CD25 is biased against anti-IL-2 antibodies.

Abstract

The present invention provides a human IL-2 (hIL-2)-specific monoclonal antibody, wherein the complex of hIL-2 with the monoclonal antibody induces IL-2 signaling preferentially through CD25 and the trimeric IL-2R. The present invention further provides a pharmaceutical composition comprising hIL-2 and the hIL-2-mAb for use in treating inflammatory diseases.

Description

[Technical Field]

[0001] This invention relates to an antibody that specifically reacts with human IL-2 and can bias the effect of IL-2 towards a tolerogenic CD25-mediated immune response rather than a CD122-mediated immune response. This invention further provides specific pharmaceutical compositions and therapeutic methods using the antibody of the present invention. [Background technology]

[0002] Interleukin-2 (IL-2) is a 15.5 kDa 4-α-helix bundle cytokine, an important T cell growth factor, and transmits signals via a specific IL-2 receptor (IL-2R). IL-2-IL-2R binding initiates downstream signaling pathways, including those involving Janus kinase signaling molecules and activators of transcription (STAT), phosphoinositide 3-kinase (PI3K)-AKT, and the mitogen-activated protein kinase (MAPK) pathway (Arenas-Ramirez, Trends Immunol. 2015, 36:763). There are two types of signaling IL-2R: dimeric and trimer (Ross, Annu. Rev. Immunol. 2018, 36:411). The dimer IL-2R is formed from IL-2Rβ (CD122) and a common γ chain (γc; CD132), and exhibits an intermediate affinity for IL-2 (dissociation constant [K]). d ]≒10 -9 M). The trimer IL-2R consists of CD122, CD132, and IL-2Rα (CD25), and CD25 plays a role in further increasing the affinity of IL-2R by 100 times, and therefore the trimer IL-2R(K d ≒10 -11 M) is also called high-affinity IL-2R (Arenas-Ramirez, 2015). As a result, fork headbox p3 (Foxp3) + CD25 high CD4 + Controllability T(T regCells such as those that strongly express high levels of trimeric IL-2R are competitive against CD25 - cells, provided that both cell subsets express CD122 and CD132 similarly. However, CD4 + T reg cells retain high levels of CD25, but the levels of CD122 remain low to moderate, while antigen-experienced (memory) CD8 + T cells and natural killer (NK) cells express high levels of CD122 at steady state, while CD25 remains at background levels. Thus, CD4 + T reg cells, CD8 + T cells and NK cells may compete for IL-2 during IL-2 immunotherapy (Arenas-Ramirez, Sci.Transl.Med.2016,8:367; Raeber, Immunol.Rev.2018,283:176).

[0003] The pleiotropic actions of IL-2 on both immunosuppressive T reg cells and immune-stimulatory effector immune cells make its therapeutic use difficult. To improve the therapeutic properties of IL-2, multiple approaches have been pursued. By introducing mutations into IL-2 (also called IL-2 mutant proteins), or PEGylating IL-2 at specific sites, or using IL-2 / anti-IL-2 monoclonal antibody (mAb) complexes (briefly, IL-2cx), IL-2 can be biased (biased) towards dimeric or trimeric IL-2R. The anti-mouse IL-2-specific mAb clone JES6-1 is a prototype antibody developed for this purpose. By complexing recombinant wild-type (WT: wild-type) mouse IL-2 with JES6-1, IL-2 / JES6-1cx is obtained, which strongly stimulates CD25 high T reg cells, while on the other hand, resting CD8 +T cells and NK cells are largely unaffected by this IL-2cx (Boyman, Science 2006, 311:1924; Letourneau, PNAS 2010, 107:2171). Based on their in vivo effects, IL-2 / JES6-1cx and similar IL-2cx are called CD25-directed or CD25-biased IL-2cx.

[0004] CD25-biased IL-2cx has been evaluated in mice in multiple models of solid allograft transplantation, as well as in chronic inflammatory and autoimmune diseases including autoimmune diabetes, experimental autoimmune encephalomyelitis (a model of multiple sclerosis), collagen-induced arthritis, inflammatory colitis, and systemic lupus erythematosus-like syndrome (Tang, Immunity 2008, 28:687; Webster, J. Exp. Med. 2009, 206:751; Lee, Immunol. 2012, 137:305; Spangler, Immunity 2015, 42:815; Yan, Kidney Int. 2017, 91:603). The receptor-biasing function of IL-2cx was thought to be achieved by the mAb cloaking the IL-2 binding site of either high-affinity or intermediate-affinity receptors. Structural analysis of IL-2 / JES6-1cx suggested that JES6-1 sterically inhibits the binding of IL-2 to CD132 and further induces mild allosteric changes in the structure of IL-2, thereby affecting the interaction between IL-2 and CD25 (Spangler, Immunity, 2015). Conversely, in CD25-biased human IL-2cx produced with the anti-human IL-2 mAb F5111.2, the epitope was covered by F5111.2 attached to the CD122 binding site, inducing mild allosteric changes in the CD25 epitope (Trotta, Nat. Med. 2018, 24:1005). It is unknown whether CD25-biased anti-IL-2 mAbs need to dissociate from IL-2cx bound to CD25 in order for IL-2 to bind to the CD122-CD132 dimer and initiate signal transduction. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Arenas-Ramirez,Trends Immunol.2015,36:763 [Non-Patent Document 2] Ross,Annu.Rev.Immunol.2018,36:411 [Non-Patent Document 3] Arenas-Ramirez, Sci.Transl.Med.2016,8:367 [Non-Patent Document 4] Raeber,Immunol.Rev.2018,283:176 [Non-Patent Document 5] Boyman, Science 2006, 311:1924 [Non-Patent Document 6] Letourneau, PNAS 2010, 107:2171 [Non-Patent Document 7] Tang,Immunity 2008,28:687 [Non-Patent Document 8] Webster,J.Exp.Med.2009,206:751 [Non-Patent Document 9] Lee, Immunol. 2012, 137:305 [Non-Patent Document 10] Spangler, Immunity 2015, 42:815 [Non-Patent Document 11] Yan, Kidney Int. 2017, 91:603 [Non-Patent Document 12] Trotta, Nat. Med. 2018, 24:1005 [Overview of the project] [Problems that the invention aims to solve]

[0006] Based on the above state of technology, the object of the present invention is to inhibit inflammation by using high-affinity IL-2 receptors, particularly T, to suppress IL-2 signaling. reg The objective is to provide improved means and methods for biasing the expression towards the receptor expressed above. [Means for solving the problem]

[0007] This objective is achieved by the subject matter of the independent claims herein. [Brief explanation of the drawing]

[0008] [Figure 1A] Figure 1(A) shows a screening design for identifying IL-2 receptor (IL-2R) biased anti-hIL-2mAbs with agonistic properties in vivo. Cell-based IL-2R binding assays and flow cytometry plots are shown, gated based on the fluorescence "barcodes" of cells expressing CD25, CD122+CD132, or CD25+CD122+CD132. The emission spectra of CD25-CyPet, CD122-YPet, and CD132-RFP were detected in the AF488, BV421, and APC channels, respectively. IL-2 complex (IL-2cx) binding was recorded in the BV605 channel using rat anti-mouse IgG-BV605. Histograms (right) show binding of IL-2 / anti-IL-2mAbCD25 (middle), IL-2 / anti-IL-2mAbCD122 (bottom), and negative control (top). [Figure 1BC]Figure 1(B) shows the quantification of IL-2cx binding, similar to (A). The readout is the geometric mean fluorescence intensity (gMFI) of BV605 quantified in cells expressing CD25 (white bars) or CD122+CD132 (gray bars), and each anti-IL-2 mAb is plotted individually. Pooled data ± SEM obtained from 3-4 experiments. gMFI background was subtracted. Unpaired two-sided t-test. Figure 1(C) shows the quantification of flow cytometry of the binding of IL-2 Rhod (PE channel) to anti-IL-2 mAb (rat anti-mouse IgG-BB605) to HEK293T cells expressing gated CD25-CyPet, CD122-YPet+CD132-RFP, or CD25-CyPet+CD122-YPet+CD132-RFP, similar to (A). Control cells were incubated without IL-2Rhod. The bar graph shows the frequency ± SEM of IL-2Rhod-positive fractions (no color) or IL-2Rhod / anti-IL-2 mAb double-positive fractions (color). Data obtained from 3-4 experiments were pooled. [Figure 1 DEFGH]Figure 1(D) shows the matrix of IL-2Rhod / anti-IL-2 mAb complexes clustered based on binding to CD25 (Y-axis) or CD122+CD132 (X-axis) (left plot) and CD25 (Y-axis) or CD25+CD122+CD132 (X-axis) (right plot). The mean percentage of the IL-2Rhod / anti-IL-2 mAb complex-positive population is obtained in (C). Figure 1(E) shows a comparison of complex IL-2Rhod and free IL-2Rhod during incubation with cells expressing trimer-high affinity IL-2R. The mean percentage of the IL-2Rhod vs. IL-2Rhod / anti-IL-2 mAb complex-positive population is obtained in (C). Figure 1(F) shows the "CD25 bias" and "IL-2 delivery" matrix of the described anti-IL-2 mAb clones. The clusters show the binding of the IL-2Rhod / anti-IL-2 mAb complex to CD25 (Y axis) versus the binding of free IL-2Rhod to CD25+CD122+CD132 (X axis). The mean percentages of the IL-2Rhod / anti-IL-2 mAb complex-positive population and the IL-2Rhod-positive population obtained in (C) are shown. Figure 1(G) shows anti-IL-2 mAb release by the difference or ratio of the binding of the IL-2Rhod / anti-IL-2 complex to CD25 and to CD25+CD122+CD132. Figure 1(H) shows an overview of the anti-IL-2 mAb clones tested. [Figure 2ABC]Figure 2(A) shows the frequency analysis of CD4+CD25+Foxp3+ T cells and CD8+CD44hiCD122+ T cells in the lymph nodes (LN) and spleen of C57BL / 6 mice that were injected with IL-2 (1.5 μg or 30 μg) and IL-2 / anti-IL-2 complex (1.5 μg / 15 μg) on ​​days 0, 1, and 2, and euthanized on day 4. (A) shows the frequency and count of CD4+CD25+Foxp3+ T cells in the spleen and LN quantified on day 4. The mean ± SEM values ​​from 2-3 experiments are shown, with n=2 for IL-2 (30 μg) and UFKA-50; n=5 for IL-2 (1.5 μg), UFKA-10, UFKA-30, UFKA-40, and NARA1; and n=6 for PBS and UFKA-20. Unpaired two-sided t-test. (B) Mean cell counts of CD4+CD25+Foxp3+ T cells, CD8+CD44hiCD122+ T cells, and CD3-NK1.1+CD122+ from the spleen are shown as a fold change compared to PBS-treated mice. (C) The ratio of CD4+CD25+Foxp3+ T cell counts and CD8+CD44hiCD122+ T cell counts from the spleen are plotted. Mean ± SEM. Unpaired two-sided t-test. The red dashed line shows the mean values ​​obtained from IL-2 (1.5 μg) treated animals. [Figure 3AB] Figure 3 shows the cell subset frequencies in the lymph nodes and spleen measured by flow cytometry after injecting wild-type (WT) C57BL / 6 mice with IL-2 (1.5 μg or 30 μg) and IL-2 / anti-IL-2 complex (1.5 μg / 15 μg) on ​​days 0, 1, and 2, and euthanizing them on day 4. (A) Frequency of CD8+CD44hiCD122+ T cells in the lymph nodes and spleen on day 4. (B) Frequency of CD3-NK1.1+CD122+ NK cells in the spleen. The values ​​are the mean ± SEM values ​​from 2-3 experiments, with n=2 for IL-2 (30 μg) and UFKA-50; n=5 for IL-2 (1.5 μg), UFKA-10, UFKA-30, UFKA-40, and NARA1; and n=6 for PBS and UFKA-20. The red dashed line shows the average values ​​obtained from animals treated with IL-2 (1.5 μg). [Figure 4AB]Figure 4 shows the results of a single injection of IL-2 (1 μg or 30 μg) or IL-2 / UFKA-20 complex (1 μg / 10 μg) into C57BL / 6. (A) Mice were euthanized, and the frequency of phosphorylated STAT5+ (pSTAT5) cells among spleen CD4+CD25+, CD8+ T cells and NK cells was measured by flow cytometry 2 hours (2hr) after injection of PBS (gray), IL-2 (1 μg, white), IL-2 (30 μg, black), or IL-2 / UFKA-20 complex (dark gray) on days 1 (d1), 2 (d2), 4 (d4), and 8 (d8). Data are shown as the mean ± SEM of three independent experiments, with n=5 mice per group. One-way ANOVA and Tukey multiple comparison test. (D) One-way ANOVA of the spleen. The number of T cells and NK cells was expressed as a ratio change relative to PBS. Data are shown as the mean ± SEM of three independent experiments, with n=5 mice per group. One-way ANOVA and Tukey's multiple comparison test were performed. [Figure 5] Figure 5 shows the results of a single injection of IL-2 (1.5 μg; colorless symbol), IL-2 / UFKA-20 complex (IL-2 / UFKA-20cx; black symbol), or IL-2 / UFKA-22 complex (IL-2 / UFKA-22cx; red symbol) into wild-type (WT) C57BL / 6 mice. IL-2 / UFKA-20cx and IL-2 / UFKA-22cx were prepared by complexing human IL-2 (1.5 μg) with UFKA-20 (15 μg) and UFKA-22 (15 μg), respectively, in a 1:1 molar ratio. After injection, blood samples were collected at the instructed time points (hours: hr), and human IL-2 in serum was detected using sandwich enzyme-linked immunosorbent assay (ELISA). The half-life (t1 / 2) value was calculated by fitting it to an exponential single-phase decay curve. IL-2 is shown as the average value for n=7, IL-2 / UFKA-20cx as the average value for n=9, and IL-2 / UFKA-22cx as the average value for n=3. [Figure 6]Figure 6 shows the quantitative analysis of the IL-2 complex stimulation of pSTAT5 in CD4+CD127lowFoxp3+ Treg cells and CD8+ T cells, measured by flow cytometry. 100 ng / ml IL-2 was compounded with UFKA-20 or UFKA-22 in a 1:1 molar ratio. The graphs show the pSTAT5 levels of the indicated human T cell subsets in response to titrated IL-2 (left graph), IL-2 / UFKA-20cx (middle graph), and IL-2 / UFKA-22cx (right graph). The maximum effective concentration (EC50) for each condition was calculated for both CD4+CD127lowFoxp3+ Treg cells (uncolored symbols) and CD8+ T cells (colored symbols). Fitted dose-response curves are shown as lines. Data are shown as the mean ± SEM of three independent experiments. [Figure 7AB] Figure 7(A) shows the experimental setup as described in Figure 2. Mice received three injections of PBS, IL-2 (1 μg) alone, or a chimeric UFKA-20 mAb (chUFKA-20), or a complex (1:1 molar ratio) of humanized UFKA-20 variant UFKA-22-00 (referred to as UFKA-22), UFKA-22-02, and UFKA-22-07. Mice were euthanized on day 4, and the frequency of CD4+CD25+Foxp3+ T cells and CD8+CD44hiCD122+ T cells was analyzed in the spleen by flow cytometry. The frequency and number of splenic CD4+CD25+Foxp3+ T cells were quantified on day 4. (B) The ratio of the number of splenic CD4+CD25+Foxp3+ T cells and CD8+CD44hiCD122+ T cells was plotted. The mean values ​​± SEM results from three experiments are shown. PBS, IL-2, and UFKA-22-07 had n=3 values, while UFKA-20, chUFKA-20, UFKA-22-02, and UFKA-22-07 had n=5 values. [Figure 8A]Figure 8(A) shows that rhesus monkeys were either infused with IL-2 daily until day 6, or with IL-2 / UFKA-22 complex or UFKA-22 mAb alone twice on days 0 and 3. Blood samples were collected and analyzed at the indicated time. The IL-2 and IL-2 / UFKA-22 complex treatment groups were subdivided into two groups: low-dose (LD) and high-dose (HD), corresponding to 10 μg / kg and 33 μg / kg of IL-2, respectively, while UFKA-22 mAb alone was infused at a dose of 330 μg / kg. The EC50 of UFKA-20 (left table) and UFKA-22 (right table) against human IL-2 and rhesus monkey IL-2 was measured using IL-2 sandwich ELISA. [Figure 8B] Figure 8(B) shows representative flow cytometry plots of pSTAT5+ cells in circulating CD4+CD25+ T cells and CD8+CD25+ T cells. The histograms show baseline pSTAT5 levels measured on day 8 (top) and pSTAT5 levels during LD IL-2 (middle) and LD IL-2 / UFKA-22 (bottom) treatment on day 1, with n=3 for each group. [Figure 8C] Figure 8(C) shows the frequencies of CD4+CD25+Foxp3+ T cell populations and CD4+CD25+ T cell populations in the blood during the test period, as measured by flow cytometry. [Figure 8DE]Figure 8(D) shows the gMFI of Foxp3, CTLA-4, and Ki-67 in CD4+CD25+ T cells in blood measured by flow cytometry at the indicated time points. For (C) and (D), the mean ± SEM is plotted as white dots (IL-2 treatment group), gray dots (IL-2 / UFKA-22 complex treatment group), and dark gray dots (UFKA-22 mAb treatment group). Significance was determined by an unpaired t-test (two-sided) on day 6. ns indicates not significant. Figure 8(E) shows the ratio of Treg (CD4+CD25+Foxp3+CD4 T) cells to the number of CD8+ T cells, NK cells, and B cells on day 6. The mean ± SEM and individual values ​​are plotted. Significance was determined by one-way ANOVA and Dunnett's multiple comparisons. ns indicates not significant. [Figure 9A] Figure 9(A) shows the front and 90° rotated side views of the IL-2 / anti-IL-2 mAb complex structure superimposed on the human quaternary IL-2R complex, which consists of IL-2 (purple), CD25 (IL-2Rα, light orange), CD122 (IL-2Rβ, white), and CD132 (IL-2Rγ, gray). Comparisons are shown for the IL-2-IL-2R complex (PDB:2B5I), IL-2 / UFKA-20 complex (PDB:6YE3), IL-2 / F5111 complex (PDB:5UTZ), IL-2 / JES6-1 complex (PDB:4YQX), and IL-2 / NARA1 complex (PDB:5LQB). [Figure 9B-1] Figure 9(B) shows the equilibrium surface plasmon resonance (SPR) quantification of the overlap between the IL-2 / anti-IL-2 complex and the IL-2-IL-2R binding epitope. The CD25 binding site, CD122 binding site, and CD132 binding site are shown. The bar graph shows the relative overlap between the antibody and the receptor epitope, calculated based on the buried surface area (Ų) using PDBePISA. [Figure 9B-2]Figure 9(B) shows the equilibrium surface plasmon resonance (SPR) quantification of the overlap between the IL-2 / anti-IL-2 complex and the IL-2-IL-2R binding epitope. The CD25 binding site, CD122 binding site, and CD132 binding site are shown. The bar graph shows the relative overlap between the antibody and the receptor epitope, calculated based on the buried surface area (Ų) using PDBePISA. [Figure 9B-3] Figure 9(B) shows the equilibrium surface plasmon resonance (SPR) quantification of the overlap between the IL-2 / anti-IL-2 complex and the IL-2-IL-2R binding epitope. The CD25 binding site, CD122 binding site, and CD132 binding site are shown. The bar graph shows the relative overlap between the antibody and the receptor epitope, calculated based on the buried surface area (Ų) using PDBePISA. [Figure 9CD] Figure 9(C) shows SPR titrations of CD25 and CD122 on IL-2 captured by immobilized UFKA-20 or NARA1, as shown. Data are representative of three experiments. RU indicates resonance units. Figure 9(D) shows IL-2 competition among HEK293T cells expressing UFKA-20 and CD25, CD122+CD132, and CD25+CD122+CD132. A constant concentration of IL-2Rhod (0.2 μg / ml) was mixed with a titration volume of UFKA-20 and incubated with IL-2R-expressing HEK293T cells. Mean ± SEM of two experiments are plotted. [Figure 10-1]Figure 10 shows the buried surface area (BSA) in square angstroms (Ų) between human IL-2 and CD25, CD122, CD132, UFKA-20-00, F5111, and NARA1. Calculations were performed using the PDBePISA server and the crystal structures of the IL-2-IL-2R quaternary complex (PDB:2B5I), IL-2 / UFKA-20-00 complex (PDB:6YE3), and IL-2 / F5111 complex (PDB:5UTZ). Three-letter amino acid codes of human IL-2 protein sequences annotated with helices A, A', B, B', C, and D (Arenas-Ramirez et al., 2015). This table shows the BSA values ​​(greater than 0.00 Ų) of amino acid residues for human IL-2 and its ligands (CD25, CD122, CD132, UFKA-20-00, F5111, NARA1). Additionally, the single-letter amino acid codes of the affected amino acid residues are also displayed. [Figure 10-2] Figure 10 shows the buried surface area (BSA) in square angstroms (Ų) between human IL-2 and CD25, CD122, CD132, UFKA-20-00, F5111, and NARA1. Calculations were performed using the PDBePISA server and the crystal structures of the IL-2-IL-2R quaternary complex (PDB:2B5I), IL-2 / UFKA-20-00 complex (PDB:6YE3), and IL-2 / F5111 complex (PDB:5UTZ). Three-letter amino acid codes of human IL-2 protein sequences annotated with helices A, A', B, B', C, and D (Arenas-Ramirez et al., 2015). This table shows the BSA values ​​(greater than 0.00 Ų) of amino acid residues for human IL-2 and its ligands (CD25, CD122, CD132, UFKA-20-00, F5111, NARA1). Additionally, the single-letter amino acid codes of the affected amino acid residues are also displayed. [Figure 10-3]Figure 10 shows the buried surface area (BSA) in square angstroms (Ų) between human IL-2 and CD25, CD122, CD132, UFKA-20-00, F5111, and NARA1. Calculations were performed using the PDBePISA server and the crystal structures of the IL-2-IL-2R quaternary complex (PDB:2B5I), IL-2 / UFKA-20-00 complex (PDB:6YE3), and IL-2 / F5111 complex (PDB:5UTZ). Three-letter amino acid codes of human IL-2 protein sequences annotated with helices A, A', B, B', C, and D (Arenas-Ramirez et al., 2015). This table shows the BSA values ​​(greater than 0.00 Ų) of amino acid residues for human IL-2 and its ligands (CD25, CD122, CD132, UFKA-20-00, F5111, NARA1). Additionally, the single-letter amino acid codes of the affected amino acid residues are also displayed. [Figure 11] Figure 11 shows the predicted buried surface area in square angstroms (Å2) representing the expected UFKA20 binding site on hIL-2. [Figure 12ABC] Figure 12 shows that the optimal affinity of UFKA-20 affects its ability to stimulate CD25+Foxp3+Treg cells in vivo. (A) The binding affinity dissociation constant (KD) of the indicated UFKA-20 variant was measured by single-cycle surface plasmon resonance (SPR) measurement. (B) C57BL / 6 wild-type mice received a single infusion of IL-2 / anti-IL-2cx (1 μg IL-2:10 μg UFKA-20 variant). The mean ± SD frequency of CD4+CD25+Foxp3+Treg cells in the spleen was measured by flow cytometry on day 4. The dotted line shows the mean value for PBS-treated mice. (C) Correlation between antibody KD and ability to induce CD4+CD25+Foxp3+Treg cells is shown (mean ± SD frequency of CD4+CD25+Foxp3+). [Figure 13ABCD]Figure 13 shows the distance between the carboxyl (C) terminus of IL-2 and the amino (N) terminus of the variable heavy chain (vH) or variable light chain (vL) of UFKA-20, measured in angstroms (Å) using PyMOL software. (A) Crystal structure of the IL-2 / UFKA-20 Fab complex (PDB: 6YE3). (B) Distance between the C terminus of IL-2 and the N terminus of UFKA-20 vH (top panel) and UFKA-20 vL (bottom panel) (black dotted line). UFKA-20 vH is shown in black, UFKA-20 vL in gray, and IL-2 in white. (C) Schematic diagram of the IL-2 / UFKA-22 fusion protein (FP), where IL-2 is bound to the UFKA-22 light or heavy chain at its N terminus. (D) Shows UFKA-22FP vL(G4S)6 in two different states. (Left) IL-2 is not associated with the binding pocket of UFKA-22, or IL-2 is associated with the binding pocket of UFKA-22 (right). [Figure 14ABCD] Figure 14 shows a comparison of IL-2 / UFKA-22 and UFKA-22FP in vivo in mice. (A) Experimental design: C57BL / 6 mice were injected with IL-2 / UFKA-22cx (12 μg [2 μg IL-2 and 10 μg UFKA-22]), UFKA-22FP vH(G4S)5 (12 μg or 24 μg), and UFKA-22FP vL(G4S)6 (12 μg or 24 μg) on ​​days 0, 1, and 2. Mice were euthanized on day 4, and the frequencies of CD4+CD25+Foxp3+Treg cells and CD8+CD122+CD44hiT cells in the spleen were analyzed by flow cytometry. (B) CD25+Foxp3+ in CD4+ T cells, (C) Ki-67+ on CD25+Foxp3+ Treg cells, and (D) the frequency of CD8+ T cells in the spleen of mice treated with PBS, IL-2 / UFKA-22, and UFKA-22FP, similar to (A), are shown, mean ± SEM. P values ​​were calculated using one-way ANOVA and Tukey's multiple comparison test; ns indicates non-significant. [Figure 15ABCD]Figure 15 shows (A) flow cytometry gating strategies for mouse spleen DC subsets including plasmacytoid DCs (pDCs) and conventional type I and type II DCs (cDC1 and cDC2). (B) shows the increase in spleen cDCs from mice treated with IL-2, CD25-biased IL-2 / 5344 complex, or CD122-biased IL-2cx (IL-2 / NARA1 complex) (n=7-9 mice per group). (C) shows the proliferation of spleen cDCs in mice treated as shown in (B), measured by BrdU uptake over 3 days (n=7-9 mice per group). (D) Representative histograms (left panel) of the abundance of CD40, CD80, CD86, MHC-I, and MHC-II on spleen cDCs in untreated and IL-2cx-treated (IL-2 / NARA1 complex) mice, and the multiplicative change in gMFI normalized to untreated (right panel). Data are shown as mean + / - SEM (n=9 mice per group). [Figure 16AB] Figure 16(A) shows the study design (top panel) of an investigator-initiated clinical trial in which 1.5 million international units (IU) of aldesleukin were subcutaneously injected daily for 5 consecutive days, with blood samples taken before and after aldesleukin injection. The corresponding gating strategy for the human cDC subset identifies CD141+cDC1 and CD1c+cDC2 (bottom panel). Figure 16(B) shows the percentage of Ki67+ proliferative cDC1 (n=8) and cDC2 (n=10) in the peripheral blood of patients before and after aldesleukin treatment. [Figure 17ABCD]Figure 17(A) shows the quantification of spleen cDCs after 3 consecutive days of treatment with inhibitory IL-2cx (IL-2 / UFKA-20 complex). Figure 17(B) shows the proliferation of spleen cDCs in mice from (A) as measured by BrdU uptake over 3 days. Data are mean + / - SEM (n=7 mice per group). Surface expression of (C) MHC-II and (D) CD80 on cDCs mediated by the UFKA-20 complex was measured by flow cytometry in spleen cDCs of untreated and UFKA-20 complex-treated mice as in (A), and is expressed as a multiple change in geometric mean fluorescence intensity (gMFI) normalized to the untreated group. Data are shown mean + / - SEM (n=5-8 mice per group). [Figure 17E] Figure 17(E) shows RNA sequencing of selected conventional DCs (cDCs) isolated from mice one day after 3 days of UFKA-20 complex treatment. A volcano plot of differentially expressed genes is shown, with genes enriched in UFKA-20 complex-treated mice on the right and genes enriched in untreated mice on the left. Each dot represents a single gene; light gray indicates genes with no significant change compared to the untreated group, and black dots indicate genes showing a significant difference (P<0.05) compared to the untreated group. Representative genes encoding pro-inflammatory or anti-inflammatory proteins are shown. The cutoff was set to a log2 ratio of 0.5. Tgfbi, transforming growth factor β-inducible; Il1rn, interleukin-1 receptor antagonist; Tab1, TGF-β activating kinase 1 / MAP3K7 binding protein 1; Il6st, interleukin-6 signal transducer; Ltb, lymphatoxin β; Tnfsf14, tumor necrosis factor (ligand) superfamily member 14; Csf1, colony-stimulating factor 1; Fas, TNF receptor superfamily member 6. [Modes for carrying out the invention]

