Antibodies against il-7r alpha subunit and uses thereof

Abstract

To provide antibodies that bind to the alpha subunit of an IL-7 receptor (IL-7Rα).SOLUTION: The present invention provides an isolated antibody or an antigen binding portion thereof, which specifically binds to an alpha-chain of a human IL-7 receptor ("anti-IL-7R antibody"), wherein the antibody is (a) capable of binding to T cells (CD4+CD45RA+, CD4+CD45RA-, CD8+CD45RA+, and / or CD8+CD45RA-) in whole blood with an EC 50 of about 5 nM or less (e.g., less than about 3 nM), (b) is not capable of binding to non-T cells in whole blood, (c) is not capable of effectively blocking thymic stromal lymphopoietin (TSLP)-mediated activation of monocytes, (d) does not agonize IL-7 receptor signaling upon binding to the IL-7 receptor, e.g., minimal pSTAT5 activation, or (e) is any combination thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] References to sequence listings submitted electronically via EFS-WEB A Sequence Listing has been submitted electronically with this application in an ASCII text file (Name: 4521.001PC02_Seqlisting_ST25.txt; Size: 100,565 bytes; Creation Date: Jan. 17, 2020), the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Interleukin-7 (IL-7) is a member of the common gamma chain (γc) cytokine family, which also includes IL-2, IL-4, IL-9, IL-15, and IL-21. Nguyen V., et al., J Immunol Res 2017:4807853 (2017). Like the other members, IL-7 signals through a ternary complex that it forms with its unique α receptor, IL-7Rα (CD127), and the common gamma receptor. Carrette, F., et al., Semin Immunol 24(3):209-17(2012). This interaction stimulates Janus kinase (JAK) and signal transducer and activator of transcription (STAT) proteins, which subsequently activate the phosphoinositol 3-kinase (PI3K) / Akt or Src pathways to promote transcription of target genes. IL-7Rα is also used by thymic stromal lymphopoietin (TSLP) as part of a complex that contains a second receptor chain (TSLPR). Durum, SK, Blood 124(1):4-5(2014).

[0003] IL-7 plays a crucial role in the development of a normal immune system, as it is essential for thymic development, peripheral homeostasis, and lymphocyte survival. Maraskovsky, E., et al., J Immunol 157(12):5315-5323(1996). IL-7 is required for the proliferation, differentiation, and survival of thymic T-cell precursors. In the periphery, IL-7 controls T-cell homeostasis by enhancing the survival and proliferation of naive and memory T cells.

[0004] IL-7 is a tissue-derived cytokine, mainly produced by stromal and epithelial cells in various tissues. For example, in the small and large intestine, IL-7 is produced by intestinal epithelial goblet cells and has been reported in animal models to be essential for the persistence of chronic colitis. Tomita, T., et al., J Immunol 180(1):383-390(2008). IL-7 has also been shown to interfere with the immunosuppressive ability of regulatory T cells (Tregs). Heninger, AK, et al., J Immunol 189(12):5649-5658(2012). Therefore, in treating inflammatory diseases (such as inflammatory bowel disease), agents that can modulate the activity of IL-7 in vivo, thereby reducing the survival / function of pathogenic T cells and / or promoting the induction of regulatory T cells, are highly desirable. Summary of the Invention

[0005] Provided herein is an isolated antibody that specifically binds to the alpha chain of the human IL-7 receptor (an "anti-IL-7R antibody"), or an antigen-binding portion thereof, which comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (a) an EC of about 5 nM or less (e.g., less than about 3 nM) 50 T cells (CD4 + CD45RA + , CD4 + CD45RA - , CD8 + CD45RA + , and / or CD8 + CD45RA - ) or (b) has no ability to bind to non-T cells in whole blood; (c) lack of ability to effectively block thymic stromal lymphopoietin (TSLP)-mediated monocyte activation; (d) does not agonize IL-7 receptor signaling upon binding to the IL-7 receptor (e.g., pSTAT5 activation is minimal), or (e) any combination thereof.

[0006] In some embodiments, the heavy chain CDR3 of an anti-IL-7R antibody (such as those disclosed herein) comprises the amino acid sequence set forth in SEQ ID NO:15 (DEYSRGYYVLDV).

[0007] In some embodiments, the anti-IL-7R antibody of the disclosure is (a) having the ability to selectively bind to the alpha chain of the human and cynomolgus monkey IL-7 receptor (IL-7R); (b) having the ability to bind to the alpha chain of soluble and membrane-bound IL-7R; (c) capable of blocking the proliferation and / or survival of pathogenic T cells when administered to a subject in need thereof; (d) capable of restoring regulatory T cell (Treg) function and / or promoting Treg survival when administered to a subject in need thereof; (e) the ability to maintain drug-free remission longer than with CTLA4-Ig (ORENCIA®); (f) having the ability to block inflammation and mucosal damage (e.g., induced by pathogenic T cells) in the intestinal tissue of a subject in need thereof; (g) having the ability to reduce the frequency of effector T cells in the mesenteric lymph nodes (MLN) and / or lamina propria (LP) in a subject in need thereof; (h) T cells (e.g., CD4+ CD45RA + ) having the ability to reduce or inhibit IL-7-mediated pSTAT activation; (i) having the ability to block the proliferation of cells that produce IL-17 and / or IFN-gamma; (j) having the ability to treat a subject having an inflammatory disease (e.g., inflammatory bowel disease); and (k) any combination thereof; The compound has one or more properties selected from the group consisting of:

[0008] In some embodiments, the anti-IL-7R antibody comprises a heavy chain CDR1, which comprises the amino acid sequence set forth in SEQ ID NO: 13 (DHAMH). In some embodiments, the anti-IL-7R antibody comprises a heavy chain CDR2, which comprises the amino acid sequence set forth in SEQ ID NO: 14 (GISWNSRGIGYADSVKG). In some embodiments, the anti-IL-7R antibody comprises a light chain CDR1, which comprises the amino acid sequence set forth in SEQ ID NO: 16 (RASQGISSALA). In some embodiments, the anti-IL-7R antibody comprises a light chain CDR2, which comprises the amino acid sequence set forth in SEQ ID NO: 17 (DASSLES). In some embodiments, the anti-IL-7R antibody comprises a light chain CDR3, which comprises the amino acid sequence set forth in SEQ ID NO: 18 (QQFNSYPLWIT).

[0009] In some embodiments, the anti-IL-7R antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to the amino acid sequence set forth in SEQ ID NO:19. In certain embodiments, the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:20.

[0010] In some embodiments, the anti-IL-7R antibodies disclosed herein are 24 SQLEVNGSQHSLTCAF 39 (SEQ ID NO: 8), 73 FIETKKFLLIGKSNIC 88 (SEQ ID NO: 9), 89 VKVGEKSLTCKKIDLTT 105 (SEQ ID NO: 10), 136 QKKYVKVLMHDVAY 149 (SEQ ID NO: 11), 181 YEIKVRSIPDHYFKGF 196 (SEQ ID NO: 12), and combinations thereof. In certain embodiments, the anti-IL-7R antibody specifically binds to the alpha chain of the human IL-7 receptor at an epitope that includes one or more amino acid residues selected from the group consisting of H33, E75, F79, I82, K84, M144, R186, H191, Y192, and combinations thereof.

[0011] In some embodiments, the anti-IL-7R antibody of the present disclosure is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and variants thereof. In certain embodiments, the IL-7R antibody is an IgG1 antibody. In some embodiments, the anti-IL-7R antibody comprises an IgG1 Fc that does not have effector function.

[0012] Also disclosed herein is an isolated antibody that specifically binds to the alpha chain of the human IL-7 receptor (an "anti-IL-7R antibody"), or an antigen-binding portion thereof, which antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:21 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:22.

[0013] In some embodiments, the anti-IL-7R antibody has a K of less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM as measured by surface plasmon resonance. D (e.g., 1.3 nM). In certain embodiments, the anti-IL-7R antibody binds to the alpha chain of the human IL-7 receptor with a K of less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM as measured by surface plasmon resonance. D (e.g., 1.7 nM). In some embodiments, binding to the alpha chain of the human IL-7 receptor or binding to the alpha chain of the cynomolgus IL-7 receptor is pH dependent. In certain embodiments, the anti-IL-7R antibody has a K of about 1.3 nM at pH 7.4. D , with a K of approximately 5.3 nM at pH 6 D In some embodiments, the anti-IL-7R antibody binds to the alpha chain of the human IL-7 receptor with a K of about 1.7 nM at pH 7.4. D , and a K of approximately 7.0 nM at pH 6. D It binds to the alpha chain of the cynomolgus monkey IL-7 receptor.

[0014] Provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein HC CDR1 comprises the amino acid sequence GX1X2FDDHAX3H (SEQ ID NO: 260), where X1 is F or Y, X2 is T, P, A, S, V, L, I, M, H, F, Y, N, D, E, or Q, and X3 is L or M. In certain aspects, X2 is D or E.

[0015] In some embodiments, HC CDR2 comprises the amino acid sequence GIX1WX2SRGX3GYX4X5X6X7X8X9 (SEQ ID NO: 261), wherein X1 is S or X is T, X2 is H or N, X3 is I or V, X4 is G, A, S, T, V, L, I, R, H, or N, X5 is P, T, N, D, E, Q, S, H, or Y, X6 is P, G, A, S, T, V, R, H, F, Y, N, D, or E, X7 is V or I, X8 is A, S, T, V, L, I, M, K, R, H, F, Y, N, D, E, or Q, and X9 is G, H, D, or Q. In certain embodiments, X1 is T.

[0016] In some embodiments, the HC CDR3 comprises the amino acid sequence DEYX1X2GYYX3LDX4 (SEQ ID NO:262), where X1 is S, T, N, D, or E, X2 is L, M, R, or S, X3 is G, A, S, T, V, M, N, E, or Q, and X4 is A, S, T, V, R, H, Y, W, N, E, Q, or M. In certain embodiments, X3 is A, S, or T. In some embodiments, X4 is E.

[0017] In some embodiments, the LC CDR1 comprises the amino acid sequence X1X2X3X4X5X6X7SX8X9A (SEQ ID NO:263), where X1 is S, T, V, K, R, H, Y, or I, X2 is A, S, T, or V, X3 is P, G, A, S, T, V, L, I, M, K, R, H, N, E, or Q, and X4 is P, G, A, S, T, V, , L, I, M, H, F, Y, N, D, E, or Q; X5 is P, G, A, S, T, H, E, Q, M, N, or D; X6 is P, G, A, S, T, V, L, I, or N; X7 is S, T, V, L, I, M, H, F, Y, N, D, E, or Q; X8 is P or A; and X9 is A, L, or V. In certain embodiments, X6 is P. In other embodiments, X8 is P. In some embodiments, X7 is D or E.

[0018] In some embodiments, the LC CDR2 comprises the amino acid sequence DX1X2X3X4X5X6 (SEQ ID NO:264), where X1 is G, A, S, M, H, N, D, E, or Q, X2 is G, A, S, T, V, M, H, F, Y, N, D, E, or Q, X3 is A, S, F, Y, W, N, D, E, or L, X4 is P, S, T, L, K, H, or N, X5 is D, E, or Q, and X6 is G, S, T, N, D, Q, P, or E.

[0019] In some embodiments, the LC CDR3 comprises the amino acid sequence X1X2FX3X4YPLX5X6X7 (SEQ ID NO:265), where X1 is M or Q, X2 is G, A, D, E, or Q, X3 is N or E, X4 is P, A, or S, X5 is T, I, M, K, W, N, E, or Q, X6 is L or I, and X7 is T, M, K, H, Y, E, or Q. In certain embodiments, X2 is A. In other embodiments, X4 is P or A.

[0020] In some embodiments, the heavy chain CDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 31 to 46. In certain embodiments, the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 43 or SEQ ID NO: 44. In some embodiments, the heavy chain CDR2 comprises one of the amino acid sequences shown in SEQ ID NOs: 47 to 96. In certain embodiments, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 47. In another embodiment, the heavy chain CDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 97 to 122. In certain embodiments, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 120. In some embodiments, the light chain CDR1 comprises one of the amino acid sequences shown in SEQ ID NOs: 123 to 194. In certain embodiments, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 172, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 192. In some embodiments, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 195 to 197. 237. In some embodiments, the light chain CDR3 comprises one of the amino acid sequences set forth in SEQ ID NOs: 238-259. In certain embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 240, SEQ ID NO: 244, or SEQ ID NO: 245.

[0021] Provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises one of the amino acid sequences set forth in SEQ ID NOs: 31-46, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain aspects, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.

[0022] Also provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) the heavy chain CDR2 comprises one of the amino acid sequences set forth in SEQ ID NOs: 47-96, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain aspects, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0023] The present disclosure provides an isolated antibody ("anti-IL-7R antibody") or antigen-binding portion thereof that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) the heavy chain CDR3 comprises one of the amino acid sequences set forth in SEQ ID NOs: 97-122, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 120.

[0024] Provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) the light chain CDR1 comprises one of the amino acid sequences set forth in SEQ ID NOs: 123-194, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain aspects, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 172, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 192.

[0025] Also provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (v) the light chain CDR2 comprises one of the amino acid sequences set forth in SEQ ID NOs: 195-237, and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0026] Provided herein is an isolated antibody ("anti-IL-7R antibody"), or an antigen-binding portion thereof, that specifically binds to the alpha chain of the human IL-7 receptor, the antibody comprising a heavy chain (HC) CDR1, HC CDR2, and HC CDR3, and a light chain (LC) CDR1, LC CDR2, and LC CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and (vi) the light chain CDR3 comprises one of the amino acid sequences set forth in SEQ ID NOs: 238-259. In certain aspects, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 240, SEQ ID NO: 244, or SEQ ID NO: 245.

[0027] The present disclosure also provides a nucleic acid encoding an anti-IL-7R antibody of the present disclosure, a vector comprising the nucleic acid, and a cell comprising the vector. In some embodiments, the cell is a CHO cell, a HEK293 cell, a HBK cell, a COS cell, an NSO cell, or a HT1080 cell.

[0028] Also provided herein are immunoconjugates comprising an anti-IL-7R antibody disclosed herein linked to an agent.

[0029] The present disclosure further provides a composition comprising an anti-IL-7R antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein and a carrier.

[0030] The disclosure also provides a kit comprising an anti-IL-7R antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein and instructions for use.

[0031] Provided herein are methods of inhibiting IL-7 activity in a subject in need thereof, the methods comprising administering to the subject an anti-IL-7R antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein.

[0032] Provided herein are methods for inhibiting the proliferation of effector T cells and / or inducing the development and / or survival of regulatory T cells in a subject in need thereof, the methods comprising administering to the subject an anti-IL-7R antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein.

[0033] Also provided herein are methods of treating an inflammatory or autoimmune disease in a subject in need thereof, comprising administering to the subject an anti-IL-7R antibody, nucleic acid, vector, cell, or immunoconjugate disclosed herein. In certain embodiments, the inflammatory or autoimmune disease is inflammatory bowel disease (IBD), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, hematopoietic syndrome, rheumatoid arthritis, ... In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of inflammatory bowel disease, sepsis, systemic inflammatory response syndrome (SIRS), type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, other autoimmune diseases that are the result of acute or chronic inflammation, and any combination thereof. In some embodiments, the inflammatory or autoimmune disease is inflammatory bowel disease. In another embodiment, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0034] In some embodiments, the subject (e.g., a subject described herein) is a subject who has not responded adequately to previous TNF-α inhibitor treatment (an anti-TNF-α poor responder).

[0035] In some embodiments, the methods disclosed herein may further include administering one or more additional therapies. In certain embodiments, the additional therapy includes an anti-TNF-α antibody.

[0036] In some embodiments, the anti-IL-7R antibody disclosed herein is administered to the subject in a flat dose or a weight-based dose. In some embodiments, the anti-IL-7R antibody is administered intravenously, subcutaneously, intramuscularly, intradermally, or intraperitoneally. In certain embodiments, the anti-IL-7R antibody is administered intravenously or subcutaneously.

[0037] Also provided herein is a method for producing an antibody that specifically binds to the alpha chain of the human IL-7 receptor (an "anti-IL-7R antibody"), the method comprising culturing a cell described herein under suitable conditions and isolating the antibody. [Brief description of the drawings]

[0038] [Figure 1]Binding curves obtained by measuring binding of anti-IL-7R antibodies to CD3- cells (i.e., non-T cells) by flow cytometry are shown, where the x-axis shows the concentration of anti-IL-7R antibodies (μg / mL) and the y-axis shows the mean fluorescence intensity (MFI) of binding. [Diagram 2] A and B show cross-reactivity of anti-IL-7R antibodies to cynomolgus IL-7Rα in cynomolgus monkeys after a single intravenous dose of the antibody. A shows the percent occupied of total IL-7Rα expressed on CD4+CD45RA+ T cells as a function of serum concentration of anti-IL-7R antibody (ng / mL). B shows pSTAT5 activation in CD4+CD45RA+ T cells as a function of serum concentration of anti-IL-7R antibody (nM). pSTAT5 activation is shown as the percent phosphorylated of total STAT5. In both A and B, serum concentration of antibody is intended to represent the measured exposure of CD4+CD45RA+ T cells to anti-IL-7R antibody. [Diagram 3] Pharmacokinetics (serum concentrations) of anti-IL-7R antibodies in NOD SCID gamma (NSG)-human PBMC engrafted mice after a single intravenous dose. Mice were administered anti-IL-7R antibodies at one of two doses: 0.5 mg / kg (solid filled squares) or 5 mg / kg (dashed open squares). Control mice were administered A3312F antibody at 0.5 mg / kg (solid filled circles) or 5 mg / kg (dashed open circles). Data are presented as mean ± standard deviation. [Figure 4] Figure 1 shows both pharmacokinetics (serum concentration) and anti-drug antibody (ADA) formation in cynomolgus monkeys after a single intravenous dose of anti-IL-7R antibody. Animals were administered the antibody at one of three doses: 0.1 mg / kg (squares), 0.5 mg / kg (circles), or 3 mg / kg (triangles). Serum concentrations of the antibody at different doses (left y-axis) are shown as solid lines. Anti-drug antibody (i.e., antibodies against the administered anti-IL-7R antibody) formation (right y-axis) is shown as dashed lines. Data are presented as mean ± standard deviation. [Diagram 5]1 shows a comparison of the pharmacokinetics (serum concentrations) of anti-IL-7R and A3312F antibodies in cynomolgus monkeys after a single intravenous dose. Animals were administered anti-IL-7R antibody at one of three doses: 0.1 mg / kg (solid filled squares), 0.5 mg / kg (solid filled circles), or 3 mg / kg (solid filled triangles). Control animals were administered A3312F antibody at one of the following doses: 0.1 mg / kg (dashed open squares), 0.5 mg / kg (dashed open circles), or 3 mg / kg (dashed open triangles). Data are presented as mean ± standard deviation. [Figure 6] Figure 1 shows pSTAT5 activation in CD4+CD45RA+ T cells in cynomolgus monkeys after a single intravenous dose of anti-IL-7R antibody (squares) or A3312F antibody (triangles). pSTAT5 activation is shown as the percent of total STAT5 that is phosphorylated. Serum concentrations of antibody are intended to represent the measured exposure of CD4+CD45RA+ T cells to the antibody. [Figure 7A] FIG. 1 shows the frequency of different T cell populations observed in the peripheral blood of different cynomolgus monkeys after a single intravenous administration of anti-IL-7R antibody (3 mg / kg). A shows the frequency of CD3+ T cells as a percentage of total lymphocytes. B and C show the frequency of CD4+ T cells and CD8+ T cells as a percentage of the total CD3+ T cell population, respectively. In A, B, and C, the frequency of different T cell populations was observed before administration (first bar from the left in each set of bars for each individual monkey), and 4 days (second bar from the left in each set of bars for each individual monkey), 8 days (third bar from the left in each set of bars for each individual monkey), and 15 days (fourth bar from the left in each set of bars for each individual monkey) after antibody administration. The x-axis shows the identification number of each monkey. [Figure 7B]FIG. 1 shows the frequency of different T cell populations observed in the peripheral blood of different cynomolgus monkeys after a single intravenous administration of anti-IL-7R antibody (3 mg / kg). A shows the frequency of CD3+ T cells as a percentage of total lymphocytes. B and C show the frequency of CD4+ T cells and CD8+ T cells as a percentage of the total CD3+ T cell population, respectively. In A, B, and C, the frequency of different T cell populations was observed before administration (first bar from the left in each set of bars for each individual monkey), and 4 days (second bar from the left in each set of bars for each individual monkey), 8 days (third bar from the left in each set of bars for each individual monkey), and 15 days (fourth bar from the left in each set of bars for each individual monkey) after antibody administration. The x-axis shows the identification number of each monkey. [Figure 7C] FIG. 1 shows the frequency of different T cell populations observed in the peripheral blood of different cynomolgus monkeys after a single intravenous administration of anti-IL-7R antibody (3 mg / kg). A shows the frequency of CD3+ T cells as a percentage of total lymphocytes. B and C show the frequency of CD4+ T cells and CD8+ T cells as a percentage of the total CD3+ T cell population, respectively. In A, B, and C, the frequency of different T cell populations was observed before administration (first bar from the left in each set of bars for each individual monkey), and 4 days (second bar from the left in each set of bars for each individual monkey), 8 days (third bar from the left in each set of bars for each individual monkey), and 15 days (fourth bar from the left in each set of bars for each individual monkey) after antibody administration. The x-axis shows the identification number of each monkey. [Figure 8] Figure 1 shows the potential immunogenicity risk of different production lots of anti-IL-7R antibodies, measured in vitro using a dendritic cell:T cell proliferation assay. The different anti-IL-7R antibodies tested include (i) P1-061895-2, (ii) P1-066930-3, (iii) P1-066930-9, and (iv) antibody A RACIR. Avastin and an anti-IL-21R antibody (IL-21R mAb) are shown as negative and positive controls, respectively. [Figure 9] 1 shows the sequence coverage of human IL-7Rα when HDX-MS epitope mapping analysis was performed. Each bar represents a digested peptide. [Figure 10] Deuterium incorporation differences are shown for five epitopes of anti-IL-7R antibodies identified using HDX-MS epitope mapping analysis. The five epitopes shown include: (i) 24SQLEVNGSQHSLTCAF39, (ii) 73FIETKKFLLIGKSNIC88, (iii) 89VKVGEKSLTCKKIDLTT105, (iv) 136QKKYVKVLMHDVAY149, and (v) 181YEIKVRSIPDHYFKGF196. The black vertical bars indicate the sum of the deuteration differences for each peptide. The lines represent the deuteration differences for each peptide at different time points, i.e., 1 min ("(2)"), 10 min ("(3)"), and 240 min ("(1)"). [Figure 11A] Figure 1 shows a summary of epitope mapping analysis of anti-IL-7R antibodies. In this figure, epitopes were mapped against the linear sequence of human IL-7Rα. Epitopes identified using HDX-MS analysis are shown as solid lines. Epitopes identified using FPOP / GEE analysis are shown as dotted lines. The most conserved residues of epitopes identified using FPOP analysis are shown as circles. The most conserved residues of epitopes identified using GEE analysis are shown as triangles. The numbering shown corresponds to the mature sequence of human IL-7Rα. [Figure 11B] Figure 1 shows a summary of epitope mapping analysis of anti-IL-7R antibodies. In this figure, epitopes are mapped onto the crystal structure of human IL-7Rα. The crystal structure on the left shows epitopes identified using HDX-MS analysis. The crystal structure on the right shows epitopes identified using FPOP / GEE analysis. The sequences of the epitopes are shown below the crystal structures. [Figure 12] A and B show schematic diagrams of the PK model (A) and the PK / OD mechanistic model (B) of the anti-IL-7R antibody used to characterize the PK and PD data in monkeys and humans. [Figure 13]Shown are predicted human pharmacokinetics (based on serum concentrations of anti-IL-7R antibody) (left y-axis) and receptor occupancy (right y-axis) for an estimated human dose of anti-IL-7R antibody (110 mg dose subcutaneously every 2 weeks for a 70 kg adult). The top line ("RO") shows receptor occupancy data. The bottom line ("PK") shows pharmacokinetic data. [Figure 14] A and B show the binding of 18B1 antibody (Antibody A) to human IL-7R after reformatting as an scFv. A shows a comparison of binding of 18B1 scFv at different concentrations. B shows ka, kd, and KD values. [Figure 15] Specific amino acid residues within the heavy and light chain CDRs are indicated, which were individually mutated to perform the mutation scanning analysis described in Example 11. [Figure 16] Results from one selection round of 18B1 scFv are shown. Percent binding of 18B1 heavy chain and 18B1 light chain to human IL-7R is shown as compared to biotin-IL-7R. 18B1 heavy chain and 18B1 light chain were tested at two different concentrations (0.2 nM and 1 nM). [Figure 17A]

