Interleukin-15-Based Immunocytokines

Abstract

The invention relates inter alia to IL-15 superagonists (based on the sushi domain of IL-15 and IL-15Rα) and immunocytokines, including antibodies. The invention also provides nucleic acids, vectors, methods and medical uses.

Description

[Technical field]

[0001] The present invention relates inter alia to IL-15 superagonists (based on the sushi domain of IL-15 and IL-15Rα) and immunocytokines, including antibodies. [Background technology]

[0002] Interleukin 15 (IL-15) regulates cytotoxic lymphocytes and memory phenotype CD8 + It is a naturally occurring cytokine that induces the generation of T cells and stimulates the proliferation and maintenance of natural killer (NK) cells, but in contrast to interleukin-2, it does not mediate activation-induced cell death, does not consistently activate Tregs, and is less likely to cause capillary leak syndrome (Waldmann, Dubois et al. 2020). Extensive preclinical and clinical studies have been conducted that demonstrate the efficacy and limitations of IL-15 and the growing number of IL-15 analogues / supergonists, particularly in the treatment of cancer, and are reviewed by Robinson and Schluns (Robinson and Schluns 2017).

[0003] IL-15, like interleukin 2 (IL-2), acts through a heterotrimeric receptor with α, β, and γ subunits, but they share a common γ chain receptor (γ cor gamma) and IL-2 / IL-15Rβ (IL-2Rβ, also known as CD122), with a gamma chain receptor also shared with IL-4, IL-7, IL-9 and IL-21. This heterotrimeric receptor contains specific subunits for IL-2 or IL-15, namely IL-2Rα (CD25) or IL-15Rα (CD215), as a third subunit. The downstream IL-2 and IL-15 heterotrimeric receptors share JAK1 (Janus kinase 1), JAK3, and STAT3 / 5 (signal transducer and activator of transcription 3 and 5) molecules for intracellular signaling, resulting in similar functions, although both cytokines also have distinct roles, as reviewed in Waldmann (2015, see e.g. Table 1) and Conlon (2019).

[0004] Therefore, activation of different heterotrimeric receptors by binding of IL-2, IL-15 or their derivatives may result in differential regulation of the immune system and potential side effects. Recently, NK cells and CD8 + Novel compounds containing IL-15 or IL-15 variants have been designed to specifically target T cell activation. These target the intermediate affinity IL-2 / IL-15Rβγ, i.e., NK cells, CD8 + IL-2 / IL-15Rβ and γ, expressed on T cells, NKT cells, and γδ T cells cThese compounds target receptors composed of IL-15 subunits. This is important for safe and potent immune stimulation mediated by IL-15 transpresentation, but the designed compounds SOT101 (SO-C101, RLI-15), ALT-803 and hetIL-15 already contain (part of) the IL-15Rα subunit and therefore mimic the transpresentation of the α subunit by antigen-presenting cells. SO-C101 contains a covalently linked sushi+ domain of IL-15Rα and therefore binds only to the intermediate affinity IL-15Rβγ. For this reason, SO-C101 does not bind to either IL-15Rα or IL-2Rα. Similarly, ALT-803 and hetIL-15 possess the IL-15Rα sushi domain or soluble IL-15Rα, respectively, and therefore bind to the intermediate affinity IL-15Rβγ receptor. Therefore, IL-15 and IL-15 analogs / supergonists are promising clinical stage development candidates for the treatment of cancer and infectious diseases (infections).

[0005] Antibody-cytokine fusion molecules, called immunocytokines, have been developed as a method of targeted cytokine delivery. Such proteins retain both antigen-binding properties and cytokine activity. By targeting the antibody moiety to tumor-associated antigens, angiogenic antigens, tumor microenvironment antigens, or immune checkpoints, the immunocytokine is sequestered in the tumor microenvironment, and the cytokine moiety can signal through its cognate receptor expressed on immune cells to induce an anti-tumor response. For example, if the antibody is a checkpoint inhibitor (CPI), the combination of the antibody and cytokine will enhance the immune response to cancer by lifting the "brakes" on the immune system through the CPI and stimulating immune cells through the cytokine. When the antibody targets a tumor antigen, the antibody effector function may be enhanced by the presence of cytokines that activate immune cells involved in the antibody effector function.

[0006] However, no approved IL-15-based immunocytokines are yet commercially available, and recently, IL-2-based immunocytokines targeted against CEA and FAP appear to have been discontinued.

[0007] Thus, there is a continuing need to improve the design of immunocytokines. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Waldmann, TA, S. Dubois et al. (2020). Frontiers in Immunology 11(868) [Non-Patent Document 2] Robinson, T.O. and K.S. Schluns (2017). Immunol Lett 190:159-168 [Non-Patent Document 3] Waldmann,TA (2015). Cancer Immunol Res 3(3):219-227 [Non-Patent Document 4] Conlon, KC et al., (2019). J Interferon Cytokine Res 39(1):6-21 Summary of the Invention [Means for solving the problem]

[0009] In the present invention, the inventors have developed a mutation / protein modification toolbox that allows the modulation of IL-15 superagonist (based on the sushi domain of IL-15 and IL-15R) activity and antibodies forming immune cytokines. The inventors have developed a mutation / protein modification toolbox that allows the modulation of IL-15 superagonist IL-2 / IL-15Rβ and / or γ in order to minimize target mediated drug deposition due to too high affinity for immune effector cells and to provide increased half-life of immune cytokines.c Suitable single or double mutations that reduce binding to the receptor were identified. Different mutations make it possible to adjust the level of reduced binding. Other mutations in the IL-15 superagonist may improve the homogeneity of the IL-15 variant with respect to post-translational modifications. Modulating the activity of the IL-15 superagonist may also include altering the presence of one or two cytokines fused to the antibody. Thus, the toolbox also includes mutations that allow heterodimeric antibodies. Toolbox mutations for modulating antibody effector functions may include Fc mutations that enhance or reduce antibody-dependent cellular cytotoxicity and / or mutations that increase the in vivo half-life or stability of the antibody. The toolbox also includes enhancing antibody-dependent cellular cytotoxicity by producing afucosylated antibodies. The toolbox further includes different formats of antibodies adapted to specific needs, such as IgG1 or IgG4 antibodies. The inventors have also designed exemplary immunocytokines in the present invention that aim to combine CPI activity or tumor antigen targeting antibody and IL-15 superagonist activity. An immunocytokine based on heterodimerized pembrolizumab with reduced ADCC activity fused to an RLI molecule with reduced binding to IL-2 / IL-15Rβ is a combination of CPI and cytokine. An immunocytokine based on heterodimerized hCl1a, anti-CLDN18.2 antibody (which may have enhanced ADCC activity) fused to an RLI molecule with reduced binding to IL-2 / IL-15Rβ is a combination of tumor antigen targeting antibody and cytokine. [Brief description of the drawings]