[0009] Table 1 shows the SPR analysis of the anti-IL-2 mAbs UFKA-20, UFKA-22-00 (abbreviated as UFKA-22), UFKA-22-02, and UFKA-22-07 in comparison with previously reported anti-IL-2 mAbs JES6-1, F5111, and NARA1.

[0010] Table 2 shows the IL-2 binding properties of UFKA-22 variants with framework mutations, as measured by surface plasmon resonance (SPR).

[0011] Table 3 shows the modifications to VH (sequence number 019) and VL (sequence number 020) in the UFKA-20 variant.

[0012] Table 4 shows the predicted rules for amino acid substitutions in the UFKA20 variant according to Table 3.

[0013] Summary of the Invention The present invention provides an anti-human IL-2 (hIL-2) mAb that is particularly effective in delivering IL-2 to cells expressing high abundances of CD25 (also called IL-2Rα), based on the results of a novel cell-based in vitro screening method that enables selection based on the binding of IL-2cx-forming IL-2 antibodies to CD25 and subsequent delivery of IL-2 to the CD122-CD132 dimer IL-2R: where these cells also need to possess CD122 (also called IL-2Rβ) and CD132 (also called IL-2Rγ) to initiate intracellular signaling pathways. Administration of human IL-2 in conjugate with a human interleukin-2 (hIL-2) specific monoclonal antibody (mAb) has been shown in vivo in mice and monkeys (macaque). reg It leads to preferential growth of cells.

[0014] A first aspect of the present invention is an anti-hIL-2 mAb or antibody fragment that is specific to an epitope containing a defined amino acid residue of the hIL-2 molecule, but leaving other residues uncovered. In certain embodiments, the binding of the anti-hIL-2 mAb to hIL-2 is controlled by a dissociation constant (K). D ) ≤ 5.51 × 10-9 , especially ≤ 5.13 × 10 -9 , binding rate (on rate) (K on )≧4.12×10 5 Lmol -1 s -1 , especially ≥4.66 × 10 5 Lmol -1 s -1 and dissociation rate (off rate) (K off ) ≤ 2.83 × 10 -3 s -1 , especially ≤2.39 × 10 -3 s -1 Characterized by, or the anti-hIL-2 mAb-hIL-2 complex according to the present invention preferentially binds to CD25 compared to CD122, and the anti-hIL-2 mAb dissociates from IL-2 when the complex interacts with cells that express CD25 in high abundance in addition to expressing CD122 and CD132. Due to these characteristics, when binding to hIL-2, IL-2Rα+T reg EC50 binds to it at 0.154 ng / ml, but IL-2Rβ+CD8 + CD44 hi CD122 + T cells are provided with an anti-hIL-2 mAb that forms a complex with an EC50 of 442.9 ng / ml.

[0015] Another aspect of the present invention is V H Complementarity Determination Region CDR H 1. CDR H 2, and CDR H 3 is included in the heavy chain variable (V H ) region, and V L Complementarity Determination Region CDR L 1. CDR L 2, and CDR L Variable light chain (V) including 3 L An anti-hIL-2 mAb having a region, and in particular an anti-hIL-2 mAb having the characteristics of the first aspect of the present invention, where the CDR H 1. CDR H 2. CDR H 3. CDR L 1. CDR L2, and CDR L 3 includes or is identical to sequence numbers 001, 002, 003, 004, 005, and 006, respectively. In some embodiments, CDR is V of sequence number 007. H In the array, and V of sequence number 015 L It is included in the sequence, or in a sequence that is specifically functionally similar.

[0016] Another aspect of the present invention is an hIL-2 fusion protein comprising an hIL-2 protein domain and an anti-hIL-2 mAb domain linked by a peptide linker, particularly a peptide linker of about 30 amino acids in length.

[0017] Further embodiments provide an anti-hIL-2 mAb or antibody fragment thereof according to the present invention, a nucleic acid molecule encoding an hIL-2 fusion protein, or a vector containing said nucleic acid molecule, or a cell or hybridoma strain that contains or can produce an anti-hIL-2 mAb or fusion protein according to the present invention.

[0018] Further aspects of the present invention relate to pharmaceutical compositions comprising an hIL-2-specific mAb or antigen-binding fragment optionally non-covalently associated with hIL-2, particularly as a pharmaceutical for treating immunoinflammatory conditions such as allograft-related diseases, chronic inflammation, allergies, or autoimmunity. The pharmaceutical composition comprising an anti-hIL-2 mAb may also comprise further immunosuppressants or pharmaceutically acceptable carriers.

[0019] Another distinct aspect of the present invention is a pharmaceutical composition for use in patients with conditions that would benefit from enhanced dendritic cell (DC) function, comprising an IL-2 complex. The IL-2 complex comprises both a human IL-2 (hIL-2) polypeptide and an hIL-2 specific monoclonal antibody (mAb). Examples of suitable hIL-2 specific mAbs are disclosed in U.S. Patent Application Publication No. 20170114130 (A1), the contents of which are incorporated herein by reference, or are hIL-2 specific mAbs according to any one of the above aspects and embodiments. The IL-2 complex according to this aspect of the present invention preferentially binds to CD25 and / or high-affinity IL-2 receptors comprising CD122, CD132 and CD25, compared to intermediate-affinity IL-2R comprising CD122 and CD132.

[0020] Detailed description of the invention Terms and Definitions For the purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular form also include the plural form, and vice versa. In the event of any conflict between the following definitions and any documents incorporated herein by reference, the defined definition shall prevail.

[0021] The terms “comprising,” “having,” “containing,” “including,” and other similar forms, as used herein, and their grammatical equivalents, are intended to be semantically equivalent and open-ended, in that one or more items following any one of these words do not exhaustively list or limit to the one or more items listed. For example, an item “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only components A, B, and C), or it may contain one or more other components in addition to components A, B, and C. Thus, it is intended and understood that “comprise” and its similar forms, and their grammatical equivalents, include disclosures of embodiments that “consisting essentially of” or “consisting of.”

[0022] Where a range of values ​​is provided, unless otherwise explicitly indicated in the context, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range, and each of the other stated or intervening values ​​within that range, are understood to be included in this disclosure, subject to the limits specifically excluded within the stated range. If the stated range includes one or both of the limiting values, the range excluding one or both of the limiting values ​​that they include is also included in the disclosure.

[0023] In this specification, any reference to a value or parameter using the term "about" includes (and describes) variations relating to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0024] As used herein, including in the attached claims, the singular “a,” “or,” and “the” include plural references unless otherwise clearly indicated by the context.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally Sambrook et al., Molecular Cloning: A Laboratory Manual, Part 4 (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002), Part 5, John Wiley & Sons, Inc.) and chemical methods.

[0026] In this specification, the term “positive,” when used in the context of marker expression, refers to the expression of an antigen assayed with a fluorescently labeled antibody, and the fluorescence of the label on a structure (e.g., a cell) considered “positive” is characterized by a median fluorescence intensity that is at least 30% (≧30%), and especially ≧50% or ≧80%, compared to staining with a fluorescently labeled antibody of a matching isotype that does not specifically bind to the same target. Such expression of a marker is, for example, CD25 + Like this, a superscript "plus" is added after the marker name. + It will be displayed as ).

[0027] In this specification, the term “negative,” when used in the context of marker expression, refers to the expression of an antigen assayed with a fluorescently labeled antibody, where the median fluorescence intensity is less than 30%, and particularly less than 15%, higher than the median fluorescence intensity of an isotype-matched antibody that does not specifically bind to the same target. Such expression of a marker is indicated by the superscript “minus” following the marker's name. - ) is shown, for example CD25 - That is the case.

[0028] High expression of a marker, such as CD25, refers to the expression level of such a marker in a clearly distinguishable cell population detected by FACS that exhibits the highest fluorescence intensity per cell compared to other populations characterized by lower fluorescence intensity per cell. High expression of, for example, CD44 high As shown above, the marker name is followed by a superscript "high" or "hi". The term "highly expressed" refers to the same characteristic.

[0029] Low expression of a marker, such as CD25, refers to the expression level of such a marker in a clearly distinguishable cell population detected by FACS, which exhibits the lowest fluorescence intensity per cell compared to other populations characterized by higher fluorescence intensity per cell. low It is indicated by the superscript "low" or "lo" after the marker name, as shown above. The term "lowly expressed" refers to the same characteristic.

[0030] Marker expression can be evaluated through techniques such as fluorescence microscopy, flow cytometry, ELISPOT, ELISA, or multiplex analysis.

[0031] The sequence of amino acid residues is written from the amino terminus to the carboxyl terminus. Uppercase letters indicating the sequence position refer to the single-letter code for the L-amino acid (Stryer, Biochemistry, Vol. 3, p. 21). Lowercase letters indicating the position of the amino acid sequence represent the corresponding D- or (2R)-amino acid. The sequence is written from left to right, from the amino terminus to the carboxyl terminus. Following standard nomenclature, the sequence of amino acid residues is represented by either a three-letter or one-letter code, as follows: Alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0032] The term "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that, after being transcribed and translated, can encode a specific polypeptide or protein. Polynucleotide sequences can be used to identify larger fragments or full-length coding sequences of the gene they associate with. Methods for isolating larger fragment sequences are known to those skilled in the art.

[0033] The terms “gene expression” or “expression,” or “gene product,” may refer to either or both the process of generating nucleic acids (RNA) or peptides or polypeptides, and their products, also known as transcription and translation, respectively, or to any intermediate process that modulates the processing of genetic information to produce polypeptide products. The term “gene expression” may also apply to the transcription and processing of RNA gene products, such as regulatory RNA or structural (e.g., ribosomal) RNA. If the expressed polynucleotides originate from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. Expression can be evaluated at the levels of both transcription and translation, i.e., mRNA and / or protein products.

[0034] In the context of this specification, the term “nucleotide” refers to a constituent unit of a nucleic acid or nucleic acid analog, whose oligomers can selectively hybridize with RNA or DNA oligomers based on base pairing. Examples of “nucleotide” in this context include the classical ribonucleotide constituents adenosine, guanosine, uridine (and ribosylthymine), cytidine, and the classical deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Furthermore, it includes nucleic acid analogs such as phosphothioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide bonds, with a nucleic acid base attached to the α-carbon of glycine), or LOK nucleic acids (LNA; 2'O,4'C methylene-bridged RNA constituent units).

[0035] Sequences similar to or homologous (e.g., with at least about 70% sequence identity) to those disclosed herein are also part of the present invention. In some embodiments, sequence identity at the amino acid level may be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. At the nucleic acid level, sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. Alternatively, substantial identity exists if the nucleic acid segment hybridizes to the chain complement under selective hybridization conditions (e.g., very stringency hybridization conditions). Nucleic acids may exist in whole cell form, cell lysates, or in partially purified or substantially pure form.

[0036] In the context of this specification, the terms “sequence identity” and “sequence identity ratio” mean a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for sequence alignment for comparison are well known in the art. Sequence alignment for comparison is performed by Smith and Waterman’s local homology algorithm, Adv.Appl.Math.2:482 (1981), Needleman and Wunsch’s global alignment algorithm, J.Mol.Biol.48:443 (1970), Pearson and Lipman’s similarity search method, Proc.Nat.Acad.Sci.85:2444 (1988), or computerized implementations of these algorithms, and includes, but is not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analysis is publicly available, for example, through the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0037] One example of amino acid sequence comparison is the BLASTP algorithm, which uses default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for nucleic acid sequence comparison is the BLASTN algorithm, which uses default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless otherwise noted, the sequence identity values ​​provided herein refer to values ​​obtained using the BLAST program suite, using the default parameters identified above for protein and nucleic acid comparison, respectively (Altschul, J.Mol.Biol.215:403-410(1990)).

[0038] When referring to the same array without specifying a percentage value, it means that the arrays are 100% identical (i.e., the same array).

[0039] In the context of this specification, the term “antibody” refers to all antibodies, including but not limited to immunoglobulins G type (IgG), A type (IgA), D type (IgD), E type (IgE), or M type (IgM), any antigen-binding fragment or single chain thereof, and associated or derived constructs. All antibodies are glycoproteins in which at least two heavy chains (H) and two light chains (L) are interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (V H ) and heavy chain constant region (C H It is composed of ) and . The heavy chain constant region of IgG is C H 1. C H 2, C H It consists of three domains. Each light chain has a light chain variable region (V in this specification). L(abbreviated as) and the light chain steady region (C L It is composed of ). The light chain constant region is a single domain called C L It is composed of the following: The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. The term "antibody" may also refer to "antibody-like molecules."

[0040] Similarly, the terms "antigen-binding antibody fragment" or "antigen-binding fragment" encompass a portion of an antibody molecule that retains antigen-binding ability, such as monovalent or bivalent antibody fragments (F(ab) or F(ab)2, respectively), so-called nanobody or single-domain antibodies, or V H and V L Selected from, but not limited to, antibody fragments consisting of one or more single monomer variable antibody domains including [specific component].

[0041] In the context of this specification, “humanized antibody” refers to an antibody originally produced by immune cells of a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. The term “humanized antibody” as used herein includes antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted onto a human framework sequence. Further modifications to the framework region may be made not only within the human framework sequence but also within the CDR sequence derived from the germline of another mammalian species.

[0042] The term "antibody-like molecule" in the context of this specification refers to a molecule that can specifically bind to another molecule or target with high affinity / Kd ≤ 10E-8 mol / l. Antibody-like molecules bind to their targets in the same way as the specific binding of antibodies. The term "antibody-like molecule" includes repeat proteins, for example, designed ankyrin repeat proteins (Molecular Partners, Zurich), modified antibody mimic proteins that exhibit high specificity and high affinity for target protein binding (see US Patent Application Publication No. 2012 / 142611, US Patent Application Publication No. 2016 / 250341, US Patent Application Publication No. 2016 / 075767, and US Patent Application Publication No. 2015 / 368302. All of these are incorporated herein by reference), and the like. The term "antibody-like molecule" further includes, but is not limited to, polypeptides derived from armadillo repeat proteins, polypeptides derived from leucine-rich repeat proteins, and polypeptides derived from tetratricopeptide repeat proteins.

[0043] The term "antibody-like molecule" further includes specific binding polypeptides derived from the following: - Protein A domain, - Fibronectin domain FN3, - Consensus fibronectin domain, - Lipocalin (Skerra, Biochim. Biophys. Acta 2000, 1482(1-2):337-50), - Polypeptides derived from zinc finger proteins (Kwan, Structure 2003, 11(7):803-813), - Src homology domain 2 (SH2) or Src homology domain 3 (SH3), - PDZ domain, - γ-Crystallin, - Ubiquitin, - Cystine knot polypeptides or knottins, - Cystatin, - Sac7d, - Triple helix coiled coil (also called alpha body), - Knitz domain or Knitz-type protease inhibitors, and - Carbohydrate binding module 32-2.

[0044] The term "protein A domain-derived polypeptide" refers to a derivative of protein A that can specifically bind to the Fc and Fab regions of immunoglobulins.

[0045] The term "armadillo repeat protein" refers to a polypeptide containing at least one armadillo repeat, characterized by a pair of α-helices that form a hairpin structure.