[0036] Figure 1 shows a schematic of the overall process for constructing a mutation scanning library and using next generation sequencing (NGS) as described in Example 11 to identify 1) CDR positions that are important for binding, 2) CDR positions that are tolerant to mutations, and 3) mutations that may improve binding of the antibody to hIL-7R. The figure shows various aspects of the process itself. [Figure 17B] Schematic of the overall process for constructing a mutation scanning library and using next generation sequencing (NGS) as described in Example 11 to identify 1) CDR positions important for binding, 2) CDR positions that are tolerant to mutations, and 3) mutations that may improve binding of the antibody to hIL-7R. The figure provides a brief explanation of the interpretation of the results obtained from the heatmap. [Figure 18A]17B shows heatmap results for 18B1 antibody variants with one mutation in LC CDR3. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 18B] 17B shows heatmap results for 18B1 antibody variants with one mutation in HC CDR3. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 18C]17B shows heatmap results for 18B1 antibody variants with one mutation in LC CDR1. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 18D] 17B shows heatmap results for 18B1 antibody variants with one mutation in LC CDR2. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 18E]17B shows heatmap results for 18B1 antibody variants with one mutation in HC CDR1. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 18F] 17B shows heatmap results for 18B1 antibody variants with one mutation in HC CDR2. Antibody variants were tested at two different concentrations (0.2 nM and 1 nM). The left-most column indicates the specific mutations introduced at specific amino acid residues. The associated boxes show the enrichment scores for the different mutations. As illustrated in FIG. 17B, an enrichment score above 1 indicates that the mutation is favorable (i.e., improves binding) and a higher enrichment score indicates that the mutation is more favorable. An enrichment score of 1 indicates that the mutation is neutral (i.e., has no significant effect on binding). An enrichment score below 1 indicates that the mutation is unfavorable (i.e., impairs binding). [Figure 19-1] (i) KD values ​​(shown as ratio of parental 18B1 antibody to variant antibody) and (ii) enrichment ratios obtained from NGS heatmap analysis for different 18B1 antibody variants are shown. [Figure 19-2] (i) KD values ​​(shown as ratio of parental 18B1 antibody to variant antibody) and (ii) enrichment ratios obtained from NGS heatmap analysis for different 18B1 antibody variants are shown. [Figure 20] A and B show an overview of the Biacore kinetic format used to assess the binding of 18B1 (Antibody A) and 18B1 alanine variants as described in Example 11. The specific conditions used are shown in the left part of A. The right part of A shows the Biacore sensorgram of the 18B1 antibody. B shows a schematic of the Biacore assay used. [Figure 21] Figure 1 shows data measuring binding of different 18B1 antibody variants to human IL-7R by Biacore analysis. [Figure 22] Figure 1 shows surface plasmon resonance (SPR) profiles of exemplary 18B1 antibody variants. For each antibody variant, the ka, kd, and KD values ​​are shown below the SPR profile. [Figure 23] A graphical summary of the distribution of KD values ​​for different 18B1 antibody variants is shown. The antibody variants are separated based on which CDRs have been mutated. Each circle represents an individual antibody. [Figure 24] Correlation between KD values ​​(squares) and heatmap results obtained using NGS (open circles) for different alanine variants is shown, with the specific mutations (in the light chain CDR or in the HC CDR) indicated on the X-axis. [Diagram 25] A and B show the crystal structure of the Fab fragment of the 18B1 antibody. In A, the heavy chain (dark grey) and light chain (light grey) of the 18B1 antibody are shown. In B, residues important for binding to IL-7Rα are shown in dark grey. Residues that can be modified to improve binding are shown in black. [Figure 26]IL-7Rα expression on CD3+ leukocytes isolated from monkeys subcutaneously administered 18B1 antibody. 18B1 antibody was administered at one of the following doses: (i) 2 mg / kg ("squares"), (ii) 10 mg / kg ("triangles"), and (iii) 50 mg / kg ("diamonds"). Animals not administered 18B1 antibody were used as controls ("circles"). IL-7Rα expression is shown as mean fluorescence intensity (MFI) measured using flow cytometry. IL-7Rα expression was measured on the following days after antibody administration: day 1, 4 hours; day 5; day 8; day 22; day 36; day 36, 4 hours; day 40; day 43; day 64; and day 99. [Figure 27A] Figure 1 shows a comparison of keyhole limpet hemocyanin (KLH)-induced IgM (A) and IgG (B) antibody responses in monkeys subcutaneously administered 18B1 antibody. 18B1 antibody was administered at one of the following doses: (i) 2 mg / kg ("squares"), (ii) 10 mg / kg ("triangles"), and (iii) 50 mg / kg ("diamonds"). Animals not administered 18B1 antibody were used as controls ("circles"). KLH-induced IgM and IgG responses are shown by listing the endpoint titers of KLH-specific IgM and IgG antibodies measured in the animals' serum using ELISA. As shown in A, KLH-specific IgM was measured on days 15, 22, and 29 after administration of 18B1 antibody (i.e., days 0, 7, and 14, respectively, after immunization with KLH). As shown in B, KLH-specific IgG was measured on days 15, 29, 36, and 43 after administration of 18B1 antibody (i.e., days 0, 14, 21, and 28, respectively, after immunization with KLH). [Figure 27B]Figure 1 shows a comparison of keyhole limpet hemocyanin (KLH)-induced IgM (A) and IgG (B) antibody responses in monkeys subcutaneously administered 18B1 antibody. 18B1 antibody was administered at one of the following doses: (i) 2 mg / kg ("squares"), (ii) 10 mg / kg ("triangles"), and (iii) 50 mg / kg ("diamonds"). Animals not administered 18B1 antibody were used as controls ("circles"). KLH-induced IgM and IgG responses are shown by listing the endpoint titers of KLH-specific IgM and IgG antibodies measured in the animals' serum using ELISA. As shown in A, KLH-specific IgM was measured on days 15, 22, and 29 after administration of 18B1 antibody (i.e., days 0, 7, and 14, respectively, after immunization with KLH). As shown in B, KLH-specific IgG was measured on days 15, 29, 36, and 43 after administration of 18B1 antibody (i.e., days 0, 14, 21, and 28, respectively, after immunization with KLH). [Figure 28A]1 shows the deuterium incorporation differentials of different epitopes identified using HDX-MS epitope mapping analysis for three reference anti-IL-7R antibodies (4A8, 13A10, and PFE A3312F, respectively). Epitopes shown in A for the 4A8 antibody include: (i) 57LVEVKCLNF65, (ii) 73FIETKKFLLIGKSNIC88, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in B for the 13A10 antibody include: (i) 73FIETKKFLLIGKSNIC88, (ii) 89VKVGEKSLTCKKIDLTT105, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in C for PFE A3312F include: (i) 24SQLEVNGSQHSLTCA38, (ii) 52EICGALVEVKCLNF65, (iii) 73FIETKKFLLIGKSNIC88, (iv) 89VKVGEKSLTCKKIDLTT105, (V) 104TTIVKPEAPFDLSV117, (vi) 109PEAPFDLSVIYRE121, (vii) 136QKKYVKVLMHDVAY149, (viii) 169TLLQRKLQPAAM180, and (ix) 181YEIKVRSIPDHYFKGF196. Black vertical bars indicate the sum of the deuteration differences for each peptide. The lines represent the deuteration difference of each peptide at different time points, i.e., 1 min ("(2)"), 10 min ("(3)"), and 240 min ("(1)"). [Figure 28B]1 shows the deuterium incorporation differentials of different epitopes identified using HDX-MS epitope mapping analysis for three reference anti-IL-7R antibodies (4A8, 13A10, and PFE A3312F, respectively). Epitopes shown in A for the 4A8 antibody include: (i) 57LVEVKCLNF65, (ii) 73FIETKKFLLIGKSNIC88, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in B for the 13A10 antibody include: (i) 73FIETKKFLLIGKSNIC88, (ii) 89VKVGEKSLTCKKIDLTT105, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in C for PFE A3312F include: (i) 24SQLEVNGSQHSLTCA38, (ii) 52EICGALVEVKCLNF65, (iii) 73FIETKKFLLIGKSNIC88, (iv) 89VKVGEKSLTCKKIDLTT105, (V) 104TTIVKPEAPFDLSV117, (vi) 109PEAPFDLSVIYRE121, (vii) 136QKKYVKVLMHDVAY149, (viii) 169TLLQRKLQPAAM180, and (ix) 181YEIKVRSIPDHYFKGF196. Black vertical bars indicate the sum of the deuteration differences for each peptide. The lines represent the deuteration difference of each peptide at different time points, i.e., 1 min ("(2)"), 10 min ("(3)"), and 240 min ("(1)"). [Figure 28C]1 shows the deuterium incorporation differentials of different epitopes identified using HDX-MS epitope mapping analysis for three reference anti-IL-7R antibodies (4A8, 13A10, and PFE A3312F, respectively). Epitopes shown in A for the 4A8 antibody include: (i) 57LVEVKCLNF65, (ii) 73FIETKKFLLIGKSNIC88, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in B for the 13A10 antibody include: (i) 73FIETKKFLLIGKSNIC88, (ii) 89VKVGEKSLTCKKIDLTT105, (iii) 136QKKYVKVLMHDVAY149, and (iv) 181YEIKVRSIPDHYFKGF196. Epitopes shown in C for PFE A3312F include: (i) 24SQLEVNGSQHSLTCA38, (ii) 52EICGALVEVKCLNF65, (iii) 73FIETKKFLLIGKSNIC88, (iv) 89VKVGEKSLTCKKIDLTT105, (V) 104TTIVKPEAPFDLSV117, (vi) 109PEAPFDLSVIYRE121, (vii) 136QKKYVKVLMHDVAY149, (viii) 169TLLQRKLQPAAM180, and (ix) 181YEIKVRSIPDHYFKGF196. Black vertical bars indicate the sum of the deuteration differences for each peptide. The lines represent the deuteration difference of each peptide at different time points, i.e., 1 min ("(2)"), 10 min ("(3)"), and 240 min ("(1)"). [Figure 29] A, B, and C show the mapping of epitopes of three reference anti-IL-7R antibodies (4A8, 13A10, and PFE A3312F, respectively) onto the crystal structure of human IL-7Rα protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] In order that the description of this disclosure may be readily understood, certain terms are defined first. Additional definitions are set forth throughout the detailed description.

[0040] It should be noted that the term "a" entity or "an" entity refers to one or more of that entity. For example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0041] Furthermore, "and / or" as used herein should be construed as specifically disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A (alone)," and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0042] It will be understood that wherever an embodiment is described herein using the word "comprising," the same embodiments described in conjunction with "consisting of" and / or "consisting essentially of" are also provided, except that the word "comprising" is not used.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For many of the terms used in this disclosure, a common dictionary for those of ordinary skill in the art can be found, for example, in the Concise Dictionary of Biomedicine and Molecular Biology, ed. lecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press.

[0044] Units, prefixes, and symbols are indicated in the form in which they are accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleotide sequences are written left to right in a 5' to 3' orientation. Amino acid sequences are written left to right in an amino-terminus to carboxy-terminus orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which aspects may be incorporated herein by reference in their entireties. Accordingly, the terms defined below are more fully defined herein by reference in their entireties.

[0045] The term "about" is used herein to mean approximately, roughly, roughly, or approximately. When used in conjunction with a numerical range, the term "about" modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated value by an upward or downward variance (e.g., a variance of 10 percent) (up or down).

[0046] The terms "IL-7R" and "IL-7 receptor" as used herein refer to any form of IL-7R, including variants of IL-7R that retain at least some of the activity of IL-7R. IL-7R is a heterodimer consisting of an α subunit (IL-7Rα, i.e., CD127) and a cytokine receptor common gamma chain (γc). The IL-7Rα subunit is expressed on a variety of cell types, including naive and memory T cells, as well as developmental B cells. As used herein, the term IL-7R, in some embodiments, refers to the α subunit and is used interchangeably with IL-7Rα.

[0047] Four isoforms of human IL-7Rα have been identified. Isoform 1 (Accession No. NP_002176.2 (SEQ ID NO: 1)) consists of 459 amino acids and is the canonical sequence. Isoform 2 (Accession No. P16871-4 (SEQ ID NO: 2)) consists of 252 amino acids and is soluble. Isoform 2 lacks amino acid residues 253 to 459, which code for part of the transmembrane domain and the entire cytoplasmic domain. Amino acids 237 to 252 also differ from the canonical sequence (i.e., isoform 1). Isoform 3 (Accession No. P16871-2 (SEQ ID NO: 3)) consists of 298 amino acids and has a truncated cytoplasmic domain. Isoform 3 differs from the canonical sequence by amino acid residues 293 to 459. Isoform 4 (Accession No. P16871-3 (SEQ ID NO: 4)) consists of 261 amino acids and is soluble. Isoform 4 differs from the standard sequence at amino acid residues 237-459.

[0048] Shown below are the amino acid sequences of the four known human IL-7Rα isoforms. [ka] [ka] [ka] [ka]

[0049] The signal sequences of isoforms 1-4 correspond to amino acid residues 1-20 (underlined). Thus, the mature forms (e.g., of the standard sequence (isoform 1)) consist of amino acids 21-459. The extracellular domain of mature human IL-7Rα (e.g., isoform 1) consists of amino acid residues 21-239 and has the following amino acid sequence: [ka]

[0050] The cynomolgus monkey IL-7Rα protein consists of the following amino acid sequence (including the signal sequence (underlined)): [ka]

[0051] As used herein, "TSLP" refers to a growth factor that closely resembles IL-7 and plays a role in the maturation and activation of myeloid cells (e.g., monocytes and dendritic cells). TSLP is produced by a variety of cell types, including fibroblasts, epithelial cells, and stromal cells. Elevated levels of TSLP are associated with diseases, including asthma, atopic dermatitis, and inflammatory arthritis, which are also known to be associated with dysregulation of IL-7. Nguyen V., et al., J Immunol Res 2017:4807853 (2017).

[0052] The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region is composed of a hinge and three domains (CH1, CH2, and CH3). In certain antibodies (e.g., naturally occurring IgG antibodies), each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)), which are sandwiched between more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system. By way of example, "antibodies" include both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0053] As used herein, an "IgG antibody" (e.g., a human IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody) has, in certain embodiments, the structure of a naturally occurring IgG antibody, i.e., has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, anti-IL-7R IgG1 antibody, anti-IL- An anti-IL-7R IgG2 antibody, an anti-IL-7R IgG3 antibody, or an anti-IL-7R IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), which are linked by the same number and position of disulfide bridges as occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively, unless the antibody carries mutations that alter these disulfide bridges.

[0054] Antibodies typically bind specifically to their corresponding antigens with high affinity, which is determined by the dissociation constant (K D ) is 10 -5 ~10 -11 This reflects that the value is less than 10 -4 K exceeds M D As used herein, an antibody that "specifically binds" to an antigen is one that binds to the antigen and has high affinity (K D 10 -7 M or less, 10 -8 M or less, 5×10 -9 M or less, or 10 -8 M~10 -10"Substantially identical" refers to an antibody that binds to a given antigen with a high affinity (meaning that the affinity is at least M or less) but does not bind to unrelated antigens with high affinity. An antigen is "substantially identical" to a given antigen if it shares a high degree of sequence identity with the given antigen (e.g., at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the sequence of the given antigen). As an example, an antibody that specifically binds to human IL-7Rα may, in certain embodiments, also have cross-reactivity with IL-7Rα antigens from certain primate species (e.g., cynomolgus IL-7Rα), but may not cross-react with IL-7Rα antigens from other species or antigens other than IL-7Rα.

[0055] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) encoded by heavy chain constant region genes. IgG isotypes are divided into subclasses in certain species, IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-IL-7R antibodies described herein are of the IgG1 isotype. Immunoglobulins (e.g., IgG1) exist in several allotypes, which differ from each other by at most a few amino acids.

[0056] The term "allotype" as used herein refers to naturally occurring variants within a particular isotype group, where such variants differ by several amino acids (see, e.g., Jefferis et al. (2009) mAbs 1:1). The anti-IL-7R antibodies described herein can be of any allotype. As used herein, antibodies designated as "IgG1f", "IgG1.1f", or "IgG1.3f" isotypes are IgG1, IgG1.1, and IgG1.3 antibodies with no effector function, respectively, of allotype "f" (i.e., those having 214R, 356E, and 358M according to the EU index as set forth in Kabat (e.g., those set forth in SEQ ID NO: 21)).

[0057] As used herein, the term "antigen-binding portion" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., human IL-7Rα). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody (e.g., an anti-IL-7R antibody described herein) include (i) a Fab fragment (a fragment obtained from cleavage with papain) or a V L Domain, V H (ii) a F(ab')2 fragment (a fragment obtained by pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H (iv) an Fd fragment consisting of the V domain and the CH1 domain of a single arm of an antibody; L Domain and V H (v) an Fv fragment consisting of a V H dA consisting of domains b fragment (Ward et al., (1989) Nature 341:544-546), (vi) an isolated complementarity determining region (CDR), and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, the two domains of the Fv fragment (V L and V H ) are encoded by separate genes, but using recombinant methods, the two domains can be isolated into their respective V L Area and V H The regions can be linked by synthetic linkers that allow them to pair into a single protein chain (known as single-chain Fv (scFv)) forming a monovalent molecule. See, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term "antigen-binding portion" of an antibody is also intended to encompass such single-chain antibodies. Such antibody fragments are obtained using conventional techniques known to those of skill in the art, and such fragments are screened for utility in the same manner as is applied to intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0058] A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0059] The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising such a population are substantially similar and bind to the same epitope(s) (e.g., such antibodies exhibit a single binding specificity and affinity), except for possible variants that may arise during the production of the monoclonal antibody (such variants are generally present to a certain extent). The modifier "monoclonal" indicates that the antibody has the character of being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. The term "human monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies that exhibits a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas in which B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome containing a human heavy chain transgene and a human light chain transgene are fused with an immortalized cell.

[0060] The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, or from hybridomas prepared from such animals, (b) antibodies isolated from host cells (e.g., transfectomas) transformed to express such antibodies, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that are encoded by germline genes but utilize certain human germline immunoglobulin sequences that subsequently undergo rearrangements and mutations (e.g., those that occur during antibody maturation). As is known in the art (see, e.g., Lonberg (2005) Nat. Immunol. 1999:1311-1323), the present invention relates to a method for producing recombinant human antibodies. In the conventional immunoglobulin sequences, the constant regions are encoded by different genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable regions may be further modified by multiple single amino acid changes (termed somatic mutations or hypermutations) to improve the affinity of the antibody for the foreign antigen. The constant regions may further change in response to antigen (i.e., isotype switching). Thus, the nucleic acid molecules encoding the light and heavy chain immunoglobulin polypeptides may not have sequence identity to the original nucleic acid molecules, but instead have substantial identity or similarity (i.e., at least 80% identity), having undergone rearrangement and somatic mutation in response to antigen.

[0061] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if such an antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The anti-IL-7R antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutated by random or site-specific in vitro mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as mouse) have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0062] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids contained in one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not impair the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0063] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a mouse antibody and whose constant region is derived from a human antibody.

[0064] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0065] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other proteins and cellular material.

[0066] As used herein, an antibody that "inhibits the binding of IL-7 to IL-7Rα" refers to an antibody that has an EC of about 1 μg / mL or less (such as about 0.9 μg / mL or less, about 0.85 μg / mL or less, about 0.8 μg / mL or less, about 0.75 μg / mL or less, about 0.7 μg / mL or less, about 0.65 μg / mL or less, about 0.6 μg / mL or less, about 0.55 μg / mL or less, about 0.5 μg / mL or less, about 0.45 μg / mL or less, about 0.4 μg / mL or less, about 0.35 μg / mL or less, about 0.3 μg / mL or less, about 0.25 μg / mL or less, about 0.2 μg / mL or less, about 0.15 μg / mL or less, about 0.1 μg / mL or less, or about 0.05 μg / mL or less) in an art-recognized method (e.g., a FACS-based binding assay as described herein). 50 and is intended to refer to an antibody that inhibits IL-7Rα from binding to its ligand, e.g., interleukin-7 (IL-7), which inhibition may occur, for example, in For example, it is tested in a binding assay using whole blood derived T cells expressing IL-7Rα.

[0067] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or to a biochemical event resulting from such an interaction. Examples of "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC and antibody-dependent cellular phagocytosis (ADCP)), and down-regulation of cell surface receptors (e.g., B cell receptor (BCR)). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

[0068] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies constitute the FcγR family of receptors (including allelic variants and alternatively spliced ​​forms of these receptors). The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice, and FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Different properties of human FcγRs are known in the art. While the majority of natural effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, natural killer (NK) cells in mice and humans selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) without expressing inhibitory FcγRIIB. Human IgG1 binds to most human Fc receptors and appears to be equivalent to mouse IgG2a with respect to the type of activating Fc receptor it binds.

[0069] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of an antibody except for the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region includes two identical protein fragments derived from the second constant (CH2) and third constant (CH3) domains of the antibody's two heavy chains. The IgM Fc region and the IgE Fc region include three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region includes the immunoglobulin domains (CH2 and CH3) and includes a hinge between the CH1 and CH2 domains. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be defined differently, as defined herein, the human IgG heavy chain Fc region is defined as the interval from amino acid residue D221 to the carboxy terminus of the heavy chain for IgG1, from amino acid residue V222 to the carboxy terminus of the heavy chain for IgG2, from amino acid residue L221 to the carboxy terminus of the heavy chain for IgG3, and from amino acid residue P224 to the carboxy terminus of the heavy chain for IgG4, where the numbering is according to the EU index as set forth in Kabat. The CH2 domain of the human IgG Fc region spans from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain in the Fc region, i.e., spans from amino acid 341 to amino acid 447 or amino acid 446 (if there is no lysine residue at the C-terminus) or amino acid 445 (if there is no glycine or lysine residue at the C-terminus) of IgG. As used herein, an Fc region may be a native sequence Fc (including any allotypic variant) or a variant Fc (eg, an Fc of non-natural origin).Fc refers to any of the above regions, whether in isolation or in the context of an Fc-containing protein polypeptide, such as a "binding protein comprising an Fc region" (also referred to as an "Fc fusion protein") (e.g., an antibody or immunoadhesin). It can refer to an area.