[0010] [Figure 1A]LMW SDS-PAGE and Western blot (anti-RLI-15) analysis of RLI2 (RLI2 wt), RLI2 with a G78A substitution (RLI2 A) and RLI2 with a G78A / N79Q substitution (RLI2 AQ) under non-reducing conditions. For Coomassie staining, 0.5 or 2 μg of protein were used (lanes 2, 4, 6, 8, 10 and 12) and for Western blotting, 25 ng of protein was used (lanes 3, 7, 11). [Figure 1B] Capillary electrophoresis, denaturation and analysis of RLI2 (RLI2 wt), RLI2 with G78A substitution (RLI2 A) and RLI2 with G78A / N79Q substitutions (RLI2 AQ) under reducing (R) and non-reducing (NR) conditions. Dashed box 1 represents the band at glycosylation site number 2 (major), box 2 represents the band at glycosylation site number 1 (minor), and dashed box 3 represents the de novo glycosylation site in RLI2A. Unnamed lanes are markers for 16, 21, 30, 48 and 68 kDa. [Diagram 2] Analysis of three deglycosylated RLI variants expressed in CHO cells by SDS-PAGE (7.5–18%) stained with Coomassie blue (left pane), silver nitrate (middle pane) and detected by anti-IL15 Western blot (right pane): lane 1: molecular weight marker; lane 2: RLI2N176Q, lane 3: RLI2N168S / N176Q / N209S, lane 4: RLI1N168S / N176Q / N209S. [Diagram 3] Potency of RLI2 and RLI2AQ from supernatants determined by activation of 32Db or kit225 cells: (A) 32Db cells, 21 hours (h), (B) kit225 cells, 4 hours. [Figure 4] Relative potency of purified RLI2 or RLI2 from supernatant compared to RLI2AQ from supernatant as determined by activation of kit225 cells. [Figure 5A]Comparison of hyperglycosylated and hypoglycosylated RLI2. CPI HIC elution profiles depending on the concentration of buffer B measured at 280 nm. The left box shows pooled fractions 2B1 1-3 for hyperglycosylated RLI2 ("RLI-15-HG") and the right box shows pooled fractions 4B1 1-3 for hypoglycosylated RLI2 ("RLI-15-LG"). [Figure 5B] Comparison of hyperglycosylated and hypoglycosylated RLI2. SDS PAGE of fractions 2B1 1-3 of RLI-15-HG, RLI2 reference standard and molecular weight ladder at the given kDa. [Figure 5C] Comparison of hyperglycosylated and hypoglycosylated RLI2. SDS PAGE of fractions 4B1 1-3 of RLI-15-LG, RLI2 reference standard and molecular weight ladder at the given kDa. [Figure 6] Capillary electrophoresis of selected PEM-RLI constructs. [Figure 7] In vivo therapeutic efficacy of PEM-RLI NA x1 compared to pembrolizumab in treating HuCell MC38-hPD-L1 tumor cell line implanted in female hPD-1 single KI HuGEMM mice. [Figure 8] Mixed lymphocyte reaction (hPBMC donor): INFγ secretion in pg / ml for control (PBMC only), pembrolizumab and PEM LE-RLI2AQ NA x1. [Figure 9A]Pharmacokinetics and pharmacodynamics of PEM-RLI constructs with reduced IL-2Rβγ binding compared to PEM-RLI with normal binding following IV administration of 10 or 30 μg / kg PEM-RLI x1 and 30 or 90 μg / kg PEM LE / YTE-RLI NA x1 on days 1 and 15 in cynomolgus monkeys, respectively. Concentration of constructs in serum dependent on time in hours after administration. 10 μg / kg PEM-RLI x1 (solid grey line), 30 μg / kg PEM LE / YTE-RLI NA x1 (dotted grey line), 30 μg / kg PEM LE / YTE-RLI NA x1 (dotted black line), or 90 μg / kg PEM LE / YTE-RLI NA x1 (solid black line); LLOQ refers to lower limit of quantification: PEM-RLI x1 (grey), PEM LE / YTE-RLI NA x1 (black). Two animals per group (filled and open circles for individual animals in the group). [Figure 9B] Pharmacokinetics and pharmacodynamics of PEM-RLI constructs with reduced IL-2Rβγ binding compared to PEM-RLI with normal binding following IV administration of 10 or 30 μg / kg PEM-RLI x1 and 30 or 90 μg / kg PEM LE / YTE-RLI NA x1 on days 1 and 15, respectively, in cynomolgus monkeys. Daily time-dependent lymphocyte counts (fold change): PEM-RLI x1 (grey), PEM LE / YTE-RLI NA x1 (black). 2 animals per group (filled and open circles for individual animals in groups). [Figure 9C] Pharmacokinetics and pharmacodynamics of PEM-RLI constructs with reduced IL-2Rβγ binding compared to PEM-RLI with normal binding following IV administration of 10 or 30 μg / kg PEM-RLI x1 and 30 or 90 μg / kg PEM LE / YTE-RLI NA x1 on days 1 and 15, respectively, in cynomolgus monkeys. % Ki67+ NK cells: PEM-RLI x1 (grey), PEM LE / YTE-RLI NA x1 (black). 2 animals per group (filled and open circles for individual animals in groups). [Figure 9D]Pharmacokinetics and pharmacodynamics of PEM-RLI constructs with reduced IL-2Rβγ binding compared to PEM-RLI with normal binding following IV administration of 10 or 30 μg / kg PEM-RLI x1 and 30 or 90 μg / kg PEM LE / YTE-RLI NA x1 on days 1 and 15, respectively, in cynomolgus monkeys. % Ki67+ CD8+ T cells: PEM-RLI x1 (grey), PEM LE / YTE-RLI NA x1 (black). 2 animals per group (filled and open circles for individual animals in groups). [Figure 10A] Comparison of the pharmacokinetics and pharmacodynamics of PEM-RLI NA x1 and PEM-RLI NA x2 in cynomolgus monkeys after a single IV dose of 30 μg / kg. Concentration of construct in serum dependent on time in hours; LLOQ refers to lower limit of quantification. PEM-RLI NA x1 is represented in black with two individual animals represented by solid and open circles, PEM-RLI NA x2 is represented in grey with two individual animals represented by solid and open circles. [Figure 10B] Comparison of pharmacokinetics and pharmacodynamics of PEM-RLI NA x1 and PEM-RLI NA x2 in cynomolgus monkeys following a single IV dose of 30 μg / kg. Time dependent lymphocyte counts (fold change) in days. PEM-RLI NA x1 is represented in black with two individual animals represented as solid and open circles, PEM-RLI NA x2 is represented in grey with two individual animals represented as solid and open circles. [Figure 10C] Comparison of pharmacokinetics and pharmacodynamics of PEM-RLI NA x1 and PEM-RLI NA x2 in cynomolgus monkeys following a single IV dose of 30 μg / kg. % Ki67+ NK cells. PEM-RLI NA x1 is represented in black with two individual animals represented as solid and open circles, PEM-RLI NA x2 is represented in grey with two individual animals represented as solid and open circles. [Figure 10D]Comparison of pharmacokinetics and pharmacodynamics of PEM-RLI NA x1 and PEM-RLI NA x2 in cynomolgus monkeys following a single IV dose of 30 μg / kg. % Ki67+ CD8+ T cells. PEM-RLI NA x1 is represented in black with two individual animals represented as solid and open circles, PEM-RLI NA x2 is represented in grey with two individual animals represented as solid and open circles. [Figure 11] Comparison of the pharmacokinetics of PEM LE / YTE-RLI NA x1 and PEM LE-RLI NA x1 in cynomolgus monkeys after a single IV dose of 600 μg / kg. Concentrations of constructs in serum are shown as a function of time in hours, where LLOQ refers to the lower limit of quantification: PEM LE / YTE-RLI NA x1 is represented by black circles / dotted line and PEM LE-RLI NA x1 is represented by grey circles and solid line. [Figure 12] Comparison of the pharmacokinetics of PEM LE-RLI NA x1 and PEM-RLI NQD x1 in cynomolgus monkeys after a single IV dose of 600 μg / kg. Concentrations of constructs in serum are shown as a function of time in hours, where LLOQ refers to the lower limit of quantification: PEM LE-RLI NA x1 is represented by grey circles / lines and PEM-RLI NQD x1 is represented by black circles / lines. [Figure 13] Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector function to immunocytokines and antibodies hCl1a and zolbetuximab with reduced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). [Figure 14A] Comparison of ADCC activity of immunocytokines based on hCl1a antibody with unmodified effector function to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (A) DLE mutations. [Figure 14B] Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector function to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (B) DE mutations. [Figure 14C] Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector function to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (C) AAA mutation. [Figure 14D] Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector functions to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (D) TL mutations. [Figure 14E] Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector function to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (E) IE mutation. [Figure 14F]Comparison of ADCC activity of immunocytokines based on hCl1a antibodies with unmodified effector functions to immunocytokines and antibodies hCl1a and zolbetuximab with enhanced ADCC activity. ADCC target cells were A549 cells overexpressing CLDN18.2 (A549-CLDN18.2) or PA-TU-8988S cells endogenously expressing CLDN18.2 (PATU). (F) Defucosylated immunocytokines. [Figure 15] PD activity of RTX-RLI2AQ immunocytokines in Balb / c mice: The percentage of CD8+ T cells or NK cells (left panel) or activated CD8+ T cells or NK cells (Ki67+ - right panel) was determined by flow cytometry. [Figure 16] Anti-metastatic activity of RTX-RLI2AQ immunocytokine in an in vivo Renca mouse metastasis model as determined by lung weight. [Figure 17] Antitumor efficacy of RTX-RLI immunocytokines in A20-hCD20 / Balb / c mice: Tumor growth is shown for individual mice depending on time. A: Buffer control injected subcutaneously on days 1-4; B: RLI2 control injected subcutaneously at 1 mg / kg on days 1-4; C: RTX-RLI2AQ injected iv at 0.15 mg / kg on days 1 and 8. [Figure 18] ADCC activity of RTX-RLI immunocytokines based on Rituximab compared to Rituximab alone: ​​% of dead tumor cells determined by DAPI positivity was determined depending on the concentration of the polypeptide tested: black circles: Rituximab, grey circles: Rituximab + NK92 cells; black triangles with dotted line: RTX-RLI2AQ x2; black triangles with solid line: RTX-RLI2AQ x2 + NK92 cells; grey squares: RTX-RLI2AQ x1; black squares: RTX-RLI2AQ x1 with NK92 cells. [Figure 19A] The % of PD-1 / PD-L1 blockade is shown depending on increasing concentrations of Keytruda and SOT201 in pM. [Figure 19B]Percentage of Ki67+ NK cells and CD8+ T cells determined by flow cytometry after 7 days of in vitro stimulation of human PBMCs from healthy donors with increasing amounts of SOT201 or SOT201 wt carrying an IL-15 moiety without reducing binding to IL-2 / IL-15Rβγ. [Figure 19C] Cell proliferation (Ki67+) of CD8+ T cells or NK cells detected by flow cytometry in the spleens of healthy C57BL / 6 mice (n=2 / group) 5 days after IV injection of equimolar amounts of compound to 5 mg / kg of the murine surrogate molecule mSOT201 (anti-mouse PD-1 antibody RMP1-14 fused RLI-15AQA) compared to anti-mouse PD-1 antibody alone or anti-human PD1 mouse IgG1-RLI-15AQA (hPD1-mSOT201) as single activity controls. [Figure 20A] Tumor volume in mm3 over a 17-day time course in C57BL / 6 mice bearing syngeneic MC38 tumor cells treated IV on day 1 (randomization day with tumor volume between 80 and 100 mm3) with a single injection of control (NaCl), mSOT201, mSOT201 (5 mg / kg) and an equimolar amount of hPD1-mSOT201 or mPD1 (n=10 mice / group). [Figure 20B] Corresponding percentage of MC38 tumor-bearing mice surviving up to 100 days after treatment. [Figure 21A(1)] Relative expression levels of indicated adaptive and innate immune cell and cancer-associated fibroblast (CAF)-related gene sets across mSOT201-treated tumor samples (N=3) and control samples (n=4) of MC38 tumor-bearing mice as determined by metagene on RNA-seq