[0046] In the context of this specification, the term “humanized camel antibody” refers to an antibody consisting of a heavy chain only or a variable domain (VHH domain) of a heavy chain, whose amino acid sequence has been modified to increase its similarity to antibodies naturally produced in humans, resulting in reduced immunogenicity when administered to humans. General strategies for humanizing camel antibodies are described below: Vincke et al., “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” J Biol Chem. January 30, 2009; 284(5):3273-3284, and U.S. Patent Application Publication No. 2011165621(A1).

[0047] In the context of this invention, the term "specific binding" refers to the properties of a ligand, which binds to its target with a certain affinity and target specificity. Such ligand affinity is indicated by the ligand's dissociation constant. A ligand that reacts specifically has a dissociation constant of 10 when bound to its target. -7Although the dissociation constant is less than mol / L, interactions with molecules that have the same overall chemical composition as the target but different three-dimensional structures exhibit a dissociation constant at least three orders of magnitude higher.

[0048] In the context of this specification, the term “dissociation constant (KD)” is used in the sense known in the arts of chemistry and physics: it refers to an equilibrium constant that measures the tendency of a complex consisting of [mainly two] different components to reversibly dissociate into its (two) components. This complex can be, for example, an antibody-antigen complex AbAg, consisting of an antibody Ab and an antigen Ag. D This is expressed as a molar concentration [mol / l] and corresponds to the concentration of [Ab] that occupies half of the binding sites of [Ag]. In other words, the concentration of unbound [Ab] is equal to the concentration of the [AbAg] complex. The dissociation constant can be calculated using the following formula:

number

[0049] In the context of this specification, the off-rate (dissociation rate) (K off ;[1 / sec]) and on-rate (binding rate) (K on ;[L / sec * The term (K) is used in the sense known in the fields of chemistry and physics: these refer to the dissociation of an antibody with its target antigen (K). off ) or meeting (K on This refers to the rate constant used to measure (K). off and K on This can be determined experimentally using methods well established in the art. The K of the antibody off and K on Surface plasmon resonance is employed as a method for measuring this. This is the principle behind biosensor systems such as the Biacore® or ProteOn® systems. These also use the following equation to determine the dissociation constant K D It can be used to find:

number

[0050] As used herein, the term “pharmaceutical composition” refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, accompanied by at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present invention is provided in a form suitable for topical, parenteral, or infusion administration.

[0051] As used herein, the term “pharmaceutically acceptable carrier” includes, as is known to those skilled in the art, any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic, absorption retarder, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, etc., and combinations thereof (see, for example, Remington: The Science and Practice of Pharmacy, ISBN 0857110624).

[0052] As used herein, the terms “treatment” or “to treat” any disease or disorder (e.g., cancer) mean, in one embodiment, improving the disease or disorder (e.g., delaying, stopping, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, “treatment” or “to treat” means alleviating or improving at least one physical parameter, including one that may not be identifiable to the patient. In yet another embodiment, “treatment” or “to treat” means modulating the disease or disorder physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for evaluating the treatment and / or prevention of a disease are generally known in the art unless specifically described below.

[0053] In the context of the complexes provided herein, the terms "interleukin-2", "IL-2", or "hIL-2" refer to human IL-2 polypeptide, unless otherwise indicated. The numbering of human IL-2 residues throughout refers to that shown in FIG. 10 (UniProt P60568). This term encompasses recombinantly produced IL-2 proteins such as the substance teselokin (0094218-75-4), aldesleukin (CAS 110942-02-4), or its variant BAY 50-4798. Nuclear magnetic resonance spectroscopy suggests that the IL-2 protein contains four major helices (A - D) with two short helices disposed on either side (Arkin M.R et al., PNAS 2003, 100:1603 - 1608).

[0054] As used in the context of this specification, the term "trimeric IL-2 receptor" or "high-affinity IL-2 receptor" refers to a multimeric receptor composed of CD122 (IL-2Rβ), CD132, and CD25 (IL-2Rα). When CD25 is present, the affinity of IL-2R for IL-2 increases 100-fold compared to the dimeric IL-2R, so the trimeric IL-2R (K d ≒10 -11 M) is also called the high-affinity IL-2R.

[0055] In the context of this specification, the term "intermediate-affinity IL-2 receptor" or "dimeric IL-2 receptor" refers to an IL-2 receptor that includes CD122 (IL-2Rβ) and the common γ-chain (γc; CD132). This dimeric IL-2R exhibits an intermediate affinity for IL-2 with a dissociation constant [K -9 M of approximately 10 d , which is decreased by the steric hindrance of the antibody bound to the binding site of IL-2 when IL-2 interacts with the receptor in the context of a complex with an anti-hIL-2 mAb according to the present invention.

[0056] In the context of this specification, the term "peptide linker" refers to a polypeptide of variable length used to connect two polypeptides to generate a single-chain polypeptide. Exemplary embodiments of linkers useful in the practice of the invention as defined herein are oligopeptide chains consisting of 30, 40 or 50 amino acids.

[0057] The terms "regulatory T cells" and "T reg " in the context of this specification refer specifically to CD3 + CD4 + CD25 + immune cells. Intracellular expression of forkhead box P3 (Foxp3), the master transcription factor of T reg cells, identifies T cells with immunosuppressive function. Additional surface and intracellular markers associated with T reg cells are known in the art (Miyara M. et al., Autoimmun Rev 2011, 10:744). Also, instead of measuring the expression of T reg cell surface markers, it is also possible to estimate the number of T reg in a sample by measuring its suppressive function. Suppression assays useful for this purpose include combining a T reg [[ID=le containing sample with an activated immune cell sample, particularly a CD8 + T cell, and measuring the suppression of its function in terms of cell death, proliferation, or production of effector molecules selected from, but not limited to, granzyme, perforin, interferon γ (IFN) or tumor necrosis factor (TNF).

[0058] A first aspect of the invention is to provide an hIL-2 mAb, or an hIL-2 binding antibody fragment, wherein the hIL-2 specific mAb interacts with a specific epitope of hIL-2 amino acid residues comprising: - H16, D20 of the α helix; - Q57, E60, E61, L63, K64, E67, E68 of the B and B' helices; and - The L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104 of the C helix and C-D loop.

[0059] Crystal structure analysis reveals that when these two molecules associate, the following rhIL-2 residues, which are covered by the hIL-2 specific mAb antibody, are (> )5A 2 exceeded: - H16, D20; - Q57, E60, E61, L63, K64, E67, E68; and - L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104.

[0060] When these two molecules associate, the following rhIL-2 residues, which are covered by the hIL-2 specific mAb antibody, are (> )15A 2 exceeded, thus highly likely mediating important biological effector functions that are characteristics of the antibody as outlined below: - H16; - Q57, E60, E61, K64, and - L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, K97, T101, M104.

[0061] The data provided in the examples suggest that the hIL-2 specific mAb according to the present invention is different from the previously described IL-2 mAb F5111.2 clone because the epitope of the antibody according to the present invention does not include M23, G27, N71, Q74, S75, K76, N77, F78, P82 of the hIL-2 residue.

[0062] The crystal structure analysis provided in the examples shows that the interaction between anti-hIL-2 mAbs and these specific residues blocks or interrupts the interaction site between IL-2 and the intermediate affinity IL-2 receptor consisting of CD122 and CD132. The complex of anti-hIL-2 mAbs with IL-2 polypeptide preferentially or biasedly associates with CD25, or with the high affinity trimer IL-2R consisting of CD25, CD122, and CD132, and as a result, T reg IL-2 signaling is increased in cells possessing this receptor, such as [examples of cells]. The anti-hIL-2 mAb epitope according to the present invention contains the hIL-2 residues Q57, E60, E61, L64, and K64. The 6.3% overlap of IL-2 CD25 binding obtained by the anti-hIL-2 mAb epitope provided herein (and summarized in Figures 9 and 10 of the Examples) helps release IL-2 from the anti-hIL-2 mAb complex and deliver it to the receptor unimpeded, resulting in improved IL-2 signaling compared to other antibody clones that do not contain these residues in the epitope.

[0063] In the context of this invention, an "epitope" of hIL-2 refers to an amino acid residue of the hIL-2 polypeptide to which an hIL-2-specific mAb or antibody fragment according to the present invention specifically binds or adheres. The hIL-2 epitope was defined by structural analysis of hIL-2 crystallized as a complex with a Fab variant of the parental mouse antibody UFKA-20, and its CDR was used to generate humanized UFKA-22 clones and other hIL-2-specific mAbs according to the present invention (Table 1, Figures 9, 10, and 11). Equilibrium surface plasmon resonance (SPR) was performed on IL-2 captured by immobilized UFKA-20 to determine the crystal structure of the complex. The buried surface area (square angstroms or Å) of the amino acid residues containing the epitopes according to the present invention was calculated using the software PDBePISA (Krissinel, J Mol Biol 2007 372, 774-797). 2It is defined as having a value greater than 0 (measured by). In other words, an hIL-2 epitope contains, or consists of, IL-2 amino acids that the heavy and light chain polypeptides of the UFKA-20 antibody, particularly the complementary determination region (CDR), contact, interact with, cover, or overlap, as well as residues that the antibody does not cover or that do not interfere with receptor interaction.

[0064] The data from the examples demonstrate the usefulness of a complete antibody molecule containing the hIL-2 epitope identified above for forming a complex with hIL-2 that enhances hIL-2 signaling to the trimer IL-2R. Previous studies have shown that F(ab')2 antibody fragments lacking the Fc portion of the antibody can also form hIL-2 / mAb complexes with specific signaling bias; however, these fragments have short half-lives and require more frequent administration. Repeated injection of an IL-2-specific F(ab')2 complex with IL-2, rather than IL-2 alone, mimicked the potent activity of the IL-2 / mAb CD122 target complex, indicating that the CDR-containing portion of the specifically binding anti-IL-2 mAb is important for in vivo activity (Letourneau, PNAS 2010, 107(5):2171). Furthermore, in related studies using the IL-7 / anti-IL-7 (clone M25) mAb complex, the inventors demonstrated that the F(ab')2 antibody fragment functions similarly to the whole antibody molecule (Boyman, J Immunol 2008, 180(11):7265). Therefore, antibody fragments lacking the Fc portion, such as the Fab and F(ab')2 fragments, as well as the scFv fragment, are included in the term "antibody" or "antibody fragment" according to the present invention.

[0065] A second aspect of the present invention is an anti-hIL-2 mAb, or antigen-binding antibody fragment, defined by the characteristics of the interaction between an anti-hIL-2 antibody and an hIL-2 polypeptide. This aspect of the present invention provides an anti-hIL-2 mAb, or hIL-2-binding antibody fragment, wherein the binding of the hIL-2-specific mAb to hIL-2 is characterized as follows: - Dissociation constant (K D ) ≤ 5.51 × 10 -9 molL -1 , especially K D ≤5.13 × 10 -9 molL -1 (K of clone UFKA-22-00) D ); - On-rate (K on )≧4.12×10 5 Lmol -1 s -1 (K of clone UFKA-22-02) on ), especially K on ≥4.66 × 10 5 Lmol -1 s -1 (K of clone UFKA-22-00) on ); - Off rate (K off ) ≤ 2.83 × 10 -3 s -1 (K of clone UFKA-22-10) off ), especially K off ≤2.39 × 10 -3 s -1 (K of clone UFKA-22-00) off ).

[0066] The hIL-2 mAb according to this embodiment of the present invention is K D Value ≤ 5.51 × 10 -9 molL -1 This has the highest KD value of anti-hIL-2 mAb UFKA-22-02 in the selection of UFKA-20-derived clones that have similar hIL-2 binding properties and have been shown to share the same CDR sequence. In certain embodiments, the anti-hIL-2 mAb according to the present invention has K D Value ≤ 5.13 × 10 -9 molL -1 This has the K of the hIL-2 specific clone UFKA-22-00. D The value does not have a reverse mutation in the mouse framework region that could generate an immune response in patients administered this mAb as a pharmaceutical component. Secondly, the anti-hIL-2 mAb according to this aspect of the present invention is Kon The value is (≧) 4.12 × 10 5 Lmol -1 s -1 That is all (derived from clone UFKA-22-02), especially K on The value is (≧) 4.66 × 10 5 Lmol -1 s -1 The above (derived from UFKA-22-00). Finally, the anti-hIL-2 mAb according to this embodiment of the present invention is K off ≤2.83 × 10 -3 s -1 (Derived from clone UFKA-22-05) In particular, K off ≤2.39 × 10 -3 s -1 (K of UFKA-22-00) off) The present inventors have found that antibodies that bind to IL-2 having the characteristics outlined above have similar IL-2 binding and receptor delivery dynamics to those of the mouse UFKA-20 clone (sequence number 019 for HC and sequence number 020 for LC) used in the preclinical studies of the examples. Antibodies having the above characteristics that form a complex with hIL-2 will preferentially deliver the IL-2 signal to high-affinity IL-2R. Importantly, upon binding to the receptor, the antibody will also dissociate from the complex, in other words, separate from the IL-2 polypeptide, to enable unhindered and optimal delivery of signaling stimuli without steric hindrance by the antibody.

[0067] In other embodiments, the interaction between hIL-2 and the hIL-2 mAb or fragment thereof according to the present invention is K D ≤5.51 × 10 -9 molL -1 , especially K D ≤5.13 × 10 -9 molL -1 Characterized by, this is also 1.856 -10 This exceeds the upper limit of affinity. In the 5+9 antibody of the mutant heavy and light chains that binds to IL-2 with the highest affinity, the decrease in in vivo activity is CD4 + CD25 + Foxp3 + T regObserved with respect to cell stimulation. In certain embodiments, the K of the interaction D It is about 10 -10 It is M.

[0068] In some embodiments, the anti-hIL-2 mAb according to the present invention is characterized by the biological function of the complex between hIL-2 and the anti-hIL-2 mAb, as measured by in vitro or in vivo experimental methods. The hIL-2 and anti-hIL-2 mAb complex according to the present invention comprises an hIL-2 polypeptide non-covalently associated with the anti-hIL-2 mAb according to the present invention. These hIL-2 mAb complexes have been demonstrated to function effectively when these elements are combined in a 2:1 ratio (Boyman, Science 2006, 311:1924; Krieg, PNAS 2010, 107:11906; Arenas-Ramirez, Sci Transl Med 2016, 8,:367ra166) or in a 1:1 ratio (Letourneau, PNAS 2010, 107:11906; Arenas-Ramirez, Sci Transl Med 2016, 8,:367ra1660). The binding (combination) of the two components of the complex takes place in solution, and the time, temperature, and conditions of this combination procedure are not particularly limited by the present invention. The complex can be formed, for example, by binding hIL-2 and an anti-hIL-2 mAb in a physiological solution such as phosphate-buffered saline at room temperature for 15 minutes. It shifts IL-2 signaling towards IL-2Rα or IL-2Rβ, and therefore T reg or CD8 + Preparation and activation of IL-2 mAb complexes using other mAbs that increase STAT5 phosphorylation in T cells have been achieved (Letourneau, PNAS 2010, 107:11906; Krieg, PNAS 2010, 107:11906; Trotta, Nat Med 2018, 24:1005).

[0069] In certain embodiments, the complex of hIL-2 and the anti-hIL-2 mAb according to the present invention showed increased binding affinity to high-affinity IL-2R consisting of CD25, CD122, and CD132 compared to binding affinity to intermediate-affinity IL-2R consisting of CD122 and CD132. In other words, the ratio of binding to the high-affinity receptor was greater than 1 compared to binding to intermediate-affinity IL-2R. In particular, this ratio was greater than 2, 4, or even 8. More specifically, binding to the high-affinity receptor was 20 to 121 times greater than binding to the intermediate-affinity receptor, and even more particularly, 71 times greater. Data from examples evaluating the interaction between the receptor and the hIL-2 complex showed a difference of 71 in magnification and an error range of ±50.

[0070] In certain embodiments, the hIL-2 and anti-hIL-2 mAb complex according to the present invention showed increased binding affinity to CD25 alone compared to the intermediate affinity receptor, with a particularly high affinity multiplier change of 277 to 483 times for CD25, and even higher than 380 times for the intermediate affinity receptor. Data from examples evaluating the interaction between the receptor and the hIL-2 complex showed a multiplier change of 380 and an error range of ±103.

[0071] In the data shown in the examples, IL-2 Rhod / UFKA-20cx preferentially associates with CD25, and approximately two-thirds of the measured interactions are with IL-2. Rhod / UFKA - This was done by 20cx, while IL-2 Rhod Less than one-third were detected by CD25 when used alone. IL-2 Rhod The coupling of / UFKA-20cx to CD122+CD132 is IL-2 Rhod Compared to this, the presence of UFKA-20 puts it at a disadvantage (Figure 1D), and as a result UFKA-20 is at a disadvantage in IL-2 Rhod It was demonstrated that "CD25 bias" could be imparted to it.

[0072] In certain embodiments of anti-hIL-2 mAbs, when the complex binds to a high-affinity hIL-2 receptor, the anti-hIL-2 mAb dissociates from the hIL-2-anti-hIL-2 mAb complex. In other words, the two components associate before binding to the receptor, and upon binding, free hIL-2 is released, enabling optimal signal transduction via the receptor.

[0073] In Figure 1D of the example, CD25 deflection and dissociation of the anti-hIL-2 mAb from hIL-2 are carried out by deflecting the delivery of the complex to CD25 / CD122 / CD132 or CD25 expressed by HEK cells, as observed using flow cytometry in which each component is fluorescently labeled, leaving the receptor-bound IL-2 in place. Rhod / UFKA-20cx rapidly dissociates when it interacts with the trimer CD25+CD122+CD132, and this interaction is IL - 2 Rhod The interaction formed by / UFKA-20cx is less than 8.4%, and the free IL-2 is not complexed. Rhod This is evident from the fact that 91.6% of the compounds bind to CD25+CD122+CD132 (Figures 1D-1F). "Delivery" refers to the fact that when delivered as a complex of hIL-2 with the anti-hIL-2 mAb according to the present invention, free IL2 rhodamine binds to CD25 / CD122 / CD132. + This refers to binding to more than 40% of cells. UFKA-30 and UFKA-40 are IL-2 Rhod Although the bias toward CD25 was increased, IL-2 could not be dissociated or delivered to the trimer IL-2R (Figure 1D to H).

[0074] In certain embodiments, when the anti-hIL-2 mAb according to the present invention is delivered to cells in complex with hIL-2, it has an EC50 < 0.154 ng / ml of human CD3 + CD4 + Foxp3 + T reg Activates cells and human CD8 with EC50 > 442.9 ng / ml + Activate T cells. To calculate the EC50 of the complex, Treg or CD8 + The methodology for measuring T cell activation is not particularly limited by the present invention. The data shown in the examples herein demonstrate the activation of T cells by the hIL-2 complex with an anti-hIL-2 mAb. reg Activation is measured by phosphorylated STAT5 (pSTAT5) levels induced in human T cells, as shown in Figure 6, measured by flow cytometry after cell culture. However, it may also be measured by other activation characteristics such as proliferation or effector cytokine production.

[0075] Alternatively, in other embodiments of the anti-hIL-2 mAb, T reg Preferential promotion of proliferation is achieved by CD8 after treatment with the complex. + CD44 hi CD122 + CD3 against memory T cells + CD4 + CD25 + T reg This is determined by checking the cell ratio. In the example, this treatment resulted in CD8 levels in monkey spleen or lymph nodes (Figure 8) or in human cells cultured in vitro (Figure 6). + Compared to the increase in memory T cells, reg This resulted in a 2-3 times increase. In other words, when the anti-hIL-2 mAb according to the present invention was delivered to human or monkey cells in a complex with IL-2, CD8 + CD44 hi CD122 + T cells compared to the rate of increase in T cells reg The ratio of cell growth is greater than 1 compared to either untreated or previously treated samples. reg The activation ratio is alternatively limited to, but is not limited to, CD8 + This can be calculated by comparing it with the total number or proportion of IL2Rα- cells, such as T cells, natural killer cells, and / or B cells.

[0076] A third aspect of the present invention is a CDR H 1. CDR H 2 and CDR H V characterized by 3H Area and CDR L 1. CDR L 2 and CDR L Light chain variable region including 3, especially kappa light chain (V L The hIL-2 specific mAb or its antigen-binding antibody fragment includes the ) region. According to this aspect of the present invention, CDR H 1. CDR H 2. CDR H 3. CDR L 1. CDR L 2 and CDR L 3 includes, or is identical to, SEQ ID NO: 001, SEQ ID NO: 002, SEQ ID NO: 003, SEQ ID NO: 004, SEQ ID NO: 005, and SEQ ID NO: 006, respectively. The anti-hIL-2 mAb according to this aspect of the present invention may optionally be characterized by an epitope or binding property according to any of the above aspects of the present invention.

[0077] In another embodiment, an anti-hIL-2 mAb according to the present invention, or its antigen-binding fragment, in particular an antibody characterized by any of the above embodiments, is characterized by: a. Select V from sequence numbers 007, 008, 009, 010, 011, 012, 013, or 014. H CDR included in the array H 1. CDR H 2, and CDR H 3, and also, b. V selected from sequence number 015 or sequence number 016 L CDR included in the array L 1. CDR L 2, and CDR L 3. In particular, the CDR L This is included in sequence number 015.

[0078] In the data shown in the examples, Table 2 shows that the same K in the series of hIL-2 antibodies corresponding to the above sequence off , K on , K DThe values ​​are shown, demonstrating that these are functional substitutes. These biochemical values ​​are predictive indicators of function and performance based on other in vitro or in vivo measurements described in the preceding aspects of the present invention. The UFKA-22-00 clone, with the heavy chain of SEQ ID NO: 007 and the light chain of SEQ ID NO: 015, is considered most desirable only if other options contain “reverse” mutations similar to the mouse Ig molecule and may have a higher risk of generating anti-drug immunity in human patients.