[0070] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG 1 Fc region, native sequence human IgG 2 Fc region, native sequence human IgG 3 Fc region, and native sequence human IgG 4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes the Fcs of the various allotypes (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0071] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., IL-7Rα) to which an immunoglobulin or antibody specifically binds, the site being defined, for example, by the particular method used to identify it. Epitopes can be formed from contiguous amino acids (usually linear epitopes) or from non-contiguous amino acids that are brought together by tertiary folding of a protein (usually structural epitopes). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically include at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids in a unique spatial arrangement. Methods for determining which epitope a given antibody binds (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation, in which overlapping or consecutive peptides (e.g., from IL-7Rα) are tested for reactivity with a given antibody (e.g., an anti-IL-7R antibody). Methods for determining the spatial location of epitopes include techniques in the art as well as those described herein (e.g., x-ray crystallography, antigenic mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0072] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0073] The term "binds to the same epitope" in relation to two or more antibodies means that the antibodies bind to the same amino acid residue segment as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on IL-7Rα" in relation to the antibodies described herein include, for example, epitope mapping methods, such as x-ray crystallography of antigen:antibody complexes (which provide atomic resolution of the epitope) and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of an antibody to an antigen fragment or mutant antigen variation, and loss of binding due to alteration of amino acid residues in the antigen sequence is often taken as an indication of an epitope element. Additionally, computational combinatorial methods for epitope mapping can also be used. These methods utilize the ability of an antibody of interest to isolate specific short peptides with affinity from a combinatorial phage display peptide library. Antibodies with the same VH and VL or the same CDR1, CDR2, and CDR3 sequences are expected to bind to the same epitope.

[0074] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to a target. When two antibodies compete with each other for binding to a target, Whether or not an antibody competes with another antibody, i.e., whether and to what extent it inhibits the binding of another antibody to a target, can be determined using known competition experiments (e.g., BIACORE™ surface plasmon resonance (SPR) analysis). In certain embodiments, an antibody competes with another antibody and inhibits the binding of the other antibody to a target by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277, or Ed Harlow and David Lane, Cold This can be performed as described in Chapter 11 of "Uses of Antibodies" by Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other by at least 50% in both modes, i.e., if this blocking occurs regardless of which antibody first contacts the antigen in the competition experiment.

[0075] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include those that examine competition for binding to T cells expressing IL-7Rα, e.g., by flow cytometry (such as those described in the Examples). Other methods include SPR (e.g., BIACORE™), solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)), solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)), solid phase direct label assays, solid phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid phase direct label RIA using I-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)), solid phase direct biotin-avidin EIA (Cheung et al., J. Immunol. 25(1):7 (1988)), solid phase direct label RIA using I-125 label (see Cheung et al., J. Immunol. 25(1):7 (1988)), solid phase direct biotin-avidin EIA (see Cheung et al., J. Immunol. 25(1):7 (1988)), solid phase direct label RIA using I-125 label ... al., Virology 176:546 (1990)), and direct label RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0076] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a given antigen. Typically, an antibody binds to an epitope that is greater than or equal to about 10 mAb, as determined, for example, by surface plasmon resonance (SPR) technology on a BIACORE™ 2000 instrument using a given antigen (e.g., recombinant human IL-7Rα) as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M (approx. 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 Equilibrium dissociation constant (KD ) and (ii) binds to a given antigen with an affinity that is at least 2-fold stronger than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human IL-7Rα" has an affinity that is at least 10 -7 M or less (about 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 M or even lower) D The term "antibody that cross-reacts with cynomolgus monkey IL-7Rα" refers to an antibody that binds to soluble human IL-7Rα or cell-bound human IL-7Rα at 10 -7 M or less (about 10 -8 Less than M, about 10 -9 Less than M or about 10 -10 M or even lower) D In certain embodiments, antibodies that do not cross-react with IL-7Rα from non-human species are essentially incapable of detecting binding to such proteins in standard binding assays. It is possible.

[0077] As used herein, "k assoc " or "k a The term "k" is intended to refer to the binding rate of a particular antibody-antigen interaction and is used herein. dis " or "k d The term "K" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "k a k for d (i.e., k d / k a ) and is expressed as a molar concentration (M). D Values ​​can be determined using methods well established in the art. DMethods available for determining include surface plasmon resonance, biosensor systems (such as the BIACORE® system or flow cytometry), and Scatchard analysis.

[0078] As used herein with respect to IgG antibodies, the term "high affinity" refers to the K D 10 -8 M or less, 10 -9 M or less, or 10 -10 However, "high affinity" binding may be different for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to binding that is greater than or equal to the K D 10 -10 M or less or 10 -8 It means that it is M or less.

[0079] "EC" in the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof 50 The term "antibody or antigen-binding portion thereof" refers to the concentration at which an antibody or antigen-binding portion thereof induces a response that is 50% of the maximal response (ie, halfway between the maximal response and the baseline).

[0080] The term "natural origin" as used herein refers to the fact that an object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by the hand of man is of natural origin.

[0081] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of such a chain. One or more of the amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0082] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0083] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, a predicted non-essential amino acid residue in an anti-IL-7R antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not impair antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10 ):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997).

[0084] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared with appropriate insertions and deletions of nucleotides, are identical in at least about 80% of the nucleotides, at least about 90%-95% of the nucleotides, or at least about 98%-99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the strand.

[0085] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, are identical in at least about 80% of the amino acids, at least about 90%-95% of the amino acids, or at least about 98%-99.5% of the amino acids, when appropriately adjusted for amino acid insertions and deletions and optimally aligned and compared.

[0086] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100), and such gaps should be introduced so that the two sequences are optimally aligned. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as those described in the non-limiting examples below.

[0087] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix with a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGNMENT program (version 2.0) using a PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0088] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, use the Al Gapped BLAST can be used as described in Tschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0089] Nucleic acids may be present in whole cells, cell lysates, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when they have been purified to remove other cellular components or other contaminants (e.g., other cellular nucleic acids (e.g., other parts of chromosomes)) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. F. Ausubel, et al., ed. Current Protocols in Molecular See Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0090] Gene sequences can be obtained by introducing mutations into nucleic acids (e.g., cDNA) according to standard techniques. For coding sequences, such mutations can affect the amino acid sequence as desired. Specifically contemplated are DNA sequences that are substantially homologous to or derived from naturally occurring V, D, J, constant, switch, and other such sequences described herein (where "derived" indicates that the sequence is identical to or has been modified from another sequence).

[0091] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated and introduced. Another type of vector is a viral vector, in which additional DNA segments can be ligated and introduced into the viral genome. Certain vectors have the ability to autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors have the ability to cause expression of a gene to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since the most commonly used form of vector is the plasmid, "plasmid" and "vector" may be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0092] The term "recombinant host cell" (or simply "host cell") as used herein is intended to refer to a cell that contains a non-naturally occurring nucleic acid therein, and such a cell may be a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain changes may occur over generations due to mutations or environmental influences, but are still included within the scope of the term "host cell" as used herein.

[0093] The term "immune response" as used herein is understood as understood in the art and generally refers to a biological response that occurs in a vertebrate to a foreign substance or abnormal cell (e.g., a cancerous cell) that protects the organism against such substances and the diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body an invading pathogen, a pathogen-infected cell or tissue, a cancerous or other abnormal cell, or, in the case of autoimmune or pathological inflammation, a normal human cell or tissue. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4 T ... + cells, CD8 + This includes the activation or inhibition of T cells, T cells, or Treg cells, or the activation or inhibition of any other immune system cell (eg, NK cells).

[0094] The term "abnormal immune response" as used herein refers to the failure of a subject's immune system to distinguish self from non-self or to respond to foreign antigens. The term also encompasses an exaggerated immune response to foreign antigens, such as in the case of allergic disorders. Thus, such responses are present in both autoimmune and allergic disorders. Abnormal immune responses include, but are not limited to, tissue injury and inflammation caused by the production of antibodies against the organism's own tissues, impaired production of cytokines, and tissue damage caused by cytotoxic or non-cytotoxic mechanisms of action. In some embodiments, an abnormal immune response is an immune response that is inappropriately controlled, leading to patient symptoms. Typically, an autoimmune response occurs when a subject's immune system recognizes self-antigens as foreign, which results in the production of autoreactive effector immune cells. Autoreactive effector immune cells include cells from a variety of lineages, including, but not limited to, cytotoxic T cells, helper T cells, and B cells. Although the exact mechanism differs, the presence of autoreactive effector immune cells in patients suffering from autoimmune disorders can result in destruction of the patient's tissues and cells, resulting in pathological symptoms. Similarly, the presence of cells that undergo a hypersensitive reaction to a foreign antigen that would cause a normal individual to respond in a more suppressed manner is a sign of hypersensitivity (allergy). Examples include, but are not limited to, food allergies, hay fever, and allergic asthma. Many assays are known to those skilled in the art for determining the presence of such cells in a patient, and thus the presence of an autoimmune disorder (such as an antigen-specific autoimmune disorder in a patient) or an allergic disorder, and such assays can be readily used in the subject method.

[0095] As used herein, the term "autoimmune disease" refers to a disease or disorder in which tissues are damaged as a result of the immune system generating an immune response (e.g., a B cell response or a T cell response) against an antigen that is part of the normal host (i.e., a self-antigen). The self-antigen may be derived from host cells or from commensal organisms, such as microorganisms that usually colonize mucosal surfaces (known as commensals).

[0096] Examples of autoimmune diseases that affect mammals include rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and the like. .

[0097] As used herein, the term "inflammatory bowel disease" (IBD) refers to a heterogeneous group of chronic inflammatory disorders of the gastrointestinal tract. In some embodiments, IBD includes Crohn's disease (CD) and ulcerative colitis (UC).

[0098] The term "immunosuppressant" as used herein refers to a molecule (such as a compound, small molecule, steroid, nucleic acid molecule, or other biological agent) that can reduce an immune response (such as an inflammatory response). Immunosuppressants include, but are not limited to, agents used in the treatment of arthritis (anti-arthritic agents). Specific examples of immunosuppressants include, but are not limited to, nonsteroidal anti-inflammatory agents, cyclosporin A, FK506, and anti-CD4. In additional examples, the agent is a biological response modifier (such as KINERET® (anakinra), ENBREL® (etanercept), or REMICADE® (infliximab)), a disease-modifying antirheumatic drug (DMARD) (such as ARAVA® (leflunomide)), a nonsteroidal anti-inflammatory drug (NSAID), specifically a cyclooxygenase-2 (COX-2) inhibitor, (such as CELEBREX® (celecoxib) and VIOXX® (rofecoxib)), or another product (such as HYALGAN® (hyaluronan) and SYNVISC® (hylan G-F20)). Rapamycin is an additional example of an immunosuppressant.

[0099] The term "inflammation" or "inflammatory process" as used herein refers to a complex series of events (including dilation of arterioles, capillaries, and venules) involving increased permeability and blood flow, exudation of fluid (including plasma proteins), and migration of leukocytes to the inflammatory focus. Inflammation can be measured by many methods well known in the art, such as leukocyte counts, polymorphonuclear neutrophil (PMN) counts, measures of PMN activation (such as luminol-enhanced chemiluminescence), or the abundance of proinflammatory cytokines (e.g., IL-6 or TNF-α).

[0100] "Immunomodulatory agent" or "immunoregulatory agent" refers to an agent (e.g., an agent that targets an element of a signaling pathway that may be involved in the regulation, regulation, or modification of an immune response). "Modulation," "regulation," or "modulation" of an immune response refers to any change in the cells of the immune system or any change in the activity of such cells (e.g., effector T cells, such as Th1 cells). Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of such cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulatory agents have been identified, some of which may enhance the function of the tumor microenvironment. In some embodiments, the immunomodulatory agent targets a molecule on the surface of a T cell. An "immunoregulatory target" or "immunoregulatory target" is a molecule (e.g., a cell surface molecule) that is targeted for binding of a substance, agent, moiety, compound, or molecule, and whose activity is altered by such binding. Immunomodulatory targets include, for example, receptors on the surface of cells ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0101] "Immunotherapy" refers to treating a subject suffering from a disease or at risk of suffering from a recurrence of a disease by methods involving inducing, enhancing, suppressing, or otherwise modulating the immune system or immune response.

[0102] As used herein, the term "pathogenic T cells" refers to T cells (e.g., CD4 T cells) that cause the causative symptoms and / or damage associated with a disease or disorder (e.g., an inflammatory disease (e.g., inflammatory bowel disease)). + T cells or CD8 + In some embodiments, the term refers to "effector T cells" (T eff ) can be used interchangeably with T ef f are T cells with cytolytic activity (e.g., CD4 + T cells and CD8 +The term "effector T cells" refers to CD4+ T cells and helper T (Th) cells (e.g., Th1 cells), which secrete inflammatory cytokines (e.g., TNF-α, IFN-γ, or IL-17) and can also activate and induce other immune cells (e.g., monocytes) to produce proinflammatory mediators, thereby causing the causative symptoms and / or damage associated with a disease or disorder. In some embodiments, effector T cells are Th17 cells, which are CD4+ T cells that produce the proinflammatory cytokine IL-17. + In some embodiments, effector T cells are cytotoxic T lymphocytes (CTLs), which are CD8 T cells that have the ability to kill other cells (e.g., via the release of perforin or granzyme B). + Refers to T cells.

[0103] The term "regulatory T cells" (Tregs) as used herein refers to a population of T cells that have the ability to reduce or suppress the induction and proliferation of effector T cells, thereby regulating immune responses. In some embodiments, Tregs can suppress immune responses by secreting anti-inflammatory cytokines (such as IL-10, TGF-β, and IL-35) that can interfere with the activation and differentiation of naive T cells into effector T cells. In some embodiments, Tregs can also produce cytolytic molecules (such as granzyme B) that can induce apoptosis of effector T cells. In some embodiments, the regulatory T cells are endogenous regulatory T cells (nTregs) (i.e., those that develop in the thymus). In some embodiments, the regulatory T cells are inducible regulatory T cells (iTregs) (i.e., naive T cells that differentiate into Tregs in peripheral tissues upon exposure to certain stimuli). Methods for identifying Tregs are known in the art. For example, Tregs express certain phenotypic markers (e.g., CD25, Foxp3, or CD39) that can be measured using flow cytometry. See, e.g., International Publication No. WO2017 / 062035A1, Gu J, et al., Cell Mol Immunol 14(6):521-528(2017). In some embodiments, Tregs express CD45RA - CD39 + T cells.

[0104] The term "linked" as used herein refers to the association of two or more molecules. The association can be covalent or non-covalent. The association can also be genetic (i.e., recombinantly fused). Such association can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0105] As used herein, "administering" refers to physically introducing a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Different routes of administration of the anti-IL-7R antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other injection routes of administration (e.g., by injection or infusion). As used herein, the phrase "injection administration" refers to modes of administration other than enteral and topical administration (usually by injection), including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein can be administered via a non-injection route of administration, such as a topical, epidermal, or mucosal route of administration (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). Administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0106] As used herein, the terms "inhibit" or "block" (e.g., with respect to inhibiting / blocking the binding of IL-7 to IL-7Rα on a cell) are used interchangeably and are intended to refer to a moiety. This term encompasses both partial and complete inhibition / blocking. In some embodiments, the anti-IL-7R antibody inhibits IL-7 binding to IL-7Rα by at least about 50% (e.g., about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%), where the inhibition is determined, for example, as further described herein. In some embodiments, the anti-IL-7R antibody inhibits IL-7 binding to IL-7Rα by no more than 50% (e.g., about 40%, about 30%, about 20%, about 10%, about 5%, or about 1%), where the inhibition is determined, for example, as further described herein.

[0107] As used herein, the terms "treat", "treating" and "treatment" refer to any type of intervention or process performed on a subject or administration of an active agent to a subject with the goal of ameliorating, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with a disease, or prolonging overall survival. Treatment can be for subjects with a disease or for subjects without a disease (e.g., for prophylaxis).

[0108] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount that, when used alone or in combination with another therapeutic agent, promotes disease relief as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional or disability impairments resulting from disease affliction. A therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or "prophylactically effective dose", which is any amount that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of a disease, the drug inhibits the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease remission or inhibit the onset or recurrence of disease can be assessed using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of such agents in in vitro assays.

[0109] The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0110] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals (non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.).

[0111] The term "weight-based" dose or administration as referred to herein means that the dose to be administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-IL-7R antibody, the appropriate amount of anti-IL-7R antibody to be administered (i.e., 180 mg) can be calculated and used.

[0112] The use of the term "fixed dose" with respect to the methods of the present disclosure means that two or more different antibodies (e.g., an anti-IL-7R antibody and a second antibody (e.g., an anti-TNFR antibody)) in a single composition are present in the composition in a specific (fixed) ratio to each other. In some embodiments, the fixed dose is based on the weight (e.g., mg) of the antibodies. In certain embodiments, the fixed dose is based on the concentration (e.g., mg / ml) of the antibodies. In some embodiments, the ratio of the two antibodies (e.g., anti-IL-7R and anti-TNFR) is based on the weight (e.g., mg / ml) of the first antibody (e.g., anti-IL-7R at least about 1:1, at least about 1:2, at least about 1:3, at least about 1:4, at least about 1:5, at least about 1:6, at least about 1:7, at least about 1:8, at least about 1:9, at least about 1:10, at least about 1:15, at least about 1:20, at least about 1:30, at least about 1:40, at least about 1:50, at least about 1:60, at least about 1:70, at least about 1:80, at least about 1:90, at least about 1:100, at least about 1:120, at least about 1:140, at least about 1:160, at least about 1:180, at least about 1:200, at least about 200:1, at least about 180:1, at least about 160:1, at least about 140:1, at least about 120:1, at least about 100:1, at least about 90:1, at least about 80:1, at least about 70:1, at least about 60:1, at least about 50:1, at least about 40:1, at least about 30:1, at least about 20:1, at least about 15:1, at least about 10:1, at least about 9:1, at least about 8:1, at least about 7:1, at least about 6:1, at least about 5:1, at least about 4:1, at least about 3:1, or at least about 2:1. For example, a 2:1 ratio of anti-IL-7R antibody to anti-TNF receptor antibody (such as atrosab) could mean that a vial or injection could contain about 480 mg of anti-IL-7R antibody and 240 mg of anti-TNFR antibody, or about 2 mg / ml of anti-IL-7R antibody and 1 mg / ml of anti-TNFR antibody.

[0113] The use of the term "flat dose" with respect to methods and doses described herein refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). Thus, a flat dose is not provided as a mg / kg dose, but rather as an absolute amount of agent (e.g., anti-IL-7R antibody). For example, a 60 kg person and a 100 kg person will receive the same dose of antibody (e.g., 480 mg of anti-IL-7R antibody).

[0114] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" or "μM," respectively.

[0115] The following subsections provide further details about various aspects described herein.

[0116] I. Anti-human IL-7R antibody Described herein are antibodies (e.g., fully human antibodies) that are characterized by particular functional features or properties. For example, the antibodies specifically bind to human IL-7Rα, and more specifically, to a particular domain (e.g., a functional domain) within the extracellular domain of human IL-7Rα. In some embodiments, the antibodies specifically bind to the IL-7 binding site on IL-7Rα. In certain embodiments, the antibodies are antagonistic antibodies, i.e., antibodies that bind to pathogenic T cells (e.g., effector CD8 + T cells) from IL-7-mediated proliferation and / or survival. In certain embodiments, the anti-IL-7R antibodies disclosed herein cross-react with IL-7Rα from one or more non-human primates, such as cynomolgus IL-7Rα. In some embodiments, the antibodies specifically bind to the extracellular domain of human IL-7Rα and the extracellular domain of cynomolgus IL-7Rα. In certain embodiments, the antibodies bind to human IL-7Rα with high affinity.

[0117] The anti-IL-7R antibodies described herein exhibit one or more of the following functional properties: (a) having the ability to bind to soluble and / or membrane-bound human IL-7Rα; (b) having the ability to bind to soluble and / or membrane-bound cynomolgus IL-7Rα; and (c) having the ability to bind to T cells (CD4 + CD45RA + , CD4 + CD45RA - , CD8 + CD45RA + , and / or CD8 + CD45RA - ) having the ability to bind to IL-7Rα, (d) does not have the ability to bind to IL-7Rα expressed on non-T cells (e.g., monocytes) in whole blood (e.g., human whole blood); (e) lack of ability to effectively block thymic stromal lymphopoietin (TSLP)-mediated monocyte activation; (f) has no ability to agonize IL-7 receptor signaling upon binding to IL-7Rα (e.g., has minimal pSTAT5 activation); (g) having the ability to restore regulatory T cell (Treg) function and / or promote Treg survival; (h) having the ability to block the proliferation of cells that produce IL-17 and / or IFN-gamma; (i) have the ability to maintain drug-free remission (e.g., longer than CTLA4-Ig (ORENCIA®)); (j) having the ability to block inflammation and mucosal damage in intestinal tissue (e.g., induced by pathogenic T cells); (k) having the ability to reduce the frequency of effector T cells (e.g., in the mesenteric lymph nodes (MLN) and / or lamina propria (LP)); (l) capable of treating a subject having an inflammatory disease (e.g., inflammatory bowel disease); (m) 24 SQLEVNGSQHSLTCAF 39 (SEQ ID NO: 8), 73 FIETKKFLLIGKSNIC 88 (SEQ ID NO: 9), 89 VKVGEKSLTCKKIDLTT 105 (SEQ ID NO: 10), 136 QKKYVKVLMHDVAY 149 (SEQ ID NO: 11), 181 YEIKVRSIPDHYFKGF 196 (SEQ ID NO: 12), and combinations thereof, as determined, for example, by hydrogen / deuterium exchange mass spectrometry (HDX-MS); and / or (n) has the ability to bind to human IL-7Rα at one or more amino acid residues selected from the group consisting of H33, E75, F79, I82, K84, M144, R186, H191, Y192, and combinations thereof (as determined, for example, by mass spectrometry-based protein footprinting techniques, such as photochemical oxidation of proteins (FPOP) and glycine ethyl ester (GEE) labeling).

[0118] In some embodiments, the anti-IL-7R antibodies described herein bind to human IL-7Rα with high affinity and have a K D For example, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7 In certain embodiments, the anti-IL-7R antibody binds to soluble human IL-7Rα and has a K D is, for example, 10 as determined by BIACORE™. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 12 M~10 -7 M, 10 -11 M~10 -7 M, 10 10 M~10 -7 M, 10 -9 M~10 -7 M, or 10 -8 M~10 -7 In certain embodiments, the anti-IL-7R antibody binds to bound (e.g., cell membrane-bound) human IL-7Rα (such as on human T cells) but not on human non-T cells, and the K Dis 10 as determined, for example, by flow cytometry and Scatchard plots. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 5×10 -10 M or less, 10 -10 M or less, 10 12 M~10 -7 M, 10 -11 M~10 -8 M, 10 -10 M~10 -8 M, 10 -9 M~10 -8 M, 10 -11 M~10 -9 M, or 10 -10 M~10 -9 In some embodiments, the anti-IL-7R antibodies disclosed herein bind to bound (e.g., cell membrane-bound) human IL-7Rα (such as on human T cells) but not on human non-T cells, and the EC 50 For example, as determined by flow cytometry, In some embodiments, the anti-IL-7R antibodies described herein bind to cyno IL-7Rα and have a K of 0.9 ug / mL or less, 0.8 ug / mL or less, 0.7 ug / mL or less, 0.6 ug / mL or less, 0.5 ug / mL or less, 0.4 ug / mL or less, 0.3 ug / mL or less, 0.2 ug / mL or less, 0.1 ug / mL or less, 0.05 ug / mL or less, or 0.01 ug / mL or less. D For example, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7In certain embodiments, the anti-IL-7R antibody binds to soluble cyno IL-7Rα and has a K D is, for example, 10 as determined by BIACORE™. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10 -9 M~10 -7 M, or 10 -8 M~10 -7 The anti-IL-7R antibodies of the present disclosure can bind to bound (e.g., membrane-bound) cynomolgus IL-7Rα (such as that on cynomolgus T cells) but not on cynomolgus non-T cells, and the EC 50 is, for example, 100 nM or less, 10 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, as determined, for example, by flow cytometry. In certain embodiments, the anti-IL-7R antibody binds to bound (e.g., cell membrane bound) cynomolgus IL-7Rα (on cynomolgus T cells) but not on cynomolgus non-T cells, and the K D is 10 as determined, for example, by flow cytometry and Scatchard plots. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 5×10 -10 M or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -8 M, 10 -10 M~10 -8 M, 10 -9 M~10 -8 M, 10 -11 M~10 -9 M, or 10 -10 M~10-9 It's M.