data. Box plots: minimum, median, maximum. [Figure 21A(2)] Continued from Figure 21A(1). [Figure 21A(3)] Continuation of Figures 21A(1) and 21A(2). [Figure 21B]Cell proliferation determined by % Ki67+ cells by flow cytometry of the indicated cells in the spleen or lymph nodes of MC38 tumor-bearing mice 7 days after mSOT201 (5 mg / kg) IV treatment of established tumors (80–100 mm3) (n=2). [Figure 22A] Tumor volume in mm3 over a 21-day time course in C57BL / 6 mice bearing MC38 tumors treated IV with a single injection of control (NaCl), mSOT201, mPD1-IL-2βγ agonist that abolishes CD25 binding (IL-2v fused to anti-mouse PD-1 antibody RMP1-14), or the combination of RLI-15AQA and anti-mouse PD-1 antibody mPD1 (RMP1-14). [Figure 22B] Cell proliferation as determined by % Ki67+ cells of CD8+ T cells and NK cells detected by flow cytometry on days 5 and 8 after IV administration in healthy C57 / BL6 mice. [Figure 22C] %Ki67+ cells of CD8+ T cells in spleen or lymph nodes of MC38 tumor-bearing C57BL / 6 mice treated IV with mSOT201, mPD1-IL-2v or a combination of RLI-15AQA and mPD-1 on day 7. Randomization day 1, tumor volume 100 mm3 (n=10 / group). [Figure 23A] Fold change in % and absolute cell counts of Ki67+ NK and CD8+ T cells in blood of cynomolgus monkeys following a single IV dose of 0.6 mg / kg SOT201 on day 1, as determined by flow cytometry and hematology on the days indicated. Each graph curve represents one animal. [Figure 23B] Percent Ki67+ of NK cells and CD8+ T cells in blood of cynomolgus monkeys following administration of 0.3 mg / kg IV SOT201 on days 1 and 21 (indicated by arrows) as determined by flow cytometry on the days indicated. Each graph curve represents one animal. [Figure 24A]Proliferation of NK cells and CD8+ T cells after treatment with murine SOT201 surrogates in vivo. Proliferation of CD8+ T cells and NK cells in the spleen of healthy C57BL / 6 mice on days 5 and 8 after treatment with hPD1-mSOT201, mPD-1, mSOT201, mSOT201 wt, and mPD1-IL2v. Expression of Ki67 in CD8+ T cells and NK cells was detected by flow cytometry. The molecules were administered iv on day 1 at 5.37 mg / kg hPD1-mSOT201, 4.51 mg / kg mPD-1, equimolar to 5 mg / kg mSOT201, and 0.26 mg / kg mPD1-IL2v, equimolar to 0.25 mg / kg mSOT201 wt. Flow cytometry analysis was performed on days 5 and 8. Data represent the mean±SEM of two individuals per group per day. [Figure 24B] Proliferation of NK cells and CD8+ T cells after treatment with murine SOT201 surrogates in vivo. Proliferation of CD8+ T cells and NK cells in the spleen of healthy C57BL / 6 mice on days 5 and 8 after treatment with hPD1-mSOT201, mPD-1, mSOT201, mSOT201 wt, and mPD1-IL2v. Expression of Ki67 in CD8+ T cells and NK cells was detected by flow cytometry. The molecules were administered iv on day 1 at 10.74 mg / kg hPD1-mSOT201, 9.02 mg / kg mPD-1, equimolar to 10 mg / kg mSOT201, and 0.1 mg / kg mPD1-IL2v, equimolar to 0.1 mg / kg mSOT201 wt. Flow cytometry analysis was performed on days 5 and 8. Data represent the mean±SEM of two individuals per group per day. [Diagram 25]Mouse SOT201 surrogates in PD-1-sensitive and PD-1-resistant tumor models in vivo. (A) Anti-PD-1-sensitive tumor models. MC38 / C57BL / 6 mouse model: single iv administration of 4.51 mg / kg mPD-1 (a suboptimal dose compared to the literature, chosen as equimolar to mSOT201), 5 mg / kg mSOT201 or 5.37 mg / kg hPD1-mSOT201 (equimolar to mSOT201) on day 0; DO = randomization day when tumor volume was approximately 80-100 mm3, 10 mice / group. CT26 / BALB / c mouse model: 9.02 mg / kg mPD-1 (effective dose compared to literature), 10 mg / kg mSOT201, 10.74 mg / kg hPD1-mSOT201 (equimolar amount to mSOT201) ip administered four times on days 0, 3, 6 and 9; DO = randomization day when tumor volume was approximately 100 mm3, 10 mice / group. (B) Anti-PD-1 resistant tumor model. CT26 STK11 ko mouse model: 4 doses of ip administration on days 0, 3, 6 and 9 with 9.02 mg / kg mPD-1 (effective dose compared to literature), 10 mg / kg mSOT201, 10.74 mg / kg hPD1-mSOT201 (equimolar amount to mSOT201); DO = randomization day when tumor volume is approximately 100 mm3, 10 mice / group. B16F10 / C57BL / 6 mouse model: 4 doses of ip administration on days 0, 3, 6 and 9 with 9.02 mg / kg mPD-1 (effective dose compared to literature), 10 mg / kg mSOT201, 10.74 mg / kg hPD1-mSOT201 (equimolar amount to mSOT201); DO = randomization day when tumor volume is approximately 100 mm3, 10 mice / group. Cut-off date for all mice present in the control group, CR = complete response. [Figure 26]Comparison of mSOT201 vs. RLI-15AQA mutein + anti-PD-1 in vivo in a MC38 / C57BL / 6 mouse model with the following groups: G1 sham control G4: Single sc dose of 0.64 mg / kg RLI-15AQA on day 0 + single ip dose of 4.51 mg / kg mPD-1 on day 0. G2 single dose of mSOT201 5 mg / kg iv on day 0 G3 single dose of mSOT201 2 mg / kg iv on day 0 G6 single dose of mPD1 alone 4.51 mg / kg ip on day 0 (selected as equimolar to mSOT201, suboptimal dose compared to literature), G11 single dose of hPD1-mSOT201 5 mg / kg iv on day 0 + single dose of mPD-1 4.36 mg / kg ip on day 0, day 0 = randomization day with tumor volume ≈80-100 mm3; cutoff day for all mice present in control group with 10 mice / group, CR = complete response. [Figure 27] MC38 / C57BL / 6 mouse model - D0 = randomization day, approximately 80-100 mm3, 10 mice / group. CR = complete response G1 Sham control G2 A single dose of mSOT201 5 mg / kg iv on day 0 G3 A single dose of mSOT201 2 mg / kg iv on day 0 G7 4 doses of RLI2AQ 1 mg / kg sc on days 0, 1, 2 and 3 G5 A single dose of RLI2AQ 1 mg / kg sc on day 0 + a single dose of mPD1 5 mg / kg ip on day 0 G8 4 doses of RLI2AQ 1 mg / kg sc on days 0, 1, 2 and 3 + a single dose of mPD1 5 mg / kg ip on day 0 G9 4 doses of RLI2AQ 1 mg / kg sc on days 0, 1, 2 and 3 + 4 doses of mPD1 5 mg / kg ip on days 0, 3, 6 and 9 G6 A single dose of mPD1 5 mg / kg ip on day 0 G10 Four ip doses of 5 mg / kg mPD1 on days 0, 3, 6, and 9. Cut-off date for all mice in the control group. [Figure 28A]Comparison of mSOT201 vs. RLI2AQ + anti-PD-1 tumor growth in vivo. MC38 / C57BL / 6 mouse model. Mean tumor volumes in mm3 shown for individual animals depending on time and on day 16. Horizontal lines indicate mean tumor volumes. G1 mock control G2 single dose iv of mSOT201 2 mg / kg on day 0, G3 two sc doses of RLI2AQ 2 mg / kg on days 0 and 1 + four ip doses of mPD1 2 mg / kg on days 0, 3, 6 and 9. One experiment only. D0 = day of randomization with tumor volume approx. 80-100 mm3; 10 mice / group. CR = complete response. Relative proliferation of NK cells, CD8+ T cells and cells expressing αβTCR and γδTCR (T cells) was examined in spleen, lymph nodes and tumors on day 7 after SOT201 (G2 from above) and RLI2AQ + anti-PD-1 (G3 from above) treatment using flow cytometry. Three tumor samples were pooled and three spleen and lymph node samples were analyzed separately. [Figure 28B] Comparison of mSOT201 vs. RLI2AQ + anti-PD-1 tumor growth in vivo in MC38 / C57BL / 6 mouse model. Parental frequencies in % (relative percentage compared to the parental population) are shown for CD8+ T cells (top) and NK cells (bottom) from lymph nodes, spleen and tumors. [Figure 28C] Comparison of mSOT201 vs. RLI2AQ + anti-PD-1 tumor growth in vivo. MC38 / C57BL / 6 mouse model. Parental frequencies in % for αβTCR+ CD3+ T cells (top) and βγTCR+ CD3+ T cells (bottom) from lymph nodes, spleen and tumor are shown. [Figure 29](A) Immunogenicity in DC-T cell based assay. T cell response to PEM-RLI-15 candidate molecules shown as %CFSElow stained CD4+ T cells after loading the candidate molecules into iDCs, incubating with autologous CD4+ T cells pre-stained with CFSE, and detecting CFSE staining. CFSElow is used as a surrogate for cycling cells. Mean ± SEM of 11 donors is shown. Significant differences compared to control DCs incubated without protein, thus inducing non-specific T cell proliferation, are shown. *p≦0.05, ***p≦0.001. (B) FluoroSpot assay for IFN-γ and TNF-α of RLI-15 peptides across the introduced substitutions N65A and G175A / N176Q. Estimation with 95% confidence interval (CI) of the effect of Mut2 or Mut3 peptides versus the respective wild type peptide on the mean dSFU in a study population of 40 donors. SFU = spot forming units, dSFU = SFU of restimulated wells - SFU of non-restimulated wells. [Diagram 30] Comparison of the ability of SOT202 molecules with altered effector functions to induce proliferation of hPBMCs. Proliferation of isolated hPBMCs was assessed for SOT202-DANA, SOT202-afuc-DANA, SOT202-DLE-DANA, SOT202-DE-DANA and SOT202-LALAPG-DANA. Cells were stimulated in vitro for 7 days. Means ± SEM of 6 donors are shown. Proliferation of NK cells (top) and CD8+ T cells (bottom) was measured by counting Ki67+ cells by flow cytometry. [Diagram 31] Comparison of the ability of SOT202 molecules and SOT201 to induce proliferation of hPBMC. Proliferation of isolated hPBMC was assessed for SOT202, SOT202-afuc, SOT201-DANA, SOT202-DANA and SOT202-afuc-DANA. Proliferation of NK cells (top) and CD8+ T cells (bottom) was measured by counting Ki67+ cells by flow cytometry. [Diagram 32]Comparison of the ability of SOT202-DANA molecules with altered effector functions and SOT201-DANA to induce proliferation of hPBMCs. Proliferation of isolated hPBMCs was assessed for SOT201-DANA, SOT202-DANA, SOT202-afuc-DANA, SOT202-LALAPG-DANA and hCl1a (also referred to as SOT202-mab). Proliferation of NK cells (top) and CD8+ T cells (bottom) was measured by counting Ki67+ cells by flow cytometry. [Figure 33A] Cell proliferation (Ki67+) of CD8+ T cells or NK cells detected in the spleen of healthy C57BL / 6 mice after stimulation with mSOT202. Cell proliferation was detected by Ki67 staining and measured by flow cytometry 5 days after IV injection of 5, 10 or 20 mg / kg of mSOT202 (hCl1a-mIgG2a-NA 1x) or hCl1a-mIgG2a compound. [Figure 33B] Percentages of NK cells and CD8+ T cells under the same experimental conditions as in (A). [Figure 34A] Cell proliferation of NK cells detected in the spleen of healthy C57BL / 6 mice after stimulation with mSOT202, mSOT202-LALAPG and hCl1a-mIgG2a. Top: Cell proliferation was detected by Ki67 staining and measured by flow cytometry 5 and 10 days after IV injection of 5 mg / kg of the compounds. Bottom: Percentage of NK cells. [Figure 34B] Cell proliferation of CD8+ T cells detected in the spleen of healthy C57BL / 6 mice after stimulation with mSOT202, mSOT202-LALAPG and hCl1a-mIgG2a. Top: Cell proliferation was detected by Ki67 staining and measured by flow cytometry 5 and 10 days after IV injection of the compounds at 5 mg / kg. Bottom: Percentage of CD8+ T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition "Antibodies", also known as immunoglobulins (Ig), are large Y-shaped proteins that, in humans and most mammals, consist of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. The light chains contain one variable domain, V L and one constant domain C L The heavy chain consists of one variable domain, V H and three constant domains C H 1. C H 2. C H Structurally, antibodies each contain one V L , V H , C L , and C H Two antigen-binding fragments (Fab) containing one domain, as well as two C domains of two heavy chains H 2 and C H It is also divided into an Fc fragment or domain comprising 3.