[0079] The hIL-2 specific mAb according to the present invention, and in particular the fourth aspect of the hIL-2 specific mAb according to any of the above embodiments, has the same (≧)96% or more V as SEQ ID NO: 007. H It is an anti-hIL-2 mAb characterized by its regional sequence, and in particular, - Position 74 and / or 84 is serine, and / or - The 93rd position is methionine, and / or - Alanine is ranked 122nd. Sequence ID 007 and V are (≥) 96% identical. H This is an anti-hIL-2 mAb characterized by its regional sequence.

[0080] Furthermore, the mAb according to this aspect of the present invention is (≧)99% or more identical to that of SEQ ID NO: 015. L Characterized by domain, in particular, - Isoleucine is ranked 69th. Sequence ID 015 and (≧) 99% or more identical V L Characterized by region.

[0081] The data in Table 2 of the Examples show clones with essential CDR regions and further framework mutations that interact with hIL-2 similarly to SEQ ID NOs. 007 and 015 of the primary heavy chain and light chain sequences, tested in the widest range of functional assays. When all positions of the heavy chain described in a. are different, the sequence differs from SEQ ID NOs. 007 by 5.74%, in other words, it is more than 96% identical to SEQ ID NOs. 007. When position 69 of the light chain is replaced with isoleucine, the resulting SEQ ID NOs. 016 sequence differs from the parent SEQ ID NOs. 15 sequence by 0.88%, in other words, it is more than 99% identical to SEQ ID NOs. 015.

[0082] A fifth aspect of an hIL-2 specific mAb, or its antigen-binding fragment, particularly as characterized by any of the foregoing aspects or embodiments of the present invention, is a V having at most two, or particularly one, conservative amino acid substitutions. H Array or V L It is an anti-hIL-2 mAb with a specific sequence. In other words, the V of the anti-hIL-2 mAb. H The region is sequence number 007(V H 1) Sequence ID 008(V H 2) Sequence ID 009(V H 3) Sequence ID 010(V H 4), Sequence ID 011(V H 5), Sequence ID 012(V H 6), Sequence ID 013(V H 7), or sequence number 014(V H 8) or a sequence selected from any one of these reference sequences, or a functionally similar sequence derived from one of these reference sequences by the substitution rules shown below. Furthermore, the V of anti-hIL-2 mAb L The region is sequence number 015(V L 1) Sequence ID 016(V L 2) Includes a sequence selected from these reference sequences, or a functionally similar sequence derived from these reference sequences according to substitution rules. The rules for this possible conservation of amino acids describe amino acids that can be replaced and result in sequences functionally similar to their respective reference sequences, possessing similar biochemical properties. These substitution rules are: I. Glycine (G) and alanine (A) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable, and A and V are interchangeable; II. Tryptophan (W) and phenylalanine (F) are interchangeable, and tyrosine (Y) and F are interchangeable; III. Serine (S) and threonine (T) are interchangeable; IV. Aspartic acid (D) and glutamic acid (E) are interchangeable; V. Asparagine (N) and glutamine (Q) are interchangeable, N and S are interchangeable, N and D are interchangeable, and E and Q are interchangeable; VI. Methionine (M) and Q are interchangeable; VII. Cysteine ​​(C), A, and S are interchangeable; VIII. Proline (P), G, and A are interchangeable; IX. Arginine (R) and lysine (K) are interchangeable;

[0083] A further aspect of the present invention is to provide an anti-hIL-2 mAb, wherein the antibody or antibody fragment is characterized by an epitope according to a first aspect of the present invention or is bound to hIL-2 in a manner provided by a second aspect of the present invention, and comprises: a. Sequence ID 007(V H 1) Sequence ID 008(V H 2) Sequence ID 009(V H 3) Sequence ID 010(V H 4), Sequence ID 011(V H 5), Sequence ID 012(V H 6), Sequence ID 013(V H 7), Sequence ID 014(V H 8) A first sequence that is identical by (≧)90% or more, in particular by (≧)94% or more, (≧)96% or more, or even more by (≧)98% or more, to at least one of the above; and b. Sequence ID 015(VL 1) Sequence ID 016(V L 2) A second sequence that is (≧)90% or more identical, in particular (≧)94% or more, (≧)96% or more, or further (≧)98% or more identical, to at least one of the above, or sequence number 018(LC).

[0084] In a particular embodiment of the anti-hIL-2 monoclonal antibody, the antibody comprises a heavy chain having the sequence specified in SEQ ID NO: 017, which associates with a light chain having the sequence specified in SEQ ID NO: 018.

[0085] A subsequent aspect of the present invention provides an hIL-2 fusion protein comprising an hIL-2 specific mAb according to any one aspect of the present invention and an hIL-2 polypeptide, wherein the two components are linked by a peptide linker.

[0086] The hIL-2 fusion protein contains an hIL-2 specific mAb, which consists of an antibody heavy chain having an N-terminus and a C-terminus, and an antibody light chain having an N-terminus and a C-terminus. The antibody heavy chain has sequences designated as SEQ ID NO: 001, SEQ ID NO: 002, and SEQ ID NO: 003, respectively, from the N-terminus to the C-terminus. H 1. CDR H 2, and CDR H Includes 3. In certain embodiments, these CDRs are selected from SEQ ID NOs: 007, 008, 009, 010, 011, 012, 013, or 014. H It is included in the sequence. In a more specific embodiment, these three CDRs H This is included in SEQ ID NO: 007. Similarly, the antibody light chain has complementary determination region CDRs with sequences of SEQ ID NO: 004, SEQ ID NO: 005, and SEQ ID NO: 006, respectively. L 1. CDR L 2, and CDR L Includes 3. In certain embodiments, these are V selected from sequence number 015 or sequence number 016. L Included in the array. In a more specific embodiment, these CDRs L This is included in sequence number 015.

[0087] The hIL-2 polypeptide portion of the hIL-2 fusion protein according to the present invention may also have an N-terminus and a C-terminus and may be any natural IL-2 polypeptide or a recombinant IL-2 protein such as tesseleukin or aldesleukin. In a particular embodiment, the polypeptide sequence of the hIL-2 portion of the fusion protein is the sequence of the hIL-2 protein P60568.

[0088] The peptide linker that conjugates the antibody to the IL-2 portion of the fusion protein according to the present invention preferably has an amino acid length between 30 and 50. In a particular embodiment, the peptide linker has an amino acid length between 30 and 40. In a more particular embodiment, the peptide linker has an amino acid length between 30 and 35. In an even more particular embodiment, the peptide linker has an amino acid length of about 30. In a particular embodiment, the peptide linker of the hIL-2 fusion protein according to the present invention conjugates the C-terminus of the hIL-2 polypeptide to either the N-terminus of the antibody heavy chain or the N-terminus of the antibody light chain. In a more particular embodiment, the peptide linker conjugates the C-terminus of the hIL-2 polypeptide to the N-terminus of the antibody light chain, as shown in the embodiment of the LC component of the hIL-2 fusion protein mAb specified in SEQ ID NO: 028.

[0089] In certain embodiments of the hIL-2 fusion protein according to the present invention, the peptide linker is composed of approximately 85% glycine and approximately 15% serine amino acid residues, which are the residues that result in reduced immunogenicity. In certain embodiments of the hIL-2 fusion protein, the peptide linker has the sequence of SEQ ID NO: 027. In even more specific embodiments, the hIL-2 fusion protein is a bivalent molecule in which each heavy chain or each light chain is independently fused to hIL-2 by the peptide linker. In other embodiments, the hIL-2 fusion protein includes a signal peptide that allows the secretion of recombinant protein, for example, a signal peptide having the sequence of SEQ ID NO: 027. In even more specific embodiments, the hIL-2 fusion protein is V HIt consists of an LC fused to hIL-2 that provides a sequence designated as sequence number 028, which further associates with sequence number 017 of the chain.

[0090] A subsequent aspect of the present invention provides a nucleic acid molecule encoding an hIL-2 specific mAb or antigen-binding antibody fragment as described in any one of the above aspects or embodiments. Another aspect of the present invention relates to a nucleic acid molecule encoding an hIL-2 fusion protein according to the present invention. Another aspect of the present invention provides a vector comprising the nucleic acid, while a further aspect provides a cell or monoclonal antibody-producing hybridoma strain comprising an anti-hIL-2 mAb or fragment thereof, an hIL-2 fusion protein, a nucleic acid, or a vector as described in the above aspects of the present invention.

[0091] Another aspect of the present invention is a pharmaceutical formulation for use as a medicine, particularly for use in the treatment of patients with adverse immune-mediated inflammation, more particularly chronic inflammation such as allograft-related disorders, vasculitis, or adverse immune-mediated infiltration resulting from allergies or autoimmune diseases. The pharmaceutical formulation according to this aspect of the present invention comprises at least two components: a. an hIL-2 specific mAb or antigen-binding fragment thereof as described in any one of the above embodiments or models of the present invention, and b. hIL-2, Includes.

[0092] The inventors believe that the improved IL-2Rα deflection delivered by the IL-2-specific mAb according to the present invention, conjugated with the hIL-2 polypeptide, can provide improved therapeutic efficacy in medical indications that can be treated with current IL-2 administration approaches. IL-2 immunotherapy has been shown in human clinical trials to alleviate rheumatoid arthritis, ankylosing spondylitis, psoriasis, inflammatory bowel disease, autoimmune hepatitis, amyotrophic lateral sclerosis, HCV-associated vasculitis, type 1 diabetes, chronic graft-versus-host disease (GVHD), lupus, alopecia areata, and systemic lupus erythematosus, and to improve liver transplant protocols (Ye, Signal Transduct Target Ther 2018, 3:2; Sharabi, Nat. Rev. Drug Discov. 2018, 17:823). Mouse models of human diseases have shown that IL-2-based immunotherapy that improves IL-2Rα signaling can improve the clinical features of metabolic diseases such as multiple sclerosis, inflammatory or autoimmune myopathy, inflammatory colitis, lupus, xenoglossal GVHD, allergic asthma, obesity-related inflammation and insulin resistance that characterize type 1 and type 2 diabetes, as well as atherosclerosis and Duchenne muscular dystrophy (Arenas-Ramirez, Trends Immunol 2015, 36:763; Tang, Immunity 2008, 28:687; Webster, J. Exp. Med. 2009, 206:751; Lee, Immunol. 2012, 137:305; Spangler, Immunity 2015, 42:815; Yan, Kidney Int. 2017, 91:603; Trotta, Nat Med 2018, 24:1005).

[0093] For example, immune-mediated conditions whose clinical outcomes have been improved by IL-2-enhanced immunotherapy, achieved by low-dose IL-2, recombinant IL-2 molecules, or IL-2-containing pharmaceutical formulations, may also be considered for treatment with hIL-2 mAbs and hIL-2 complexes according to the present invention. Such immune-mediated medical indications include, for example, chronic inflammatory diseases, allergies, or autoimmune diseases, and metabolic diseases that are suitable for IL-2 immunotherapy. Furthermore, the treatment of allograft-related disorders may include, for example, whole organ transplants, tissue transplants, or bone marrow transplants, and may include, as a coordination approach, the application of IL-2-specific mAbs that associate with IL-2 before and / or after the transplant procedure. In certain embodiments, allograft-related disorders are whole organ transplants, e.g., kidney transplants or lung transplants.

[0094] The data provided in the examples show that anti-hIL-2 mAbs and IL-2 used as pharmaceuticals in vivo associate in a 1:1 ratio and substantially do not contain free hIL-2, although the ratio of the combined components may vary, for example, 2:1, 1:1, or even 1:2. By injecting this type of complex parenterally or topically, T is produced against inflammatory cells. reg It increases the ratio and suppresses immune activation that causes harmful histopathology in allergies, infections, or autoimmune diseases.

[0095] In some embodiments, the hIL-2 and anti-hIL-2 mAbs contained in the pharmaceutical formulation are covalently associated. The data from the examples suggest that the dissociation of IL-2 from the anti-hIL-2 mAb is a distinctive feature of the mAb according to the present invention, and that any linkage between hIL-2 and the hIL-2 mAb according to these embodiments should not inhibit the ability of hIL-2 to deliver optimal signals via high-affinity IL-2R. In certain embodiments, the covalently associated hIL-2 and anti-hIL-2 mAb have the form of an hIL-2 fusion protein according to the present invention.

[0096] In other embodiments, the hIL-2-containing pharmaceutical composition according to the above-described aspect of the present invention is a compound pharmaceutical further comprising the following: - mTOR inhibitors, particularly mTOR inhibitors selected from rapamycin (sirolimus) and everolimus. - Anti-inflammatory mAbs, particularly mAbs selected from anti-TNF, anti-IL-6, or anti-OX40L blockers, - Corticosteroid drugs, - Sphingosine-1-phosphate (S1P) pathway inhibitors, particularly S1P pathway inhibitors selected from FTY720 or S1P receptor blockers. - Anti-inflammatory antioxidants, particularly anti-inflammatory antioxidants selected from metformin or N-acetylcysteine, - Calmodulin kinase type II or type IV inhibitors, - PI3K inhibitors, or pyrazopyramidine derivatives, - HDAC6 and other T reg Cell-specific histone deacetylase, - T reg Cell therapy, for example, chimeric antigen receptor or transgenic T cell receptor T reg Therapy, and / or - Low-dose IL-2, Ig-fused IL-2, or pegylated IL-2.

[0097] The synergistic effect of the hIL-2 and anti-hIL-2 mAb complex according to the present invention and the above-mentioned pharmaceutical product is attributed to their complementary mechanisms of action, according to a review by experts in the fields of IL-2 biology and oncology (Sharabi A. et al., Nat. Rev. Drug Discov. 2018, 17:823).

[0098] Another aspect of the present invention is a method for treating immune inflammation, the method comprising: i. Selecting patients diagnosed with adverse inflammation, particularly allograft-related disorders, chronic inflammation, allergies, or autoimmune conditions, and ii. Administering an anti-hIL-2 mAb according to any one of claims 1 to 8 and hIL-2 in a complex in a 2:1 or 1:2 ratio, particularly a complex in a 1:1 ratio.

[0099] Further embodiments provide the use of an hIL-2 specific mAb, antigen-binding fragment, or hIL-2 fusion protein according to any of the above embodiments of the present invention in the manufacture of a pharmaceutical product for use in the treatment of immune-mediated diseases, particularly allograft-related diseases, chronic inflammation, allergies, or autoimmune diseases.

[0100] Another aspect of the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to an hIL-2 epitope, comprising hIL-2 residues H16, D20, Q57, E60, E61, L63, K64, E67, E68, L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, and M104, but excluding hIL-2 residues M23, G27, N71, Q74, S75, K76, N77, F78, and P82.

[0101] The final aspect of the present invention provides an isolated antibody or an antigen-binding fragment thereof, comprising an antigen-recognizing surface having epitope recognition properties equivalent to those of an antibody or molecule as described in any of the above descriptions.

[0102] Medical supplies, dosage forms, and salts Similarly, the scope of the present invention includes treating or a method thereof for treating inflammatory disorders in patients in need, including administering to a patient a pharmaceutical composition comprising hIL-2 and an anti-hIL-2 mAb as described above.

[0103] In certain embodiments, the anti-hIL-2 mAb is an antibody, an antibody fragment, an antibody-like molecule, or a polypeptide derived from the protein A domain.

[0104] In some embodiments, the anti-hIL-2 mAb is an immunoglobulin comprising two heavy chains and two light chains. In some embodiments, the anti-hIL-2 mAb is a single-domain antibody comprising a variable domain isolated from the heavy chain or light chain.

[0105] In certain embodiments, the anti-hIL-2 mAb is an antibody fragment. In certain embodiments, the anti-hIL-2 mAb is a Fab fragment, i.e., an antigen-binding fragment of an antibody, or a single-chain variable fragment, i.e., a fusion protein in which the variable regions of the heavy and light chains of an antibody are linked by a peptide linker. Multiple single-chain variable fragments with the same or different antigen specificities may be linked in a multimer format to form two or more separate epitope-binding regions.

[0106] In other embodiments, the composition comprises covalently linked hIL-2 and anti-hIL-2 mAbs. In specific embodiments, the composition comprises an hIL-2 fusion protein.

[0107] Similarly, a dosage form for the prevention or treatment of inflammatory symptoms is provided, comprising an anti-hIL-2 mAb and IL-2 conjugate according to any of the above aspects or embodiments of the present invention.

[0108] Those skilled in the art will recognize that any of the drugs specifically mentioned may exist as pharmaceutically acceptable salts thereof. Pharmaceutically acceptable salts include ionized drugs and counterions with the opposite charge. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetates, benzoates, besilates, bitatrates, bromides, carbonates, chlorides, citrates, edetates, edisylates, embonates, estolates, fumarates, gluceptates, glucons, hydrobroms, hydrochlorides, iodides, lactates, lactobionates, malates, maleates, mandelates, mesylates, methyl bromides, methyl sulfates, mucinates, napsylates, nitrates, pamosates, phosphates, diphosphates, salicylates, disalicylates, stearates, succinates, sulfates, tartrates, tosylates, triethiozides, and valersates. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0109] The Il-2 complex has been successfully administered via subcutaneous, intravenous, and intraperitoneal routes in experimental models; therefore, parenteral administration, such as subcutaneous, intravenous, intrahepatic, or intramuscular injection, is possible. However, the inventors anticipate local administration or enteral administration, such as nasal, oral, rectal, transdermal, or oral administration. Alternatively, administration in the form of inhalation or suppository may also yield desirable physiological results. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0110] Topical administration is also within the scope of the advantageous uses of the present invention. Those skilled in the art will be familiar with a wide range of possible formulations for providing topical formulations, as exemplified by the following: Benson and Watkinson (eds.), Topical and Transdermal Drug Delivery: Principles and Practice (Part 1, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (Part 2, CRC Press 2002, ISBN-13: 978-0824708610); and Osborne and Amann (eds.), Topical Drug Delivery Formulations (Part 1, CRC Press 1989; ISBN-13: 978-0824781835).

[0111] Pharmaceutical composition and administration Another aspect of the present invention relates to a pharmaceutical composition comprising the compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0112] In certain embodiments of the present invention, the compounds of the present invention are typically formulated into pharmaceutical dosage forms to provide a controlled dose of the drug.

[0113] In embodiments of the present invention relating to the topical use of the compounds of the present invention, the pharmaceutical composition is formulated in a manner suitable for topical administration, such as an aqueous solution, suspension, ointment, cream, gel, or sprayable formulation, for delivery by an aerosol, and contains the active ingredient together with one or more solubilizers, stabilizers, isotonic enhancers, buffers, and preservatives known to those skilled in the art.

[0114] The pharmaceutical composition can be formulated for oral, parenteral, or rectal administration. Furthermore, the pharmaceutical composition of the present invention may be in solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or liquid form (including, but not limited to, solutions, suspensions, or emulsions).

[0115] The administration plan for the compounds of the present invention will vary depending on known factors such as the pharmacodynamic properties of the particular drug and the mode and route of administration: the recipient's species, age, sex, health status, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect. In certain embodiments, the compounds of the present invention may be administered in a once-daily dose, or the total daily dose may be divided into two, three, or four doses per day.

[0116] In certain embodiments, the pharmaceutical compositions or combinations of the present invention may have a unit dose of approximately 1 to 1000 mg of active ingredient(s) per subject weighing approximately 50 to 70 kg. The therapeutically effective dose of a compound, pharmaceutical composition, or combination thereof depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian with ordinary skill can easily determine the effective amount of each active ingredient necessary for the prevention, treatment, or inhibition of the progression of the disorder or disease.

[0117] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical procedures such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. These can be produced by standard processes, such as conventional mixing, granulation, dissolution, or freeze-drying processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art; see, for example, L. Lachman et al., The Theory and Practice of Industrial Pharmacy, Vol. 4, 2013 (ISBN 8123922892).

[0118] Where, in this specification, alternative forms of a single separable feature, such as an isotype protein or coding sequence, ligand type, or medical indication, are described as “embodiments,” it should be understood that such alternative forms may be freely combined to form separate embodiments of the invention disclosed herein. Accordingly, any alternative embodiment relating to an antibody may be combined with any medical indication described herein.