[0119] Standard assays are known in the art for evaluating the binding ability of antibodies to IL-7Rα of various species, including, for example, ELISA, Western blot, and RIA. Suitable assays are also described in the Examples. Antibody binding kinetics (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as by Biacore analysis. Assays for evaluating the effect of antibodies on immune cell functional properties (e.g., ligand binding, STAT5 phosphorylation / activation, cytokine production) are further detailed below and in the Examples.

[0120] In some embodiments, the anti-IL-7R antibodies disclosed herein inhibit T cells (e.g., CD4 + CD45RA + , CD4 + CD45RA - , CD8 + CD45RA + , CD8 + CD45RA -), but not IL-7Rα expressed on non-T cells (e.g., monocytes). Thus, in some embodiments, an anti-IL-7R antibody may effectively block IL-7 binding to IL-7Rα on T cells, but not TSLP binding to IL-7Rα expressed on non-T cells (e.g., monocytes). In some embodiments, an anti-IL-7R antibody may inhibit or reduce IL-7-mediated phosphorylation of STAT5 in T cells ("pSTAT5 activation"). In some embodiments, pSTAT5 activation is inhibited or reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference (e.g., a matched T cell not treated with an anti-IL-7R antibody disclosed herein). In some embodiments, the anti-IL-7R antibodies disclosed herein do not reduce or block TSLP-mediated monocyte activation.

[0121] In some embodiments, the anti-IL-7R antibodies described herein inhibit effector T cells. In some embodiments, the anti-IL-7R antibody is capable of modulating (e.g., increasing) the ratio of regulatory T cells to Tregs in a subject in need thereof. - CD39 +In certain embodiments, the anti-IL-7R antibodies of the present disclosure increase Treg function (e.g., ability to suppress T cell proliferation and / or IL-10 production) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference (e.g., matched Tregs in a subject not administered the anti-IL-7R antibody). In some embodiments, the frequency of Tregs in a subject is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference (e.g., matched Tregs in a subject not administered the anti-IL-7R antibody).

[0122] In some embodiments, the anti-IL-7R antibodies disclosed herein modulate the ratio of regulatory T cells to effector T cells by blocking the proliferation of effector T cells (e.g., T cells that produce IL-17 and / or IFN-γ) in a subject. In certain embodiments, the frequency of effector T cells in a subject is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference (e.g., matched Tregs in a subject not administered an anti-IL-7R antibody). In certain embodiments, the anti-IL-7R antibodies disclosed herein modulate the ratio by both restoring function and / or promoting survival of Tregs and blocking proliferation of effector T cells in a subject.

[0123] In some embodiments, the ratio of regulatory T cells to effector T cells is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a reference (e.g., a corresponding ratio in a subject not administered an anti-IL-7R antibody).

[0124] In some embodiments, the anti-IL-7R antibodies described herein bind to an epitope (e.g., a structural epitope) in the extracellular portion of human IL-7Rα (e.g., the Ig-like domain of the extracellular region, i.e., amino acids 21-239 of SEQ ID NO:1). In certain embodiments, the anti-IL-7R antibodies bind to or an epitope within the region consisting of amino acid residues 44-59 of SEQ ID NO:1 (i.e., residues 24-39 of SEQ ID NO:6) (SQLEVNGSQHSLTCAF, "Epitope 1" (SEQ ID NO:8)). In some embodiments, the anti-IL-7R antibodies bind to or an epitope within the region consisting of amino acid residues 93-108 of SEQ ID NO:1 (i.e., residues 73-88 of SEQ ID NO:6) (FIETKKFLLIGKSNIC, "Epitope 2" (SEQ ID NO:9)). In other embodiments, the anti-IL-7R antibody binds to a region consisting of amino acid residues 109-125 of SEQ ID NO:1 (i.e., residues 89-105 of SEQ ID NO:6) (VKVGEKSLTCKKIDLTT, "Epitope 3" (SEQ ID NO:10)) or an epitope within that region. In some embodiments, the anti-IL-7R antibody binds to a region consisting of amino acid residues 156-169 of SEQ ID NO:1 (i.e., residues 136-149 of SEQ ID NO:6) (QKKYVKVLMHDVAY, "Epitope 4" (SEQ ID NO:11)) or an epitope within that region. In other embodiments, the anti-IL-7R antibody binds to a region consisting of amino acid residues 201-216 of SEQ ID NO:1 (i.e., residues 181-196 of SEQ ID NO:6) (YEIKVRSIPDHYFKGF, "Epitope 5" (SEQ ID NO:12)) or an epitope within that region.

[0125] In some embodiments, an anti-IL-7R antibody of the disclosure binds to an epitope (or region of human IL-7Rα) that includes one or more of amino acid residue H53, amino acid residue E95, amino acid residue F99, amino acid residue I102, amino acid residue K104, amino acid residue M164, amino acid residue R206, amino acid residue H211, and amino acid residue Y212 of SEQ ID NO:1 (i.e., H33, E75, F79, I82, K84, M144, R186, H191, and Y192 of SEQ ID NO:6). In some embodiments, the anti-IL-7R antibodies disclosed herein do not significantly bind to human IL-7Rα in which one or more of amino acid residues H53, E95, F99, I102, K104, M164, R206, H211, and Y212 of SEQ ID NO:1 have been altered to another amino acid (e.g., by non-conservative amino acid substitutions), or if they do bind, they do so with significantly reduced binding affinity.

[0126] In some embodiments, the anti-IL-7R antibodies disclosed herein are not naturally occurring or naturally occurring antibodies. For example, in certain embodiments, the anti-IL-7R antibodies have post-translational modifications that differ from those of naturally occurring antibodies, such as by having more, fewer, or different types of post-translational modifications.

[0127] In some embodiments, the anti-IL-7R antibodies do not have agonist activity and such antibodies do not enhance activity beyond that of the anti-IL-7R antibody alone, as determined, for example, by measuring pSTAT activation after culturing T cells with the anti-IL-7R antibody. In certain embodiments, the anti-IL-7R antibodies block the interaction of IL-7Rα with IL-7 without promoting agonist activity.

[0128] In some embodiments, the anti-IL-7R antibodies of the present disclosure do not have the ability to block thymic stromal lymphopoietin (TSLP)-mediated monocyte activation.

[0129] In some embodiments, the anti-IL-7R antibody has the ability to maintain drug-free remission (e.g., longer than CTLA4-Ig (ORENCIA®)).

[0130] In some embodiments, the anti-IL-7R antibody has the ability to block inflammation and mucosal damage in intestinal tissue (e.g., induced by pathogenic T cells). In some embodiments, the anti-IL-7R antibody has the ability to reduce the frequency of effector T cells (e.g., in the mesenteric lymph nodes (MLN) and / or lamina propria (LP)). In some embodiments, the anti-IL-7R antibody has the ability to treat a subject with an inflammatory disease (e.g., inflammatory bowel disease).

[0131] In some embodiments, an anti-IL-7R antibody of the disclosure comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, where the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16. In other embodiments, the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0132] In some embodiments, the anti-IL-7R antibody comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are respectively set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. and the light chain CDR1, light chain CDR2, and light chain CDR3 comprise the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and the anti-IL-7R antibody has one or more of the properties disclosed herein.

[0133] In some embodiments, an anti-IL-7R antibody of the disclosure comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the heavy chain CDR3 comprises one or more amino acid modifications (e.g., one, two, three, or four substitutions (e.g., as shown in FIG. 18B, which results in an enrichment ratio of 1.00 or greater)) compared to SEQ ID NO: 15. In some embodiments, the heavy chain CDR1 comprises one or more amino acid modifications (e.g., one, two, or three substitutions (e.g., as shown in FIG. 18E, which results in an enrichment ratio of 1.00 or greater)) compared to SEQ ID NO: 13. In certain embodiments, the heavy chain CDR2 comprises one or more amino acid modifications compared to SEQ ID NO: 14 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions (e.g., as shown in FIG. 18F, resulting in an enrichment ratio of 1.00 or more)). In another embodiment, the light chain CDR1 comprises one or more amino acid modifications compared to SEQ ID NO: 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions (e.g., as shown in FIG. 18C, resulting in an enrichment ratio of 1.00 or more). In some embodiments, the light chain CDR2 comprises one or more amino acid modifications compared to SEQ ID NO: 17 (e.g., 1, 2, 3, 4, 5, or 6 substitutions (e.g., as shown in FIG. 18D, resulting in an enrichment ratio of 1.00 or more). In certain embodiments, the light chain CDR3 comprises one or more amino acid modifications compared to SEQ ID NO: 18 (e.g., 1, 2, 3, 4, 5, 6, or 7 substitutions (e.g., those shown in Figure 18A that result in an enrichment ratio of 1.00 or greater)).

[0134] In some embodiments, the heavy chain CDR1 of the anti-IL-7R antibodies disclosed herein comprises the amino acid sequence GX1X2FDDHAX3H (SEQ ID NO: 260), where X1 is F or Y, X2 is T, P, A, S, V, L, I, M, H, F, Y, N, D, E, or Q, and X3 is L or M. In certain embodiments, X2 is D or E. In another embodiment, X2 is D. In other embodiments, X2 is E. Non-limiting examples of heavy chain CDR1 sequences of the IL-7R antibodies disclosed herein are shown in Table 13.

[0135] In some embodiments, the heavy chain CDR2 of an anti-IL-7R antibody disclosed herein comprises the amino acid sequence GIX1WX2SRGX3GYX4X5X6X7X8X9 (SEQ ID NO: 261), where X1 is S or T, X2 is H or N, X3 is I or V, X4 is G, A, S, T, V, L, I, R, H, or N, X5 is P, T, N, D, E, Q, S, H, or Y, X6 is P, G, A, S, T, V, R, H, F, Y, N, D, or E, X7 is V or I, X8 is A, S, T, V, L, I, M, K, R, H, F, Y, N, D, E, or Q, and X9 is G, H, D, or Q. In certain embodiments, X1 is T. Non-limiting examples of heavy chain CDR2 sequences for IL-7R antibodies disclosed herein are shown in Table 14.

[0136] In some embodiments, the heavy chain CDR3 of the anti-IL-7R antibody disclosed herein comprises the amino acid sequence DEYX1X2GYYX3LDX4 (SEQ ID NO: 262), in which X1 is S, T, N, D, or E, X2 is L, M, R, or S, X3 is G, A, S, T, V, M, N, E, or Q, and X4 is A, S, T, V, R, H, Y, W, N, E, Q, or M. In certain embodiments, X3 is A, S, or T. In some embodiments, X3 is A. In other embodiments, X3 is S. In another embodiment, X3 is T. In some embodiments, X4 is E. Non-limiting examples of heavy chain CDR3 sequences for the indicated IL-7R antibodies are shown in Table 15.

[0137] In some embodiments, the light chain CDR1 of an anti-IL-7R antibody of the disclosure comprises the amino acid sequence of X1X2X3X4X5X6X7SX8X9A (SEQ ID NO: 263), where X1 is S, T, V, K, R, H, Y, or I, X2 is A, S, T, or V, X3 is P, G, A, S, T, V, L, I, M, K, R, H, N, E, or Q, and X4 is , P, G, A, S, T, V, L, I, M, H, F, Y, N, D, E, or Q; X5 is P, G, A, S, T, H, E, Q, M, N, or D; X6 is P, G, A, S, T, V, L, I, or N; X7 is S, T, V, L, I, M, H, F, Y, N, D, E, or Q; X8 is P or A; and X9 is A, L, or V. In certain embodiments, X6 is P. In some embodiments, X8 is P. In another embodiment, X7 is D or E. In certain embodiments, X7 is D. In some embodiments, X7 is E. Non-limiting examples of light chain CDR1 sequences of IL-7R antibodies disclosed herein are shown in Table 16.

[0138] In some embodiments, the light chain CDR2 of an anti-IL-7R antibody disclosed herein comprises the amino acid sequence DX1X2X3X4X5X6 (SEQ ID NO: 264), where X1 is G, A, S, M, H, N, D, E, or Q, X2 is G, A, S, T, V, M, H, F, Y, N, D, E, or Q, X3 is A, S, F, Y, W, N, D, E, or L, X4 is P, S, T, L, K, H, or N, X5 is D, E, or Q, and X6 is G, S, T, N, D, Q, P, or E. Non-limiting examples of light chain CDR2 sequences of IL-7R antibodies disclosed herein are shown in Table 17.

[0139] In some embodiments, the light chain CDR3 of the anti-IL-7R antibodies disclosed herein comprises the amino acid sequence X1X2FX3X4YPLX5X6X7 (SEQ ID NO: 265), where X1 is M or Q, X2 is G, A, D, E, or Q, X3 is N or E, X4 is P, A, or S, X5 is T, I, M, K, W, N, E, or Q, X6 is L or I, and X7 is T, M, K, H, Y, E, or Q. In certain embodiments, X2 is A. In some embodiments, X4 is P or A. In certain embodiments, X4 is P. In other embodiments, X4 is A. Non-limiting examples of light chain CDR3 sequences of the IL-7R antibodies disclosed herein are shown in Table 18.

[0140] In some embodiments, the anti-IL-7R antibodies disclosed herein comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises one of the amino acid sequences set forth in SEQ ID NOs: 31-46; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0141] In some aspects, an anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises one of the amino acid sequences set forth in SEQ ID NOs: 47 to 96; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0142] In some aspects, an anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises one of the amino acid sequences set forth in SEQ ID NOs: 97 to 122; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0143] In some aspects, an anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises one of the amino acid sequences set forth in SEQ ID NOs: 123 to 194; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0144] In some aspects, an anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises one of the amino acid sequences set forth in SEQ ID NOs: 195 to 237; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0145] In some aspects, an anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises one of the amino acid sequences shown in SEQ ID NOs: 238 to 259.

[0146] In some embodiments, the anti-IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein (i) the heavy chain CDR1 comprises any one of the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NOs: 31 to 46; (ii) the heavy chain CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 14 and SEQ ID NOs: 47 to 96; (iii) Heavy chain CDR3 is the amino acid sequence shown in SEQ ID NO: 15 and SEQ ID NOs: 97 to 122 Contains one of the sequences, (iv) the light chain CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NOs: 123 to 194; (v) the light chain CDR2 comprises any one of the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NOs: 195 to 237, and / or (vi) The light chain CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 18 and SEQ ID NOs: 238 to 259.

[0147] In some embodiments, the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. In some embodiments, the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:47. In some embodiments, the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, or SEQ ID NO:120. In some embodiments, the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO:172, SEQ ID NO:189, SEQ ID NO:190, or SEQ ID NO:192. In some embodiments, the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:240, SEQ ID NO:244, or SEQ ID NO:245.

[0148] In some embodiments, an IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0149] In some embodiments, an IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 47; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0150] In some embodiments, an IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 120; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0151] In some embodiments, an IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 172, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 192; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:18.

[0152] In some embodiments, an IL-7R antibody disclosed herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 240, SEQ ID NO: 244, or SEQ ID NO: 245.

[0153] In some embodiments, the IL-7R antibodies disclosed herein comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3, where one of the heavy or light chain CDRs comprises an amino acid modification disclosed herein, and the other heavy or light chain CDR is unaltered compared to the corresponding CDR of the 18B1 antibody disclosed herein (also referred to herein as "Antibody A"). In some embodiments, two of the heavy or light chain CDRs comprise an amino acid modification disclosed herein, and the other heavy or light chain CDR is unaltered compared to the corresponding CDR of the 18B1 antibody. In another embodiment, three of the heavy or light chain CDRs comprise an amino acid modification disclosed herein, and the other heavy or light chain CDR is unaltered compared to the corresponding CDR of the 18B1 antibody. In certain embodiments, four of the heavy or light chain CDRs comprise the amino acid modifications disclosed herein, and the other heavy or light chain CDRs are unaltered compared to the corresponding CDRs of the 18B1 antibody. In another embodiment, five of the heavy or light chain CDRs comprise the amino acid modifications disclosed herein, and the other heavy or light chain CDRs are unaltered compared to the corresponding CDRs of the 18B1 antibody. In some embodiments, all six of the heavy and light chain CDRs comprise the amino acid modifications disclosed herein, compared to the corresponding CDRs of the 18B1 antibody. The 18B1 antibody described herein comprises a heavy chain CDR1 as set forth in SEQ ID NO: 13, a heavy chain CDR2 as set forth in SEQ ID NO: 14, a heavy chain CDR3 as set forth in SEQ ID NO: 15, a light chain CDR1 as set forth in SEQ ID NO: 16, a light chain CDR2 as set forth in SEQ ID NO: 17, and a light chain CDR3 as set forth in SEQ ID NO: 18.

[0154] In some embodiments, the anti-IL-7R antibodies disclosed herein comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 20, and wherein the anti-IL-7R antibodies have one or more of the properties disclosed herein.

[0155] In some embodiments, the anti-IL-7R antibody comprises a VH and a VL, wherein the VH comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of SEQ ID NO: 19, and the VL comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 of SEQ ID NO: 20, and the anti-IL-7R antibody has one or more of the properties disclosed herein.

[0156] In some embodiments, an anti-IL-7R antibody of the present disclosure cross-competes for binding to human IL-7Rα with a reference anti-IL-7R antibody, which comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 19 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the epitope on human IL-7Rα to which an anti-IL-7R antibody disclosed herein binds is the same as that bound by a reference anti-IL-7R antibody, which comprises a VH and a VL, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 19 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the anti-IL-7R antibody and the reference antibody both bind to an epitope selected from the group consisting of SQLEVNGSQHSLTCAF (SEQ ID NO: 8), FIETKKFLLIGKSNIC (SEQ ID NO: 9), VKVGEKSLTCKKIDLTT (SEQ ID NO: 10), QKKYVKVLMHDVAY (SEQ ID NO: 11), YEIKVRSIPDHYFKGF (SEQ ID NO: 12). In some embodiments, the anti-IL-7R antibody and the reference antibody both bind to an epitope that includes one or more of amino acid residue H53, amino acid residue E95, amino acid residue F99, amino acid residue I102, amino acid residue K104, amino acid residue M164, amino acid residue R206, amino acid residue H211, and amino acid residue Y212 of SEQ ID NO: 1 (i.e., H33, E75, F79, I82, K84, M144, R186, H191, and Y192 of SEQ ID NO: 6).

[0157] In some embodiments, the anti-IL-7R antibodies disclosed herein inhibit the binding of a reference anti-IL-7R antibody to human IL-7Rα by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. Competing antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art, such as RIA and EIA.

[0158] A VH domain described herein, or one or more CDRs thereof, may be linked to a constant domain to form a heavy chain (e.g., a full-length heavy chain). Similarly, a VL domain described herein, or one or more CDRs thereof, may be linked to a constant domain to form a light chain (e.g., a full-length light chain). A full-length heavy chain (with the exception that the C-terminal lysine (K) or C-terminal glycine and lysine (GK) may be absent) and a full-length light chain are combined to form a full-length antibody.

[0159] In some embodiments, a VH domain described herein may be fused to a constant domain of naturally occurring or modified human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). This may be done, for example, as further described herein. For example, the VH domain may comprise the amino acid sequence of any VH domain described herein, which is fused to a constant region of human IgG (e.g., IgG1), which constant region has the following wild-type human IgG1 constant domain amino acid sequence: [ka] or an allotypic variant of SEQ ID NO: 23, such as having the amino acid sequence: [ka]

[0160] In some embodiments, the VH domain of an anti-IL-7R antibody can comprise the amino acid sequence of any VH domain described herein, where the VH domain is fused to a constant region that has no effector function, such as the amino acid sequence of a human IgG1 constant domain that has no effector function, such as: [ka] or [ka]

[0161] For example, allotypic variants of IgG1 include K97R, D239E, and / or L241M (shown in bold and underlined above), where the numbering is according to that of SEQ ID NOs: 24-26. Within the full length heavy region, these amino acid substitutions are numbered K214R, D356E, and L358M according to EU numbering. In some embodiments, the constant region of the anti-IL-7R antibody may further comprise one or more mutations or substitutions at amino acid positions L117, A118, G120, A213, and P214 (shown in underlined above) according to the numbering of SEQ ID NOs: 24-26, or at amino acid positions L234, A235, G237, A330, and P331 according to EU numbering. In another embodiment, the constant region of the anti-IL-7R antibody comprises one or more mutations or substitutions at amino acid positions L117A, A118E, G120A, A213S, and P214S of SEQ ID NO: 23, or at amino acid positions L234A, L235E, G237A, A330S, and P331S according to EU numbering. The constant region of the anti-IL-7R antibody may also comprise one or more mutations or substitutions at L117A, A118E, and G120A of SEQ ID NO: 23, or at L234A, L235E, and G237A according to EU numbering.

[0162] The VL domains described herein may be fused to a human kappa or lambda light chain constant domain. For example, the VL domain of an anti-IL-7R antibody may comprise the amino acid sequence of any of the VL domains described herein, which is fused to the human IgG1 kappa light chain amino acid sequence: [ka]

[0163] In certain embodiments, the heavy chain constant region comprises a lysine or another amino acid at the C-terminus, e.g., the amino acid sequence LSPGK (SEQ ID NO: 28) at the end of the heavy chain. In certain embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus, e.g., has the C-terminal sequence LSPG (SEQ ID NO: 29) or LSP.

[0164] In some embodiments, an anti-IL-7R antibody of the disclosure comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO:21, and the LC comprises the amino acid sequence set forth in SEQ ID NO:22.

[0165] II. Engineered and Modified Antibodies Engineered and modified antibodies are also provided that can be prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as a starting material to engineer an engineered antibody, which can have altered properties from those of the starting antibody. The antibody can be engineered by modifying one or more residues in one or both variable regions (i.e., VH and / or VL) (e.g., residues in one or more CDR regions and / or one or more framework regions). Additionally or alternatively, the antibody can be engineered by modifying residues in the constant region(s), for example, to alter the effector function(s) of the antibody.

[0166] One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Because the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to construct expression vectors that contain CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties. It is possible to express recombinant antibodies that mimic the properties of those particular naturally occurring antibodies by using recombinant human antibodies (see, for example, Riechmann, L. et al. (1998) Nature 1998:1331-1335). 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter; and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.

[0167] Accordingly, one embodiment described herein relates to an isolated monoclonal antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and the VL comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and wherein the antibody has one or more of the properties disclosed herein. Thus, the anti-IL-7R antibodies disclosed herein may comprise the VH and VL CDR sequences set forth above and may further comprise different framework sequences.

[0168] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase) as well as in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "The Human Germline Sequences of Human Heavy and Light Chain Variable Region Genes." Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops” / .Mol.Biol.227:776-798, and Cox, JPLet al. (1994) “A Directory of Human Germ-line V H "Eur. J. Immunol. 24:827-836," the contents of each of which are expressly incorporated herein by reference.