[0012] As used herein, "antibody variants" or "antibody functional variants" refer to antibodies with modifications, for example, to modulate the effector functions of the antibody, to modulate the antibody stability and in vivo half-life, and / or to induce heterodimerization of the antibody Fc domain. Such variants may be achieved by mutation and / or post-translational modifications. Antibody variants also include antibody heavy chains with shortening (truncating) of the N-terminal lysine. Other included variations are N- or C-terminal tags of the heavy and / or light chains for chemical or enzymatic coupling to other moieties, such as dyes, radionuclides, toxins, or other binding moieties. Furthermore, antibody variants may include chemical modifications, modifications of their glycosylation, or substitutions with artificial amino acids for chemical linking to other moieties.

[0013] Antibody variants as used herein also refer to immunoglobulin gamma (IgG)-based bispecific antibodies that potentially recognize two or more different epitopes. Various formats of bispecific antibodies are known in the art and are reviewed, for example, by Godar et al. (2018) and Spiess et al. (2015). The bispecific format according to the present invention comprises an Fc domain. For the immunocytokines of the present invention, the two RLI conjugates may be fused either to the C-terminus of both light chains or to the C-terminus of both heavy chains, if not otherwise linked to a moiety, or one RLI conjugate may be fused to the C-terminus of one heavy chain for a heterodimeric bispecific format, or to the heavy chain or one light chain of a heterodimeric bispecific format with different light chains. An antibody functional variant is capable of binding to the same epitope or target as the corresponding unmodified antibody. The term "antibody" when used generically includes antibody variants as defined herein.

[0014] "Conjugate" as used herein relates to either a non-covalent or covalent complex of interleukin 15 (IL-15) or a derivative thereof with the sushi domain of interleukin 15 receptor alpha (IL-15Rα) or a derivative thereof. The non-covalent complex may be formed by co-expression of the two polypeptides or by separate expression, (partial) purification and subsequent combination of such polypeptides to form such a complex by affinity. Preferably, the conjugate is a fusion protein, where the two polypeptides are genetically fused and recombinantly expressed resulting in a single polypeptide chain to form an intact complex.

[0015] "Immunocytokine" as used herein relates to a polypeptide comprising an antibody or a functional variant thereof genetically fused to a conjugate according to the invention.

[0016] When RLI is referred to within a particular immunocytokine construct, it is RLI2.

[0017] The EU numbering scheme has been applied to the disclosed antibody or partial antibody sequences.

[0018] "In vivo half-life" or T 1 / 2 refers to the (terminal) plasma half-life, or T 1 / 2 is the half-life of elimination or the half-life of the terminal phase, i.e., after administration, the in vivo half-life is the time required for the plasma concentration / blood concentration to decrease by 50% after reaching the pseudo-equilibrium of distribution (Toutain and Bousquet-Melou, 2004). Measurement of drugs, here immunocytokine agonists, which are polypeptides, in blood / plasma is typically performed by polypeptide-specific ELISA. The in vivo half-life of a particular drug can be determined in any mammal. For example, the in vivo half-life can be determined in humans, primates or mice. Although the in vivo half-life determined in humans can be significantly different from the in vivo half-life in mice, i.e., the in vivo half-life in mice for a particular drug is usually shorter than the in vivo half-life determined for the same drug in humans, such in vivo half-life determined in mice still gives an indication of the particular in vivo half-life in humans. Therefore, the in vivo half-life of a particular drug determined in mice can be used to estimate the in vivo half-life of that drug in humans.This is particularly important because direct determination of the in vivo half-life of a particular drug in humans is rarely possible due to the prohibition of experiments involving humans for purely scientific purposes.Alternatively, half-life can be determined in primates (e.g., cynomolgus monkeys) that are more similar to the half-life in humans.

[0019] When described as "administered in combination," this typically does not mean that two agents are co-formulated and administered simultaneously, but rather that one agent has a label that identifies its use in combination with the other agent. So, for example, an immunocytokine is for use in the treatment or management of cancer, and this use includes administering the immunocytokine and an additional therapeutic agent simultaneously, separately, or sequentially, or vice versa. However, nothing in this application should preclude the two combined agents from being provided as a package or kit, or even from being co-formulated and administered together when the dosing schedules match. So, "administered in combination" includes (i) the agents are administered together, such as by joint infusion, joint injection, etc.; (ii) the agents are administered separately but in parallel according to the given administration method for each agent; and (iii) the agents are administered separately and sequentially. Concurrent administration in this context preferably means that both treatments are started together, e.g., the first administration of each drug in the treatment regimen is administered on the same day. Considering the potential different treatment schedules, it is clear that administration does not always have to occur on the same day during the following days / weeks / months. Generally, concurrent administration aims for both drugs to be present in the body at the same time at the beginning of each treatment cycle. Sequential administration in this context preferably means that both treatments are started sequentially, e.g., the first administration of the first drug is performed at least one day, preferably several days or a week before the first administration of the second drug, to allow the body's pharmacodynamic response to the first drug before the second drug becomes active. Thereafter, the treatment schedules may overlap or be intermittent with each other, or may directly follow each other.

[0020] The term "about" when used in conjunction with a value means ±10% of that value, preferably ±5% and especially ±1% of that value.

[0021] Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". In the following, when a group is defined as comprising at least a certain number of embodiments, this is to be understood as also disclosing a group that preferably consists only of these embodiments.

[0022] Where an indefinite or definite article such as "a", "an" or "the" is used when referring to a singular noun, this includes a plural of that noun, unless something else is specifically stated.

[0023] Thus, the terms "at least one," "at least one," as in "at least one chemotherapeutic agent," may be meant to mean one or more chemotherapeutic agents. The term "a combination thereof," in the same context, refers to a combination including more than one chemotherapeutic agent.

[0024] Technical terms are used with their common meaning. Where a specific meaning is conveyed to a particular term, the definition of the term is given below in the context in which the term is used.

[0025] "qxw", derived from the Latin quaque / each, every, means every x weeks, for example q2w means every two weeks. "sc" or "SC" stands for subcutaneous. "iv" or "IV" stands for intravenous. "ip" or "IP" stands for intraperitoneal. C max represents the maximum concentration. AUC stands for area under the curve.

[0026] [Table 1(1)] [Table 1(2)] [Table 1(3)] [Table 1(4)] [Table 1(5)] [Table 1(6)] [Table 1(7)]

[0027] In a first aspect, the present invention relates to an immunocytokine comprising a cytokine conjugate and an antibody or a functional fragment thereof. The cytokine conjugate comprises a polypeptide comprising the amino acid sequence of the sushi domain of interleukin 15 (IL-15) or a derivative thereof and interleukin 15 receptor alpha (IL-15Rα) or a derivative thereof. The antibody or functional variant thereof comprised in the immunocytokine is characterized by having a heterodimeric Fc domain, an altered effector function (compared to the same immunocytokine with a wild-type Fc domain of the same IgG class) and / or an increased in vivo half-life (compared to the same immunocytokine with a wild-type Fc domain of the same IgG class). The conjugate may be fused directly or indirectly to the C-terminus of both antibody heavy or light chains or, in the case of a heterodimeric Fc domain, to the C-terminus of one antibody heavy chain. The increased in vivo half-life of the immunocytokine may be achieved by Fc mutations that increase FcRn binding.

[0028] The Fc domain of said antibody or functional variant thereof may also comprise further modifications such as truncation of the C-terminal lysine of the heavy chain or, in the case of a heterodimeric Fc domain, one or both heavy chains. In addition, for indirect fusion, a flexible linker composed of residues such as glycine and serine may be introduced at the C-terminus of the antibody heavy or light chain to allow the adjacent conjugate to move freely relative to the antibody Fc domain.

[0029] In one embodiment, the antibody or functional variant thereof comprised in the immunocytokine is not antibody hCl1a, hCl1b, hCl1c, hCl1d, hCl1e, hCl1f, hCl1g, hCl1h, hCl1i, or hCl1j disclosed in Table 4.

[0030] In one embodiment, the antibodies or functional variants thereof comprised in the immunocytokine are antibodies hCl1a, hCl1b, hCl1c, hCl1d, hCl1e, hCl1f, hCl1g, hCl1h, hCl1i and hCl1j disclosed in Table 4.

[0031] In one embodiment, the present invention provides an antibody or functional variant thereof that binds to a target, the antibody being an IgG1, IgG2, IgG4, synthetic IgG or bispecific antibody, or an Fc-engineered version thereof. In a preferred embodiment, the antibody is an IgG1 or IgG4 class antibody. If the target is present on a tumor cell, the preferred antibody format is IgG1. If the target is present on an immune cell, the preferred antibody format is IgG4. If the antibody format is IgG1, the Fc region of the immunoglobulin preferentially interacts with multiple Fcγ receptors (FcγR) and complement proteins (e.g., C1q) to mediate immune effector functions such as elimination of target cells via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). For therapeutic approaches, it may be beneficial to enhance or silence Fc-mediated effector functions. Fc-mediated effector functions, such as ADCC, may be enhanced if the antibody targets tumor cells, or silenced if the antibody targets checkpoint inhibitors present on immune cells, such as PD-1 or CTLA-4. If the antibody targets checkpoint inhibitors present on immune cells, such as PD-1 or CTLA-4, the antibody may be in IgG4 format, which is a weak inducer of Fc-mediated effector functions. In another embodiment, an antibody targeting a checkpoint inhibitor, such as PD-1 or CTLA-4, may be in IgG1 format engineered to strongly reduce or silence ADCC and / or CDC activity, e.g., reduced FcγR and C1q binding. Many guidelines on how to select IgG subclasses in the development of anti-tumor therapeutic antibodies may be found in Yu J. et al. (Yu, Song et al., 2020).