[0119] The present invention further includes the following: Item 1. A human interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or an antigen-binding fragment thereof, wherein the hIL-2 specific mAb interacts with an amino acid residue of hIL-2 that provides an epitope, and The aforementioned epitope is an hIL-2 residue: - H16, D20, - Q57, E60, E61, L63, K64, E67, E68, and - L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104, including, The aforementioned human interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or its antigen-binding fragment. Item 2. The binding of the aforementioned hIL-2-specific mAb to hIL-2 is: - Dissociation constant (K D ) is (≦) 4.3 × 10 -9 The following, in particular K D (≦) 5.13 × 10 -9 The following: - On-rate (binding rate) (K on ) is (≧)4.12 × 10 5 Ms -1 That's all, especially K on (≧)4.66 × 10 5 Ms -1 The above, - Off-rate (dissociation rate) (K off) is (≦)2.20 × 10 -3 s -1 The following, in particular K off (≦)2.39 × 10 -3 s -1 The following: Characterized by, hIL-2 specific mAbs, or antigen-binding fragments thereof, in particular the hIL-2 specific mAbs, or antigen-binding fragments thereof, as described in item 1. Item 3. Complexes obtained by combining the aforementioned hIL-2-specific mAb and hIL-2 in a ratio between 2:1 and 1:2, and especially complexes obtained by combining them in a 1:1 ratio: - The binding ratio to high-affinity hIL-2 receptors compared to intermediate-affinity hIL-2 receptors is between 20 and 121, in particular the ratio is between 71 and 121, and / or - The ratio of CD25 binding affinity to intermediate affinity hIL-2 receptor is between 277 and 483, in particular the ratio is between 380 and 483, and / or - Dissociation of hIL-2 mAb from hIL-2 in the binding of the complex to the high-affinity hIL-2 receptor, and / or - Human CD3 + CD4 + CD127 low Foxp3 + T reg Cells with an EC50 of ≤ 0.154 and human CD8 + Activating T cells when EC50 is (≧) 442.9 or higher. An hIL-2 specific mAb, or its antigen-binding fragment, as characterized by item 1 or 2. Item 4.V H Complementarity Determination Region CDR H 1. CDR H 2, and CDR H 3 is included in the heavy chain variable (V H ) region and V L Complementarity Determination Region CDR L 1. CDR L 2, and CDR L Variable light chain (V) including 3 L ) including the region and here a. CDR H 1 includes or is identical to sequence number 001; and b. CDR H 2 includes or is identical to sequence number 002, and c. CDR H 3 includes or is identical to sequence number 003; and d. CDR L 1 includes or is identical to sequence number 004; and e. CDR L 2 includes or is identical to sequence number 005; and f. CDR L 3 includes or is identical to sequence number 006. hIL-2 specific mAb, or an antigen-binding fragment thereof, particularly the hIL-2 specific mAb described in any one of items 1 to 3. Item 5. a. CDR H 1. CDR H 2, and CDR H 3 is selected from sequence numbers 007, 008, 009, 010, 011, 012, 013, and 014. H Included in the array, in particular the CDR H This is included in sequence number 007, and b. CDR L 1. CDR L 2, and CDR L 3 is V selected from sequence number 015 and sequence number 016. L Included in the array, in particular the CDR L This is included in sequence number 015, hIL-2 specific mAbs, or antigen-binding fragments thereof, particularly hIL-2 specific mAbs as described in any one of items 1 to 4. Item 6. a. - Position 74 and / or 84 is serine, and / or - The 93rd position is methionine, and / or - Alanine is ranked 122nd; V is (≧)96% or more identical to sequence number 007. H Region array, and b. - Isoleucine is ranked 69th. V is (≥) 99% identical to sequence number 015. L region, including, hIL-2 specific mAbs, or antigen-binding fragments thereof, particularly hIL-2 specific mAbs as described in any one of items 1 to 5. Item 7. a. V H The region includes sequences selected from sequence numbers 007, 008, 009, 010, 011, 012, 013, and 014, or functionally similar sequences derived from any one of these reference sequences by the substitution rules shown below; and b. V L The region includes sequences selected from sequence numbers 015 and 016, or functionally similar sequences derived from any one of these reference sequences by the substitution rules shown below. The substitution rules that derive functionally similar sequences from each reference sequence are: i. Glycine (G) and alanine (A) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable; and A and V are interchangeable; ii. Tryptophan (W) and phenylalanine (F) are interchangeable, and tyrosine (Y) and F are interchangeable; iii. Serine (S) and threonine (T) are interchangeable; iv. Aspartic acid (D) and glutamic acid (E) are interchangeable; v. Asparagine (N) and glutamine (Q) are interchangeable, N and S are interchangeable, N and D are interchangeable, and E and Q are interchangeable; vi. Methionine (M) and Q are interchangeable; vii. Cysteine ​​(C), A, and S are interchangeable; viii. Proline (P), G, and A are interchangeable; ix. Arginine (R) and lysine (K) are interchangeable; In particular, the above substitution rules result in the exchange of at least two amino acids, and more specifically, at least one amino acid. hIL-2 specific mAbs, or antigen-binding fragments thereof, particularly hIL-2 specific mAbs as described in any one of items 1 to 5. Item 8. a. A first sequence that is (≧)90% or more identical, in particular (≧)94% or more, (≧)96% or more, or even (≧)98% or more identical, to at least one of sequence numbers 007, 008, 009, 010, 011, 012, 013, 014, and 017; and b. A second sequence that is (≧)90% or more identical to at least one of sequence numbers 015, 016, and 018, particularly (≧)94% or more, (≧)96% or more, or further (≧)98% or more identical. Furthermore, having the characteristics described in any one of items 1 to 3, hIL-2 specific mAb, or its antigen-binding fragment. Item 9. The hIL-2 specific mAb is: a. A heavy chain comprising or consisting of Sequence ID No. 017; and b. A light chain, which includes or consists of Sequence ID No. 018, including, An hIL-2 specific mAb as described in any one of items 1-8. Item 10. hIL-2 fusion protein: a. i. Antibody heavy chain; and ii. Antibody light chain; A human interleukin-2 (hIL-2) specific monoclonal antibody (mAb) described in any one of items 1 to 9, which includes or consists of; and b. hIL-2 polypeptide; c. Peptide linkers with amino acid lengths between 25 and 50, especially those with amino acid lengths between 25 and 35, and more particularly those with amino acid lengths of 30. hIL-2 fusion protein, which includes, The peptide linker has the C-terminus of the hIL-2 polypeptide linked to either the N-terminus of the antibody heavy chain or the N-terminus of the antibody light chain, and in particular the peptide linker has the C-terminus of the hIL-2 polypeptide linked to the N-terminus of the antibody light chain. The aforementioned hIL-2 fusion protein. Item 11. The hIL-2 fusion protein described in Item 10, wherein the peptide linker is composed of approximately 85% glycine and approximately 15% serine, and in particular the peptide linker has the sequence of Sequence ID No. 026. Item 12. A nucleic acid molecule encoding an hIL-2 specific mAb according to any one of claims 1 to 9, or an antigen-binding fragment thereof, or an hIL-2 fusion protein according to item 10 or 11. Item 13. a. An hIL-2 specific mAb or its antigen-binding fragment described in any one of items 1 to 9, and b. hIL-2, A pharmaceutical composition for use as a pharmaceutical, particularly suitable for IL-2 immunotherapy, for use in the treatment of immune-mediated diseases, more particularly selected from allograft-related disorders, chronic inflammation, allergies, autoimmune and metabolic diseases, for use in the treatment of immune-mediated diseases. Item 14. A pharmaceutical composition for use according to Item 13, wherein the IL-2 and the hIL-2-specific mAb are covalently associated, and in particular the IL-2 and the hIL-2-specific mAb are contained within the hIL-2 fusion protein according to Item 10 or 11. Item 15. A pharmaceutical composition comprising an hIL-2 specific mAb for use as described in Item 13 or 14, wherein the autoimmune disease is selected from systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, autoimmune hepatitis, amyotrophic lateral sclerosis, type 1 diabetes mellitus, type 2 diabetes mellitus, arteriosclerosis, multiple sclerosis, inflammatory and autoimmune myopathy, alopecia areata, psoriasis, or inflammatory bowel disease. Item 16. A pharmaceutical composition comprising an hIL-2 specific mAb for use as described in any one of items 13-15, wherein the allograft-related disorder is diagnosed in a patient undergoing (or receiving) a solid organ transplant. Item 17. An isolated antibody or antigen-binding fragment thereof that binds to an hIL-2 epitope comprising the hIL-2 residues H16, D20, Q57, E60, E61, L63, K64, E67, E68, L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104, wherein the epitope does not contain the hIL-2 residues M23, G27, N71, Q74, S75, K76, N77, F78, P82. Item 18. An isolated antibody, or an antigen-binding fragment thereof, comprising an antigen-recognizing surface having epitope recognition properties equivalent to those of an antibody or molecule described in any one of items 1 to 11.

[0120] Technical background of IL-2-mediated dendritic cell stimulation Dendritic cells (DCs) are a subgroup of professional antigen-presenting cells that are considered essential for organizing T cell responses to intracellular pathogens and tumors (Mildner A. et al., Immunity, 2014, 30:1; Durei V. and Murphy KM, Immunity 2014, 40:642). Human blood dendritic cells (DCs) were previously subdivided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). However, single-cell RNA and protein analysis revealed that in mice and humans, differentiation into type 1 cDCs (cDC1), regulated by interferon-regulatory factor 8 (IRF8) and basic leucine zipper transcriptional factor ATF-like 3 (BATF3), and type 2 cDCs (cDC2), regulated by IRF4, was identified (Villani AC et al., Science 2017, 356:6335; Dutertre CA et al., Immunity 2019, 51:573, Schraml BU and Reis e Souse C. Curr Opin Immunol 2015, 32:13). DC subsets in non-lymphoid tissues, including the tumor microenvironment (TME), are highly distinct in terms of phenotypic and functional characteristics (Worbs T. et al., Nat. Rev. Immunol 2017, 17:30; Broz ML et al., Cancer Cell 2014, 26:638). However, the upstream molecular and cellular factors that promote the on-demand generation and growth of cDCs in antitumor responses remain unclear.

[0121] Two studies suggest that NK cells promote dendritic cell (DC) invasion in tumors and correlate with extended human survival (Bottcher JP et al., Cell 2018, 172:1022; Barry KC et al., Nat Med 2018, 24:1178). NK cells are lymphoid cells whose survival and homeostasis depend on signaling mediated through a common gamma-chain cytokine receptor (γc, also known as CD132) encoded by Il2rg. Members of this CD132 cytokine family include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (Raeber ME et al., Immunol Rev 2018, 283:176). IL-2 transmits signals via either an intermediate-affinity dimer, IL-2R, composed of IL-2Rβ (CD122) and CD132, or a trimer, IL-2R, further containing IL-2Rα (CD25). The receptor for this dimer is primarily memory CD8. + IL-2 is present on T cells and NK cells, while its trimer receptor is primarily found on Treg cells under steady state, and its expression is transiently upregulated in recently activated effector T cells (Arenas-Ramirez J. et al., Trends Immunol 2015, 36:763). In addition to its effects on T cells and NK cells, IL-2 can also stimulate innate lymphoid cells (ILCs), particularly type 2 ILCs (ILC2s), NKT cells, activated B cells, and certain non-immune cells (Malek RT and Castro I., Immunity 2010, 33:153; Abbas AK et al., Sci Immunol 2018, 3(25):eaat1482). However, IL-2 is not known to affect dendritic cell homeostasis in vivo.

[0122] Overview of dendritic cell stimulation with hIL-2 mAb compositions Example 5 relates to a clinical trial studying the immune response of systemic lupus erythematosus patients receiving IL-2 therapy to induce immune tolerance. The inventors were surprised to observe a significant increase in multiple DC subsets. Studies of IL-2 immunotherapy conducted in both mice and humans demonstrated the enlargement and activation of DCs by a complex containing IL-2 and either a trimer IL-2R-biased mAb or an IL-2Rα-biased mAb. This pathway is driven by IL-2 and stimulates an increase in both the DC population and the DC process.

[0123] A first aspect of the present invention is an IL-2 complex pharmaceutical composition for use in patients requiring enhancement of dendritic cell function, wherein the IL-2 complex comprises both human hIL-2 and an hIL-2 specific mAb, and the IL-2 complex preferentially binds to high affinity IL-2R or CD25 rather than intermediate affinity IL-2R.

[0124] In some embodiments, the pharmaceutical composition for use is V H Complementarity Determination Region CDR H 1. CDR H 2 and CDR H 3 heavy chain variable (V H ) region and V L Complementarity Determination Region CDR L 1. CDR L 2 and CDR L Variable light chain (V) having 3 L ) comprises an hIL-2 specific mAb including the region, where the CDR H 1. CDR H 2. CDR H 3. CDR L 1. CDR L 2, and CDR L 3 includes or is identical to sequence numbers 001, 002, 003, 004, 005, and 006, respectively. In a further embodiment, the CDR is V of sequence number 007. H V of sequence and sequence number 015 LIt is included in the sequence, or in a functionally similar sequence. In other embodiments, a pharmaceutical composition for use in a patient to enhance DC function is T reg It is biased towards increased activation and promotes the activation or proliferation of DCs in patients.

[0125] The present invention further provides a method for treating patients diagnosed with autoimmune diseases or inflammatory diseases using the IL-2 complex according to the present invention.

[0126] Detailed description of dendritic cell stimulation with hIL-2 mAb composition In interpreting this specification, the definitions set forth in the "Terms and Definitions" section shall continue to apply, and where appropriate, terms used in the singular form shall also include their plural forms, and vice versa.

[0127] A first aspect of the present invention is a pharmaceutical composition for use in patients having a condition that would benefit from enhanced DC function, the pharmaceutical composition comprising an IL-2 complex itself, comprising both a human IL-2 (hIL-2) polypeptide and an hIL-2 specific monoclonal antibody (mAb). An example of a suitable hIL-2 specific mAb is disclosed in U.S. Patent Application Publication No. 2017 / 0114130 (A1), which is incorporated herein by reference. The IL-2 complex according to this aspect of the present invention preferentially binds to CD25 and / or to high-affinity IL-2 receptors comprising CD122, CD132 and CD25, compared to intermediate-affinity IL-2R comprising CD122 and CD132.

[0128] The data in Figures 15 and 17 of the Examples demonstrate that both the commercially available CD25-biased anti-IL-2 antibody clone 5344, or the UKFA-20 clone described herein, when conjugated with IL-2, can increase the total number of spleen DCs to a level comparable to that of IL-2 conjugates prepared using the CD122-biased anti-IL-2 NARA1 clone. This data suggests that the pharmaceutical composition may be beneficial to patients diagnosed with inflammatory or autoimmune diseases by increasing the number of DCs with tolerogenic phenotypes.

[0129] IL-2 complexes containing CD25-biased mAbs secrete immunotolerogenic molecules such as IL-10 and transforming growth factor β. reg It will preferentially deliver IL-2 signaling to CD25-rich cells such as those mentioned above. Therefore, this pharmaceutical composition is expected to be particularly useful in patients diagnosed with diseases that would benefit from enhanced immunotolerogenic DC function. Tolerogenic DCs can be identified by an immunotolerogenic signature, for example, which includes the expression of the following genes or their products: CD274, PDCD1LG2, CD200, CD205, FAS, ALDH1A2, SOCS1, SOCS2, IL4R, IL4I1, IL10, CCL17, CCL22, TNFRSF4, and BCL2L1 (Maier, Nature 2020, 580:257).

[0130] In certain embodiments, the hIL-2-anti-hIL-2 mAb complex according to the present invention contains an hIL-2 polypeptide non-covalently associated with the anti-hIL-2 mAb according to the present invention. According to the present invention, the ratio of hIL-2 and hIL-2-specific mAbs to be combined in the IL-2 complex is not particularly limited. These hIL-2 mAb complexes have been demonstrated to function effectively when the elements are combined in a 2:1 ratio (Boyman, Science 2006, 311:1924; Krieg, PNAS 2010, 107:11906; Arenas-Ramirez, Sci Transl Med 2016, 8, :367ra166) or in a 1:1 ratio (Letourneau, PNAS 2010, 107:11906; Arenas-Ramirez, Sci Transl Med 2016, 8, :367ra1660). The binding (combination) of these two components of the complex takes place in solution, and the time, temperature, and conditions of this combination procedure are also not particularly limited by the present invention. The complex can be formed, for example, by binding hIL-2 and anti-hIL-2 mAb in a physiological solution such as phosphate-buffered saline at room temperature for 15 minutes. This deflects IL-2 signaling to IL-2Rα or IL-2Rβ, and therefore Treg or CD8 + Preparation and activation of IL-2 mAb complexes using mAbs that increase STAT5 phosphorylation in T cells have been carried out (Letourneau, PNAS 2010, 107:11906; Krieg, PNAS 2010, 107:11906; Trotta, Nat Med 2018, 24:1005). In other embodiments, the hIL-2 and anti-hIL-2 mAb of the pharmaceutical composition according to the present invention are covalently associated, particularly by a peptide linker.

[0131] In certain embodiments of pharmaceutical compositions containing IL-2 complex for use, activation and / or proliferation, as measured by STAT5 phosphorylation, for example, CD8 + Regulatory T cells (T) reg ) significantly increases in cells. In certain embodiments, when the pharmaceutical composition for use specified above is applied to human or primate immune cells, CD8 + T cells, natural killer (NK) cells, innate lymphoid cells (ILCs), and / or B cells reg Increase the cell ratio.

[0132] The inventors have developed UFKA-20, a novel hIL-2-specific mAb, and UFKA-22, its humanized derivative. IL-2 complexes formed using these mAbs efficiently and specifically stimulate CD25-expressing T cells. The inventors have demonstrated that the hIL-2-specific mAb according to this embodiment of the invention efficiently increases the immunotolerogenic DC population compared to other previously described CD122-targeted antibody clones. In certain embodiments, the hIL-2-specific mAb of an IL-2 complex pharmaceutical composition for patient use is used in CDR H 1. CDR H 2 and CDR H V including 3 H Area, and CDR L 1. CDR L 2 and CDR L V including 3 L Includes the region. According to this embodiment: CDR H 1 includes or is identical to sequence number 001; and CDR H 2 includes or is identical to sequence number 002; and CDR H 3 includes or is identical to sequence number 003; and CDR L 1 includes or is identical to sequence number 004; and CDR L 2 includes or is identical to sequence number 005; and CDR L 3 includes or is identical to sequence number 006.

[0133] In another embodiment, a CDR of hIL-2 mAb contained in an IL-2 complex pharmaceutical conjugate for use in patients requiring DC enhancement. H 1. CDR H 2 and CDR H 3 is included in at least one VH sequence selected from sequence numbers 007, 008, 009, 010, 011, 012, 013, or 014, or 017, in particular the CDR H This is included in sequence number 007. Also, the CDR of hIL-2 mAb L 1. CDR L 2, and CDR L 3 is included in at least one VL sequence selected from sequence number 015, sequence number 016, or sequence number 0018, and in particular the CDR LThis is included in Sequence ID No. 015. The isolated antibody hIL-2-specific antibody or its antigen-binding fragment according to this embodiment of the present invention contains the hIL-2 residues H16, D20, Q57, E60, E61, L63, K64, E67, E68, L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, and M104, but excludes the hIL-2 residues M23, G27, N71, Q74, S75, K76, N77, F78, and P82, and binds to the hIL-2 epitope. The antibody having the above characteristics, when complexed with hIL-2, will preferentially deliver the IL-2 signal to high-affinity IL-2R. Importantly, upon binding to the receptor, the antibody will also dissociate from the complex, in other words, separate from the IL-2 polypeptide, in order to allow for optimal signal transduction stimulation delivery without steric hindrance by the antibody.

[0134] According to another embodiment, a pharmaceutical composition comprising an IL-2 complex for use according to the present invention promotes the proliferation and / or activation of dendritic cells (DCs) in a patient. DC proliferation can be measured by the uptake of detectable DNA insertors such as Brdu, 7AAD, or by an increase in their number over time, or by the upregulation of surface markers indicating cell cycle entry, such as Ki67. Activation can be defined as an increase in measured values ​​of factors produced by mature dendritic cells, such as MHC or co-stimulatory molecules. Alternatively, activation may be defined by an increase in molecules that have been shown in examples to drive dendritic cell activation and proliferation, particularly tumor necrosis factor (TNF, TNFA, TNFSF2, UniProt P01375), Fms-related tyrosine kinase 3 ligand (Flt3l, UniProt P49771), and granulocyte-macrophage colony-stimulating factor (CSF2, UniProt P04141).

[0135] The data shown in Figures 15 and 17 demonstrate that parenteral administration of an IL-2 complex containing a CD25-biased antibody increases the dendritic cell (DC) population in the spleen of mouse recipients, and that IL-2 complex stimulation induces both the proliferation of mature dendritic cells, as measured by BrdU incorporation, and the maturation of DC precursors to inhibitory states, as measured by the modulation of activity markers (see Figures 15 and 17).

[0136] In certain embodiments of the pharmaceutical compositions comprising the IL-2 complex for use according to the present invention, the composition is administered to patients diagnosed with immune-mediated diseases. Autoimmune and autoinflammatory diseases in which DC-based therapy may benefit patients include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's disease, type 1 diabetes, psoriasis, vitiligo, inflammatory bowel disease, multiple sclerosis, and hemophagocytic lymphohistiocytosis. Furthermore, the importance of dendritic cells in priming and sustaining T-cell responses suggests that patients diagnosed with allergic or atopic diseases, such as asthma or atopic dermatitis, may also benefit from administration of pharmaceutical compositions comprising the IL-2 complex according to the present invention.

[0137] In another embodiment, a pharmaceutical composition comprising the IL-2 complex for use according to the present invention is administered to a patient diagnosed with an allograft-related disorder (e.g., acute and chronic graft-versus-host disease, or vasculitis such as granulomatous vasculitis with polyangiitis). Furthermore, it may be administered to promote the acceptance of a future allograft in transplantation. Administration of the pharmaceutical composition comprising the IL-2 complex may therefore be performed before or concurrently with allograft or organ transplantation procedures, or after the patient has previously undergone a tissue graft or organ transplantation procedure.

[0138] Figure 16 of the examples demonstrates that IL-2 immunotherapy using aldethleukin increases dendritic cells in human systemic lupus erythematosus patients, particularly enhancing the number of cDC1 and cDC2. Figures 15 and 17 show that treatment with an IL-2 complex containing NARA1, or a CD25-biased pharmaceutical composition containing the IL-2 complex according to the present invention, achieves similar acute DC enlargement in mice. Furthermore, the IL-2 complex provided by the present invention is known to be superior to IL-2 immunotherapy in terms of efficacy, reduced side effects, and response lifetime (Arenas-Ramirez J. et al., Trends Immunol 2015, 36:763). CD25-biased hIL-2-specific mAb complexes have been demonstrated to enhance DCs with an immunotolerogenic phenotype characterized by decreased CD80 and MHC II expression and increased expression of the PD-1 family of checkpoint inhibitor molecules.

[0139] Further aspects of the present invention are methods for treating patients requiring enhancement of DC function, for example, cancer, or autoimmune, inflammatory, or allograft-related conditions, the methods comprising administering a pharmaceutical composition comprising an effective amount of hIL-2 polypeptide associated with an hIL-2 specific mAb according to the specification of the present invention.