[0169] In some embodiments, framework sequences for use in the anti-IL-7R antibodies described herein are structurally similar to the framework sequences used by the anti-IL-7R antibodies described herein. The VH CDR1, VH CDR2, and VH CDR3 sequences, and the VL CDR1, VL CDR2, and VL CDR3 sequences, CDR3 sequences can be grafted into framework regions that have the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions that contain one or more mutations compared to those of the germline sequences. For example, in certain cases, it has been found to be beneficial to mutate residues in framework regions so that the antigen-binding ability of the antibody is maintained or enhanced (see, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0170] The engineered anti-IL-7R antibodies described herein include those in which modifications have been made to framework residues in the VH and / or VL, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody was derived. Such residues may be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was derived. To return the framework region sequence to its germline configuration, the somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed. Another type of framework modification is to mutate one or more residues in the framework region, or even one or more residues in one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also called "deimmunization" and is described by Carr et al. This is further described in U.S. Patent Publication No. 20030153043 by al.

[0171] Another type of variable region modification is to alter amino acid residues in the VH CDR1, VH CDR2, and / or VH CDR3, and / or VL CDR1, VL CDR2, and / or VL CDR3 regions, thereby improving one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays described herein and provided in the Examples. In some embodiments, conservative modifications (as discussed above) are introduced. Such mutations can be amino acid substitutions, amino acid additions, or amino acid deletions. Moreover, typically no more than one, no more than two, no more than three, no more than four, or no more than five residues in the CDR regions are altered.

[0172] Accordingly, also provided herein is an isolated anti-IL-7R antibody comprising a VH and a VL, wherein the VH comprises (i) a CDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 13 (DHAMH), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 13, (ii) a CDR2 region comprising the amino acid sequence set forth in SEQ ID NO: 14 (GISWNSRGIGYADSVKG), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 14, and (iii) a CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 15 (DEYSRGYYVLDV), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 15. and the VL comprises (iv) a CDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 16 (RASQGISSALA), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 16; (v) a CDR2 region comprising the amino acid sequence set forth in SEQ ID NO: 17 (DASSLES), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 17; and (vi) a CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 18 (QQFNSYPLWIT), or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 18; and the antibody has one or more properties disclosed herein. Examples of such VH and VL CDR sequences include, but are not limited to, those shown in Table 13 (VH CDR1), Table 14 (VH CDR2), Table 15 (VH CDR3), Table 16 (VL CDR1), Table 17 (VL CDR2), and Table 18 (VL CDR3). can be.

[0173] Oxidation of methionine residues in the CDRs of an antibody can lead to chemical degradation, which can result in reduced efficacy of the antibody. Thus, anti-IL-7R antibodies are also provided in which one or more methionine residues in the heavy and / or light chain CDRs are replaced with an amino acid residue that is not susceptible to oxidative degradation.

[0174] Similarly, deamidation sites can be removed from the anti-IL-7R antibodies disclosed herein, specifically, deamidation sites in the CDRs can be removed.

[0175] The anti-IL-7R variable regions described herein include Fc (e.g., IgG1 Fc, IgG2 The antibody may be linked (e.g., covalently linked or fused) to an IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc. Such Fc can be of any allotype or isoallotype, e.g., for IgG1, G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z), for IgG2, G2m, G2m23(n), for IgG3, G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v), and for K, Km1, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs). 1:1).

[0176] In certain embodiments, the anti-IL-7R variable regions described herein are linked to an Fc with no or little effector function (e.g., IgG1.1f or IgG1.3f).

[0177] Generally, the variable regions described herein may be linked to an Fc that contains one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be added to the antibody) or modified to alter its glycosylation to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0178] The Fc region encompasses domains derived from the constant region of an immunoglobulin, including fragments, analogs, variants, mutants, or derivatives of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes, such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide that is homologous to the immunoglobulin C-terminal region, and may include the CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, either separately or in combination.

[0179] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) (i.e., FcγRI, FcγRII, and FcγRIII) that are specific for IgG class antibodies. It has been reported that the sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to Fc receptors (FcR).

[0180] In certain embodiments, the Fc region is a variant Fc region, e.g., These have an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to produce the variant) to obtain structural characteristics and / or biological activity.

[0181] In general, a variant of a constant region or a portion thereof (e.g., a CH1 domain, a CL domain, a hinge domain, a CH2 domain, or a CH3 domain) comprises or corresponds to one, two, three, four, five, six, seven, eight, nine, ten or more mutations, and / or up to ten, up to nine, up to eight, up to seven, up to six, up to five, up to four, up to three, up to two, or up to one mutation, or 1-10 or 1-5 mutations. The heavy chain constant region may comprise an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of the wild-type region or domain (the CH1 domain, CL domain, hinge domain, CH2 domain, or CH3 domain, respectively), provided that the heavy chain constant region containing the particular variant retains the requisite biological activity.

[0182] For example, modifications can be made to the Fc region to generate Fc variants that have (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors, as compared to the parent Fc. Such Fc region variants will generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications may be particularly desirable. For example, the variant Fc region may contain two, three, four, five, etc. substitutions therein (e.g., substitutions at specific Fc region positions as indicated herein).

[0183] The variant Fc region may contain sequence changes in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cells used to generate the anti-IL-7R antibodies described herein. Even if the cysteine ​​residues are removed, the single chain Fc domain may still form a non-covalently held together dimeric Fc domain. In other embodiments, the Fc region may be modified to improve its compatibility with the host cell of choice. For example, the PA sequence near the N-terminus of a typical native Fc region may be removed, which may be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g., asparagine) may confer a cytolytic response. Such residues may be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement (such as the C1q binding site) may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or replaced. In certain embodiments, sites that affect binding to Fc receptors (preferably sites other than the salvage receptor binding site) may be removed. In other embodiments, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art. For ADCC sites in IgG1, see, for example, Molec. Immunol. 29(5):633-9 (1992). Specific examples of variant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0184] In one embodiment, the hinge region of the Fc is modified to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region. The method is described in further detail in U.S. Patent No. 5,677,425 by Ward et al. The number of cysteine ​​residues in the hinge region of the Fc can be altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced in the interface region between the Fc hinge fragment and the CH2-CH3 domain to weaken the binding of the antibody to Staphylococcus aureus protein A (SpA) compared to the binding of the native hinge domain to SpA. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0185] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue such that the effector function(s) of the antibody are altered. For example, one or more amino acids selected from amino acid residue 234, amino acid residue 235, amino acid residue 236, amino acid residue 237, amino acid residue 297, amino acid residue 318, amino acid residue 320, amino acid residue 322, amino acid residue 330, and / or amino acid residue 331 may be replaced with a different amino acid residue such that the affinity of the antibody for the effector ligand is altered but the antibody retains the antigen-binding ability of the parent antibody. The effector ligand to which the affinity is altered may be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0186] In another example, one or more amino acids selected from amino acid residue 329, amino acid residue 331, and amino acid residue 322 may be replaced with a different amino acid residue such that the antibody's C1q binding is altered and / or complement dependent cytotoxicity (CDC) is reduced or eliminated. This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0187] In another example, one or more amino acid residues within amino acid positions 231-239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described in further detail in PCT Publication WO 94 / 29351 by Bodmer et al.

[0188] In yet another example, the Fc region can be modified to decrease antibody-dependent cellular cytotoxicity (ADCC) and / or decrease affinity for Fcγ receptors by modifying one or more of the amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. Examples of substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Examples of variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications to enhance interaction with FcγR and with complement include, but are not limited to, the following substitutions: 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications include: Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0189] Fc modifications that increase binding to an Fcγ receptor include those at amino acid position 238, amino acid position 239, amino acid position 248, amino acid position 249, amino acid position 252, amino acid position 254, amino acid position 255, amino acid position 256, amino acid position 258, amino acid position 265, amino acid position 267, amino acid position 268, amino acid position 269, amino acid position 270, amino acid position 272, amino acid position 279, amino acid position 280, amino acid position 283, amino acid position 285, amino acid position 298, amino acid position 289, amino acid position 290, amino acid position 292, amino acid position 293, amino acid position 294, amino acid position 295, amino acid position 296, amino acid position 298, amino acid position 301, amino acid position 303, amino acid position 305, amino acid position 307, and any one or more of amino acid modifications at amino acid position 312, amino acid position 315, amino acid position 324, amino acid position 327, amino acid position 329, amino acid position 330, amino acid position 335, amino acid position 337, amino acid position 338, amino acid position 340, amino acid position 360, amino acid position 373, amino acid position 376, amino acid position 379, amino acid position 382, ​​amino acid position 388, amino acid position 389, amino acid position 398, amino acid position 414, amino acid position 416, amino acid position 419, amino acid position 430, amino acid position 434, amino acid position 435, amino acid position 437, amino acid position 438, or amino acid position 439, where the numbering of these residues in the Fc region is that of the EU index as set forth in Kabat (WO00 / 42072).

[0190] Other Fc modifications that can be made to the Fc are those to reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331), numbering according to the EU index. Exemplary substitutions include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), numbering according to the EU index. Fc variants can include 236R / 328R. Other modifications to reduce interaction with FcγR and complement include substitutions of 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309F, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutagenesis or enzymatic means, or by producing the protein in a non-glycosylated organism, such as a bacterium. These and other modifications are described in Strohl, 2009, Current Reviewed in Opinion in Biotechnology 20:685-691.

[0191] Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720, PCX Patent Publication WO 00 / 42072, WO 00 / 42073, and WO 00 / 42074). (See WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0192] Fc variants that enhance affinity to the inhibitory receptor FcγRIIb may also be used. Such variants may confer immunomodulatory activity to the Fc fusion protein in association with FcγRIIb cells (including, for example, B cells and monocytes). In one embodiment, the Fc variants selectively enhance affinity to FcγRIIb relative to one or more activating receptors. Modifications to alter binding to FcγRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 (according to the EU index). Examples of substitutions for enhancing affinity to FcγRIIb include, but are not limited to, 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 331S, and 332E. Examples of substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcγRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0193] The affinity and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assays (biochemical or immunological based assays) known in the art, including, but not limited to, equilibrium (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic (e.g., BIACORE™ analysis) methods, as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label for one or more of the entities being examined and / or may utilize a variety of detection methods. Such labels and detection methods include, but are not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0194] In certain embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn, e.g., by mutating one or more of the following residues: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific examples of substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase biological half-life, the antibody can be altered to include a salvage receptor binding epitope in the CH1 or CL region, which is obtained from two loops of the CH2 domain of the Fc region of IgG. This is done as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other examples of variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434 (e.g., 2591, 308F, 428L, 428M, 434S, 43411, 434F, 434Y, and 434X1). Other variants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8):6213-6216; Hinton et al. 2006 Journal of Immunology 176 :346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K434F / 436H, 308T / 309P / 311S (Dall'Acqua et al.Journal of Immunology,2002,169:5171-5180, Dall'Acqua et al.,2006,Journal of Biological Chemistry 281:23514-23524). Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0195] In certain embodiments, hybrid IgG isotypes with specific biological characteristics may be used. For example, IgG1 / IgG3 hybrid variants may be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with IgG3-derived amino acids at positions that differ between IgG1 and IgG3 isotypes. Thus, hybrid variant IgG antibodies may be constructed that contain one or more substitutions (e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F). In other embodiments described herein, IgG1 / IgG2 hybrid variants may be constructed by substituting IgG2 positions in the CH2 and / or CH3 regions with IgG1-derived amino acids at positions that differ between IgG2 and IgG1 isotypes. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions (e.g., one or more of the following amino acid substitutions): 233E, 234L, 235L, -236G (referring to the insertion of a glycine at position 236), and 327A.

[0196] Additionally, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutations have been shown to improve binding to FcγRIII: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which have been shown to enhance binding to FcγRIIIa and ADCC activity (Shields et al. (2001) J. Biol. Chem. 276:6591-6604). (Lazar et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have also been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest affinity for FcγRIIIa, reduced binding to FcγRIIb, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introducing these triple mutations into antibodies, such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific), greatly enhanced in vitro ADCC activity, with the S239D / I332E variant showing enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). Furthermore, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations have been identified, and these IgG1 mutants have been shown to inhibit the IgG1-related phenotype in human FcγRIIIa-expressing transgenic mice, which are models of B cell malignancies and breast cancer. The Fc variants have been shown to enhance binding to FcγRIIIa and concomitantly enhance ADCC activity (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc variants that can be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0197] In certain embodiments, an Fc is selected that has reduced binding to FcγR, for example, an IgG1 Fc that contains three amino acid substitutions, L234A, L235E, and G237A, and has reduced binding to FcγR.

[0198] In certain embodiments, an Fc is selected that has reduced complement binding. An example of an Fc is an IgG1 Fc that has two amino acid substitutions, e.g., A330S and P331S, that have reduced complement binding.

[0199] In certain embodiments, an Fc is selected that has essentially no effector function, i.e., it has reduced binding to FcγR and reduced complement fixation. An example of an Fc is an IgG1 Fc that contains five mutations, e.g., L234A, L235E, G237A, A330S, and P331S, and has no effector function.

[0200] When an IgG4 constant domain is used, it may contain the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule.

[0201] In yet other embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., an aglycosylated antibody) can be prepared. Glycosylation can be altered, for example, to improve the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more variable region framework glycosylation sites can be removed, thereby making one or more amino acid substitutions that prevent glycosylation at that site. Such aglycosylation can improve the affinity of the antibody for the antigen. Such techniques are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0202] Glycosylation of the constant region at N297 can be prevented by mutating the N297 residue to another residue (e.g., N297A) and / or mutating the adjacent amino acid (e.g., 298), thereby reducing glycosylation at N297.

[0203] Additionally or alternatively, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such alterations in glycosylation patterns have been demonstrated to improve the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art, and such cells can be used as host cells to express the recombinant anti-IL-7R antibodies described herein, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes cell lines with a functional disruption of the FUT8 gene (encoding fucosyltransferase), which results in reduced fucosylation of antibodies expressed in such cell lines. In PCT Publication WO03 / 035835, Presta described a variant CHO cell line (Led3 cell) that had reduced ability to add fucose to Asn(297)-linked carbohydrates. (See also Shields, RR et al. (2002) J. Biol. Chem. 277:26733-26740). Umana et al., PCT Publication WO 99 / 54342, describes cell lines engineered to express glycoprotein-modifying glycosyltransferases, such as beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), which result in increased bisecting GlcNac structures in antibodies expressed in the engineered cell lines, and therefore increased ADCC activity of the antibodies (See also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0204] Another modification of the anti-IL-7R antibodies described herein is pegylation. Pegylation of an antibody can, for example, increase the biological half-life (e.g., serum half-life) of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically subjected to reaction with polyethylene glycol (PEG) (such as a reactive ester or reactive aldehyde derivative of PEG) under conditions in which one or more PEG groups are added to the antibody or antibody fragment. In some embodiments, pegylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). The term "polyethylene glycol" as used herein is intended to encompass any of the PEG forms that have been used to derivatize other proteins (such as mono(CI-CIO)alkoxy-polyethylene glycol or mono(CI-CIO)aryloxy-polyethylene glycol, or polyethylene glycol-maleimide). In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art, and such methods can be applied to the anti-IL-7R antibodies described herein. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0205] III. Physical Properties of Antibodies Anti-IL-7R antibodies (eg, those described herein) have some or all of the physical characteristics described herein (such as those described in the Examples).

[0206] The anti-IL-7R antibodies described herein may contain one or more glycosylation sites in either the light chain variable region or the heavy chain variable region. The presence of such glycosylation sites may increase the immunogenicity of the antibody due to altered antigen binding or may alter the pK of the antibody (Marshall et al., (1972) Anna Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168:1099-109, Spiro (2002) Glycobiology 12:43R-56R, Parekh et al., (1985) Nature 316:452-7, Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some embodiments, the variable regions of the anti-IL-7R antibodies are not glycosylated. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region.

[0207] In some embodiments, the anti-IL-7R antibodies described herein do not contain an asparagine isomerism site. Deamidation of asparagine can occur at NG or DG sequences, resulting in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain, reducing its stability (the isoaspartic acid effect).

[0208] Each antibody will have a unique isoelectric point (pi), which generally falls within a pH range of 6-9.5. The pi of an IgG1 antibody typically falls within a pH range of 7-9.5, and the pi of an IgG4 antibody typically falls within a pH range of 6-8. It is expected that antibodies with pi outside the normal range may experience some unfolding and instability under in vivo conditions. Thus, an anti-IL-7R antibody may have a pi value that falls within the normal range. This can be achieved by selecting an antibody with a pi within the normal range or by mutating charged surface residues.

[0209] Each antibody will have a characteristic melting temperature, with a higher melting temperature indicating a higher overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). M i (the temperature at which unfolding begins) can be greater than 60° C., greater than 65° C., or greater than 70° C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20:1952-60; Ghirlando et al., (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al., (2002) J. Chromatogr Sci 40:343-9).

[0210] In one embodiment, an antibody is selected that does not degrade rapidly. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0211] In some embodiments, antibodies are selected that have minimal aggregation effects, which may result in the induction of undesirable immune responses and / or altered or poor pharmacokinetic properties. Generally, acceptable antibody aggregation is 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation may be measured by several techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0212] IV. Antibody Engineering Methods As discussed above, anti-IL-7R antibodies having VH and VL sequences disclosed herein can be used to create new anti-IL-7R antibodies by modifying such VH and / or VL sequences or modifying the constant region(s) added thereto. Thus, in another aspect described herein, the structural features of the anti-IL-7R antibodies described herein are used to create structurally related anti-IL-7R antibodies that retain at least one functional property of the anti-IL-7R antibodies described herein, such as binding to human IL-7Rα and cynomolgus IL-7Rα. For example, one or more of the CDR regions of the anti-IL-7R antibodies disclosed herein can be recombinantly combined with known framework regions and / or other CDRs to create additional recombinantly engineered anti-IL-7R antibodies as described above. Other types of modifications include those described in the preceding sections. The starting material for the engineering method is one or more of the VH and / or VL sequences provided herein, or one or more of their CDR regions. The creation of an engineered antibody does not require the actual preparation (i.e., expression as a protein) of an antibody having one or more of the VH and / or VL sequences provided herein, or one or more of their CDR regions. Instead, the information contained in the sequence(s) is used as a starting material to create "second generation" sequence(s) derived from the original sequence(s), and the "second generation" sequence(s) are then prepared and expressed as a protein.

[0213] Accordingly, a method for preparing an anti-IL-7R antibody is provided herein, the method comprising the steps of: teeth, (a) providing (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence as set forth in SEQ ID NO: 13 or any one of the sequences as set forth in Table 13, a CDR2 sequence as set forth in SEQ ID NO: 14 or any one of the sequences as set forth in Table 14, and / or a CDR3 sequence as set forth in SEQ ID NO: 15 or any one of the sequences as set forth in Table 15, and (ii) a light chain variable region antibody sequence comprising a CDR1 sequence as set forth in SEQ ID NO: 16 or any one of the sequences as set forth in Table 16, a CDR2 sequence as set forth in SEQ ID NO: 17 or any one of the sequences as set forth in Table 17, and / or a CDR3 sequence as set forth in SEQ ID NO: 18 or any one of the sequences as set forth in Table 18; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein; Includes.

[0214] Standard molecular biology techniques can be used to prepare and express the altered antibody sequence. In some embodiments, the antibody encoded by the altered antibody sequence(s) retains one, some, or all of the functional properties of the anti-IL-7R antibodies described herein. The functional properties of the altered antibodies can be assessed using standard assays available in the art and / or described herein, such as those shown in the Examples (e.g., ELISA, FACS).

[0215] In some embodiments, mutations may be introduced randomly or selectively along all or part of the sequence encoding the anti-IL-7R antibody. The resulting modified anti-IL-7R antibodies may be screened for binding activity and / or other functional properties as described herein. Mutagenesis methods are described in the art. For example, PCT Publication WO02 / 092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation construction, or a combination thereof. Alternatively, PCT Publication WO03 / 074679 by Lazar et al. describes methods for optimizing physiochemical properties of antibodies using computational screening methods.

[0216] V. Nucleic Acids, Vectors, and Cells Also described herein are nucleic acid molecules encoding the anti-IL-7R antibodies described herein. The nucleic acid may be present in whole cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified to remove other cellular components or other contaminants (e.g., other cellular nucleic acids (e.g., chromosomal DNA (e.g., chromosomal DNA linked to the isolated DNA in nature))) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. F. Ausubel, et al., ed. (1987) Current Protocols in Molecular See, Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein can be, for example, DNA or RNA, and may or may not contain intron sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

[0217] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), cDNAs encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.

[0218] In some embodiments, the nucleic acids described herein encode the VH and VL sequences of an anti-IL-7R antibody of the present disclosure. In some embodiments, the nucleic acids described herein encode VH and VL sequences that are homologous to an anti-IL-7R antibody of the present disclosure. In some embodiments, the nucleic acids encode VH and VL sequences that are at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to a nucleic acid molecule encoding the VH and VL sequences of an anti-IL-7R antibody of the present disclosure.

[0219] In some embodiments, the nucleic acid molecules described herein may be modified to delete specific sequences (e.g., restriction enzyme recognition sequences). In some embodiments, the nucleic acid molecules contain conservative substitutions (i.e., substitutions that do not change the resulting amino acid sequence upon translation of the nucleic acid molecule) (e.g., for codon optimization).

[0220] Methods for preparing anti-IL-7R antibodies disclosed herein may include expressing the heavy and light chains in a cell line that contains nucleotide sequences encoding the heavy and light chains with signal peptides (e.g., SEQ ID NO:21 and SEQ ID NO:22, respectively). The present specification encompasses host cells that contain such nucleotide sequences. In some embodiments, the host cell is derived from the CHOZN cell line.

[0221] Once a DNA fragment encoding a VH segment and a DNA fragment encoding a VL segment are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In such manipulations, the VL-encoding or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0222] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Internal Medicine, 1999). (See, for example, the National Institute of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing such regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, an IgG4 constant region, an IgA constant region, an IgE constant region, an IgM constant region, or an IgD constant region (e.g., an IgG1 region). For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0223] The isolated DNA encoding the VL region is a clone encoding another D gene encoding the light chain constant region (CL). The VF-encoding DNA can be operably linked to the NA molecule to convert it into a full-length light chain gene (as well as a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 2002-002363). (See, for example, US Pat. No. 91-3242) and a DNA fragment containing such a region can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region.

[0224] The present disclosure further provides cells (e.g., host cells) that express (e.g., recombinantly) the anti-IL-7R antibodies described herein, as well as related polynucleotides and expression vectors. Also provided herein are vectors encoding polynucleotides that include a nucleotide sequence that encodes an anti-IL-7R antibody or a fragment thereof. In some embodiments, the vectors may be used to recombinantly express the anti-IL-7R antibodies described herein in a host cell (e.g., a mammalian cell (e.g., a CHOZN cell)). In some embodiments, the vectors may be used for gene therapy. Vectors suitable for the present disclosure include expression vectors, viral vectors, and plasmid vectors.

[0225] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence, or, in the case of an RNA viral vector, for replication and translation upon introduction into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0226] An expression vector of the present disclosure may include a polynucleotide encoding an antibody or antigen-binding portion thereof as described herein. In some embodiments, the coding sequence of the antibody or antigen-binding portion thereof is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in a manner that allows each constituent nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a manner that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked when induction by a promoter in the 5' gene expression sequence results in transcription of the coding sequence, and the nature of the binding between the two DNA sequences is such that (1) it does not introduce a frameshift mutation, (2) it does not interfere with the ability of the promoter region to result in transcription of the coding sequence, or (3) it does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence is capable of affecting the transcription of the coding nucleic acid sequence such that the resulting transcript is translated into the desired antibody or antigen-binding portion thereof.