[0032] The Fc-mediated functions of antibodies may be modulated using Fc-engineered immunoglobulins. Table 2 shows examples of such Fc engineering.

[0033] [Table 2(1)] [Table 2(2)]

[0034] There are several laboratory methods to determine the effectiveness of antibodies or effector cells in inducing ADCC. Typically, a target cell line expressing a surface-exposed antigen is incubated with an antibody or immunocytokine specific for that antigen. After incubation, effector cells expressing the Fc receptor CD16 (Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b)) are co-incubated with the antibody or immunocytokine-labeled target cells. The effector cells are typically PBMCs (peripheral blood mononuclear cells), of which a small percentage are NK cells. Alternatively, purified NK cells may be used. A further alternative is the use of the human NK cell line NK92 (ATCC CRL-2407) that exogenously expresses human CD16 (NK92-hCD16). Over the course of several hours, complexes form between the antibody, the target cells, and the effector cells, which results in lysis of the target cell membrane. If the target cells are preloaded with a label of some kind, the label is released in proportion to the amount of cell lysis. Cytotoxicity can be quantified by measuring the amount of label in solution relative to the amount of label remaining in healthy intact cells. The label can be a radioactive label as described in Perussia and Loza (Perussia and Loza 2000). 51Cr. Instead of using preloaded target cells, ADCC activity may also be measured using the LDH cytotoxicity assay. The LDH cytotoxicity assay is a colorimetric assay that provides a simple and reliable method for determining cellular cytotoxicity. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme present in many different cell types that is released into cell culture medium upon damage to the plasma membrane, such as the plasma membrane damage that occurs during ADCC. The LDH assay protocol is based on an enzyme coupling reaction. LDH released from cells oxidizes lactate to generate NADH, which can then react with water-soluble tetrazolium salts (WST) to generate a yellow color. The intensity of the color generated directly correlates with the number of lysed cells. ADCC activity may also be measured as disclosed in Example 9.

[0035] As an alternative to ADCC assays using target and effector cells, Fc receptor binding of immunocytokines can also be tested by surface plasmon resonance (SPR), as described in Example 24.

[0036] In one embodiment, the altered effector function of the antibody or functional variant thereof comprised in said immunocytokine is a reduction in antibody-dependent cellular cytotoxicity compared to the same immunocytokine having a wild-type Fc domain of the same IgG class. The antibody effector function may be reduced by reducing FcγR and C1q binding via (by) the mutations listed in the corresponding sections of Table 2.

[0037] In one embodiment, when the antibody or functional variant thereof is an IgG1, reduced ADCC may be achieved by mutations selected from L234A / L235A, P329G, L234A / L235A / P329G, G236R / L328R, D265A, N297A, N297Q, N297G or L234A / L235A / G237A / P238S / H268A / A330S / P331S, preferably L234A / L235A / P329G.

[0038] In another embodiment, when the antibody or functional variant thereof is IgG4, reduced ADCC may be achieved by mutations selected from L235E, F234A / L235A, F234A / L235A / P329G, P329G, S228P / L235E, S228P / F234A / L235A or E233P / F234V / L235A / D265A / R409K, preferably L235E.

[0039] In yet another embodiment, where the antibody or functional variant thereof is an IgG2, reduced ADCC may be achieved by mutations selected from H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S.

[0040] In one embodiment, reduced ADCC may be achieved when the antibody or functional variant thereof is a hybrid of IgG2 (IgG2a or IgG2b) and IgG4 or a functional variant thereof and comprises the CH1 + hinge region from IgG2 and the CH2 + CH3 region from IgG4 (IgG2 amino acids 118-260 and IgG4 amino acids 261-447).

[0041] In a preferred embodiment, reduced ADCC of an IgG1 antibody is achieved via L234A / L235A ("LALA") mutations and may comprise the IgG1 Fc region of SEQ ID NO:26.

[0042] In another preferred embodiment, reduced ADCC of an IgG1 antibody is achieved via L234A / L235A / P329G ("LALAPG") mutations and may comprise the IgG1 Fc region of SEQ ID NO:27.

[0043] Example 23 and Figure 13 show that the immunocytokine hCl1a LALAPG-RLI DANA nearly abolished ADCC activity compared to the hCl1a-RLI DANA immunocytokine with the hCl1a antibody alone when tested in A549-CLDN18.2 cells or PA-TU-8988 in the presence of NK92 cells. The hCl1a LALA antibody also reduced ADCC activity compared to the hCl1a antibody, but did not completely abolish ADCC activity.

[0044] In another preferred embodiment, when an IgG4 antibody is selected, the already low induction of Fc-mediated effector functions may be further reduced via L235E mutations, F234A / L235A or E233P / F234V / L235A / D265A / L309V / R409K mutations.

[0045] In a preferred embodiment, reduced ADCC of an IgG4 antibody is achieved via the L235E mutation and may comprise the IgG4 FC region of SEQ ID NO:43.

[0046] In another embodiment of the invention, the altered effector function of the antibody or functional variant thereof comprised in the immunocytokine is enhanced ADCC.

[0047] In one embodiment, the above antibodies may be modified to enhance ADCC by increased FcγRIIIa binding via the mutations listed in the corresponding section of Table 2 and / or by defucosylation.

[0048] In one embodiment, ADCC is enhanced in an IgG1 antibody or variant thereof via a mutation selected from F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A.

[0049] In a preferred embodiment, ADCC is enhanced in an IgG1 antibody or variant thereof, preferably via mutations selected from S239D / I332E ("DE"), S239D / I332E / A330L ("DLE"), S298A / E333A / K334A ("AAA"), K392T / P396L ("TL") or V264I / I332E ("IE").

[0050] In one embodiment, ADCC is enhanced in an IgG1 antibody via DE mutations and may comprise the IgG1 Fc region of SEQ ID NO:30.

[0051] In another embodiment, ADCC is enhanced in an IgG1 antibody via DLE mutations and may comprise an IgG1 Fc region of SEQ ID NO:31.

[0052] In another embodiment, ADCC is enhanced in an IgG1 antibody via AAA mutations and may comprise an IgG1 Fc region of SEQ ID NO:34.

[0053] In yet another embodiment, ADCC may be enhanced in an IgG1 antibody via TL mutations, comprising the IgG1 Fc region of SEQ ID NO:36.

[0054] In another embodiment, ADCC is enhanced in an IgG1 antibody via IE mutations and may comprise the IgG1 Fc region of SEQ ID NO:37.

[0055] In yet another embodiment, ADCC may also be enhanced by reducing the fucose content of antibodies via defucosylation (Pereira, Chan et al., 2018). Fucose (6-deoxy-L-galactose) is a common component of many N- and O-linked glycans produced in mammalian cells. The absence of core fucose on the Fc N-glycan of IgG1 at the conserved N-glycosylation site Asn297 (N297) in each of the CH2 domains has been shown to increase IgG1 Fc binding affinity to FcγRIIIa present on immune effector cells such as natural killer cells, resulting in enhanced ADCC activity. Fucosyltransferases (FUTs) transfer fucose residues from GDP-fucose to acceptor substrates. FUT8 is the only α1,6-fucosyltransferase that transfers fucose via an α1,6 linkage to the innermost N-acetylglucosamine on the N-glycan for core fucosylation of IgG1. Defucosylated antibodies may be produced in CHO cells in which the FUT8 gene has been knocked out (POTELLIGENT® technology). Antibodies produced in such cell lines have shown enhanced ADCC compared to the same antibodies produced in conventional CHO cells (Yamane-Ohnuki, Kinoshita et al. 2004). Alternatively, antibodies may be produced in glycoengineered cell lines (GlymaX®, ProBioGen) in which the fucose synthesis pathway is biased, resulting in defucosylated antibodies as well (Rosenlocher, Bohrsch et al. 2015; Dekkers, Plomp et al. 2016).

[0056] In one embodiment, the antibody may be modified to enhance ADCC by increased FcγRIIIa binding via defucosylation of the antibody.

[0057] In another embodiment, the antibody may be modified to enhance ADCC by increased FcγRIIIa binding via one of the mutations listed in the corresponding section of Table 2 in combination with defucosylation of the antibody.

[0058] In a preferred embodiment, an IgG1 antibody may be modified to enhance ADCC through increased FcγRIIIa binding via AAA mutations in combination with defucosylation of the antibody.

[0059] The ADCC activity of immunocytokines with different Fc mutations, or defucosylation, or mutations combined with defucosylation that enhance ADCC activity, as measured by cell-based ADCC assay, is shown in Example 23 and Figure 14. All mutations increased the ADCC activity of the immunocytokines tested to a similar extent compared to the heterodimeric immunocytokines or antibodies alone without Fc domain mutations. Similarly, defucosylation also enhanced the ADCC activity of the immunocytokines. The combination of defucosylation and AAA mutations also enhanced ADCC, while the combination of DE or DLE with defucosylation further reduced ADCC activity compared to DE, DLE or defucosylation alone.

[0060] Fc receptor binding of immunocytokines with mutations modulating effector function was also tested by SPR, as described in Example 24. SPR allowed evaluation of antibody Fc binding to the ADCC activating receptors FcγRIIIa V158 and FcγRIIIa F158, and to the ADCC inhibitory receptor FcγRIIb. SPR studies confirmed that, overall, immunocytokines with mutations enhancing ADCC exhibited higher A / I ratios than immunocytokines without mutations enhancing ADCC, as long as antibody glycosylation was not affected by the mutations.

[0061] The introduction of mutations into the Fc domain may also affect the stability and developability of the immunocytokine and may depend on each particular antibody used in the immunocytokine. More specifically, the melting temperature and glycosylation of immunocytokines with Fc mutations were examined (see Example 25). Overall, for hCl1a-based immunocytokines, the TL and IE mutations introduced unfavorable glycosylation, while the DE and DLE mutations reduced C HAlthough lowering the melting temperature of the 2 domains and affecting their stability, the AAA mutations, optionally in combination with defucosylation, did not affect the stability and developability of hCl1a-based immunocytokines.

[0062] In another embodiment, the modification made to the Fc domain of an antibody to improve antibody stability may be the S228P mutation in an IgG4 antibody to avoid Fab arm exchange (Silva, Vetterlein et al. 2015) (SEQ ID NO: 39).