[0140] The present invention further encompasses the following: Item A. A pharmaceutical composition comprising an IL-2 complex for use in patients with conditions that would benefit from enhanced dendritic cell (DC) function, The IL-2 complex comprises a human IL-2 (hIL-2) polypeptide associated with an hIL-2-specific monoclonal antibody (mAb), and The IL-2 complex preferentially binds to CD25 and / or high-affinity IL-2 receptors including CD122, CD132, and CD25, compared to intermediate-affinity IL-2 receptors including CD122 and CD132. The aforementioned pharmaceutical composition. Item B. The IL-2 complex is, a. CD8 +Rather than increasing STAT5 phosphorylation in T cells, regulatory T(T) reg ) to significantly increase STAT5 phosphorylation in cells, and / or b. CD8 + Rather than increasing T cell proliferation, reg To significantly increase cell proliferation, and / or c. CD8 + T cells, natural killer (NK) cells, innate lymphoid cells (ILCs), and / or B cells reg Increase the cell ratio. A pharmaceutical composition comprising an IL-2 complex for use as described in item A. Item C. The hIL-2 specific mAb is V H Complementarity Determination Region (CDR) H )CDR H 1. CDR H 2 and CDR H 3 is included in the heavy chain variable (V H ) region, and V L Complementarity Determination Region (CDR) L )CDR L 1. CDR L 2 and CDR L 3 including light chain variable (V L ) including the region, where: a. CDR H 1 includes or is identical to sequence number 001; and b. CDR H 2 includes or is identical to sequence number 002; and c. CDR H 3 includes or is identical to sequence number 003; and d. CDR L 1 includes or is identical to sequence number 004; and e. CDR L 2 includes or is identical to sequence number 005; and f. CDR L 3 includes or is identical to sequence number 006. A pharmaceutical composition comprising an IL-2 complex for use as described in item A or B. Item D. c. CDR of the aforementioned hIL-2 mAb H 1. CDR H 2 and CDR H 3 is V selected from sequence number 007, sequence number 008, sequence number 009, sequence number 010, sequence number 011, sequence number 012, sequence number 013, or sequence number 014. H It is included in the sequence, and in particular the CDR H This is included in sequence number 007, and d. CDR of the aforementioned hIL-2 mAb L 1. CDR L 2 and CDR L 3 is V selected from sequence number 015 or sequence number 016. L It is included in the sequence, and in particular the CDR L This is included in sequence number 015. A pharmaceutical composition comprising an IL-2 complex for use as described in any one of items A to C. Item E. The pharmaceutical composition comprises an IL-2 complex for use according to any one of items A to D, which promotes the proliferation and / or activation of DCs in a patient. Item F. The pharmaceutical composition is a pharmaceutical composition comprising an IL-2 complex for use according to any one of items A to E, administered to a patient diagnosed with an immune-mediated condition. Item G. A pharmaceutical composition comprising an IL-2 complex for use as described in any one of items A to F, administered to a patient before a procedure for allogeneic tissue graft or organ transplantation, or to a patient who has previously undergone a procedure for tissue graft or organ transplantation. Item H. A method for treating a patient with cancer, autoimmune disease, inflammatory disease, or allograft-related disease, comprising administering an effective amount of a pharmaceutical composition containing an hIL-2 polypeptide associated with an hIL-2 specific mAb described in any one of items A to G.

[0141] The present invention is further illustrated by the following embodiments and figures, from which further embodiments and advantages can be derived. These embodiments are for illustrative purposes only and do not limit the scope of the present invention. [Examples]

[0142] Materials and methods Cell lines and primary cells HEK293T cells obtained from the American Type Culture Collection (ATCC) were maintained in Dulbecco's Modified Eagle Medium supplemented with fetal calf serum (10% v / v, Thermo Scientific) and penicillin-streptomycin (100 U / ml, Thermo Scientific). Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood collected from healthy individuals by Ficoll-Paque PLUS (GE Healthcare) gradient centrifugation, after obtaining prior informed consent and approval from the Zurich Cantonal Ethics Committee (BASEC number 2016-01440).

[0143] Generation of fluorescently tagged IL-2R subunits The IL-2R subunit was C-terminus-linked to a fluorescent protein with a flexible 15-amino acid (GGGGS)3 spacer (motif sequence number 021). Sequences encoding CyPet, YPet, or RFP657 (RFP) were derived from the plasmids pCEP4CyPet-MAMM and pCEP4yPET-MAMM (Donated by P. Daugherty, Addgene plasmids 14033 and 14032, respectively) and pSG4OC-RFP657 (Donated by D. Hecki, Hanover Medical School). CyPet was amplified using the following specific primers: forward 5'-CGTCTCGTGGTGGTGGTTCTGGTGGTGGTGGTTC-TGTGACAAGG-3' (Sequence Number 022) and reverse 5'-GGTGGTCTCGAGTTATTTGTACA-GTTCGTCCATGCCG TG-3' (Sequence Number 023). The human CD25 gene sequence was amplified from human PBMC RNA (RNeasy Plus mini-kit, Qiagen), and after PCR amplification using a human CD25-specific primer pair, it was transcribed to complementary DNA (cDNA) using the QuantiTect reverse transcription kit (Qiagen): forward 5'-CTAGGAAGCTTATCTATGGATTCATACCTGCTG-3' (SEQ ID NO: 024) and reverse 5'-ACCAGAACCACCACCACCAGAACCACCACCACCGATTGTTCTTCTA-CTCTTCCTCTG-3' (SEQ ID NO: 025). The PCR products were purified by gel extraction (New England BioLabs) on a 0.5-1% agarose gel, and the fragments were annealed using overlap extension PCR and cloned into the mammalian expression vector pCSCMV (Addgene plasmid 30530, donated by G. Ryffel). Human CD122-CyPet and human CD132-RFP657 (referred to as CD132-RFP) were synthesized using the GeneArt service (Thermo Scientific) and cloned into the mammalian expression vector pcDNA3.1.

[0144] Cell-based IL-2R binding assay 0.75 × 10 6HEK293T cells were co-transfected in Opti-MEM (Thermo Scientific) in 6-well plates using 1.3 μg each of pCD25-CyPet, pCD122-YPet, and pCD132-RFP in a DNA:ViaFect (Promega) ratio of 1:3. When transfecting with one or two IL-2R subunits and culturing at 37°C and 5% CO2, the total DNA amount was adjusted to 3.9 μg using the empty vector pcDNA3.1. 48 hours after transfection, cells were detached using enzyme-free cell dissociation buffer (Thermo Scientific) and recovered in FACS buffer (PBS containing 2% FBS and 2 mM EDTA). Rhodamine-labeled IL-2 (IL-2 Rhod ) and anti-IL-2 mAb were mixed in a 1:1 molar ratio and incubated at room temperature (RT) for 15 minutes. IL-2 Rhod To produce IL-2, human IL-2 was reconstituted with sterile water, dialyzed with 50 mM phosphate buffer (pH=6.5) to optimize the preferred N-terminal rhodamine bond, and subsequently incubated with N-hydroxysuccinimidyl (NHS)-rhodamine (Thermo Scientific) on ice for 2 hours. Unreacted NHS-rhodamine was removed by gel filtration (Zeba Spin desalting column, 7K MWCO, Thermo Scientific). IL-2 Rhod The anti-IL-2 mAb complex was found in a V-bottom, 96-well plate at a concentration of 0.3 × 10⁶ units. 6 HEK293T cells (expressing IL-2R subunit or mock control) were incubated at 37°C for 10 minutes, washed twice with cold FACS buffer, and incubated with BV605 rat anti-mouse IgG1 (BD Biosciences, clone X56) for 20 minutes in the refrigerator. After surface staining, cells were washed with PBS, fixed with 2% paraformaldehyde, acquired with BD LSRFortessa, and analyzed with FlowJo software (both BD Biosciences).

[0145] mouse C57Bl / 6J mice were purchased from Charles River Laboratories. Female mice were used in the experiment at 2–5 months of age. The experiment was approved by the Zurich Cantonal Veterinary Office (license 246 / 2016) and conducted in accordance with Swiss federal and cantonal laws. Mice were randomized by the open-label principal investigator and housed in a designated sterile facility at Zurich University Hospital in accordance with institutional guidelines.

[0146] Rhesus macaque The study using rhesus macaques (Macaca mulatta) was conducted at the Biomedical Primate Research Centre (BPRC) with 15 healthy adult females aged 4–15 years and weighing 5–15 kg. The animals did not show circulating antibodies specific to STLV or SRV and had not received immunosuppressive or antibody therapy prior to the study. All procedures and protocols complied with all relevant ethical guidelines for animal experimentation set forth by the BPRC's Animal Experimentation Committee. The animals were randomly divided into five groups of three: Group 1: LD IL-2 (10 μg / kg); Group 2: HD IL-2 (33 μg / kg); Group 3: LD IL-2 / UFKA-22cx (10 / 100 μg / kg); Group 4: HD IL-2 / UFKA-22cx (33 / 330 μg / kg); and Group 5: UFKA-22 (330 μg / kg). IL-2 was administered daily by subcutaneous injection, and IL-2 / UFKA-22cx and UFKA-22 were administered intravenously on days 0 and 3. Animals were sedated during injection and bleeding.

[0147] Clinical trials and human samples Human samples were collected in the clinical trial "An open-label, monocentric, phase II, investigator-initiated clinical trial on the fair characterization of immunological parameters in interleukin-2-treated systemic lupus erythematosus" (Charact-IL-2, clinical trial identifier: NCT03312335) and the "Basic research project on the characterization of phenotypic and functional differences in leukocyte subsets from healthy and diseased individuals" (PFCL-1, BASEC number 2016-01440). Both projects were reviewed and approved by the Swiss regulatory authorities and conducted in accordance with the latest version of the Declaration of Helsinki, the Good Clinical Practice guidelines, and Swiss legal requirements. Written informed consent was obtained prior to enrollment in clinical trials or sample collection. Human blood was collected in EDTA vacuum tubes (BD Biosciences), and peripheral blood mononuclear cells (PBMCs) were subsequently isolated by gradient centrifugation using a Ficoll-Paque PLUS (GE Healthcare) system. Isolated PBMCs were frozen in 10% dimethyl sulfoxide (Sigma)-containing fetal calf serum (FCS, Gibco) and stored in liquid nitrogen for less than one year prior to analysis. Serum was isolated from blood collected using Clot Activator vacuum tubes (BD Biosciences) and stored at -80°C for less than 18 months prior to analysis. To evaluate IL-2-mediated cDC and lymphocyte growth, blood from patients with systemic lupus erythematosus (SLE) was collected before and after a 5-day course of 1.5 million international units (IU) of aldezleukin (Proleukin®, Novartis Pharma) daily, according to the study protocol.

[0148] IL-2 mAb complex formation In the HEK cell-based assay, IL-2 RhodThe IL-2 was mixed with anti-hIL-2 antibody in a 1:1 ratio in FACS buffer (1×PBS, 2% FBS, 2 mM EDTA) and incubated at room temperature for at least 15 minutes. For in vivo application, hIL-2 was mixed with anti-hIL-2 antibody in a 1:1 ratio in sterile PBS and incubated at room temperature for at least 15 minutes. The infusion volume was 200 microliters per intraperitoneal infusion. Recombinant human IL-2 (teceleukin, Roche) was obtained from the National Cancer Institute of the National Institutes of Health. Antibody conjugates were prepared as previously described (Arenas-Rameriz N.Sci Transl Med 2016,8:367ra166) by mixing 15,000 IU of IL-2 with 15 μg of anti-IL-2 monoclonal antibody (mAb) per infusion. IL-2cx, or 200,000 IU of IL-2, was infused daily for 3 consecutive days. BrdU-intake cells were measured using the FITC BrdU Flow Kit (BD Biosciences) according to the manufacturer's instructions.

[0149] Flow cytometry Single-cell suspensions of lymph nodes (LN) and spleen were prepared, and surface markers and intracellular Foxp3 and Ki-67 were stained using the Foxp3 / transcription factor intracellular staining kit (Thermo Fisher) according to the manufacturer's instructions. To detect pSTAT5 in mice or monkeys, cells were immediately fixed using Phosflow Lyse / Fix Buffer (BD Biosciences) or lysis solution (Becton Dickinson), and further intracellular staining was performed according to the manufacturer's instructions. To measure pSTAT5 in vitro, 10 6 Individual magnetically purified human CD3 +T cells (BioLegend) were seeded in 96-well, V-bottom plates and stimulated with IL-2, IL-2 / UFKA-20cx, or IL-2 / UFKA-22cx at 37°C for 15 minutes. Intracellular pSTAT5 was stained using anti-STAT5 (pY694) mAb (Thermo Fisher) as described above. For surface staining of monkey cells, the mAb was incubated in 200 μl of EDTA blood according to a standard protocol, followed by erythrocyte lysis, fixation, and permeabilization, and intracellular staining for Foxp3 and Ki-67. Samples were acquired using BD LSRFortessa and analyzed using FlowJo. Antibodies and fluorescent dyes used for flow cytometry were purchased from ebioscience, BD Biosciences, Biolegend, or Miltenyi.

[0150] ELISA Flat-bottom Nunc MaxiSorp 96-well plates (Thermo Scientific) were coated overnight at 4°C with NARA1 anti-human IL-2 mAb (capture). After washing the plates with PBS and 0.1% Tween 20 (Sigma-Aldrich), the wells were incubated in a solution of PBS, 1% BSA (Sigma-Aldrich), and 0.1% Tween 20 with shaking at 450 rpm for over 1 hour, then blocked at room temperature. Cell supernatant or purified UFKA mAb was incubated on the plates for 1-2 hours to directly coat IL-2 or capture it with plate-coated NARA1. After washing the plates, IL-2 or competitive binding was evaluated by incubation with anti-mouse IgG (BioLegend) or biotinylated anti-IL-2 detection mAb (clone 5344.111, BD Biosciences) at 450 rpm for 1 hour at room temperature. After further washing, the plates were incubated with streptavidin-labeled wasabi peroxidase (BD Biosciences) in the dark at room temperature for 45 minutes. Finally, after the final wash, the plates were spread with TMB peroxidase EIA substrate (BioRad) for 2–5 minutes, and then stopped with the addition of H2SO4 (1.8M, Sigma-Aldrich). The absorbance at 450 nm was read using an iMark microplate reader (BioRad). The serum half-life of IL-2 or IL-2 / UFKA-20cx was measured using a sandwich ELISA with NARA1 as the capture agent and biotinylated anti-IL-2 mAb (clone 5334, R&D Systems) as the detection mAb, followed by spreading in the same manner as above.

[0151] Surface plasmon resonance In the SPR (Sample-Progressive Reflux) test, UFKA-20 or NARA1 was directly immobilized on a CMD200 tip (XanTec bioanalytics), and titrated IL-2 concentrations were injected starting at 300 nM, followed by injection of a 2-fold dilution. To measure the binding of CD25 and CD122, IL-2 (1000 nM) was captured on an anti-IL-2 mAb coated tip for 60 seconds, followed by continuous injection of recombinant CD25 or CD122 (R&D Systems) starting at 333 nM, followed by injection of a 3-fold dilution. The tip surface was regenerated with glycine buffer pH 1.5 after each cycle. Measurements were acquired at 20°C and analyzed using a Biacore T100 (GE Healthcare).

[0152] Structural analysis of IL-2 / UFKA-20cx The Fab fragment of UFKA-20 was prepared by cleaving the full-length mAb with papain and then purifying it with Protein A. 1.5 ml of UFKA-20 (15.3 mg / ml in a 50 mM solution containing 90 mM NaCl at pH 7.0) was mixed with dichlorodiphenyltrichloroethane (DDT) and papain (Roche) to final concentrations of 5 mM and 1.5 mg / ml, respectively. After digestion at room temperature for 16 hours, papain was inactivated with 56 mM E64 solution (Roche), and the mixture was diluted 10-fold with Tris / NaCl buffer (25 mM Tris, 25 mM NaCl, pH 8.0). This mixture was loaded onto a Protein A column equilibrated with Tris / NaCl buffer, and the flow-through fraction containing the Fab fragment was collected and further purified by size exclusion chromatography (SEC). The IL-2 / UFKA-20 Fab complex, formed by mixing purified UFKA-20 Fab with a 10-fold molar excess of human IL-2 dissolved in water, was purified by SEC using an Akta pure chromatography system (GE Healthcare) with a Superdex 200 10 / 300 GL column. The fraction containing the complex was pooled, dialyzed overnight at 4°C in Tris / NaCl buffer (pH 7.4), and concentrated using an Amicon ultracentrifuge filter unit (10-kDa, Merck Millipore) to a final protein concentration of 10 mg / ml, which was measured by absorption at 280 nm. Various crystallization buffers were screened, and purification was performed to find the optimal crystallization conditions. Finally, the IL-2 / UFKA-20 Fab complex solution was mixed 1:1 with a crystallization buffer containing 10.86% (v / v) PEG 8000, 5.76% (v / v) ethylene glycol, and 100 mM HEPES (pH 7.48). Crystals were grown in a 96-well plate at 20°C by sitting drop vapor diffusion, collected and cryoprotected using a reservoir solution with 30% (v / v) ethylene glycol, and immediately frozen in liquid nitrogen. Diffraction data were collected at a wavelength of 1 Å at beamline X06DA (Swiss light source, Paul Scherrer Institute, Wirrigen, Switzerland) equipped with a Pilatus 2M detector (Dectris, Baden-Wattville, Switzerland).Data processing was performed using XDS and Aimless. The IL-2 / UFKA-20 Fab complex structure was initially solved using the structure of the Fab fragment of an anti-leukotriene antibody (PDB: 5B6F), and then the structure of human IL-2 (PDB: 1M47) as search models, using MOLREP by molecular substitution (Arkin MR et al., PNAS 2003 100:1603). Model construction was performed using Coot, and refinement was carried out using REFMAC5, BUSTER, and PHENIX. TLS refinement was used, defining each domain as a separate TLS group. The final structure contained three IL-2 / UFKA-20 complexes of asymmetric units. The epitope overlap between the IL-2R subunit and anti-IL-2 mAbs was quantified using the Protein Interfaces, Surfaces and Assemblies' Service (PISA) at the European Bioinformatics Institute (http: / / www.ebi.ac.uk / pdbe / prot_int / pistart.html), and further calculated using Excel (Microsoft).

[0153] RNA sequencing (RNA-seq) 40,000 spleen mouse cDCs obtained from untreated and UFKA20 complex-treated wild-type mice were separated by FACS in RLT Plus lysis buffer (Qiagen) containing 1% 2-mercaptoethanol (Sigma-Aldrich). RNA was then isolated using the RNeasy Plus microkit (Qiagen). RNA extracted from the sorted cells was quantified for quality and concentration using the TapeStation RNA Sensitivity Kit (Agilent). cDNA was prepared by universal priming (including 3 minutes of fragmentation) using the SMARTer Stranded Total RNA Seq Kit v2 (Takara Bio), and ribosomal cDNA was depleted using ZapR v2 and R Probes v2. This library was quantified using Tapestation D1000 (Agilent) and sequenced using a HiSeq 4000 platform with approximately 40M reads per sample, using 125 cycles of single read sequencing. Before read alignment, adapters and low-quality tails were removed from the reads. Alignment of the RNA-seq dataset to Ensembl genome build GRCh38.p10 (Release 91) was performed using STAR aligner (v2.5.4b). Gene expression counts were calculated using feature count in the Bioconductor package Rsubread (v1.32.1). A gene was considered expressed if it exceeded 10 counts in at least one comparison group where more than half of the samples were expressed. Differential expression genes were detected using the Bioconductor package EdgeR (v3.20.6). Gene set enrichment analysis was performed using Gene Ontology analyzer for RNA-seq and other length biased data (goseq, v1.30.0).

[0154] Quantitative and statistical analysis Statistical tests were performed using Prism software (GraphPad). As shown in the legend of the figures, most experiments were analyzed using one-way ANOVA with Tukey or Dunnett's multiple comparisons, or two-tailed, unpaired Student's t-test. For datasets where the counts were too small for normality testing, a normal distribution was assumed based on the data distribution. p<0.05 was considered statistically significant.