[0227] Viral vectors include, but are not limited to, nucleic acid sequences derived from the following viruses: retroviruses (such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus); lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses (such as retroviruses). Other vectors well known in the art can also be readily used. Certain viral vectors are based on non-cytotoxic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytotoxic The pathogenic viruses include retroviruses, whose life cycle includes reverse transcription of genomic viral RNA into DNA followed by integration of the provirus into host cell DNA. Retroviruses are approved for human gene therapy trials. Replication-defective retroviruses (i.e., those capable of directing the synthesis of desired proteins but not of producing infectious particles) are the most useful. Such genetically modified retroviral expression vectors are widely useful for high-efficiency gene transfer in vivo. Standard protocols for generating replication-defective retroviruses, including the steps of incorporating the exogenous genetic material into a plasmid, transfecting the plasmid into a packaging cell line, producing recombinant retrovirus by the packaging cell line, harvesting the viral particles from tissue culture medium, and infecting target cells with the viral particles, are set forth in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990), and Murry, E.J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ (1991).

[0228] VI. Generation of Antibodies The anti-IL-7R antibodies described herein can be produced using various known techniques (Kohler and Monoclonal antibodies can be obtained using standard somatic cell hybridization techniques, such as those described by Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures are preferred, in principle other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes, and phage display techniques using libraries of human antibody genes.

[0229] The preferred animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0230] The chimeric or humanized anti-IL-7R antibodies described herein can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and manipulated using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, mouse CDR regions can be inserted into a human framework using methods known in the art (see, e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0231] In one embodiment, the anti-IL-7R antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed against IL-7Rα can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include those referred to herein as HuMAb mice and KM mice, respectively, and such mice are collectively referred to herein as "human Ig mice."

[0232] HUMAB-MOUSE® (Medarex, Inc.) is an unmodified human It contains a human immunoglobulin gene minilocus encoding heavy (μ and γ) and κ light chain immunoglobulin sequences, and carries targeted inactivating mutations of the endogenous μ and κ chain loci (see, e.g., Lonberg, et al., (1994) Nature 368(6474):856-859). Thus, these mice have reduced expression of murine IgM or κ and, in response to immunization, undergo class switching and somatic mutation of the introduced human heavy and light chain transgenes to produce high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994) supra (reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101); Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. al. (1994) Immunol. 152:2912-2920, Taylor, L. et al. (1994) International Immunology 6:579-591, and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851. U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429 (all to Lonberg and Kay), Surani et al. No. 5,545,807 to Lonberg et al., PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962 (all to Lonberg and Kay), and Korman et al. See also PCT Publication WO 01 / 14424 to al.

[0233] In certain embodiments, the anti-IL-7R antibodies described herein are generated using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM mice," and are described in detail in PCT Publication WO02 / 43478 to Ishida et al.

[0234] Additionally, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-IL-7R antibodies as described herein. For example, an alternative transgenic system called XENOMOUSE® (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0235] Additionally, the art provides alternative transchromosomic animals that express human immunoglobulin genes. Systems are also available, and such transchromosomic animal systems can be used to generate anti-IL-7R antibodies as described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used. Such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy chain transchromosomes and human light chain transchromosomes have also been reported in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894), and such cows can be used to generate anti-IL-7R antibodies as described herein.

[0236] Additional mouse systems reported in the art for the generation of human antibodies (e.g., human anti-IL-7R antibodies) include: (i) the VELOCLMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions are replaced with human heavy and light chain variable regions via homologous recombination and operably linked to endogenous mouse constant regions, resulting in the generation of chimeric antibodies (human V / mouse C) in the mouse that are subsequently converted to fully human antibodies using standard recombinant DNA techniques; and (ii) the MEMO® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions and also contains a single rearranged human common light chain variable region. Such mice and their use for antibody generation are described, for example, in WO2009 / 15777, US2010 / 0069614, WO2011 / 072204, WO2011 / 097603, WO2011 / 163311, WO2011 / 163314, WO2012 / 148873, US2012 / 0070861, and US2012 / 0073004.

[0237] Human monoclonal anti-IL-7R antibodies described herein can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well established in the art. See, for example, U.S. Pat. Nos. 5,223,409, 5,403,484, and 5,571,698 to Ladner et al., U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al., U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al., and U.S. Pat. Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0238] Human monoclonal anti-IL-7R antibodies described herein can also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0239] VI.A. Immunization To generate fully human antibodies against IL-7Rα, transgenic or transchromosomic mice containing human immunoglobulin genes (e.g., HCol2, HCo7, or KM mice) can be immunized with purified or enriched preparations of IL-7Rα antigen and / or cells expressing IL-7Rα or a fragment thereof, as described, for example, in Lonberg et al., (1994) Nature 36. 8(6474):856-859, Fishwild et al., (1996) Nature Biotechnology 14:845-851, and WO98 / 24884 for other antigens. Alternatively, mice can be immunized with DNA encoding human IL-7Rα or a fragment thereof. In some embodiments, mice can be 6-16 weeks old at the time of the first injection. For example, a purified or concentrated preparation of recombinant IL-7Rα antigen (5-50 μg) can be used for intraperitoneal immunization of HuMAb mice. If antibodies are not obtained using a purified or concentrated preparation of IL-7Rα antigen for immunization, mice can also be immunized with cells (e.g., cell lines) expressing IL-7Rα to promote the immune response. An example of a cell line is the IL-7Rα-overexpressing stable CHO cell line (CHOZN).

[0240] Accumulating experience with various antigens has demonstrated that initial intraperitoneal (IP) or subcutaneous (SC) immunizations with antigen in Ribi adjuvant followed by IP / SC immunizations (up to a total of 10) every 2 weeks with antigen in Ribi adjuvant yields the best response in HuMAb transgenic mice. Immune responses can be monitored over the course of the immunization protocol with plasma samples obtained by bleeding from the retro-orbital plexus. Plasma can be screened by ELISA and FACS (as described below) and mice with sufficient titers of anti-IL-7R human immunoglobulin can be used for fusions. Booster immunizations are performed by intravenously administering antigen to the mice 3 days before sacrifice and removal of the spleen and lymph nodes. It is expected that 2-3 fusions may need to be performed for each immunization. Typically, 6-24 mice are immunized for each antigen. HCo7, HCol2 and KM strains are usually used. Additionally, both the HCo7 and HCol2 transgenes can be introduced together into one mouse by cross breeding to carry two different human heavy chain transgenes (HCo7 / HCol2).

[0241] VI.B. Generation of Hybridomas Producing Monoclonal Antibodies to IL-7Rα To generate hybridomas producing human monoclonal anti-IL-7R antibodies as described herein, spleen cells and / or lymph node cells can be isolated from immunized mice and fused with a suitable immortalized cell line (such as a mouse myeloma cell line). The resulting hybridomas can be screened for production of antigen-specific antibodies. For example, splenic lymphocytes from immunized mice can be fused as single cell suspensions with Sp2 / 0 non-secreting mouse myeloma cells (ATCC, CRL1581) using PEG. After seeding the cells in flat-bottom microtiter plates, the cells can be incubated in selective medium. After several weeks, the cells can be cultured in medium. Individual wells can then be screened by ELISA for human monoclonal IgM and human monoclonal IgG antibodies. As the hybridomas grow, the medium can be monitored, usually after 10-14 days. Antibody-secreting hybridomas can be replaced and screened again. If the antibody-secreting hybridomas are still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution and the stable subclones can then be cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

[0242] To purify human monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated, then subjected to affinity chromatography using protein A-sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be examined by gel electrophoresis and high performance liquid chromatography to ensure purity. A buffer exchange into PBS can be performed. The concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored. It is possible.

[0243] Generation of transfectomas producing monoclonal antibodies against IL-7Rα Antibodies can be produced in host cell transfectomas, which can be obtained, for example, by a combination of recombinant DNA techniques and gene transfection methods, which are well known in the art (Morrison, S. (1985) Science 229:1202).

[0244] For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing the antibody of interest) and such DNA can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. The term "operably linked" in this context is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended antibody gene transcriptional and translational control functions. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector(s) by standard methods (e.g., ligation at complementary restriction sites present on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present). The Cs in the vector can be inserted into the expression vector(s) by standard methods (e.g., ligation at complementary restriction sites present on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present). H V on segment(s) H The segments are operably linked and expressed in a vector L V in segment LThe light and heavy chain variable regions of the anti-IL-7R antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting these variable regions into an expression vector that already encodes heavy and light chain constant regions of the desired isotype such that the segments are operably linked.

[0245] Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein). Examples of signal peptide sequences that may be used are known in the art. See, for example, International Publication No. WO2018 / 013818A2.

[0246] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the desired expression level of protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, and polyoma (e.g., adenovirus major late promoter (AdMLP)). Alternatively, Alternatively, non-viral regulatory sequences (such as the ubiquitin promoter or the β-globin promoter) may be used. In addition, regulatory elements composed of sequences of different origins also exist (such as the SRa promoter), which contains sequences derived from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

[0247] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0248] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The term "transfection" in its various forms is intended to encompass the various techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0249] Although it is theoretically possible to express the anti-IL-7R antibodies described herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, most preferably mammalian host cells, is most preferred since such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be inefficient for obtaining high yields of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today 6:12-13).

[0250] Certain mammalian host cells for expressing the recombinant anti-IL-7R antibodies described herein include Chinese Hamster Ovary (CHO) cells (including dhfr-CHO cells (used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 759:601-621) as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), NSO myeloma cells, COS cells, and SP2 cells. Specifically for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium that the host cell is grown in. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0251] VII. Immunoconjugates, Antibody Derivatives, and Diagnostics The anti-IL-7R antibodies described herein can be used in a variety of applications, including sample testing and in vivo imaging. It can be used for diagnostic purposes, where the antibody (or binding fragment thereof) can be conjugated with a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels including radioisotopes (for whole body imaging), as well as radioisotopes, enzymes, fluorescent labels, and other antibody tags suitable for sample examination.

[0252] Detectable labels that may be linked to any of the IL-7R antibodies described herein may be of any of a variety of types currently used in the area of ​​in vitro diagnostics, including particle labels (including metal sols (such as gold colloids)), isotopes (I 125 or Tc 99Enzyme labels include enzyme labels that convert a given substrate into a detectable marker (such as those provided with N2S2, N3S, or N4 type peptidic chelators), chromophores (including fluorescent, luminescent, phosphorescent, and the like), and polynucleotide tags that appear after amplification (such as by polymerase chain reaction). Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label can be an alkaline phosphatase enzyme, which reacts with a 1,2 dioxetane substrate (adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 The presence or formation of chemiluminescence following conversion of a luminescent substrate such as disodium 3,7}decane}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-STAR®, or other luminescent substrates well known to those skilled in the art (e.g., appropriate lanthanide (such as terbium(III) and europium(III) chelates). The detection means will be determined by the label selected. The appearance of the label or its reaction products can be determined by the naked eye (if the label is particulate and accumulates at appropriate levels) or by using an instrument (such as a spectrophotometer, luminometer, fluorometer, and the like), all performed according to standard practices.

[0253] In some embodiments, the conjugation method produces bonds that are substantially (or nearly) non-immunogenic, such as peptide bonds (i.e., amide bonds), sulfide bonds, (sterically hindered) disulfide bonds, hydrazone bonds, and ether bonds. Such bonds are nearly non-immunogenic and exhibit reasonable stability in serum (see, e.g., Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244, WO2009 / 059278, WO95 / 17886).

[0254] Depending on the biochemical properties of the moiety and the antibody, different conjugation strategies can be used. If the moiety is of natural origin or recombinant origin of 50-500 amino acids, standard procedures exist in textbooks describing the chemistry for the synthesis of protein conjugates and can be easily followed by the skilled artisan (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the reaction of a maleimide moiety with a cysteine ​​residue contained in the antibody or moiety is utilized. This is a coupling chemistry that is particularly suitable when, for example, Fab or Fab' fragments of an antibody are used. Alternatively, in one embodiment, coupling to the C-terminus of the antibody or moiety is performed. C-terminal modification of proteins (e.g., Fab fragments) can be performed as reported (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0255] In general, site-specific reactions and covalent couplings are based on converting natural amino acids into amino acids with reactivity distinct from that of other functional groups present. For example, specific cysteines in low frequency sequence contexts can be enzymatically converted to aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to make the desired amino acid modifications by utilizing certain enzymes with specific enzymatic reactivity for the natural amino acids in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; and Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403, which utilizes protease-catalyzed C-N bond formation). Site-specific reactions and covalent couplings can also be achieved by selective reaction of terminal amino acids with appropriate modification reagents.

[0256] The reactivity of the N-terminal cysteine ​​with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be exploited to achieve site-specific covalent coupling.

[0257] Native chemical ligation can also rely on a C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0258] US6437095B1 describes a conjugation method based on the faster reaction of cysteines in stretches of negatively charged amino acids with cysteines located in stretches of positively charged amino acids.

[0259] The moiety may also be a synthetic peptide or peptidomimetic. If the polypeptide is chemically synthesized, amino acids with independent chemical reactivity may be incorporated during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). A wide variety of independently reactive functional groups have been discussed and can be incorporated into synthetic peptides, making it standard chemistry to couple such peptides with linkers.

[0260] To obtain mono-labeled polypeptides, the 1:1 stoichiometric complexes can be separated from other complexation by-products by chromatography. This procedure can be facilitated by the use of dye-labeled binding pair members and charged linkers. The use of such labeled binding members and highly negatively charged binding members facilitates the separation of mono-complexed polypeptides from unlabeled polypeptides and polypeptides with multiple linkers, because the charge and molecular weight differences can be exploited for separation. Similar to labeled monovalent binding agents, fluorescent dyes can also be useful for purification of the complexes from non-binding components.

[0261] In one embodiment, the moiety attached to the anti-IL-7R antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0262] The anti-IL-7R antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or topoisomerase II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and mitotic inhibitors. In an ADC, the antibody and the therapeutic agent is preferably conjugated via a cleavable linker, such as a peptidyl linker, a disulfide linker, or a hydrazone linker. In other embodiments, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 30), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600, 6,989,452, and 7,129,261, PCT Publication Nos. WO02 / 096910, WO07 / 038658, WO07 / 051081, WO07 / 059404, WO08 / 083312, and WO08 / 103693, U.S. Patent Publication Nos. 20060024317, 20060004081, and 20060247295.

[0263] Anti-IL-7R antibodies (e.g., those described herein) can also be used to detect IL-7Rα (such as human IL-7Rα (e.g., human IL-7Rα in a tissue or tissue sample)). The antibodies can be used, for example, in an ELISA assay or flow cytometry. In certain embodiments, the anti-IL-7R antibody is contacted with cells (e.g., cells in a tissue) for a suitable time for specific binding to occur, and then a reagent (e.g., an antibody that detects the anti-IL-7R antibody) is added. Exemplary assays are provided in the Examples. The anti-IL-7R antibody can be a fully human antibody or a chimeric antibody (such as an antibody having a human variable region and a mouse constant region or portion thereof). A washing step can be included after incubation with the antibody and / or detection reagent. The anti-IL-7R antibody for use in such methods does not need to be labeled or linked to a detection agent, as the detection agent can be used separately.

[0264] Other uses of anti-IL-7R antibodies (eg, as monotherapy or in combination therapy) are provided elsewhere herein (eg, in the section regarding therapeutic uses).

[0265] VIII. Bispecific molecules The antibodies described herein can be used to form bispecific molecules. The anti-IL-7R antibodies disclosed herein can be derivatized or linked to another functional molecule (e.g., another peptide or protein (e.g., another antibody or ligand for the receptor)) to generate bispecific molecules that bind at least two different binding sites or target molecules. It has been reported that various cytokines play an important role in the development of many inflammatory diseases (such as inflammatory bowel disease). Examples of such cytokines include, but are not limited to, TNF-α, TL1α, IL-1β, IL-6, IL-18, IL-12, IL-23, IL-17, and IL-27. Sanchez-Munoz F., et al., World J Gastroenterol 14(27):4280-4288(2008). As mentioned above, regulatory T cells are also believed to be important in the treatment of inflammatory diseases. Several cytokines are known to be important in the induction of regulatory T cells, including TGF-β, IL-10, and IL-2. Hoeppli RE, et al., Front Immunol 6:61(2015). Thus, the anti-IL-7R antibodies disclosed herein can be linked to antibodies that specifically bind to any of the above cytokines, thereby controlling the development of inflammatory diseases and / or the induction of regulatory T cells. In some embodiments, the anti-IL-7R antibodies can be linked to antibodies that treat diseases or disorders (e.g., inflammatory diseases). Examples of such antibodies include, but are not limited to, natalizumab (TYSABRI®), infliximab, adalimumab, ustekinumab, golimumab, tocilizumab, vedolizumab, and secokinumab.

[0266] The antibodies described herein may in fact be derivatized or linked to multiple other functional molecules to generate multispecific molecules that bind to two or more different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecules" described herein. To create the bispecific molecules described herein, the antibodies described herein may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise linked) to one or more other binding molecules (such as another antibody, its antibody-binding portion, peptide, or binding mimetics), resulting in a bispecific molecule.

[0267] Thus, provided herein are bispecific molecules comprising at least one first binding specificity for IL-7Rα and a second binding specificity for a second target epitope, hi some embodiments, the bispecific molecule may further comprise a third binding specificity.

[0268] In some embodiments, the bispecific molecules described herein comprise at least one antibody as a binding specificity, including, for example, a Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv). The antibody can also be a light or heavy chain dimer or any smallest fragment thereof, such as an Fv, or a single chain construct as described in U.S. Patent No. 4,946,778 to Ladner et al., the contents of which are expressly incorporated by reference.

[0269] Human monoclonal antibodies are preferred, although other antibodies, including murine, chimeric, and humanized monoclonal antibodies, can be used in the bispecific molecules described herein.

[0270] The bispecific molecules described herein can be prepared by combining the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be produced separately and then combined with each other. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Exemplary cross-linkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (1985) Behring Ins. Mitt. No. 78, 118-132, Brennan et al. (1985) Science 229:81-83, and Glennie et al. (1987) J. Immunol. 139:2367-2375. Some complexing agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0271] When the binding specificities are antibodies, the antibodies can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge regions are modified to contain an odd number of sulfhydryl residues (preferably one) prior to conjugation.

[0272] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method allows the bispecific molecule to be expressed as mAb x mAb, mAb x mAb, or mAb x mAb. It is particularly useful when it is a Fab, mAb x (scFv)2, Fab x F(ab')2, or ligand x Fab fusion protein. Bispecific antibodies can include antibodies that contain scFv at the C-terminus of each heavy chain. The bispecific molecules described herein can be single chain molecules containing one single chain antibody and one binding determinant, or single chain bispecific molecules containing two binding determinants. Bispecific molecules can include at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. No. 5,260,203, U.S. Pat. No. 5,455,030, U.S. Pat. No. 4,881,175, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513, U.S. Pat. No. 5,476,786, U.S. Pat. No. 5,013,653, U.S. Pat. No. 5,258,498, and U.S. Pat. No. 5,482,858.

[0273] Binding of a bispecific molecule to its specific target may be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest using a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0274] IX. Kit Provided herein are kits comprising one or more of the anti-IL-7R antibodies or antigen-binding portions thereof, bispecific molecules, or immunoconjugates thereof described herein. In some embodiments, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein (such as the antibodies or antigen-binding portions thereof provided herein), and optionally instructions for use. In some embodiments, the kits comprise the pharmaceutical compositions described herein and any prophylactic or therapeutic agents (such as those described herein).

[0275] X. Pharmaceutical Compositions Physiologically acceptable carriers, excipients, or stabilizers (see Remington's Provided herein are compositions comprising an antibody or antigen-binding portion thereof described herein in a desired degree of purity in Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers (such as phosphates, citrates, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polysaccharides, and the like. These include peptides, proteins (such as serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0276] In certain embodiments, the pharmaceutical composition comprises an antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, and optionally one or more additional prophylactic or therapeutic agents. and in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or antigen-binding portion thereof described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for modulating (e.g., reducing or inhibiting) IL-7 activity in T cells (e.g., pathogenic T cells) and treating a disease or disorder, such as an inflammatory disease (e.g., inflammatory bowel disease).

[0277] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, injectable, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody, immunoconjugate, or bispecific molecule) may be coated and placed in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0278] Accordingly, one object of the present disclosure is to provide a pharmaceutical formulation that enhances the stability of an anti-IL-7R antibody, thereby allowing for its long-term storage. In some embodiments, the pharmaceutical formulation disclosed herein comprises (a) an anti-IL-7R antibody, (b) a buffering agent, (c) a stabilizing agent, (d) a salt, (e) a bulking agent, and / or (f) a surfactant. In some embodiments, the pharmaceutical formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or more. In some embodiments, the formulation is stable when stored at 4° C., 25° C., or 40° C.

[0279] Buffer Buffers useful in the present invention can be weak acids or weak bases used to maintain the acidity (pH) of a solution near a selected value even after another acid or base is added. A suitable buffer can maximize the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also maintain physiological compatibility or optimize solubility. Rheology, viscosity, and other properties can also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer includes histidine (e.g., L-histidine) along with a tonicity agent, and pH adjustments can be made with acids or bases known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained at about 2 to about 10 or about 4 to about 8.

[0280] Stabilizers Stabilizing agents are added to pharmaceutical products to stabilize them. Such agents can stabilize proteins in many different ways. Common stabilizers include, but are not limited to, amino acids (such as glycine, alanine, lysine, arginine, or threonine), carbohydrates (such as glucose, sucrose, trehalose, raffmose, or maltose), polyols (such as glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight), or PEG. In one aspect of the invention, the stabilizer is selected to maximize the stability of the FIX polypeptide in the lyophilized preparation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0281] Bulking Agent To increase the volume and mass of a pharmaceutical product, thereby facilitating accurate weighing and its handling. Bulking agents may be added to the product to provide bulking. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0282] Surfactants Surfactants are amphiphilic substances with lyophilic and lyophobic groups. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols (such as cetyl alcohol or oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0283] In some embodiments, the pharmaceutical formulation of the present disclosure comprises: (a) about 0.25 mg / mL to 250 mg / mL (e.g., 10 to 200 mg / mL) of an anti-IL-7R antibody; (b) about 20 mM histidine; (c) about 260 mM sucrose; (d) about 0.5 mM DTPA, and (e) about 0.05% Tween-80.

[0284] The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and / or a surfactant, and various combinations thereof. The use of preservatives, tonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those of skill in the art. References include, for example, Remington: The Science and Practice of Pharmacy, 1999; th edition, 1995.

[0285] In some embodiments, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation that contains at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution that contains at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension that contains at least 50% w / w water.

[0286] In some embodiments, the pharmaceutical formulation is a freeze-dried formulation, to which solvents and / or diluents are added by the physician or the patient prior to use.

[0287] The pharmaceutical compositions described herein may also be administered in combination therapy, i.e., in conjunction with other agents. For example, the combination therapy may include the use of an IL-7R antibody described herein in combination with at least one other therapeutic agent. Examples of therapeutic agents that may be used in combination therapy may include other compounds, drugs, and / or agents used to treat diseases or disorders (e.g., inflammatory disorders). Such compounds, drugs, and / or agents may include, for example, anti-inflammatory agents or antibodies that block or reduce the production of inflammatory cytokines. In some embodiments, the therapeutic agent includes an anti-IP-10 antibody, an anti-TNF-α antibody (e.g., adalimumab (HUMIRA®), golimumab (SIMPONI®), infliximab (REMICADE®), certolizumab pegol (CIMZIA®), interferon beta-1a (e.g., AV Therapeutic agents may include, but are not limited to, TGF-β, IL-10, IL-2, and combinations thereof. In some embodiments, therapeutic agents may include compounds, drugs, and / or agents that can induce the development of regulatory T cells (e.g., inducible regulatory T cells). Examples of such therapeutic agents include, but are not limited to, TGF-β, IL-10, IL-2, and combinations thereof.