[0063] In one embodiment, the antibody Fc domain may be a heterodimer to have only one heavy chain fused to a cytokine. Heterodimerization occurs when two Fc domains (C H 3A chain and C H This may be achieved by mutation of each of the three A chains. Table 3 below summarizes the design of heterodimeric Fc variants (Ha, Kim et al., 2016).

[0064] [Table 3]

[0065] In one embodiment, antibody heterodimerization may be achieved by using any one of the heterodimeric Fc variants of KiH, KiHS-S, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTS-S, SEED or A107.

[0066] In a preferred embodiment, antibody heterodimerization is achieved via a T366W mutation (SEQ ID NO: 28) in the CH3 domain of one heavy chain and a T366S / L368A / Y407V mutation (SEQ ID NO: 29) in the CH3 domain of the other heavy chain, resulting in a "Knobs-into-Holes (KiH)" Fc variant. In Example 2, the potency of the immunocytokine homodimerized with two RLI2 conjugates is compared to the potency of the heterodimerized immunocytokine. Table 11 shows that only one RLI2 conjugate is present in the heterodimeric immunocytokine (RTX-RLI x1), yet surprisingly, its potency is still greater than 50% of the potency of the homodimeric immunocytokine (RTX-RLI 2x).

[0067] A method for producing heterodimeric immunocytokines can be found in Example 3. Measurements of the potency of such heterodimeric immunocytokines compared to homodimeric immunocytokines can be found in Example 5 and Table 15. Since one objective of the present invention is to reduce the potency of the conjugates, the inventors show herein that a heterodimeric immunocytokine with only one RLI2 conjugate showed approximately a 10-fold reduction in potency against kit225 cells, whereas a homodimeric immunocytokine with two RLL2 conjugates fused to the C-terminus had a slight reduction in potency.

[0068] Preferably, the RLI2 conjugate is fused to a knob heavy chain.

[0069] Exemplary sequences of the heterodimeric immunocytokines of the present invention are SEQ ID NO: 21 ("HC knob-RLI2") for SOT201 (pembrolizumab variant-based heterodimeric immunocytokine): AQNA”), SEQ ID NO: 22 or SEQ ID NO: 101 (“HC hole”, with or without terminal lysine deletion) and SEQ ID NO: 23 (LC), preferably SEQ ID NO: 21, SEQ ID NO: 101 and SEQ ID NO: 23, and for hCl1a variant-based heterodimeric immunocytokine, SEQ ID NO: 85 (“HC knob-RLI2 AQ DANA"), SEQ ID NO: 87 ("HC hole") and SEQ ID NO: 88 (LC), and for another hCl1a variant-based heterodimeric immunocytokine, SEQ ID NO: 111 ("HC knob-RLI2 AQ NA"), SEQ ID NO: 110 ("HC hole") and SEQ ID NO: 88 ((LC), and for rituximab-based heterodimeric immunocytokine SEQ ID NO: 97 ("HC knob AAA-RLI2 AQ NA"), SEQ ID NO: 98 ("HC holeAAA") and SEQ ID NO: 99 (LC), or for cetuximab-based heterodimeric immunocytokine SEQ ID NO: 94 ("HC knob-RLI2 AQ DANA), SEQ ID NO:95 ("HC hole") and SEQ ID NO:92 (LC).

[0070] In one embodiment, the heterodimeric Fc domain results in a higher yield of the immunocytokine upon expression in cell culture compared to immunocytokines with homodimeric Fc domains. Although heterodimeric antibody formats are generally expected to have lower expression due to mispairing of heavy and light chains, it has surprisingly been observed that for heterodimeric immunocytokines, heterodimeric constructs using KiH technology have higher expression compared to the respective homodimeric constructs (see Example 3).

[0071] If the immunocytokine is not a heterodimer, the conjugate may also be fused to the C-terminus of the light chain of the antibody (e.g., SEQ ID NO: 45). A linker consisting of glycine or serine and glycine may be present between the C-terminus of the light chain and the N-terminus of the conjugate to allow flexibility of the fusion conjugate relative to the antibody.

[0072] Alternatively, RLI2 AQ to the C-terminus of one or both heavy chains. Such a linker is preferably composed of glycine or glycine and serine, more preferably composed of a GGGGS unit or 30 to 50 amino acids in length, and is in particular the L40 linker of SEQ ID NO: 100.

[0073] In another embodiment, the invention relates to an immunocytokine, wherein the in vivo half-life of the immunocytokine is increased, and wherein the antibody or functional variant thereof is an IgG1 or IgG4 antibody or functional variant thereof and comprises mutations selected from M252Y / S254T / T256E, M428L / N434S or T250Q / M428L.

[0074] The Fc domain plays a central role in the stability and serum half-life of antibodies. The in vivo half-life of antibodies may be increased through M252Y / S254T / T256E or M428L / N434S mutations in the Fc domain that increase FcRn binding (Dall'Acqua, Woods et al. 2002; Zalevsky, Chamberlain et al. 2010).

[0075] In one embodiment, the half-life of an antibody of IgG1 or IgG4 type is increased via M252Y / S254T / T256E ("YTE") mutations in the Fc domain of SEQ ID NO: 35 and SEQ ID NO: 44, respectively.

[0076] In another embodiment, the present invention relates to an immunocytokine, wherein the antibody or functional variant thereof has reduced ADCC, the antibody or functional variant thereof is an IgG4 antibody or functional variant thereof, and comprises an L235E mutation and a KiH-heterodimer Fc domain. When the antibody target is a checkpoint inhibitor present on immune cells, such as PD-1 or CTLA-4 on the surface of T cells, reducing the ADCC may be beneficial to avoid NK cell-induced cytotoxicity against these immune cells. The IgG4 antibody may also optionally contain a S228P mutation to stabilize the antibody. The IgG4 Fc domain with the L235E mutation may be of the sequence of SEQ ID NO: 43. The IgG4 CH1-hinge domain with the S228P mutation may be of the sequence of SEQ ID NO: 39. The KiH-heterodimer Fc domain of the IgG4 antibody may be of the sequence of SEQ ID NO: 41 ("knob") and SEQ ID NO: 42 ("hole"). Optionally, one or preferably both heavy chains may have a terminal lysine deletion (dK), i.e., the sequences of SEQ ID NO: 41 ("knob") and SEQ ID NO: 42 ("hole"). In another embodiment, both heavy chains have a terminal lysine.

[0077] Examples 10 to 26 relate to such immunocytokines.

[0078] In one embodiment, the immunocytokine conjugate is a fusion protein comprising, in order from N-terminus to C-terminus, an IL-15Rαsushi domain or a derivative thereof, a linker, and IL-15 or a derivative thereof, preferably the IL-15Rαsushi domain comprises the sequence of SEQ ID NO: 5, more preferably the IL-15Rαsushi+fragment of SEQ ID NO: 6, the linker has a length of 18 to 22 amino acids and is preferably composed of glycine or serine and glycine, more preferably has the sequence of SEQ ID NO: 7, and IL-15 has the sequence of SEQ ID NO: 2.

[0079] A fusion protein having the IL-15Rαsushi+ fragment of SEQ ID NO:6 fused to the N-terminus of mature human IL-15 of SEQ ID NO:2 via a flexible linker of SEQ ID NO:7, called RLI2, which stands for receptor-linker-interleukin 2, or SO-C101, which has the sequence of SEQ ID NO:8, is a clinical stage IL-2 / IL-15Rβγ superagonist with low immunogenicity. This makes such a fusion protein a preferred conjugate for use in immunocytokine formats.

[0080] In a preferred embodiment, the immunocytokine comprises an IL-15 variant comprising at least one mutation that increases the homogeneity of the IL-15 variant with respect to post-translational modifications, preferably the mutation reduces deamidation at N77 and / or glycosylation at N79 of IL-15 mature human IL-15 (SEQ ID NO: 2), more preferably said mutation is selected from the mutations G78A, G78V, G78L or G78I, and N79Q, N79S or N79T, most preferably said mutation is G78A / N79Q ("AQ mutation").

[0081] IL-15 mutations and IL-2 / IL-15Rβ and / or γ that increase immune cytokine homogeneity c IL-15 mutations that reduce binding to the receptor may be used independently in the immunocytokines of the invention or may be combined in the immunocytokines of the invention.

[0082] In another preferred embodiment, the immunocytokine is IL-2 / IL-15Rβ and / or γ c The present invention includes IL-15 variants comprising at least one mutation that reduces binding to the receptor, preferably the mutated amino acid is selected from N1, N4, S7, D8, K10, K11, D30, D61, E64, N65, L69, N72, E92, Q101, Q108, I111 of IL-15 mature human IL-15 having the sequence of SEQ ID NO:2, more preferably the mutated amino acid is selected from D61, N65 and Q101, and most preferably the mutated amino acid is N65.

[0083] IL-2 / IL-15Rβ and / or γ c The mutations that reduce the binding to the receptor are preferably N1D, N1A, N1G, N4D, S7Y, S7A, D8A, D8N, K10A, K11A, D30N, D61A, D61N, E64Q, N65D, N65A, N65E, N65R, N65K, L69R, N72R, Q101D, Q101E, Q108D, Q108A, Q108E and Q108R, Preferred are substitutions selected from D8A, D8N, D61A, D61N, N65A, N65D, N72R, Q101D, Q101E and Q108A, more preferably D61A, N65A and Q101, most preferably N65A, or combined substitutions selected from D8N / N65A, D61A / N65A or D61A / N65A / Q101D.

[0084] In another embodiment, the immunocytokine comprises an antibody or functional variant thereof, which binds to a tumor antigen, preferably selected from EGFR, HER2, FGFR2, FOLR1, CLDN18.2, CEA, GD2, O-acetyl-GD-2, GM1, CAIX, EPCAM, MUC1, PSMA, c-Met, ROR1, GPC3, CD19, CD20, CD38, to a tumor extracellular matrix antigen, preferably selected from FAP, the EDA domain of fibronectin, the EDB domain of fibronectin and LRRC15, preferably FAP and the EDB domain of fibronectin, to an angiogenesis antigen, preferably VEGF, or endoglin (CD105), or is an immunomodulatory antibody or functional variant thereof, which immunomodulatory antibody is preferably a CD40 agonist, CD137 / 4-1 The immunomodulatory antibody stimulates a costimulatory receptor selected from a BB agonist, a CD134 / OX40 agonist and a TNFRSF18 / GITR agonist, or inhibits an immunosuppressive receptor, preferably selected from a PD-1 antagonist, a CTLA-4 antagonist, a LAG3 antagonist, a TIGIT antagonist, an inhibitory KIR antagonist, a BTLA / CD272 antagonist, a HAVCR2 / TIM-3 / CD366 antagonist and an ADORA2A antagonist, more preferably a PD-1 antagonist.

