST (SEQ ID NO: 24) or a substantially homologous sequence thereof, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (iii) VH CDR3 comprising the amino acid sequence of ARDGNYVSDY (SEQ ID NO: 25) or a substantially homologous sequence, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, including and / or The aforementioned light chain variable region is A variable light (VL) CDR1 comprising the amino acid sequence of (iv)ESVDSYGNSF (SEQ ID NO: 26) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, (v) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 27) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, (vi) A VL CDR3 comprising the amino acid sequence of QQNNEDPWT (SEQ ID NO: 28) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, or three amino acid substitutions, The antibody according to claim 1, comprising:

3. The antigen-binding domain comprises a heavy chain variable region containing three complementarity-determining regions (CDRs) and a light chain variable region containing three CDRs, wherein the heavy chain region a) GFTFSX ６ YX ８ A variable weight (VH) CDR1 containing the amino acid sequence of X ６ and X ８ is one of the amino acids (SEQ ID NO: 91), and X ６ is preferably N or S, and / or X ８ VH CDR1 is preferably G or A, b) X １ NSX ４ A VH CDR2 comprising the amino acid sequence of GGST, wherein X １ and X ４ is any amino acid (SEQ ID NO: 94), and X １ is preferably I or M, and / or X ４ is preferably an VH CDR2 which is N or K, c) X １ RX ３ GX ５ X ６ X ７ X ８ VH CDR3 containing the amino acid sequence DY, X １ , X ３ , X ５ , X ６ , X ７ and X ８ is one of the amino acids (SEQ ID NO: 97), and X １ is preferably A or T, and X ３ is preferably D or E, X ５ is preferably N or Y, and X ６ is preferably Y or F, and X ７ is preferably V or T, and / or X ８ It is preferably S or M, Preferably, the amino acid sequence is ARX ３ The formula includes GYYTMDY, where X ３ is any amino acid (SEQ ID NO: 100), preferably E or D, or the amino acid sequence is X １ RDGNX ６ Including VSDY, X １ and X ６ is one of the amino acids (SEQ ID NO: 103), and X １ is preferably A or T, and / or X ６ is preferably F or Y. VH CDR3 and, including and / or The aforementioned light chain variable region is d) X １ SVDSX ６ GNX ９ A variable light (VL) CDR1 containing the amino acid sequence of F, X １ , X ６ and X ９ is any amino acid (SEQ ID NO: 106), and X １ is preferably E or K, and X ６ is preferably Y or F, and / or X ９ VL CDR1 is preferably S or R, e) A VL CDR2 comprising the amino acid sequence of LAS (SEQ ID NO: 9) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one amino acid substitution, f)QQNNEDPX ８ VL CDR3 containing the amino acid sequence of T, and X ８ VL CDR3 is any amino acid (SEQ ID NO: 109), preferably Y or W, The antibody according to claim 1 or 2, comprising:

4. The antigen-binding domain comprises a heavy chain variable region containing three CDRs and a light chain variable region containing three CDRs. I. The heavy chain variable region is the amino acid sequence of SEQ ID NO: 75 or a sequence having at least 80% sequence identity thereto. including and / or The light chain variable region is the amino acid sequence of SEQ ID NO: 76 or a sequence having at least 80% sequence identity thereto. including, or II. The heavy chain variable region is the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% sequence identity thereto. including and / or The light chain variable region is the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto. Includes, or, III. The heavy chain variable region is the amino acid sequence of SEQ ID NO: 39 or a sequence having at least 80% sequence identity thereto. including and / or The light chain variable region is the amino acid sequence of SEQ ID NO: 40 or a sequence having at least 80% sequence identity thereto. including, or IV. The heavy chain variable region is the amino acid sequence of SEQ ID NO: 57 or a sequence having at least 80% sequence identity thereto. including and / or The light chain variable region is the amino acid sequence of SEQ ID NO: 58 or a sequence having at least 80% sequence identity thereto. Includes, or, V. The heavy chain variable region is the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 80% sequence identity thereto. including and / or The light chain variable region includes the amino acid sequence of SEQ ID NO: 22 or a sequence having at least 80% sequence identity thereto. The antibody according to any one of claims 1 to 3.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The antibody is a full-length antibody, as described in any one of claims 1 to 5.

7. The antibody according to any one of claims 1 to 5, wherein the antibody is an antigen-binding fragment of an antibody, preferably a Fab fragment.

8. The antibody is PEG-modified, as described in any one of claims 1 to 7.

9. An antibody that binds to the same epitope as the antibody described in any one of claims 2 to 4 in the human IL-6 receptor.

10. An antigen-binding protein comprising an antibody or antigen-binding domain as defined in any one of claims 1 to 9.

11. A composition comprising an antibody according to any one of claims 1 to 9 or an antigen-binding protein according to claim 10, and a diluent, carrier, or excipient, preferably a pharmaceutically acceptable diluent, carrier, or excipient.

12. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody according to any one of claims 1 to 9 or an antigen-binding protein according to claim 10, or a set of nucleic acid molecules each comprising a nucleotide sequence, wherein the set of nucleic acid molecules together encodes an antibody according to any one of claims 1 to 9 or an antigen-binding protein according to claim 10.

13. One or more expression vectors comprising one or more nucleic acid molecules as described in claim 12.

14. One or more host cells or viruses that express an antibody or antigen-binding protein according to any one of claims 1 to 10, comprising the expression vector according to claim 13 or the nucleic acid molecule according to claim 12.

15. A method for producing an antibody or antigen-binding protein according to any one of claims 1 to 10, (i) A step of culturing a host cell containing one or more nucleic acid molecules as defined in claim 12, or one or more expression vectors as defined in claim 13, under conditions suitable for the expression of an encoded antibody or antigen-binding protein; (ii) the step of isolating or obtaining the antibody or antigen-binding protein from the host cells or from the growth medium / supernatant, A method that includes this.

16. An antibody, antigen-binding protein, or composition as defined in any one of claims 1 to 11 for use in the treatment of therapies.

17. An antibody, antigen-binding protein, or composition for use in the treatment of a disease or disorder, as defined in any one of claims 1 to 11, wherein the disease or disorder is an inflammatory disease, a CNS disorder, or cancer, preferably the disease or disorder is one or more of cancer, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis, or Covid-19 or Covid-19 complications.

18. A method for treating a disease or disorder, the method comprising administering to a patient in need a therapeutically effective amount of an antibody, antigen-binding protein or composition as defined in any one of claims 1 to 11, wherein the disease or disorder is an inflammatory disease, a CNS disorder or cancer, preferably the disease or disorder is one or more of cancer, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis, or Covid-19 or Covid-19 complications.

19. Use of an antibody, antigen-binding protein, or composition as defined in any one of claims 1 to 11 in the manufacture of a pharmaceutical product used in therapeutic purposes.

20. The use according to claim 19, wherein the treatment is the treatment of one or more inflammatory diseases, CNS disorders or cancer, preferably cancer, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Castleman disease, cytokine release syndrome, multiple sclerosis, anemia, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), pancreatitis, acute lung injury (ALI), depression, Alzheimer's disease, sepsis or Covid-19 or Covid-19 complications.

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