[0288] The pharmaceutical compounds described herein may include one or more pharma- ceutically acceptable salts. A "pharma-ceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J.Pharm.Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as those derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, and those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0289] The pharmaceutical compositions described herein may also include pharma- ceutically acceptable antioxidants. Examples of pharma-ceutically acceptable antioxidants include (1) water-soluble antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like), (2) oil-soluble antioxidants (such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like), and (3) metal chelators (such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like).

[0290] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate).Proper fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the required particle size in the case of dispersions, or by using surfactants.

[0291] These compositions may also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Sterilization procedures (described above) may be performed and the presence of microorganisms may be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0292] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use of such conventional media or agents in the pharmaceutical compositions described herein is contemplated. The pharmaceutical compositions may or may not contain a preservative. The composition may additionally contain an active compound.

[0293] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier may be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, the compositions may include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Absorption of the composition may be prolonged by including in the injectable composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0294] Sterile injection solution can be prepared by incorporating active compound in the required amount in a suitable solvent with one or a combination of above-mentioned components as necessary, and then carrying out sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound in a sterile medium that contains a basic dispersion medium and other necessary components selected from above.In the case of sterile powder for preparing sterile injection solution, some preparation methods include vacuum drying and freeze-drying (lyophilization), which obtains active ingredient plus any desired additional components as powder from the solution that has been previously sterilized and filtered.

[0295] The amount of active ingredient that can be combined with carrier materials to obtain a single dosage form will vary depending on the subject being treated and the specific mode of administration. The amount of active ingredient that can be combined with carrier materials to obtain a single dosage form is generally that amount of composition that provides a therapeutic effect. Generally, this amount is in the range of about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent, based on the percentage of active ingredient combined with pharma-ceutically acceptable carrier being 100 percent.

[0296] The dosage regimen is adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally increased or decreased according to the exigencies of the therapeutic situation. To facilitate administration and obtain uniformity of dose, it is particularly advantageous to formulate the injectable composition in unit dosage form. Unit dosage form as used herein refers to a physically discrete unit suitable as a unitary dose to the subject to be treated, each unit containing a predetermined amount of active compound calculated to obtain the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of unit dosage forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active compounds to accommodate individual sensitivities.

[0297] For administration of anti-IL-7R antibodies (such as those described herein), the dosage range is about 0.0001 to 100 mg / kg of host body weight, more usually 0.01 to 5 or 10 mg / kg of host body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg of body weight, or within the range of 1 to 10 mg / kg. An example of a treatment regimen is once weekly administration, once every two weeks. These involve administration once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0298] In some embodiments, the anti-IL-7R antibody is administered in a flat dose (flat dose regimen). In other embodiments, the anti-IL-7R antibody is administered in a fixed dose with another antibody. In certain embodiments, the anti-IL-7R antibody is administered in a weight-based dose.

[0299] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, where the dose of each administered antibody falls within a specified range. The antibodies are usually administered multiple times. The interval between single doses can be, for example, one week, one month, three months, or one year. The interval can also be irregular, as indicated by measuring the blood level of the antibody against the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods, to achieve a plasma antibody concentration of about 25-300 μg / ml.

[0300] The antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will depend on the half-life of the antibody in the patient. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered relatively frequently over a long period of time. Some patients continue to be treated for the rest of their lives. In therapeutic applications, relatively high doses may be required at relatively short intervals until the progression of the disease is reduced or stopped and the patient is partially or completely relieved of the symptoms of the disease. A preventive regimen may then be administered to the patient.

[0301] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response without toxicity to the patient for a particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition described herein or its esters, salts, or amides used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0302] The compositions described herein may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by one of skill in the art, the route and / or mode of administration will vary depending on the desired outcome. The route of administration of the anti-IL-7R antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other injection routes of administration (e.g., by injection or infusion). The phrase "injection administration" as used herein refers to modes of administration other than enteral and topical administration (usually by injection), including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion.

[0303] Alternatively, the antibodies described herein may potentially be administered via non-injection routes of administration, such as topical, epidermal, or mucosal routes of administration (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical).

[0304] The active compound may be prepared with a carrier that protects the compound against rapid release (e.g., Controlled release formulations (including implants, transdermal patches, and microencapsulated delivery systems). Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many of the methods for preparing such formulations are patented or generally known to those skilled in the art. For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0305] The therapeutic composition can be administered using medical equipment known in the art.For example, in certain embodiments, the therapeutic composition described herein can be administered using needleless hypodermic injection equipment, such as the equipment disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implantable medical devices and modules for use with the anti-IL-7R antibodies described herein include U.S. Pat. No. 4,487,603 (disclosing an implantable microinfusion pump for controlled rate dosing), U.S. Pat. No. 4,486,194 (disclosing a therapeutic device for administering drugs through the skin), U.S. Pat. No. 4,447,233 (disclosing a drug infusion pump for delivering drugs at precise infusion rates), U.S. Pat. No. 4,447,224 (disclosing an implantable variable flow infusion device for continuous drug delivery), U.S. Pat. No. 4,439,196 (disclosing an osmotic drug delivery system with multiple chamber compartments), and U.S. Pat. No. 4,475,196 (disclosing an osmotic drug delivery system). These patents are incorporated herein by reference. Many other such implantable medical devices, delivery systems, and modules are known to those skilled in the art.

[0306] XI. Uses and Methods The anti-IL-7R antibodies of the present disclosure, as well as compositions comprising such antibodies (e.g., pharmaceutical compositions, formulations, polynucleotides, vectors, and cells), can be used to treat inflammatory diseases (e.g., by modulating the ratio of regulatory T cells to effector T cells in a subject).

[0307] Thus, in one aspect, the disclosure provides a method for treating an inflammatory disease in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective dose of an anti-IL-7R antibody. Examples of inflammatory diseases that may be treated using the anti-IL-7R antibodies of the present disclosure include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft versus host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, allergies, asthma, and other autoimmune diseases that are the result of acute or chronic inflammation.

[0308] In some embodiments, the anti-IL-7R antibodies are suitable for use in treating individuals with inflammatory bowel disease. Inflammatory bowel disease (IBD) is a disease that can affect any part of the gastrointestinal tract from the mouth to the anus, resulting in a wide variety of symptoms. IBD primarily causes abdominal pain, diarrhea (which may be bleeding), vomiting, or weight loss, but can also cause extra-gastrointestinal complications, such as rashes, arthritis, eye inflammation, fatigue, and lack of concentration. IBD patients can be divided into two main classes: those with ulcerative colitis (UC) and those with Crohn's disease (CD). CD generally affects the ileum and colon, but can affect any area of ​​the intestine and is often discontinuous (areas of disease concentration scattered throughout the intestine). UC always affects the rectum (colon) and is more continuous. In CD, inflammation is transmural, resulting in abscesses, fistulas, and strictures, while in UC, inflammation is typically localized to the mucosa. While there is no known medical or surgical cure for Crohn's disease, UC patients may be cured by surgical removal of the colon in some patients. Treatment options are limited to controlling symptoms, maintaining remission, and preventing recurrence. Clinical efficacy in inflammatory bowel disease can be measured for CD as a reduction in the Crohn's Disease Activity Index (CDAI) score, which is a scoring scale based on clinical examinations and quality of life questionnaires. In animal models, efficacy is most often measured by weight gain, but may also be assessed by the Disease Activity Index (DAI), which combines fecal consistency, body weight, and fecal occult blood.

[0309] In some embodiments, the anti-IL-7R antibodies of the present disclosure are suitable for use in treating individuals with rheumatoid arthritis. Rheumatoid arthritis (RA) is a systemic disease that affects most, if not all, of the body and is one of the most common forms of arthritis. RA is characterized by inflammation of the joints, which causes pain, stiffness, warmth, redness, and swelling. This inflammation is the result of inflammatory cells infiltrating the joints, which release enzymes that can digest bone and cartilage. As a result, this inflammation can cause severe damage to bone and cartilage, leading to joint deterioration and severe pain, among other physiological effects. Affected joints can lose their shape and alignment, resulting in pain and reduced joint mobility. Several animal models of rheumatoid arthritis are known in the art. For example, in the collagen-induced arthritis (CIA) model, mice develop inflammatory arthritis that resembles human rheumatoid arthritis. CIA shares similar immunological and pathological characteristics with RA, making it a suitable model for screening potential human anti-inflammatory compounds.Efficacy in this model is measured by reduction in joint swelling.Efficacy in clinical RA is measured by the ability to reduce symptoms in patients, which is measured as a combination of joint swelling, erythrocyte sedimentation rate, C-reactive protein level, and serum factor (such as anti-citrullinated peptide antibody) level.

[0310] In some embodiments, the anti-IL-7R antibodies disclosed herein are suitable for use in treating individuals with psoriasis. Psoriasis is a T cell-mediated inflammatory skin disorder that can cause considerable discomfort. Psoriasis is a disease for which there is currently no cure and occurs in all age groups. Individuals with mild psoriasis can often control their disease with topical preparations, but there are over one million patients worldwide who require ultraviolet light treatment or systemic immunosuppressive therapy. Unfortunately, the inconvenience and risks of ultraviolet light exposure and the toxicity of many treatments limit the long-term use of such treatments. Furthermore, patients usually experience relapses of psoriasis, which in some cases rebound soon after cessation of immunosuppressive therapy. A recently developed psoriasis model is characterized by the expression of CD4 + It is based on the transfer of T cells and mimics many aspects of human psoriasis, and can therefore be used to identify compounds suitable for use in the treatment of psoriasis (Davenport et al.,Internat.Immunopharmacol 2:653-672,2002).Efficacy in this model is measured by the reduction of skin pathology using a scoring system.Similarly, efficacy in patients is measured by the reduction of skin pathology.

[0311] In some embodiments, the anti-IL-7R antibodies are suitable for use in treating individuals with psoriatic arthritis. Psoriatic arthritis (PA) is a type of inflammatory arthritis that occurs in a subset of psoriasis patients. Arthritis. In these patients, skin symptoms / pathologies are accompanied by joint swelling similar to that seen in rheumatoid arthritis. PA is characterized by patchy, thickened, red areas of scaly skin inflammation. Psoriasis often affects the elbows and knees, scalp, navel, and around the genital or anal areas. Approximately 10% of psoriasis patients also develop associated inflammation in the joints.

[0312] In the context of this disclosure, preventive, palliative, symptomatic, and / or curative treatments may be separate aspects of the disclosure. The antibodies of the present invention may be administered by injection (intravenous, intramuscular, subcutaneous, etc.). Alternatively, the antibodies of the present invention may be administered via a non-injection route (oral or topical, etc.). The antibodies of the present invention may be administered prophylactically. The antibodies of the present invention may be administered therapeutically (if necessary).

[0313] All references cited above, as well as those cited herein, are incorporated by reference in their entirety.

[0314] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0315] Example 1: Characterization of human anti-IL-7R antibodies An anti-IL-7R antibody disclosed herein (comprising a VH set forth in SEQ ID NO: 19 and a VL set forth in SEQ ID NO: 20 (see Table 12)) ("Antibody A") was assayed for binding to IL-7Rα-expressing cells. IL-7Rα is expressed primarily on human T cells and, to a lesser extent, on monocytes.

[0316] As shown in Table 1 (below), Antibody A inhibits various T cell subsets from whole blood (CD4 + CD45RA + , CD4 + CD45RA - , CD8 + CD45RA + , and CD8 + CD45RA - ) were able to bind to IL-7Rα expressed on EC 50 (The values ​​ranged from about 1.4 to 3.3 nM.) Furthermore, Antibody A did not agonize IL-7Rα signaling (see Table 2) and did not inhibit CD4 + CD45RA +Antibody A was able to effectively block IL-7 binding to IL-7Rα expressed on T cells (as measured by pSTAT5 activation). Compared to a reference anti-IL-7R antibody ("PFE A3312F"), Antibody A blocked IL-7 binding to IL-7Rα much more potently. + CD45RA + When T cells were stimulated with IL-7 (e.g., 2.0 ng / mL) in the presence of Antibody A, pSTAT5 activation was minimal (IC50=2.1±1.4 nM). In contrast, PFE A3312F induced significantly greater pSTAT5 activation (IC50=15.8±4.0). [Table 1]

[0317] Antibody A also differed from the reference antibodies in its ability to block TSLP-mediated monocyte activation. As shown in Table 1 (last row), monocytes stimulated with TSLP in the presence of PFE A3312F failed to produce monocyte chemoattractant protein 1 (MCP-1) (IC50=0.08±0.3 nM), suggesting that PFE A3312F bound to IL-7Rα expressed on monocytes and was able to efficiently block TSLP binding to IL-7Rα. In contrast, monocytes stimulated with TSLP, even with Antibody A, produced significantly more MCP1 (IC50=24±4.5).

[0318] Next, to assess whether the inability to block TSLP-stimulated monocyte activation was due to binding issues, whole blood from human donors was incubated with different concentrations of Antibody A. Then, non-T cell (CD3 - The mean fluorescence intensity (MFI) of antibody A binding to IL-7Rα expressed on T cells was measured using flow cytometry. As shown in Figure 1, antibody A binding to non-T cells was minimal, even at concentrations of 70 nM.

[0319] Taken together, the above data demonstrate that, compared to other known antibodies (PFE A3312F), the anti-IL-7R antibody disclosed herein (Antibody A) potently blocks IL-7 binding and is selective for IL-7Rα expressed on T cells, but not on non-T cells (e.g., monocytes and other CD3s in whole blood). - These results indicate that the antibody is not selective for IL-7Rα expressed on IL-7Rα-dependent ...

[0320] Example 2: Analysis of agonist activity of anti-IL-7R antibodies Next, to evaluate whether binding of anti-IL-7R antibodies could induce IL-7Rα signaling, peripheral blood mononuclear cells (PBMCs) and whole blood were incubated with different concentrations of antibody A (100 nM, 50 nM, 25 nM, 13 nM, 6 nM, 3 nM, 2 nM, 1 nM, and 0 nM). An isotype control antibody and IL-7 (2 nM) were used as negative and positive controls, respectively.

[0321] As shown in Table 2, compared to the negative control, Antibody A did not induce pSTAT5 activation at all concentrations tested, demonstrating that Antibody A does not agonize signal transduction upon binding to IL-7Rα. [Table 2]

[0322] Example 3: Cross-reactivity of anti-IL-7R antibodies in cynomolgus monkeys To assess the cross-reactivity of Antibody A, its binding affinity to human IL-7Rα and cynomolgus IL-7Rα was assessed using surface plasmon resonance (SPR). Briefly, BIACOR® antibodies were assayed in a running buffer consisting of 10 mM NaPO4, 130 mM NaCl, 0.05% p20 (PBS-T) (pH 7.4 or pH 6.0). Experiments were performed on an E™ T200 instrument (GE Healthcare, Chicago, IL, USA). The chip surface was prepared by immobilizing Protein A to a density of 4000 RU on a CM5 sensor chip on flow cell 1, flow cell 2, flow cell 3, and flow cell 4 using ethyl(dimethylaminopropyl)carbodiimide / NHS. Antibody A was captured at 10 μl / min with a contact time of 30 s. Purified extracellular domain constructs of his-tagged human IL7R or cynomolgus IL7R (prepared in-house) were used for binding studies in two-fold serial dilutions from 500 nM to 7.8 nM with an association time of 180 s and a dissociation time of 360 s at 30 μl / min. Regeneration between cycles was performed by injection of 10 mM glycine-HCL (pH 1.5) (2x 30 s). Data were analyzed using Biacore T200 Evaluation software (GE Healthcare) and fitted to a 1:1 Langmuir model.

[0323] As shown in Table 3, Antibody A was able to bind to both human IL-7Rα and cynomolgus IL-7Rα with similar affinity. For example, at pH 7.4, Antibody A bound to human IL-7Rα and cynomolgus IL-7Rα with KD values ​​of 1.3 nM and 1.7 nM, respectively. At pH 6.0, the binding was about 4-fold weaker, suggesting that the binding of anti-IL-7R to both human IL-7Rα and cynomolgus IL-7Rα was pH-dependent. In contrast, the binding of PFE A3312F showed no pH dependence, with KD values ​​of 4.4 nM at pH 7.4 and 3.9 nM at pH 6.0. SPR analysis also demonstrated that binding to all human and cynomolgus monkey FcγRs was very weak at neutral pH and that this binding became stronger at pH 6.0, as expected for the hIgG1.3f isotype. [Table 3]

[0324] To confirm the cross-reactivity data above, cell binding and functional blocking were also evaluated. Briefly, antibody A was administered to cynomolgus monkeys at a single dose (0.1 mg / kg, 0.5 mg / kg, or 3.0 mg / kg). PBMCs were then isolated from the animals on day 8 post-administration. To measure the ability to block IL-7 binding to cynomolgus IL-7Rα, PBMCs were stimulated with recombinant cynomolgus IL-7 (5 pM) for 15 min and CD4 + pSTAT5 activity and receptor occupancy in T cells was assessed using flow cytometry.

[0325] As shown in FIG. 2A, at serum anti-IL-7R antibody concentrations of approximately 310-350 ng / mL (approximately 2.1-2.3 nM), cynomolgus monkey CD4 + More than 95% of IL-7Rα expressed on T cells was occupied. Similarly, as shown in Figure 2B, there was a direct inverse correlation between serum anti-IL-7R antibody concentrations and pSTAT5 activity. At serum concentrations of 670-2200 ng / mL (4.5-15 nM), STAT5 inhibition was greater than 90%. Based on these results, it is estimated that a trough concentration of approximately 2-15 nM would be required to completely block IL-7Rα in vivo.

[0326] Taken together, the above data demonstrate that the anti-IL-7R antibody disclosed herein (Antibody A) is fully cross-reactive in cynomolgus monkeys and can efficiently bind to cynomolgus IL-7Rα and block IL-7-mediated signaling of cynomolgus IL-7Rα.

[0327] Example 4: Additional Characterization of Anti-IL-7R Antibodies Table 4 shows the analytical and biophysical characteristics of Antibody A.

[0328] The analytical and biophysical properties of Antibody A were favorable for continued development. The identity of the antibody was confirmed by mass spectrometry (intact mass spectrometry and peptide mapping). Greater than 99% of the antibody was monomeric when tested by analytical size exclusion chromatography. One N-glycosylation site was identified at N297 on the heavy chain, and the glycan profile matched that of monoclonal antibodies expressed in CHO (G0F, G1F, and G2F). The charge variant profile determined by imaging capillary isoelectric focusing revealed a major peak (60%) at pI 8.5, with 34% acidic variants and 6% basic variants. The thermal stability (Tm1=70.0°C, Tm2=75.4°C, Tm3=84.2°C) was within the range of a typical human IgG1.3f monoclonal antibody. [Table 4]

[0329] Table 5 shows the stability characteristics of Antibody A. [Table 5]

[0330] Preliminary formulation evaluations (including searching for optimal pH, buffer composition, and excipients, as well as enhanced stability studies) of the anti-IL-7R antibodies disclosed herein were performed using HEK-derived material. A simple platform formulation (20 mM histidine, 260 mM sucrose, 0.05 mM sucrose) was performed at 150 mg / mL with or without the presence of surfactant. No physical stability issues were observed during freeze-thaw stress (5 cycles) at 10 mM DTPA, pH 6.0, or due to agitation stress. No additional surfactants were evaluated during these studies. Forced degradation studies were performed at 150 mg / mL at 4°C, 25°C, and 40°C. Chemical changes were observed in the CDR regions under the most stressful conditions (3 months at 40°C), which correlated with changes in both KD and Rmax in the SPR activity assay. Mild chemical changes were observed at the same CDR sites under storage conditions at 25°C. No changes in activity were observed in samples stored at 4°C or 25°C. The expected deamidation changes in VSNK were also observed.

[0331] Feasibility studies performed with HEK-derived material also demonstrated that the changes in both HMW and LMW variants were time- and temperature-dependent (increasing by approximately 1% / month and 1.3% / month, respectively, at 40°C). Under storage conditions at 4°C, HMW remained unchanged, whereas it increased by 0.3% / month at 25°C. The formation of LMW observed under storage conditions at 40°C was characterized by an exact match in mass with a cumulative species that had lost one Fab in addition to a Fab arm, and their generation is presumed to be the result of chemical cleavage at a conserved sequence in the upper hinge region. The HMW and LMW changes obtained from these HEK-derived studies are within the range of those expected for an IgG1.3 mAb, supporting molecular progress.

[0332] An early obstacle in lead optimization was the viscosity limitation of Antibody A. Viscosity-concentration profiles measured during formulation evaluation showed a viscosity of 8 cP at 100 mg / mL and approximately 19 cP at 140 mg / mL. Based on this viscosity profile of Antibody A, it was estimated that a concentration of approximately 125 mg / mL would be the upper limit of what could be syringed using conventional equipment.

[0333] Example 5: Pharmacokinetics (PK) of anti-IL-7R antibodies in NOD Scid gamma (NSG)-human PBMC engrafted mice Antibody A does not cross-react with mouse IL-7Rα, so it was possible to assess the impact of target-mediated clearance on PK in NSG-human PBMC-engrafted mice. Furthermore, it has been shown that antibody A (e.g., compared to PFE A3312F) has a pH-dependent binding affinity for IL-7Rα (see Example 3). For mAbs with short half-lives due to target-mediated clearance, it has been demonstrated that making antigen binding pH-dependent improves the PK profile. See Igawa T., et at, Nat Biotechnol 28(11):1203-7(2010). Therefore, this model was also used to assess whether pH-dependent binding to the target would lead to improved pharmacokinetics.

[0334] Briefly, NSG-human PBMC engrafted mice were administered a single dose of antibody A or the reference PFE A3312F antibody. Mice were administered one of two doses (0.5 mg / kg or 5 mg / kg) intravenously. Mice were then bled at various time points after dosing to determine serum antibody concentrations. As shown in Figure 3, the PK of PFE A3312F was non-linear, presumably due to target-mediated drug elimination (TMDD), as there was a 5.7-fold decrease in blood clearance (CL) between the 0.5 mg / kg and 5 mg / kg doses (see Table 6). The PK of antibody A at 5 mg / kg was similar to PFE A3312F, but a significant improvement in exposure was observed at the lower dose (0.5 mg / kg). Without being bound by any one theory, target-mediated drug clearance is generally more pronounced in the low dose range, and therefore, it was hypothesized that the improved PK observed at low doses was due to the pH-dependent binding affinity of the anti-IL-7R antibody to IL-7Rα. [Table 6]

[0335] Example 6: Pharmacokinetics (PK) of anti-IL-7R antibodies in cynomolgus monkeys To further evaluate the PK of Antibody A, a single dose of either Antibody A or the reference PFE A3312F antibody (either 0.1 mg / kg, 0.5 mg / kg, or 3 mg / kg) was administered intravenously to cynomolgus monkeys. Animals were then bled at various time points post-dosing to compare the in vivo potency of the antibodies (PK, RO, and ex vivo pSTAT5 activation).

[0336] As shown in FIG. 4, in animals administered ≧0.5 mg / kg of either antibody, anti-drug antibodies (ADA) (dotted line) were detected from day 10 onwards, resulting in a decline in serum antibody concentrations (solid line) in the animals. As shown in FIG. 5, the pharmacokinetics of antibody A and PFE A3312F were nonlinear at the doses tested, suggesting TMDD. However, as shown in Table 7, the mean exposure (AUC) of antibody A was higher (1.6-2.1-fold) compared to PFE A3312F. Similarly, the clearance values ​​of antibody A were also high: at the 0.1 mg / kg, 0.5 mg / kg, and 3 mg / kg doses, the clearance values ​​were 1.15±0.20 mL / hr / kg, 0.64±0.10 mL / hr / kg, and 0.44±0.02 mL / hr / kg, respectively. On the other hand, the clearance values ​​of A3312F were 1.76±0.22 mL / hr / kg, 1.16±0.39 mL / hr / kg, and 0.94±0.12 mL / hr / kg, respectively (Table 7). [Table 7]

[0337] There was a good correlation between pSTAT5 inhibition and serum concentrations of Antibody A in cynomolgus monkeys. See Figure 6. Increasing exposure to Antibody A (corresponding to increased serum antibody concentrations) IL-7-mediated pSTAT5 activation was associated with a decrease in IL-7-mediated pSTAT5 activation in monkeys treated with an anti-IL-7R antibody of the present disclosure as well as in monkeys treated with PFE A3312 (IC50 approx. 2 nM).