[0085] Antibodies against the targets listed above are well known in the art or can be generated by standard immunization or phage display protocols. Non-human antibodies can be humanized. Examples of anti-EGFR antibodies are cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab. Examples of anti-HER2 antibodies are trastuzumab, pertuzumab, or margetuximab. Examples of anti-CLDN18.2 antibodies are zolbetuximab and the antibodies of the present invention below. An example of an anti-CEA antibody is arcitumomab. An example of an anti-GD2 is hu14.18K322A. An example of an anti-O-acetyl-GD-2 is c.8B6. FGFR2, FOLR1, GM1, CAIX, EPCAM, MUC1, PSMA, c-Met, ROR1, GPC3, CD19. Examples of anti-CD20 antibodies are rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab, tositumomab and ublituximab. Examples of anti-CD38 antibodies are daratumumab, MOR202 and isatuximab.

[0086] Examples of anti-FAP antibodies are sibrotuzumab and B12 (US Patent Publication No. 2020-0246383A1). An example of an anti-EDA domain antibody of fibronectin is the F8 antibody (Villa, Trachsel et al., 2008), WO 2010 / 078945, WO 2014 / 174105), an example of an anti-EDB domain of fibronectin is the L19 antibody (Pini, Viti et al., 1998), WO 1999 / 058570), and an example of an anti-LRRC15 antibody is Samrotamab / huM25 (WO 2017 / 095805).

[0087] Examples of anti-VEGF antibodies are bevacizumab and ranibizumab. An example of an anti-endoglin antibody is TRC105 (WO2010039873A2).

[0088] Examples of anti-CD40 agonist antibodies are selicrelumab, APX005M, ChiLob7 / 4, ADC-1013, SEA-CD40 and CDX-1140 (Vonderheide 2020). Examples of anti-CD137 / 4-1 BB agonist antibodies are urelumab and utomilumab (Chester, Sanmamed et al. 2018). Examples of anti-CD134 / OX40 agonist antibodies are PF-04518600, MEDI6469, MOXR0916, MEDI0562, INCAGN01949 (Fu, Lin et al. 2020). An example of an anti-TNFRSF18 / GITR agonist antibody is DTA-1.

[0089] Examples of PD-1 antagonists are anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies. Examples of anti-PD-1 antagonist antibodies (antagonistic antibodies) are pembrolizumab, nivolumab, pidilizumab, toripalimab, and tislelizumab (Dolgin, 2020). Examples of anti-PD-L1 antagonist antibodies are atezolizumab and avelumab. An example of an anti-CTLA-4 antagonist antibody is ipilimumab. An example of an anti-LAG3 antagonist antibody is leratolimab. Examples of anti-TIGIT antagonist antibodies are tiragolumab, vibostolimab, domvanalimab, etigilimab, BMS-986207, EOS-448, COM902, ASP8374, SEA-TGT, BGB-A1217, IBI-939 and M6223.

[0090] An example of an anti-BTLA antagonist antibody is TAB004. Examples of anti-HAVCR2 / TIM-3 antagonist antibodies are LY3321367, MBG453 and TSR-022.

[0091] A preferred embodiment is an immunocytokine, preferably SEQ ID NO: 21, wherein the conjugate comprises the sequence of SEQ ID NO: 10 and the antibody comprises a heavy chain knob sequence from pembrolizumab of SEQ ID NO: 20, a heavy chain hole sequence from pembrolizumab of SEQ ID NO: 22, and a light chain sequence of SEQ ID NO: 16, said conjugate fused to the C-terminal heavy chain knob sequence without a linker.

[0092] In one embodiment, the immunocytokine of the invention comprises a conjugate of the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, wherein the antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of Table 4, wherein the IgG1 variant is heterodimeric via KiH mutations in Table 3 and has enhanced ADCC activity via DE, DLE, AAA, TL or IE mutations in Table 2, or via defucosylation, or via a combination of the mutations listed above and defucosylation.

[0093] In another embodiment, the immunocytokine of the invention comprises a conjugate of the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and the antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of Table 4, and the IgG1 variant is a heterodimer via the KiH mutation of Table 3.

[0094] [Table 4]

[0095] In a preferred embodiment, the immunocytokine of the present invention comprises a conjugate of the sequence of SEQ ID NO: 10 and an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, wherein the IgG1 variant is a heterodimer via the KiH mutations in Table 3.

[0096] In a preferred embodiment, the immunocytokine of the present invention comprises a conjugate of the sequence of SEQ ID NO: 10 and an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, wherein the IgG1 variant is a heterodimer via a KiH mutation in Table 3 and has enhanced ADCC activity via a DE, DLE, AAA, TL or IE mutation in Table 2, or via defucosylation, or via a combination of the above listed mutations and defucosylation.

[0097] In another preferred embodiment, the immunocytokine of the present invention comprises a conjugate of the sequence of SEQ ID NO: 10 and an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, wherein the IgG1 variant is a heterodimer via the KiH mutation in Table 3 and has enhanced ADCC activity via defucosylation.

[0098] In a preferred embodiment, the immunocytokine comprises a conjugate of the sequence of SEQ ID NO: 11 and the antibody variant is a heterodimeric IgG1 anti-CLDN18.2 antibody having a heavy chain knob sequence of SEQ ID NO: 84, a heavy chain hole sequence of SEQ ID NO: 87 and a light chain sequence of SEQ ID NO: 88.

[0099] Exemplary sequences of preferred immunocytokines may be SEQ ID NO:85 ("HC knob"), SEQ ID NO:87 ("HC hole") and SEQ ID NO:88 (LC).

[0100] Further exemplary sequences of preferred immunocytokines may be SEQ ID NO:86 ("HC knob"), SEQ ID NO:87 ("HC hole") and SEQ ID NO:88 (LC).

[0101] Further exemplary sequences of preferred immunocytokines may be SEQ ID NO:111 ("HC knob"), SEQ ID NO:110 ("HC hole") and SEQ ID NO:88 (LC).

[0102] In another preferred embodiment, the immunocytokine of the present invention comprises a conjugate of the sequence of SEQ ID NO: 10, and the antibody variant is a heterodimeric IgG1 anti-CLDN18.2 antibody having a heavy chain knob sequence of SEQ ID NO: 84, a heavy chain hole sequence of SEQ ID NO: 87 and a light chain sequence of SEQ ID NO: 88.

[0103] Surprisingly, the RLI2AQ DANA mutant fused to the anti-Claudin 18.2 antibody hCl1a exhibited higher ADCC compared to the RLI2AQ NA mutant.

[0104] In yet another embodiment, the immunocytokine of the invention comprises a conjugate of the sequence of SEQ ID NO: 10 and an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, wherein the IgG1 variant is a heterodimer via the KiH mutations of Table 3 and has the ADCC enhancing mutations S239D / I332E(DE) in the IgG1 Fc domain.

[0105] In yet another embodiment, the immunocytokine of the invention comprises a conjugate of the sequence of SEQ ID NO: 11 and an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, wherein the IgG1 variant is a heterodimer via the KiH mutations of Table 3 and has S239D / I332E(DE) ADCC enhancing mutations in the IgG1 Fc domain.

[0106] In another embodiment, the immunocytokine of the invention comprises a fusion protein having the sequence of SEQ ID NO: 10, and the antibody variant is a heterodimeric IgG1 anti-EGFR antibody having the VH sequence of SEQ ID NO: 91 and the VL sequence of SEQ ID NO: 92, the IgG1 variant being heterodimeric via KiH mutations in Table 3 and having enhanced ADCC activity via FC mutations listed in the corresponding section of Table 2, or via defucosylation, or via a combination of mutations and defucosylation.

[0107] In another embodiment, the present invention relates to a nucleic acid encoding the immunocytokines disclosed herein.

[0108] In yet another embodiment, the invention relates to a vector comprising a nucleic acid encoding said immunocytokine.

[0109] In a further embodiment, the invention relates to a host cell comprising the vector or nucleic acid encoding said immunocytokine.

[0110] Another embodiment of the invention relates to said immunocytokine, nucleic acid or vector for use in therapy.

[0111] Yet another embodiment of the invention relates to a pharmaceutical composition comprising said immunocytokine, nucleic acid or vector and a pharma- ceutically acceptable carrier.

[0112] In another embodiment, the immunocytokine, nucleic acid or vector may be for use in treating a subject suffering from, at risk of developing, and / or diagnosed with a neoplastic or infectious disease.

[0113] In another embodiment, the present invention relates to a method for treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic or infectious disease, comprising administering said immunocytokine, nucleic acid or vector.

[0114] The immunocytokines of the present invention may be administered in combination with other agents, typically approved standard therapies for a particular indication, since the typical clinical development pathway is combination with standard therapies. The immunocytokines of the present invention may be combined with checkpoint inhibitors, which may be anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG3, anti-TIM-3, anti-CTLA4 or anti-TIGIT antibodies, preferably anti-PD-L1 or anti-PD-1 antibodies. These antibodies have in common that they block / antagonize cellular interactions that block or downregulate immune cells, especially T cells, from killing cancer cells, and therefore, all of these antibodies are antagonistic antibodies. Examples of anti-PD-1 antibodies are pembrolizumab, nivolumab, cemiplimab (REGN2810), BMS-936558, SHR1210, IBI308, PDR001, BGB-A317, BCD-100 and JS001. Examples of anti-PD-L1 antibodies are avelumab, atezolizumab, durvalumab, KN035, and MGD013 (bispecific for PD-1 and LAG-3). An example of a PD-L2 antibody is sHIgM12. Examples of anti-LAG-3 antibodies are leratolimab (BMS986016), Sym022, REGN3767, TSR-033, GSK2831781, MGD013 (bispecific for PD-1 and LAG-3), and LAG525 (IMP701). Examples of anti-TIM-3 antibodies are TSR-022 and Sym023. Examples of anti-CTLA-4 antibodies are ipilimumab and tremelimumab (ticilimumab). Examples of anti-TIGIT antibodies are tiragolumab (MTIG7192A, RG6058) and etigilimab.