[0155] Example 1: Generation and selection of anti-human IL-2 monoclonal antibodies Balb / c mice were immunized with human IL-2 in complete Freund's adjuvant (Sigma-Aldrich) and boosted twice with IL-2 emulsified in incomplete Freund's adjuvant (Sigma-Aldrich). Mice were euthanized 4-5 weeks after the initial immunization and their spleens were collected. Spleen cells and myeloma cells were mixed in a 5:1 ratio using polyethylene glycol 1500 (Roche). Clones were cultured in Iskoff-modified Dulbecco medium supplemented with 10% fetal bovine serum (FBS), 50 mM mercaptoethanol, 1:100 insulin-transferrin-selenium, 2% IL-6 modified medium, penicillin-streptomycin, gentamicin (all Life Technologies), and hypoxanthine-aminopterin-thymidine (HAT) (Sigma-Aldrich). In the supernatant of B cell hybridomas, screening for IL-2 reactivity was performed using IL-2-binding ELISA, and specificity was screened using competitive ELISA. Positive hits were then subcloned. mAbs were grown in hypoxanthine-thymidine (HT) medium (LifeTechnologies). After retesting, anti-IL-2 mAbs were purified from the cell supernatant using protein G agarose purification (Thermo Fisher). Antibodies were produced using transiently transfected HEK293F cells, purified by affinity using Protein A MabSelect SuRe resin (GE Healthcare), and fractionated. Purity was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

[0156] Example 2: The polarized anti-IL-2 mAb has distinct properties of binding to IL-2R and delivering IL-2. Using competitive enzyme-linked immunosorbent assay (ELISA), the binding properties of over 10,000 anti-human IL-2 mAbs, including those produced in Example 1, patented antibodies, and publicly available clones from a mouse hybridoma library, were evaluated for their interaction with IL-2 and IL-2R. Unless otherwise specified, all IL-2 and IL-2R subunits in the examples refer to human molecules. To identify and compare CD25-biased anti-IL-2 mAbs, a novel cell-based in vitro screening platform was developed in which monomeric CD25, dimeric CD122+CD132, and trimer CD25+CD122+CD132 were expressed on human cells. Fluorescently tagged IL-2R subunits were generated and transiently expressed in human embryonic kidney (HEK) 293T cells, thereby accurately identifying cells expressing the IL-2R subset as defined by flow cytometry, and rhodamine-labeled IL-2 (IL-2) was identified either alone or in combination with an anti-IL-2 mAb. Rhod This makes it possible to quantify the binding of IL-2. The CD25-biased IL-2 complex (cx) associates with CD25 but not with CD122+CD132, while the CD122-biased IL-2cx shows the opposite pattern (Figure 1A). Five anti-IL-2 mAbs were selected based on their different binding patterns and named UFKA-10, UFKA-20 (heavy chain SEQ ID NO: 019, light chain SEQ ID NO: 020), UFKA-30, UFKA-40, and UFKA-50, which can be broadly divided into three categories: non-biased (UFKA-10), CD25-biased (UFKA-20, UFKA-30, and UFKA-40), and CD122-biased (UFKA-50) (Figure 1B). As expected, IL-2 Rhod When used alone, it showed low binding with CD25, intermediate binding with CD122+CD132, and strong binding with CD25+CD122+CD132 (Figure 1C). Next, IL-2 Rhod The inventors conjugated this compound with various anti-IL-2 mAbs in a 1:1 ratio and tested it in IL-2R subunit-expressing HEK293T cells. Rhod In comparison, UFKA-10 and IL-2 RhodThe complex showed a slight decrease in binding to CD25 and CD122+CD132, suggesting mild interference between UFKA-10 and these receptor subunits, while IL-2 binding with CD25+CD122+CD132 Rhod The meeting of IL-2 was not altered by this mAb (Figure 1C). When the mAbs UFKA-20, UFKA-30, and UFKA-40 were tested, distinct differences existed among these mAbs, but a clear pattern of CD25 deflection was clearly observed (Figures 1C and 1D). Thus, IL-2 Rhod UFKA-30cx and IL-2 Rhod UFKA-40cx preferentially bound to CD25 and CD25+CD122+CD132, remaining bound as complexes at 74.5% and 93.2% respectively, while association with CD122+CD132 was either slightly reduced (similar to UFKA-30) or unchanged (similar to UFKA-40) (Figures 1C-1E). In particular, IL-2 Rhod / UFKA-20cx preferentially associates with CD25, and approximately two-thirds of the measured interactions are with IL-2. Rhod This was done by / UFKA-20cx and IL-2 Rhod Less than one-third of the cases were detected as a standalone compound on CD25. However, IL-2 Rhod / UFKA-20cx appears to rapidly dissociate when it interacts with the trimer CD25+CD122+CD132, which is related to IL-2 Rhod The interaction formed by / UFKA-20cx is less than 8.4%, and free IL-2 is not bound to the CD25+CD122+CD132 complex. Rhod This is evident from the fact that the interaction formed by them is 91% (Fig. 1C~1E); however, IL-2 Rhod The coupling of / UFKA-20cx to CD122+CD132 is IL-2 Rhod Compared to that, it is impaired by the presence of UFKA-20 (Figure 1C), which means that UFKA-20 is IL-2 Rhod Add "CD25 bias" to it, and at the same time, IL-2 RhodThis suggests that UFKA-20 dissociates and binds to the trimer CD25+CD122+CD132, thereby enabling "IL-2 delivery" to the IL-2R complex that signals IL-2 (Figure 1F~1H). Conversely, UFKA-30 and UFKA-40 enable IL-2 Rhod This resulted in even stronger CD25 deflection, but it was unable to dissociate IL-2 from the trimer IL-2R and deliver it (Figure 1F~1H). Unlike the aforementioned CD25-deflecting anti-IL-2 mAb, IL-2 Rhod UFKA-50cx exhibits reduced CD25 binding and clearly favors the association of the dimer CD122+CD132 and trimer CD25+CD122+CD132 with IL-2R (Figure 1C-1H), making it similar to IL-2cx mediated by a well-characterized CD122-biased NARA1 mAb (Arena-Ramirez et al., Sci. Transl. Med. 2006 8:367). From these findings, we observed clear differences in the mechanism of CD25-biased mAbs in terms of two features: CD25 biasing and IL-2 delivery to the signaling IL-2R.

[0157] Example 3: Mouse T reg Selective cell stimulation requires CD25 deflection and IL-2 delivery. Next, the in vivo activity of our CD25-biased mAb in mice was evaluated. C57BL / 6 wild-type (WT) mice were injected daily for 3 days with either IL-2 alone or a complex of UFKA-10, UFKA-20, UFKA-30, UFKA-40 and NARA1, followed by CD4 in the lymph nodes (LN) and spleen of the treated animals. + CD25 + Foxp3 + T reg cells, CD8 + CD44 hi CD122 + Memory T cells and CD3 - NK1.1 + CD122 + Flow cytometry analysis of NK cells was performed (Figures 2A, 3A, and 3B). In the control group administered with physiological saline, LN cells contained an average of 8.7% CD4+ CD25 + Foxp3 + T reg Cells and 7% CD8 + CD44 hi CD122 + Memory T cells, as well as 8.6% CD4 in the spleen + CD25 + Foxp3 + T reg Cells, 13.4% CD8 + CD44 hi CD122 + Memory T cells and 2.8% NK cells were observed (Figures 2A, 3A, and 3B). Low-dose (LD) IL-2 treatment (1.5 μg per mouse daily) was observed in CD4 + CD25 + Foxp3 + T reg Cells and CD8 + CD122 + The proportion and number of memory T cells increased by approximately 2-3 times in both the LN and the spleen (Figures 2A, 2B, and 3A). IL-2cx produced with UFKA-10, UFKA-30, and UFKA-40 was found to be CD4 + CD25 + Foxp3 + T reg Although the cell stimulation was only slightly improved compared to that observed with LD IL-2 (Figures 2A and 2B), these IL-2cx were CD8 + CD44 hi CD122 + The increase in memory T cells was suppressed (Figures 2A-2C). However, IL-2 / UFKA-20cx was found to be CD4 + CD25 + Foxp3 + T reg It induces active cell growth and LN and spleen T reg The total number of cells is approximately 20 x 10 6 In contrast to animals treated with physiological saline, the result was 1.5 × 10⁻⁶. 6 In LD IL-2 treated animals, 3 × 10 6 And on the other hand, CD8 + CD44 hi CD122+ The proportion and number of memory T cells remained unchanged compared to IL-2 alone (Figures 2A-2C). As expected, CD122-biased IL-2 / NARA1cx was CD8 + CD44 hi CD122 + This induced preferential stimulation of memory T cells, but likely due to the dissociation of IL-2 from NARA1, and some CD4 + CD25 + Foxp3 + T reg Cell enlargement was observed (Figures 2A-2C) (Arenas-Ramirez N. et al., Trends Immunol. 2015 36:763). In summary, these data suggest that CD25 bias alone, as seen in vitro with IL-2 / UFKA-30cx and IL-2 / UFKA-40cx, is insufficient to achieve T in vivo. reg This demonstrates that it is insufficient to stimulate cells. Rather, the ability of IL-2cx to confuse mild CD25 bias and efficiently deliver IL-2 to the trimer IL-2R, such as IL-2 / UFKA-20cx, is considered a necessary feature for in vivo selectivity and efficacy. Therefore, these screening assays demonstrate that T is suppressing inflammation. reg As an improved method for increasing IL-2 signaling to cells, UFKA-20 was identified as a candidate mAb for further characterization.

[0158] Example 4: IL-2 / UFKA-20cx was used in vivo in mice T reg Improving signal transduction to cells In the time course experiment, the CD4 in the spleen of mice + CD25 + T cells, CD8 + We compared the ability of a single intraperitoneal infusion of LD IL-2 (1 μg) versus IL-2 / UFKA-20cx (1 μg / 10 μg) to induce signal transduction, as measured by intracellular staining of phosphorylated STAT5 (pSTAT5) in T cells and NK cells (Figure 4). In mice administered with LD IL-2, CD4 2 hours after infusion + CD25+ We observed preferential stimulation of T cells, but this effect had already disappeared by day 1 (Figure 4A). CD8 + T cells and NK cells did not phosphorylate STAT5 in response to LD IL-2, but when HD IL-2 (30 μg) was administered as a single injection, CD8 + Strong signaling occurred in all three lymphocyte subsets, including T cells and NK cells (Figure 4A). IL-2 / UFKA-20cx was detected on CD4 + CD25 + It showed high selectivity for T cells, which became apparent 2 hours after injection and persisted for at least 2 days, but CD8 + T cell and NK cell pSTAT5 levels remained unaffected by this treatment (Figure 4A). As a result, CD4 + CD25 + T cell counts increased on day 2 after a single infusion of IL-2 / UFKA-20cx, peaked on day 4, and returned to baseline on day 8, but CD8 + The number of T cells and NK cells did not change significantly during the experimental period compared to IL-2 (Figure 4B). The pSTAT5 signaling profile was similar to that of IL-2 / UFKA-20cx CD4 + CD25 + This study not only confirmed the selectivity of IL-2 for T cells but also suggested that IL-2 / UFKA-20cx has a much longer in vivo half-life than IL-2. To verify this, mice were given a single injection of either IL-2 or IL-2 / UFKA-20cx, and then free IL-2 or UFKA-20-compounded IL-2 was measured by sandwich ELISA using NARA1 as the capture mAb and MAB202 as the detection mAb. IL-2 was detected 30 minutes after injection and disappeared within 4 hours, while IL-2 / UFKA-20cx peaked 4 hours after injection and remained present for more than 24 hours (Figure 5). The in vivo half-life was estimated to be 30 hours for IL-2 / UFKA-20cx, compared to approximately 30 minutes for IL-2. The serum half-life of IL-2 / UFKA-22cx was approximately 21 hours.

[0159] Example 5: IL-2 / UFKA-20cx was used in vitro to metabolize human T reg Selectively stimulates cells The activity of IL-2 / UFKA-20cx is, as previously described, that CD4 retains the trimer IL-2R. + CD25 + T cells and CD8 cells equipped with the dimer (CD122+CD132) IL-2R + Evaluation was performed using a newly isolated resting human T cell subset, including T cells (Arena-Ramirez, 2006). CD3 was extracted from the peripheral blood of healthy human donors. + T cells were purified, stimulated for 15 minutes with titrated IL-2 and IL-2 / UFKA-20cx (with a molar ratio of IL-2 to UFKA-20 of 1:1), and then gated to CD4. + CD25 + CD127 lo Foxp3 + T reg Cells and CD8 + Intracellular pSTAT5 levels in T cells were evaluated using flow cytometry. A low concentration of IL-2 (0.1 ng / ml) was observed in CD4 cells. + T reg We were able to induce up to half the amount of STAT5 activation in cells, but CD8 + Achieving equivalent STAT5 activation in T cells requires a concentration approximately 1000 times higher (Figure 6), which is consistent with previous publications (Yu, Diabetes 2015 64:2172). IL-2 / UFKA-20cx is used in human CD4 + T reg In cell stimulation, pSTAT5 showed comparable efficiency to IL-2 (Figure 6). Conversely, 50% pSTAT5 + CD8 +To achieve T cell formation, approximately 17 times higher concentrations of IL-2 / UFKA-20cx were required, based on the half-effect concentration (EC50) (Figure 6). The improved CD25 bias selectivity and efficacy of UFKA-20 were demonstrated in in vitro measurements using human IL-2R-containing cell lines, various in vivo experiments in mice, and in vitro measurements using newly isolated primary T cell subsets from various healthy donors, after which several humanized versions of UFKA-20 were generated. L (Sequence ID 020) and V H Human germline genes sharing the highest level of identity with the framework sequence of (SEQ ID NO: 019) were identified, codon-optimized, and synthesized by GeneScript Custom Gene Synthesis, then cloned into an expression vector containing Fc-silent (N297A) human IgG1. The complementarity-determining region (CDR) of UFKA-20 was transcribed to the human immunoglobulin G1 (IgG1) backbone with the N297A mutation, thereby preventing glycosylation at this site and thus significantly reducing Fc γ receptor binding and effector function (Park HI et al., Trends Biotenchol 2016 34:895; Arnold JN et al., Annu. Rev. Immunol. 2007 25:21). The best humanization candidate was named UFKA-22-00, possessing the heavy chain of SEQ ID NO: 017 and the light chain of SEQ ID NO: 018, and is abbreviated as UFKA-22. IL-2 / UFKA-22cx, and similar clones sharing the same CDR but with framework mutations, showed comparable stimulatory activity and selectivity to IL-2 / UFKA-20cx in kinetic binding analysis (Table 2) of IL-2 in vitro (Figure 6) using newly isolated human T cells, or IL-2 injected into mice (Figure 7). Overall, the complex of IL-2 with UFKA-20 and its humanized version UFKA-22 showed comparable stimulatory activity and selectivity to human T cells. reg It shows strong in vitro activity against cells, but human CD8 + The activity against T cells was significantly reduced compared to uncomplexed free IL-2.

[0160] Example 6: Humanized IL-2 / UFKA-22cx was used in rhesus monkeys. reg It exhibits selectivity for cells in vivo. The IL-2R subunit exhibits a high degree of homology between humans and rhesus monkeys. Therefore, a homology search using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) revealed that the identity of CD25, CD122, and CD132 between these two species was 91.9% (accession number NP 001028089.1), 94.2% (NP 001244989.1), and 97.3% (NP001030606.1), respectively. Both the mouse antibody UFKA-20 and the humanized UFKA-22 clone were found to have similar binding to either monkey or human IL-2 in vitro (data not shown). To compensate for the difference in in vivo half-lives between IL-2 and IL-2 / UFKA-22cx, animals were infused daily with 10 μg / kg (LD) or 33 μg / kg (HD) of IL-2 (aldesleukin) from days 0 to 6, while IL-2 / UFKA-22cx in the form of 10 μg / kg IL-2 and 100 μg / kg mAb (LD) or 33 μg / kg IL-2 and 330 μg / kg mAb (HD) was administered on days 0 and 3 (Figure 8A). Infusion of 330 μg / kg of UFKA-22 (without IL-2) on days 0 and 3 was used to evaluate whether it bound to endogenous monkey IL-2. Parameter evaluation on day 8 served as baseline and untreated controls. pSTAT5 levels were measured 1 day after the initial injection, and CD4 was measured after IL-2 in HD and IL-2 / UFKA-22cx in LD and HD. + CD25 + A significant increase in pSTAT5 levels was observed in T cells (Figure 8B), while IL-2 or UFKA-22 alone in LD showed a decrease in CD4 + CD25 + T cell pSTAT5 levels did not change beyond the values ​​measured at baseline at day 8 (Figure 8B). Overall, CD4+ CD25 + The increase in pSTAT5 levels in T cells was more pronounced with IL-2 / UFKA-22cx than with IL-2. CD4 + CD25 + In contrast to T cells, CD4 + CD25 - T cells, CD8 + CD25 + T cells and CD8 + CD25 - pSTAT5 levels in T cells were not significantly altered by IL-2, IL-2 / UFKA-22cx, or UFKA-22 compared to baseline (Figure 8B). reg To evaluate the selectivity for cells, T in the blood of monkeys reg The dose- and time-dependent changes in cells were quantified. The strongest changes were observed on day 6, and here we see CD4 + CD25 + Foxp3 + T cells and CD4 + CD25 + The proportion of T cells was significantly higher in animals treated with LD IL-2 / UFKA-22cx compared to those treated with LD IL-2 (Figure 8C). CD4 + CD25 + Foxp3 + T cells were treated with LD IL-2 / UFKA-22cx twice, followed by CD4 + Although the overall T cell count increased to an average of 29%, even with seven daily infusions of low-dose (LD) IL-2, CD4 + CD25 + Foxp3 + Only 4.8% of T cells were converted (Figure 8C). Although HD IL-2 and HD IL-2 / UFKA-22cx did not surpass the effect seen with LD IL-2 / UFKA-22cx, they produced comparable T cells. reg This resulted in a cellular response (Figure 8C). CD4 on days 3 and 6 after injection of LD's IL-2 / UFKA-22cx. + CD25 +In T cells, levels of Foxp3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) were significantly increased compared to LD IL-2, but HD IL-2 / UFKA-22cx and HD IL-2 did not offer any further advantages (Figure 8D). Similarly, CD4 + CD25 + T cells start to show Ki-67 from day 2. + As a result, Ki-67 levels peaked on days 3 and 6 (Figure 8D), which most likely reflects the cell cycle and proliferation in which IL-2 signaling is induced. Infusion of IL-2-free UFKA-22 was found to be CD4 + CD25 + This resulted in a small but distinct decrease in T cell frequency (Figure 8C), which is likely due to the mild neutralization of endogenous monkey IL-2 by UFKA-22. Foxp3, CTLA-4, and Ki-67 expression levels remained unchanged in the UFKA-22 group (Figure 8D). CD4 + CD25 + Foxp3 + T reg CD8 in cells + When calculating the ratio to T cells, NK cells, and B cells, LD IL-2 / UFKA-22cx is the best T reg Cell selectivity was achieved (Figure 8E). In humanized IL-2 / UFKA-22cx from rhesus monkeys, CD4 + CD25 + Foxp3 + T reg We confirmed the selectivity of these IL-2cx molecules for cells and their superiority over IL-2.

[0161] Example 7: UFKA-20 sterically interferes with the binding of IL-2 to CD122 and CD25. To gain structural and further mechanistic insights into the IL-2 / UFKA-20 interaction, a fragment antigen-binding (Fab) variant of UFKA-20 was created and complexed with IL-2. Subsequently, the IL-2 / UFKA-20 Fab complex was crystallized and structurally analyzed. Crystals were grown at physiological pH (pH 7.48) and diffracted at a resolution of 2.89 Å. The structure was solved by molecular substitution and contained three IL-2 / UFKA-20 Fab complexes in an asymmetric unit. Compared to the crystal structure of the human IL-2 quaternion, UFKA-20 was bound to IL-2 dorsally at an angle of approximately 55° counterclockwise with respect to the longitudinal axis, which was significantly different from the complexes of IL-2 with F5111 (Worldwide Protein Databank (PDB): 5UTZ), JES6-1 (PDB: 4YQX), or NARA1 (PDB: 5LQB) (Figure 9A). Next, the CD25, CD122, and CD132 binding sites were meticulously analyzed. Epitope overlap between the anti-IL-2 mAb and the IL-2R subunit was evaluated and quantified based on the embedded surface area within the IL-2cx and quaternary IL-2R complexes using protein interface, surface, and assembly software tools. UFKA-20 strongly overlapped with the CD122 binding site of IL-2, with an estimated overlap of 40%, although the interference of UFKA-20 with the CD25 binding site of IL-2 was rather mild, at only 6.3% (Figure 9B). The overlap between UFKA-20 and the CD132 binding site of IL-2 was not evident. Thus, UFKA-20 exhibits variable heavy chain (V) H ) CDR1-3 and variable light chain (V L The C and B helices of IL-2 were primarily contacted via CDR1 and CDR3 of UFKA-2, forming a "clamp" around the C helice (Figure 9B). IL-2 residues D84, N88, and V91, normally involved in CD122 interactions, were closely engaged with UFKA-20 (Figure 9A). These interactions are highly likely to inhibit the binding of IL-2 to CD122, as shown by surface plasmon resonance (SPR) measurements (Figure 9C). Furthermore, the V of UFKA-20 HThe chain made small but significant contact with CD25 by being in close contact with IL-2 residues E60, E61, and K64 located at the IL-2-CD25 interface (Figure 9B). However, as measured by SPR, IL-2 / UFKA-20cx efficiently bound to recombinant human CD25 in a dose-dependent manner (Figure 9C). As a result of these multiple contacts, UFKA-20 associated with IL-2 with high affinity, approximately 10 -9 M's K d (Table 1). F5111 bound to IL-2 at a different angle than UFKA-20, and there was a significant overlap between the F5111 and CD122 epitopes (calculated as 48.5%), while this overlap for the CD132 epitope was very slight (2.5%), and there was no significant overlap with the CD25 epitope (0.85%) (Figures 9A and 9B). JES6-1 interacted with mouse IL-2 in a completely different manner than UFKA-20 and F5111. Compared to UFKA-20, JES6-1 bound to the opposite side of IL-2, mainly interfering with CD132 (18%), followed by CD25 (16%) and CD122 (8%) (Figures 9A and 9B). Since mouse IL-2R quaternary complexes are unavailable, the IL-2R overlap of mouse IL-2 / JES6-1cx was calculated using quaternary human IL-2R crystals. IL-2 / NARA1 completely overlapped with CD25, thus "mimicking" the binding of CD25 to IL-2. Detailed observations revealed that NARA1 largely overlapped with the CD25 binding site of IL-2 (52.5%), while the CD122 and CD132 binding sites remained fully accessible (0%) (Figures 9A and 9B). As a result, in SPR, IL-2 / NARA1cx bound very efficiently to recombinant human CD122 but not to CD25 (Figure 9C).

[0162] As suggested by structural analysis, to evaluate whether UFKA-20 functionally competes with the CD122 and CD25 binding sites of IL-2, HEK293T cells expressing different IL-2R subunits were subjected to a set concentration of IL-2 RhodA competitive assay was performed using UFKA-20 at the titration concentration. IL-2 to CD122+CD132 Rhod The binding was already reduced at molar ratios of IL-2 to UFKA-20 of 10:1 and 1:1, thus confirming functional interference of UFKA-20 with CD122 (Figure 9D, middle panel). Conversely, IL-2 Rhod Binding to CD25 was not interfered with at IL-2 to UFKA-20 molar ratios of 10:1 and 1:1, and a 5-50 times higher UFKA-20 concentration was required to compete with CD25 binding (9D, left panel); a similar pattern was observed in HEK293T cells expressing CD25+CD122+CD132 (Figure 9D, right panel). The results regarding the overlap of UFKA20 epitopes and the embedded surface area within the IL-2cx and IL-2R quaternary complex suggest that binding occurs at the following three sites of the hIL-2 sequence: Epitope A, including H16 and D20. Epitope B includes Q57, E60, E61, L63, K64, E67, and E68. Epitope C includes L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, and M104.