[0338] During the 3-week evaluation period, no changes were made to body weight, hematology (including WBC differential determination), serum clinical chemistry, or T / B / NK cell phenotyping (CD4 as shown in Figures 7A-7C). + and CD8 + There were no clinical findings or adverse effects associated with anti-IL-7R antibodies for either IL-7R or IL-7R-associated serotoninib.

[0339] The above results suggest that, compared to the reference antibody, the anti-IL-7R antibody disclosed herein (Antibody A) has similar in vivo efficacy but improved pharmacokinetics in cynomolgus monkeys and minimized toxicity issues.

[0340] Example 7: Analysis of immunogenicity of anti-IL-7R antibodies Potential immunogenic risk of antibody A was assessed by in silico methods as well as in vitro DC:T cell proliferation assays. In silico iDAB analysis showed some binding potential to VL CDR3 and a framework mutation from serine to phenylalanine at position 71 in VH (F71S). In vitro DC:T cell proliferation assays involved incubation of 40 PBMC donors with dendritic cells pulsed with antibody A. The assays showed that non-RACIR batches of antibody A generated significant immunogenic responses (approximately 30-50% of donors) (see P1-066930-3 and P1-066930-9 in Figure 8). Interestingly, these results correlated with the ADA severity observed in the monkey studies previously described (see, e.g., Example 6). RACIR batches of antibody A were minimally immunogenic in donors (approximately 12.5%) and comparable to the control protein (Avastin). Given the low response to the RACIR material in this assay, it is expected that Antibody A will be safe for use in clinical settings (e.g., to treat the inflammatory diseases disclosed herein).

[0341] Example 8: Analysis of the ability of anti-IL-7R antibodies to block IL-7 signaling in human T cells To further evaluate the ability of Antibody A to inhibit IL-7 binding to IL-7Rα, peripheral blood was collected from healthy volunteers (NHV), ulcerative colitis (UC) patients, and Crohn's disease (CD) patients. PBCM were then incubated ex vivo and stimulated with IL-7 for approximately 15 minutes in the presence of Antibody A. pSTAT5 activity in T cells was then analyzed using flow cytometry.

[0342] As shown in Table 8, pSTAT5 activity was minimal in T cells from all tested individuals, suggesting that Antibody A can effectively block IL-7 binding to human IL-7Rα from both healthy volunteers and disease patients. No statistical difference was observed between the responses of NHV donors and UC or CD donors. This result indicates that the anti-IL-7R antibody disclosed herein (Antibody A) may be effective in treating inflammatory diseases in vivo by effectively blocking IL-7 signaling in pathogenic T cells. [Table 8]

[0343] Example 9: Epitope mapping analysis Hydrogen / deuterium exchange mass spectrometry (HDX-MS) was used in combination with independent covalent labeling footprinting techniques, including fast photochemical oxidation of proteins (FPOP) and glycine ethyl ester (GEE) labeling, to explore the binding epitope of hIL7Rα upon interaction with Antibody A.

[0344] HDX-MX De-deuteration experiments were performed prior to epitope mapping experiments to obtain a list of peptides (generated in-house) found in recombinant human hIL7Rα (10 μM) and the protein complex of hIL7Rα with Fab of antibody A (1:1 molar ratio). Samples were injected onto a Waters Enzymate BEH pepsin enzyme column (2.1 × 30 mm) and digested at 15 °C for 3 min. The cooling chamber of the UPLC system (containing all chromatographic elements) was kept at 0.0 ± 0.1 °C throughout the run. Injected peptides were captured and desalted at 40 μL / min for 3 min, and then separated by a gradient of 5 to 40% acetonitrile in water over 6 min at 65 μL / min. The separation column used was a 1.0 mm × 100.0 mm ACQUITY UPLC BEH C18 column (Waters) containing 1.7 μm particles, with an average back pressure of 8500 psi at 0.1 °C. A Xevo G2 mass spectrometer was used for data acquisition. The instrument configuration was as follows: capillary voltage 3.2 kV, trap collision energy 6 V, sampling cone voltage 35 V, ion source temperature 80 °C, and desolvation temperature 175 °C. Mass spectra were acquired over the m / z range of 100-1900. Mass accuracy was ensured by calibration with 500 nM [Glu1]-Fibrinopeptide B and was less than 10 ppm throughout all experiments. Identification of digested peptides was performed using ProteinLynx Global This was achieved by combining accurate mass analysis and MSE using SERVER 2.5 (Waters).

[0345] For HDX-MS experiments, the labeling reaction was started by diluting 5 μL of each sample (hIL7Rα or hIL7Rα plus antibody A Fab, respectively) into 55 μL of DO buffer (10 mM phosphate buffer, DO, pH 7.0). The reaction was run for different times (1 min, 10 min, and 240 min). By the end of each labeling reaction time, the reaction was stopped by adding (1:1 (v / v)) a stop buffer (100 mM phosphate buffer, pH 2.5, containing 4 M GdnCl and 0.4 M TCEP). 50 μL of the stopped sample was subjected to on-line digestion using exactly the same conditions as in the non-deuterated experiment. All comparative experiments were performed under identical experimental conditions to avoid the need for back-exchange correction of deuterium levels. Therefore, deuterium levels are reported as relative. All experiments were performed in duplicate. The measurement error of the mass of each peptide was ±0.20 Da in this experimental setup, which was consistent with previously obtained values. Deuterium incorporation was calculated by subtracting the central mass of the isotopic distribution of peptide ions obtained from the non-deuterated protein from the central mass of the isotopic distribution of peptide ions obtained from the deuterium-labeled sample. The obtained relative deuterium levels were analyzed using the software program DYNAMX3.0 ( The data were plotted against exchange time using Waters.

[0346] FFOP FPOP experiments were performed on hIL7Rα and hIL7Rα / Fab (antibody A) complex (1:1 molar ratio, 10 μM final concentration). Hydroxyl radicals were generated by photolysis of H2O2 using a KrF excimer laser. The excitation wavelength was set at 248 nm. Just before labeling, 5 μL of histidine and H2O2 were added to the protein aliquot, respectively. The final volume of the protein solution was 50 μL, and the final concentrations of histidine and H2O2 were 500 μM and 15 mM, respectively. The laser energy was adjusted to 28 mJ / pulse (7.4 Hz). Both FPOP experiments and control experiments without laser irradiation were performed in triplicate. Each replicate was collected in a microcentrifuge tube containing 11 μL of stop solution (800 nM catalase tetramer and 200 mM methionine). Samples were subjected to denaturation, reduction, alkylation, and digestion with chymotrypsin. Data acquisition was performed on a Thermo Q Exactive Plus mass spectrometer equipped with a Waters Acquity UPLC system. The Byonic search engine was used to generate sequencing coverage and identify oxidation sites. Relative oxidation levels of tryptic peptides were calculated using Byologic software (Protein Metrics, San Carlos, CA). Only peptides with a statistically significant difference in relative oxidation between free and bound states (based on a p-value of Student's T-test of <0.01) were subjected to further analysis. Residue-level analysis was performed in Byologic software and manually verified for each replicate. Only residues with a statistically significant difference in oxidation between free hIL7Rα and hIL7Rα / Fab-Antibody A (based on a p-value of Student's T-test of <0.01) were considered as protected residues.

[0347] GEE labeling GEE labeling was initiated by mixing 10 μL of each sample (hPAI1 or hPAI1 plus mAb (1 mg / mL)) with 2 M GEE (1 μL) and 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1 μL) for 1 min at room temperature. The reaction was stopped by adding 10 μL of 1 M ammonium acetate to the sample. 17.5 μL of each GEE-labeled sample was subjected to enzymatic digestion. Samples were denatured in the presence of 0.5% Rapigest detergent, reduced with DTT for 30 min at 56 °C, alkylated with IAM for 30 min at room temperature in the dark, digested with chymotrypsin in Tris (pH 7.4) at 37 °C overnight, and then acidified to stop the digestion reaction. Digested samples were analyzed on a Thermo Q Exactive Plus mass spectrometer to monitor the level of GEE modification on hIL7Rα in the absence / presence of Antibody A.

[0348] result As shown in Figure 9, the sequence coverage of hIL7R obtained using HDX-MS analysis was 97.3%. This analysis revealed the following non-continuous epitopes (covering four distinct regions within hIL7R): (i) Region 1: 24 SQLEVNGSQHSLTCAF 39 , (ii) Region 2: 73 FIETKKFLLIGKSNIC 88 , 89 VKVGEKSLTCKKIDLTT 105 , (iii) Region 3: 136 QKKYVKVLMHDVAY 149 and (iv) Region 4: 181 YEIKVRSIPDHYFKGF 196 Based on the relative deuterium uptake differences, these peptide regions could be ranked as region 1 > region 2, region 3, region 4, with the change in deuterium uptake in region 1 being the most significant (Figure 10).

[0349] FPOP analysis identified five epitopes in hIF7R: (i) Region 1: 24 SQLEVNGSQHS35 , (ii) Region 2: 73 FIETKKF 79 and 80 LLIGKSNICVKVGEKSL 95 , (iii) Region 3: 144 MHDVA Y 149 , and (iv) Region 4: 182 EIKVRSIPDHYFKGF 193 These epitopes overlapped with the non-contiguous regions identified using HDX-MS analysis. These identified epitopes also showed reduced oxidation levels compared to the control, as shown in Table 9. The following were identified as the most conserved residues: (i) Region 1: H33, (ii) Region 2: F79, I82, and K84, (iii) Region 3: M144, and (iv) Region 4: R186, H191, and Y192. [Table 9] Residues that show a significant difference (p value < 0.01) in oxidation levels upon binding to Antibody A compared to the hIL7Rα control are considered the most protected residues. Calculations represent replicates (n = 3) per condition.

[0350] Finally, GEE labeling (used to obtain complementary data for residues with limited oxidation propensity (residues that may be unresponsive to FPOP), specifically aspartic acid and glutamic acid) identified E75 as the most protected residue on hIL7Rα upon interaction with Antibody A (Table 10). [Table 10] Residues that show a significant difference (p-value < 0.01) in GEE labeling levels upon binding with Antibody A compared to the hIL7Rα control are considered the most protected residues. Calculations represent replicates (n = 2) per condition.

[0351] Based on the above analysis, the epitope of hIL7Rα that interacts with Antibody A was mapped to the linear sequence of hIL7Rα (FIG. 11A) and further to the crystal structure (FIG. 11B).

[0352] In summary, hydrogen / deuterium exchange mass spectrometry (HDX-MS) experiments showed that the ANTIBODY A binding site on human IL7Rα maps to residues in the following peptide sequence: 24 SQLEVNGSQHSLTCAF 39 , 73 FIETKKFLLIGKSNIC 88 , 89 VKVGEKSLTCKKIDLTT 105 , 136 QKKYVKVLMHDVAY 149 , and 181 YEIKVRSIPDHYFKGF 196 Additional mass spectrometry-based protein footprinting techniques (including FPOP and GEE labeling) provided the ANTIBODY A binding site on human IL7Rα at amino acid resolution, identifying the following amino acids: H33, E75, F79, I82, K84, M144, R186, H191, and Y192.

[0353] Example 10: Human Dose Estimation A recently reported mechanistic model of target-mediated drug elimination for PFE A3312F (nbc.aapsmeeting.org / event / member / 373852) was used to estimate human PK / RO / PD for the anti-IL-7R antibodies disclosed herein (FIGS. 12A and 12B). This mechanistic model allowed us to capture the PK / RO / PD time course following administration of PFE A3312F mAb in human clinical trials.

[0354] To allow simulation of human PK and PD of the anti-IL-7R antibodies, most of the model parameters were kept similar to the published models, while a 2-fold lower clearance in humans (compared to PFE A3312F) was assumed. This was supported by the 2-fold lower CL observed in monkeys compared to A3312F at a pharmacologically relevant dose (3 mg / kg; Figure 4). Furthermore, the binding affinity of the anti-IL-7R antibodies was , were similar between monkeys and humans, especially at pH 7.4 (see Example 3). Therefore, it was assumed that the PK estimation would reflect this CL difference in humans. Given that the in vivo potency of the anti-IL-7R antibodies of the present disclosure and A3312F in monkeys was similar (Figure 5), it was assumed that the binding kinetics of these two antibodies would be similar in humans.

[0355] The effective dose in humans was estimated by ensuring a trough RO of 95% after biweekly dosing. Monkey PK / PD studies demonstrated that an RO of >95% resulted in 90% inhibition of pSTAT5. See Example 3. Therefore, RO rather than pSTAT5 coverage was used for dose estimation due to the high variability of pSTAT5 inhibition in monkeys and humans.

[0356] Combining the above information, it was estimated that a sustained SC dose of 110 mg once every two weeks (Q2W) for a 70 kg adult would achieve 95% of the RO of IL-7R during treatment with an anti-IL-7R antibody of the present disclosure. The SC loading dose that would allow achievement of this RO after the first dose was calculated to be 140 mg (Figure 13).

[0357] Table 11 shows the predicted steady-state exposure and PK parameters in humans at this dose. As expected, due to non-linear pharmacokinetics, the estimated half-life of the anti-IL-7R antibody in humans was protective with increasing dose. At an effective dose of 110 mg, the estimated half-life was 53 hours, but was expected to increase with increasing dose (e.g., 85 hours at 125 mg). [Table 11]

[0358] Example 11: Mutational Scanning Analysis of Antibody 18B1 To perform the mutational scan, antibody 18B1 (Antibody A, Table 12) was first reshaped as an scFv and confirmed to bind full-length hIL-7R via mRNA display (Xu L et al. (2002) Chemistry & Biology 9:933; Roberts RW and JW Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297; Kurz et al. (2000) Nucleic Acids Res. 28(18):E83). Binding data for the reshaped antibody is shown in Figures 14A and 14B.

[0359] Once binding was confirmed, we then constructed a mutation scanning library in which each library variant contained only a single mutation within a CDR. All six CDRs of the antibody were mutated, and each position in the CDR was mutated to all amino acids possible at that position. The specific positions mutated are shown in Figure 15. This 18B1 scFv variant library The libraries were subjected to a single round of selection against hIL-7R using mRNA display. The results from this single round of selection are shown in FIG. 16. The selection output was analyzed by next generation sequencing (NGS) and heat maps were generated to visualize the NGS data and facilitate the identification of 1) CDR positions important for binding, 2) CDR positions where mutations are tolerated, and 3) mutations that may improve binding of the antibody to hIL-7R. FIG. 17A and FIG. 17B show a schematic of the entire process. FIG. 18A-18F show the heat map results.

[0360] To confirm the above NGS data, alanine substitution variants of the 18B1 antibody were prepared. Briefly, a series of single mutation variants were designed in which a single alanine mutation was introduced at a single position in the CDR. This alanine mutation was carried out at all positions of the six CDRs, except for the CDR positions that already code for alanine. These alanine substitution variants were gene synthesized, transiently expressed in Expi293 as IgG1.3, and purified using Protein A resin.

[0361] 18B1 and alanine substituted variants were tested for their binding to hIL-7R by biacore. Briefly, 12.5 nM of antibody was captured using a CM5 chip (with Fc on the chip surface) and 30 nM, 10 nM, and 3.3 nM of hIL-7R were introduced as ligand in HBS-P (pH 7.4) at 37°C. See Figures 20A and 20B. This approach was used to determine the affinity (K D ) was obtained and compared to the enrichment ratio (ER) obtained via NGS heatmap analysis for the same variants. Figure 19 shows the values ​​for each antibody variant.

[0362] As shown in Figure 21, several of the 18B1 variants were able to bind to hIL-7R with similar affinity as the 18B1 antibody (i.e., antibody A). In addition, as shown in Figures 22 and 23, alanine mutations in the heavy and light chain CDR3 appear to have the greatest impact on binding. Finally, as shown in Figure 24, the results shown in heat maps show that the K D Figure 25 shows the crystal structure of the Fab fragment of the 18B1 antibody, showing residues important for binding (red) and residues that may improve binding (blue).

[0363] Example 12: Internalization analysis of 18B1 antibody upon binding to the IF-7 receptor alpha chain (IF-7Rα) To further characterize the anti-IL-7R antibodies disclosed herein, the 18B1 antibody (i.e., antibody A) was tested for its internalization upon binding to IL-7Rα expressed on monkey leukocytes. Briefly, the 18B1 antibody was administered subcutaneously to cynomolgus monkeys at either (i) 2 mg / kg, (ii) 10 mg / kg, or (iii) 50 mg / kg. Animals not administered the 18B1 antibody were used as controls. Peripheral blood was then collected from the animals at various time points after administration, and the mean fluorescence intensity (MFI) of IL-7Rα expression on CD3+ leukocytes was assessed using flow cytometry.

[0364] As shown in Figure 26, IL-7Rα expression on CD3+ leukocytes remained largely consistent throughout the experimental period in all animals treated with 18B1 antibody, and administration of 18B1 antibody did not decrease expression of IL-7Rα on CD3+ leukocytes compared to control animals.

[0365] This result demonstrates that the 18B1 antibody disclosed herein does not induce internalization of IL-7Rα upon binding, confirming that the antibody does not have any agonistic effect.

[0366] Example 13: Analysis of the potency of 18B1 antibody To evaluate the efficacy of the anti-IL-7R antibodies disclosed herein, the ability of 18B1 antibody to inhibit keyhole limpet hemocyanin (KLH)-induced antibody responses was measured. Briefly, 18B1 antibody was administered subcutaneously to cynomolgus monkeys (both male and female) at doses of (i) 2 mg / kg, (ii) 10 mg / kg, and (iii) 50 mg / kg. Animals not administered 18B1 antibody were used as controls. All animals were then immunized with KLH (10 mg intramuscularly in the posterior quadriceps muscle) 15 days after antibody administration. Peripheral blood was then collected from the animals (femoral, cephalic, or saphenous vein) at various time points, and the endpoint titers (EPTs) of KLH-specific IgM and IgG antibodies in the serum were measured using ELISA.

[0367] As shown in Figures 27A and 27B, an increase in KLH-specific IgM and IgG antibodies was observed in all treatment groups after immunization with KLH. There was no significant difference in KLH-specific IgM responses between treatment groups (see Figure 27A). However, in animals treated with 18B1 antibody, KLH-specific IgG responses were statistically significantly decreased (suppressed by up to 80%) compared to control animals (i.e., animals immunized with KLH but not administered 18B1 antibody) 2 weeks ("Day 29"), 3 weeks ("Day 36"), and 4 weeks ("Day 43") after immunization with KLH (see Figure 27B). The observed suppression of KLH-specific IgG responses was dose-independent, as all animals treated with 18B1 antibody showed comparable responses regardless of dose.

[0368] The above data demonstrate the efficacy of the anti-IL-7R antibodies of the present disclosure and demonstrate that even at a dose as low as 2 mg / kg, the 18B1 antibody can effectively suppress KLH-specific IgG responses. Without being bound by any one theory, the suppression of KLH-specific IgG responses, but not KLH-specific IgM responses, suggests that the 18B1 antibody can suppress antibody class switch recombination in B cells undergoing isotype switching.

[0369] Example 14: Additional epitope mapping analyses To further characterize the anti-IL-7R antibodies of the present disclosure, the binding epitopes of three reference antibodies (i.e., 4A8, 13A10, and PFE A3312F) were determined and compared to the binding epitope of antibody 18B1 (Antibody A, Table 12). The binding epitopes were determined as described above in Example 9.

[0370] As shown in Figures 28A-28C, based on HDX-MS experiments, the binding site of the 4A8 antibody was mapped to residues in the following peptide sequences: (i) 57 LVEVKCLNF 65, (ii) 73 FIETKKFLLIGKSNIC 88 , (iii) 136 QKKYVKVLMHDVAY 149 and (iv) 181 YEIKVRSIPDHYFKGF 196 The binding site of the 13A10 antibody was mapped to the following peptide sequences: (i) 73 FIETKKFLLIGKSNIC 88 , (ii) 89 VKVGEKSLTCKKIDLTT 105 , (iii) 136 QKKYVKVLMHDVAY 149 and (iv) 181 YEIKVRSIPDHYFKGF 196 The binding site of PFE A3312F was mapped to the following peptide sequences: (i) 24 SQLEVNGSQHSLTCA 38 , (ii) 52 EICGALVEVKCLNF 65 , (iii) 73 FIETKKFLLIGKSNIC 88 , (iv) 89 VKVGEKSLTCKKIDLTT 105 , (v) 104 TTIVKPEAPFDLSV 117 , (vi) 109 PEAPFDLSVIYRE 121 , (vii) 136 QKKYVKVLMHDVAY 149 , (viii) 169 TLLQRKLQPAAM 180 and (ix) 181 YEIKVRSIPDHYFKGF 196 29A-29C show the mapping of the binding sites of 4A8, 13A10, and PFE A3312F onto the crystal structure of the human IL-7Rα protein.

[0371] In comparison with the data presented in Example 9 (see also Figures 10 and 11B), the above results demonstrate that antibody 18B1 disclosed in the present application binds to a different region on the human IL-7Rα protein compared to the reference antibody described in this Example. [Table 12] [Table 13] [Table 14-1] [Table 14-2] [Table 14-3] [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 17-1] [Table 17-2] [Table 17-3] [Table 18-1]

Table 18-2

Claims

1. A composition for treating a disease or condition in a subject in need thereof by inhibiting IL-7 activity, comprising: the composition comprises an effective amount of an isolated antibody or antigen-binding portion thereof; wherein the isolated antibody, or antigen-binding portion thereof, specifically binds to the alpha chain of a human IL-7 receptor and comprises heavy chain (HC) CDR1, CDR2, and CDR3, and light chain (LC) CDR1, CDR2, and CDR3; Where: (i) the heavy chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; (ii) the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; (iii) the heavy chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; (iv) the light chain CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16; (v) the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17; and (vi) the light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; The disease or condition is inflammatory bowel disease (IBD), rheumatoid arthritis (RA), ulcerative colitis (UC), systemic lupus erythematosus (SLE), lupus nephritis, multiple sclerosis (MS), atopic dermatitis, Sjogren's syndrome, asthma, type I diabetes, coronary artery disease, graft-versus-host disease, or autoimmune nephritis; The composition.

2. The composition described in claim 1, wherein the disease or condition is systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), atopic dermatitis, Sjogren's syndrome, multiple sclerosis (MS), ulcerative colitis (UC) or lupus nephritis.

3. The composition described in claim 1, wherein the antibody is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

4. The composition described in claim 3, wherein the antibody is an IgG1 antibody.

5. The composition of claim 4, comprising an IgG1 Fc that has no effector function.

6. The composition of claim 1, wherein the antigen-binding portion is a Fab, F(ab')2, Fv, or single chain Fv (scFv).

7. The composition described in claim 1, wherein the subject is one for whom previous TNF-α inhibitor treatment has not been sufficient or is an insufficient responder to anti-TNF-α therapy.

8. The composition described in claim 1 for administration in combination with an anti-TNF-α antibody.

9. The composition of claim 1, wherein the antibody or antigen-binding portion thereof is adapted to be administered to the subject intravenously, subcutaneously, intramuscularly, intradermally or intraperitoneally.

10. The composition of claim 1, wherein the antibody is adapted to be administered intravenously or subcutaneously to the subject.