[0163] Epitopes A and C overlap with the IL-2 region (targeted by F5111) which is important for binding to CD122 and CD132, respectively. However, epitope B, which overlaps with the CD25 binding site, is uniquely targeted by UFKA-20 compared to existing antibody clones (Figures 9, 10, and 11), and is likely associated with the enhanced effect provided by this clone compared to more than 10,000 alternatives screened.

[0164] Example 8: CDR mutations that alter the binding of UFKA20 to the hIL-2 epitope UFKA-20 variants containing specific amino acid substitutions in the VH chain (SEQ ID NO: 019) and VL chain (SEQ ID NO: 020) were created, and their effects on weakening or strengthening polar and nonpolar interactions between specific CDR loops and proposed hIL-2 epitopes were investigated (Tables 3 and 4). Seven VH chain variants were included, along with VL chain variants between 1-3 and ~4 containing 2-4 amino acid substitutions. Twelve UFKA-20 variants, including the original UFKA-20 mAb, were expressed, purified, and then their affinity and in vivo activity (Figure 12) were measured. The affinity of the variant antibody was 6.403 × 10⁶. -8 M~1.856×10 -10 Within the M range, 2+9 was the lowest and 5+9 was the highest among the tested variants. Most antibody variants were 10 -10 It binds within the M range and clusters around the affinity of the unmodified original 1+6 (UFKA-20) antibody. C57BL / 6 wild-type mice were administered a single dose of the UFKA-20 variant IL-2 / anti-IL-2cx. CD4 + CD25 + Foxp3 + Cell frequencies were not significantly altered by antibodies containing variants 5+9;4+9;4+10;2+9 (Figure 12B), indicating that these variants lack optimal interaction with the residues of hIL-2 epitopes B (e.g., E61, Q57, R83) and C (L94 and E95), which are affected by residue substitutions in the chains of these variants (Table 4), and that interaction with both is essential for the high efficacy of the hIL-2 complex formed with the antibodies according to the present invention. Notably, epitope B is uniquely targeted by antibodies derived from UFKA-20 compared to known clones capable of forming CD25 target complexes (Figure 10). Most of the test antibodies containing 105+6, 105+9, 2+9, 103+6, and 103+9 are CD4 + CD25 + Foxp3 + T regThe sequence is highly similar to UFKA-20(1+6) in terms of cell stimulation, indicating tolerance to specific amino acid changes in the CDR region, provided that residues S56, M100, Y102 of the humanized antibody derived from SEQ ID NO: 019, and K36, F56, S32, A33, and A100 of SEQ ID NO: 020 maintain their optimal orientation for interaction with IL-2 epitopes B and C.

[0165] K D Value and T reg CD4 mediated by cell stimulation + CD25 + Foxp3 + T reg A correlation was observed between the ability to increase cell frequency and the activity of the antibodies (Figure 12C). Except for antibodies 2+9 and 4+10, the antibody activity clustered around the UFKA-20(1+6) antibody, which had an optimal affinity of 10. -10 This suggested that it was in the M range. However, the 5+9 antibody that bound to IL-2 with the highest affinity was CD4 + CD25 + Foxp3 + T reg The decrease in in vivo activity with respect to cell stimulation indicates that the upper limit of affinity is 1.856. -10 This suggested that...

[0166] Example 9: Fusion protein of IL-2 and UFKA-22 antibody CD25-biased immune complexes (CD25-biased immune complexes) possess excellent immunomodulatory potential, but several biological aspects have hindered their clinical development, preventing their approval for use in inhibiting human inflammatory responses. Firstly, the IL-2 antibody complex formed by incubating IL-2 and an anti-IL-2 antibody at 37°C must be prepared immediately before administration to prevent degradation into separate components. This is inconvenient in a clinical setting and can lead to slight activity differences between batches. Furthermore, this complex may dissociate in vivo, generating soluble IL-2 and producing undesirable off-target signaling. To address these issues, we developed a monotherapy compound that replaces IL-2 / UFKA-22cx therapy (a two-component immunotherapy consisting of recombinant human IL-2 and the humanized CD25-biased anti-IL-2 antibody UFKA-22) with a combined IL-2 / UFKA-22 fusion protein (UFKA-22FP) that retains optimal CD25-mediated signaling and offers improved stability.

[0167] In the UFKA-22FP design, the IL-2 protein and the UFKA-22 antibody need to be linked with a flexible linker, which facilitates not only IL-2 association but, importantly, the dissociation of the UFKA-22 antibody from the IL-2 binding groove at an optimal rate, so that IL-2 signaling via the dimer IL-2R (CD122+CD132) is not hindered by the bound antibody structure. The crystal structure of IL-2 / UFKA-20cx (PDB:6YE3) was analyzed to determine the variable heavy chain (V) of UFKA-20. H ) and variable light chain (V L The distance from the N-terminus of ) to the C-terminus of IL-2 was measured to be 32.2 Å and 43.5 Å, respectively (Figures 12A and 12B). Subsequently, to test which linker could optimize CD25 target signaling via IL-2R, the N-terminal IL-12 polypeptide (Uniprot P60568) was linked to UFKA-22 VH (SEQ ID NO: 017), or to the VL chain (SEQ ID NO: 018), or to (G4S) nUFKA-22FP was generated by linking with a flexible glycine (G) serine (S) linker consisting of (SEQ ID NO: 023) (with n being in the range of 3 to 6 repetitions) (Figures 13C and D, and Figure 14). UFKA-22FP vH(G4S)3, UFKA-22FP vH(G4S)4, UFKA-22FP vH(G4S)5, and UFKA-22FP vL(G4S)6 were obtained by suspension culture of HEK293 FreeStyle cells expressed downstream of a secretion signal (SEQ ID NO: 027) and purified using a HiTrap® Protein G column. UFKA-22FP was tested for binding to CD25 minibodies (NARA1) by ELISA. The fact that all four UFKA-22FP variants bound to NARA1 suggested that the IL-2 domain of UFKA-22FP was correctly folded. The physiological activity of IL-2 was investigated using a cell proliferation assay with mouse CTLL-2 cells expressing all three IL-2R subunits, a STAT5 signaling assay with HEK-Blue IL-2 reporter cells expressing human IL-2Rαβγ, and T in human PBMCs. reg Measurements were taken upon stimulation. The activity and T of the fusion protein were measured once. reg Selectivity was confirmed for all fusion proteins, and the most promising candidates were selected for further mouse testing.

[0168] C57BL / 6 wild-type mice were infused daily for 3 days with IL-2 / UFKA-22cx, UFKA-22FP vH(G4S)5 and UFKA-22FP vL(G4S)6 (including the hIL-2 LC fusion sequence number 028), followed by CD4 infusion in the spleen of the treated animals. + CD25 + Foxp3 + T reg cells, CD8 + CD44 hi CD122 + Memory T cells and CD3 - NK1.1 + CD122 +Flow cytometry analysis was performed on NK cells. Since the UFKA-22FP molecule consists of one UFKA-22 antibody and two IL-2 molecules, a molar ratio of 2:1 for IL-2 to UFKA-22 antibody was used in the IL-2 / UFKA-22cx formulation. Three injections of UFKA-22FP vH(G4S)5 were performed, resulting in CD25 + Foxp3 + T reg The cell fraction increased slightly, but the change was not significant at the applied dose. On the other hand, UFKA-22FP vL(G4S)6 showed CD25 + Foxp3 + T reg The frequency of cells was significantly increased to 15.2±1.2% in the spleen at a dose of 12 μg, and reached 20.6±1.4% at a dose of 24 μg. Treatment with UFKA-22FP vL(G4S)6 is effective for CD4 + CD25 + Foxp3 + T reg It induced a dose-dependent increase in Ki-67 expression in cells, and compared to 12 μg and 24 μg of UFKA-22FP vL(G4S)6, it increased CD4 expression by 42.0 ± 5.5% and 64.1 ± 3.7%, respectively. + CD25 + Foxp3 + T reg The cells upregulated Ki-67. + CD4 + CD25 + Foxp3 + T reg A significant increase in cells was observed in mice treated with 24 μg UFKA-22FP vL(G4S)6 and in mice injected with 12 μg IL-2 / UFKA-22cx, with an increase of 70.9 ± 1.1% Ki-67. + It was very comparable to that. CD8 + The absence of a significant change in T cell frequency suggests that the fusion protein is cytotoxic CD8 +We demonstrated that it does not produce off-target effects on T cells. Although the activity of UFKA-22FP was lower than that of IL-2 / UFKA-22cx, UFKA-22FP vL(G4S)6 achieved similar activity at a slightly higher dose (Figure 14). In summary, a peptide linker length of 30 amino acids relative to the light chain is preferable. Binding of IL-2 to the antibody heavy chain is also achievable. These results suggest that a fusion protein with desirable manufacturing and pharmacosafety properties can achieve comparable efficacy in vivo to two-component IL-2cx, albeit at a moderate dose increase.

[0169] Example 10: Enhancing mouse and human cDCs with IL-2 immunotherapy. DC is characterized by the absence of lineage markers (Lin) and by CD11c being intermediate (int) or high (hi), and CD11c int B220 hi pDC, CD11c hi Major histocompatibility complex class II (MHC-II) hi cDC, CD11b low XCR1 + CD8α + DNGR-1(CLEC9A) + cDC1, CD11b hi XCR1 - cDC2 can be further subdivided (Figure 15A). A short course of three injections of recombinant human IL-2 (IL-2; teseroykin) increased the total cDC count in the spleen of wild-type (WT) adult mice (Figure 15B). This increase was attributable to the active proliferation of cDCs, as evidenced by the increased uptake of the thymidine analog bromodeoxyuridine (BrdU) into cDCs (Figure 15C). This IL-2 effect is attributed to CD25 hi or CD122 hiTo assess whether the effects were caused by IL-2 binding to cells, CD25-biased IL-2 / anti-IL-2(5344) antibody conjugates (IL-2 / 5344) and CD122-biased IL-2 / anti-IL-2(NARA1) antibody conjugates (IL-2 / NARA1) were tested (Letourneau EMPNAS 2010,107:11906; Krieg C. PNAS 2010,107:11906, Arenas-Ramirez N. Sci Transl Med 2016). Both IL-2 / 5344 and IL-2 / NARA1 mouse IL-2 / antibody conjugates stimulated spleen DC enlargement and proliferation to quantitatively equivalent levels as unbiased IL-2 (Figure 15B, C): Treatment of mice with IL-2cx containing an inflammatory CD122-targeted IL-2 antibody induced upregulation of CD40, CD80, CD86, and MHC class I (MHC-I) on cDCs, but not MHC-II (Figure 15D), indicating mature cDCs with increased potential for cross-presentation and co-stimulation for T cell activation.

[0170] Human CD11c + MHC-II (HLA-DR) + DC was tested in a investigator-initiated clinical trial using recombinant hIL-2 (aldesleukin) immunotherapy (called Charact-IL-2, NCT 03312335) (Figure 16A). In clinical trials testing aldesleukin, aldesleukin immunotherapy was shown to improve CD4 + and CD8 + Proliferation of T cells and NK cells has been reported (Klatzmann D. et al., Nat Rev Immunol 2015, 15:283; Humrich JY et al., Lancet Rheumatol 2019, 1:e44). However, Ki67 on day 0 (before administration) and day 5 (1 day after the last administration) of a 5-day course of daily aldesleukin administration + Comparing the DCs, we observed an increase in the proliferation of cDC1 and cDC2 (Figure 16B).

[0171] Parenteral administration of the UFKA20 conjugate (IL-2 conjugated to the CD25-biased antibody UFKA20) also increased cDCs in the spleen of mouse recipients (Figure 17A) and induced cDC proliferation as measured by BrdU uptake (Figure 17B). Treatment with pharmaceutical compositions containing the UFKA20 conjugate downregulated MHC-II and CD80 (Figures 17C and D), indicating that the conjugate with the CD25-targeted antibody reduces the ability of cDCs to present antigens and transmit costimulatory signals to T cells. Treatment with the UFKA20 conjugate further induces upregulation of the immunoregulatory proteins programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and PD-L2 expressed in cDCs (Figure 17E). In mice treated with the UFA20 complex, immunomodulatory genes such as transforming growth factor β-inducible (Tgfbi), interleukin-1 receptor antagonist (Il1rn), and TGF-β-activated kinase 1 / MAP3K7-binding protein 1 (Tab1) were upregulated (positive values ​​on the x-axis). Immunostimulatory genes, including interleukin-6 signal transducer (Il6st), lymphatoxin β (Ltb), tumor necrosis factor (ligand) superfamily member 14 (Tnfsf14), and colony-stimulating factor 1 (Csf1), were downregulated in mice treated with the UFKA20 complex (negative values ​​on the x-axis). Furthermore, cDCs treated with the UFKA20 complex downregulated TNF receptor superfamily member 6 (Fas), suggesting an extension of survival time (Figure 17F). From the above, it was shown that pharmaceutical compounds containing UFKA20 promote the proliferation of cDCs with immunotolerative phenotypes and immunomodulatory properties.

[0172] [Table 1]

[0173] [Table 2]

[0174] Table 3

[0175] Table 4

[0176] JPEG0007849048000007.jpg212153JPEG0007849048000008.jpg228153

Claims

1. A human interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or an antigen-binding fragment thereof, wherein the hIL-2 specific mAb interacts with an amino acid residue of hIL-2 that provides an epitope, and The epitope is the hIL-2 residue: - H16, D20, - Q57, E60, E61, L63, K64, E67, E68, and - L80, R81, R83, D84, I86, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104, It consists of, The hIL-2 specific mAb, or an antigen-binding fragment thereof, has a V Ｈ complementary determining region CDR Ｈ 1, CDR Ｈ 2, and CDR Ｈ 3-containing heavy chain variable (V Ｈ ) region, and a V Ｌ complementary determining region CDR Ｌ 1, CDR Ｌ 2, and CDR Ｌ 3-containing light chain variable (V Ｌ ) region, and herein a. CD-R Ｈ 1 contains or is identical to the amino acid sequence shown in Sequence ID No. 001; and b. CD-R Ｈ 2 contains or is identical to the amino acid sequence shown in Sequence ID No. 002; and c. CD-R Ｈ 3 contains or is identical to the amino acid sequence shown in Sequence ID No. 003; and d. CD-R Ｌ 1 contains or is identical to the amino acid sequence shown in Sequence ID No. 004; and e. CD-R Ｌ 2 contains or is identical to the amino acid sequence shown in Sequence ID No. 005; and f. CD-R Ｌ 3 contains or is identical to the amino acid sequence shown in Sequence ID No.

006. The aforementioned hIL-2 amino acid residue of the epitope has a buried surface area of ​​5 square angstroms (Å) as measured by buried surface area analysis of the crystal structure of the Fab fragment of hIL-2 specific mAb that has formed a complex with the hIL-2 polypeptide. ２ ) Defined as exceeding, The human interleukin-2 (hIL-2) specific monoclonal antibody (mAb), or its antigen-binding fragment.

2. The binding of the aforementioned hIL-2-specific mAb to hIL-2 is as follows: - (≤) 4.3 × 10 －９ The following dissociation constant (K) Ｄ ), - (≧) 4.12 × 10 ５ Ms －１ The above on rate (K ｏｎ ), and - (≤) 2.20 × 10 －３ s －１ The following off rates (K ｏｆｆ ), Characterized by, The hIL-2 specific mAb according to claim 1, or its antigen-binding fragment.

3. The complex obtained by combining the aforementioned hIL-2-specific mAb and hIL-2 in a ratio between 2:1 and 1:2 is: - The binding ratio to high-affinity hIL-2 receptors compared to intermediate-affinity hIL-2 receptors is between 20 and 121, and / or - The ratio of the binding affinity of CD25 alone to that of the intermediate affinity hIL-2 receptor is between 277 and 483, and / or - Dissociation of hIL-2 mAb from hIL-2 in the binding of the complex to the high-affinity hIL-2 receptor, and / or - Human CD3 ＋ CD4 ＋ CD127 ｌｏｗ Foxp3 ＋ T ｒｅｇ Cells with an EC50 of ≤ 0.154 and human CD8 ＋ Activating T cells when EC50 is (≥) 442.9, The hIL-2 specific mAb according to claim 1 or 2, or its antigen-binding fragment, characterized by...

4. hIL-2 specific mAb, or its antigen-binding fragment, V Ｈ Complementarity Determination Region CDR Ｈ 1. CD-R Ｈ 2, and CDR Ｈ 3 is included in the heavy chain variable (V Ｈ ) region and V Ｌ Complementarity Determination Region CDR Ｌ 1. CD-R Ｌ 2, and CDR Ｌ Variable light chain (V) including 3 Ｌ ) including the region and here a. CD-R Ｈ 1 is identical to the amino acid sequence shown in Sequence ID No. 001; and b. CD-R Ｈ 2 is identical to the amino acid sequence shown in Sequence ID No. 002; and c. CD-R Ｈ 3 is identical to the amino acid sequence shown in Sequence ID No. 003; and d. CD-R Ｌ 1 is identical to the amino acid sequence shown in Sequence ID No. 004; and e. CD-R Ｌ 2 is identical to the amino acid sequence shown in Sequence ID No. 005; and f. CD-R Ｌ 3 is identical to the amino acid sequence shown in Sequence ID No.

006. The aforementioned hIL-2 specific mAb, or its antigen-binding fragment.

5. a. The aforementioned V Ｈ The sequence is selected from the amino acid sequences shown in SEQ ID NO: 007, SEQ ID NO: 008, SEQ ID NO: 009, SEQ ID NO: 010, SEQ ID NO: 011, SEQ ID NO: 012, SEQ ID NO: 013, and SEQ ID NO: 014, and b. The above V Ｌ The sequence is selected from the amino acid sequences shown in SEQ ID NO: 015 and SEQ ID NO:

016. The hIL-2 specific mAb or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. a. - Position 74 and / or 84 is serine, and / or - The 93rd position is methionine, and / or - Alanine is ranked 122nd; V is identical to the amino acid sequence shown in Sequence ID No. 007 by (≥) 96% or more. Ｈ Region array, and b. - Isoleucine is ranked 69th. V is identical to the amino acid sequence shown in Sequence ID No. 015 by more than 99%. Ｈ Region array, including, The hIL-2 specific mAb according to any one of claims 1 to 5, or the antigen-binding fragment thereof.

7. a. V Ｈ The region includes a sequence selected from the amino acid sequences shown in SEQ ID NO: 007, SEQ ID NO: 008, SEQ ID NO: 009, SEQ ID NO: 010, SEQ ID NO: 011, SEQ ID NO: 012, SEQ ID NO: 013, and SEQ ID NO: 014, or a functionally similar sequence derived from any one of these reference sequences by the substitution rules shown below; and b. V Ｌ The region includes a sequence selected from the amino acid sequences shown in SEQ ID NO: 015 and SEQ ID NO: 016, or a functionally similar sequence derived from any one of these reference sequences by the substitution rules shown below. Here, the substitution rule for deriving functionally similar sequences from each reference sequence is: i. Glycine (G) and alanine (A) are interchangeable; valine (V), leucine (L), and isoleucine (I) are interchangeable; and A and V are interchangeable; ii. Tryptophan (W) and phenylalanine (F) are interchangeable, and tyrosine (Y) and F are interchangeable; iii. Serine (S) and threonine (T) are interchangeable; iv. Aspartic acid (D) and glutamic acid (E) are interchangeable; v. Asparagine (N) and glutamine (Q) are interchangeable, N and S are interchangeable, N and D are interchangeable, and E and Q are interchangeable; vi. Methionine (M) and Q are interchangeable; vii. Cysteine ​​(C), A, and S are interchangeable; viiii. Proline (P), G, and A are interchangeable; ix. Arginine (R) and lysine (K) are interchangeable; The hIL-2 specific mAb according to any one of claims 1 to 5, or the antigen-binding fragment thereof.

8. a. A first sequence that is (≥) 90% identical to at least one of the amino acid sequences shown in SEQ ID NO: 007, SEQ ID NO: 008, SEQ ID NO: 009, SEQ ID NO: 010, SEQ ID NO: 011, SEQ ID NO: 012, SEQ ID NO: 013, SEQ ID NO: 014, and SEQ ID NO: 017; and b. A second sequence that is (≥) 90% identical to at least one of the amino acid sequences shown in SEQ ID NO: 015, SEQ ID NO: 016, and SEQ ID NO:

018. Further including, Having the features described in any one of claims 1 to 3, hIL-2 specific mAb, or its antigen-binding fragment.

9. The aforementioned hIL-2 specific mAb is: a. A heavy chain comprising or consisting of the amino acid sequence shown in Sequence ID No. 017; and b. A light chain comprising or consisting of the amino acid sequence shown in Sequence ID No. 018, including, The hIL-2 specific mAb according to any one of claims 1 to 8.

10. A nucleic acid molecule encoding an hIL-2 specific mAb or its antigen-binding fragment, as described in any one of claims 1 to 9.

11. c. The hIL-2 specific mAb according to any one of claims 1 to 9, or the antigen-binding fragment thereof, and d. hIL-2, A pharmaceutical composition for use as a medicine, including the above.

12. The pharmaceutical composition for use according to claim 11, wherein the IL-2 and the hIL-2 specific mAb are covalently associated.

13. A pharmaceutical composition comprising an hIL-2 specific mAb for use according to claim 11 or 12 in the treatment of an autoimmune disease selected from systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, autoimmune hepatitis, amyotrophic lateral sclerosis, type 1 diabetes mellitus, type 2 diabetes mellitus, arteriosclerosis, multiple sclerosis, inflammatory and autoimmune myopathy, alopecia areata, psoriasis, or inflammatory bowel disease.

14. A pharmaceutical composition comprising an hIL-2 specific mAb for use according to any one of claims 11 to 13 in the treatment of allograft-related disorders diagnosed in patients undergoing solid organ transplantation.

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