[0115] array [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] EXAMPLES

[0116] Example 1: General Materials and Methods Potency assay with kit225 The activity of both IL-2 and IL-15 can be determined by induction of proliferation of kit225 cells as described by Hori et al. (1987). kit225 cells (Hori, Uchiyama et al. 1987) were passaged in kit225 basal medium and used for potency assays at passages 4 to 7. Prior to potency assays, kit225 cells were cultured for 24 hours in kit225 basal medium without IL-2 (starvation period). 1 × 10 4 Kit225 cells were plated in 96-well plates and serial dilutions of RLI-15 and the respective molecule PEM-RLI-15 were added to the cells. The cells were incubated at 37° C., 5% CO2 for 72±3 hours. After incubation, 10 μl (10% of the volume in the well) of Alamar Blue was added to each well and after 6 hours, absorbance was measured at 560 nm with a 620 nm reference (mixing was set for 15 seconds before detection) using a Tecan Spark absorbance microplate reader. In some cases, when lower potency RLI2 variants were tested, incubation with kit225 cells was extended from 3 days (72 hours±3 hours) to 5 days.

[0117] Preferably, methods such as colorimetry or fluorescence are used to determine proliferation activation by IL-2 or IL-15 stimulation, for example as described by Soman et al. (Soman, Yang et al. 2009) using CTLL-2 cells. As an alternative to cell lines such as kit225 cells, human peripheral blood mononuclear cells (PBMCs) or buffy coats can be used. A preferred bioassay for determining IL-2 or IL-15 activity is the IL-2 / IL-15 Bioassay Kit (Promega Catalogue No. CS2018B03 / B07 / B05) using STAT5-RE CTLL-2 cells.

[0118] The concentrations of the RLI variants analyzed were as follows: RLI2 supernatant: 0.133mg / ml (ELISA, average from two experiments) RLI2 AQSupernatant: 0.0297mg / ml (ELISA, average from two experiments)

[0119] RLI2 Characteristics Purity (RP-UPLC) 99.8% Preparation 20 mM histidine, 6% (w / v) sorbitol, pH 6.5 Storage temperature -20℃

[0120] kit225 basal medium RPMI (460 mL) + FBS (30 mL) + Glutamax (5 mL) + penicillin-streptomycin (5 mL) + cytokines added to flask (75 cm2); IL-2 (5 ng / mL). Cytokines were added to the medium immediately before culture.

[0121] hPBMC potency assay Buffy coats were obtained from healthy donors. PBMCs were isolated by Ficoll Paque gradient, washed three times, and resuspended in T cell complete medium in 96-well plates. Immune cytokines were added at the indicated concentrations, and plates were incubated at 37°C, 5% CO2 for 7 days. Proliferation of immune cell populations was detected by flow cytometry.

[0122] T cell complete medium RPMI1640 medium, CTS GlutaMAX-I 1x, 100U / mL penicillin-streptomycin, 1mM sodium pyruvate, NEAA 1x (non-essential amino acid mixture), 2-mercaptoethanol 0.05mM and 10% AB human serum (heat inactivated).

[0123] [Table 5]

[0124] Isolation of human NK cells (hNK): Fresh blood from healthy donors was diluted in a 1:1 ratio with cold PBS-EDTA, ph 7.4, and PBMCs were isolated by Ficoll-Paque gradient isolation. Isolated PBMCs were resuspended in complete culture medium. hNK cells were isolated from PBMCs using the EasySep Human NK Cell Isolation kit (Stem Cell Technologies, USA) according to the manufacturer's instructions. The isolated hNK cells from each donor were diluted at 3 × 10 6 The cells were resuspended in NK medium containing 10% serum at a concentration of cells / ml.

[0125] PD-1 / PD-L1 blockade bioassay The assay was performed according to the manufacturer's instructions (Promega PD-1 / PD-L1 Blockade Bioassay J1250). Briefly, PD-L1 aAPC / CHO-K1 cells were plated in a 96-well plate and incubated for 16-20 hours in a 37°C, 5% CO2 incubator. The indicated concentrations of PEM-RLI immunocytokines and PD-1 effector cells were then added to the cells and incubated for 6 hours in a 37°C, 5% CO2 incubator. After the incubation period, Bio-Glo™ reagent was added to the wells, incubated for 15 minutes at room temperature, and luminescence measurements were performed.

[0126] Cynomolgus monkey research The pharmacokinetics of the indicated PEM-RLI molecules were studied in cynomolgus monkeys (n=2-3) on days 1 or 15 after administration of the indicated doses. Blood for serum isolation was collected 1, 4, 8, 24, 48, 60, 72, 84, 96, 120, and 168 hours after administration (some time points were omitted in some cases). Serum concentrations of immune cytokines were determined by ELISA using the antibodies in Table 5. Selected immune cell populations (NK cells and CD8 +Blood for flow cytometric assessment of T cells) was collected pre-dose, and on days 5, 8, 12, 15, 19, 22, and 26.

[0127] [Table 6]

[0128] [Table 7]

[0129] Mouse Efficacy Study The purpose of these studies was to obtain female hPD1 single KI HuGEMM mice (C57BL / 6-Pdcd1 em1(hPDCD1) / Smoc The objective of this study was to evaluate the in vivo therapeutic efficacy of PEM-RLI2 NA x1 and pembrolizumab as monotherapy in the treatment of HuCell MC38-hPD-L1 tumor cell line in mice (n=8 mice / group). Each mouse was inoculated with MC38-hPD-L1 tumor cells (1×10 6 ) was inoculated subcutaneously. The average tumor size was 108 mm 3 Randomization began when the mean age of the tumor reached 18.4 mm. Forty mice were enrolled in this study. All animals were randomly assigned to five test groups. Randomization was based on the "Matched distribution" method (StudyDirector™ software, version 3.1.399.19). The date of randomization was designated as day 0 (D0). After tumor cell inoculation, animals were checked daily (or more frequently, as necessary, at the discretion of the Study Director) for morbidity and mortality. Tumor volumes were measured three times a week in two dimensions using calipers, and volumes were calculated in mm using the formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). 3PEM-RLI2 NA x1 was administered IV at 20 mg / kg on day 0 and pembrolizumab was administered IP at 5 mg / kg on days 0, 3, 6, and 9. Tumor observation was continued for 18 days. Concurrently, PEM-RLI2 NA x1 (IL-15 with N65A and AQ mutations) was administered IV at 5, 10 on day 0. Tumor observation was continued for 6 days.

[0130] Mixed lymphocyte reaction Buffy coats were obtained from healthy donors. PBMCs were isolated by Ficoll Paque gradient and washed three times. PBMCs were isolated by Ficoll Paque gradient and washed three times. Pairs of hPBMC donors were cultured with equimolar concentrations of 1 nM pembrolizumab and PEM L-RLI NA x1 for 6 days. IFNγ production in cell supernatants was determined using a human IFN-γ DuoSet ELISA (R&D systems, no. DY258B). Data are expressed as relative response [%] of IFNγ production and represent the mean ± SEM from 12 pairs of hPBMC healthy donors.

[0131] SDS-PAGE and anti-RLI Western blot analysis The purified proteins were analyzed by SDS-PAGE and anti-RLI Western blot.

[0132] Coomassie staining: protein bands are visualized according to their molecular weight under denaturing conditions. Briefly, 1 volume of loading buffer (with or without β-mercaptoethanol) was added to 3 volumes of samples to be analyzed (then more or less diluted in 1× loading buffer), homogenized, and denatured at 95° C. for 5 min. The denatured samples were loaded onto Criterion TGX gels and run in 1× TGS buffer at constant voltage (300 V) and limiting current (75 mA or 135 mA per gel depending on gel type) in running buffer for 18 or 21 min depending on gel type. The gels were removed from the cassette, washed 3 times in water for 5 min, stained with Biosafe staining solution (Biorad) for 20 min, washed 3 times in water for 20 min, followed by a final destain wash in water for 3 h. The stained gels were then scanned on a gel scanner.

[0133] Western blot analysis: The gel is then transferred to a nitrocellulose membrane and used for Western blot analysis with different antibodies. At the end of the transfer, the gel is used for protein transfer to a nitrocellulose membrane. For the reference example (Biorad number 170-4155, Trans-BlotR Turbo™ Transfer Starter System), the transfer parameters are 2.5A, 25V, 7 minutes (for Criterion gels) or 2.5A, 25V, 3 minutes (for Mini-PROTEAN gels). After membrane saturation in iBind™ Flex solution, antibody incubation and washing steps are then performed in the iBind system. After exposure and complete drying, the membrane is scanned for analysis. The primary antibody used was anti-RLI2-PR01 antibody (Cytune, dilution 1:25000) and the secondary antibody used was donkey anti-rabbit IgG-AP antibody (Santa Cruz Biotechnology, dilution 1:5000).

[0134] Capillary electrophoresis Protein analysis by capillary electrophoresis relies on the separation of LDS-labeled protein variants through a sieving matrix in a constant electric field. The Labchip GXII instrument uses a single sipper microfluidic chip to characterize protein samples loaded onto a 96-well plate. The microfluidic chip technology allows the separation and analysis of protein samples. After detection and analysis of the laser-induced signal, the data provided are: relative protein concentration, molecular size and percent purity using ladder and marker calibration standards. The samples are denatured by mixing 5 μL of sample and 35 μL of HT Protein Sample Buffer with or without DTT at a final concentration of 35 mM. If necessary, the samples are pre-diluted in HT Protein Sample Buffer at 1 mg / mL. Denaturation is performed by heating the mixture at 100° C. for 5 minutes. 70 μL of water is then added and the samples are centrifuged at 2,000 g for 10 minutes. The samples (in a 96-well plate) are then loaded onto the LabChip GXII instrument for chip transfer and analysis.

[0135] [Table 8]

[0136] Glycosylation / deamidation mutants [Table 9]

[0137] The RLI2 molecule has a major glycosylation site at N176 (RLI numbering) and a secondary site at N168. No glycosylation is found at N209. The glycans are complex, mostly biantennary, fucosylated, and rarely sialylated G0-G2. In cell culture, about 40-50% of the protein is glycosylated, with N168 at about 5%. After purification as described above, about 14-25% of RLI2 is glycosylated. Different levels of glycosylation do not show any effect on potency, stability, and only a minor effect on pharmacokinetics, and glycosylated RLI2 has a shorter half-life, but the heterogeneity of the active pharmacological moiety remains problematic from a regulatory point of view.

[0138] A potential hotspot for deamidation identified in IL-15 expressed in E. coli (Nellis, Michiel et al. 2012) is N77 (IL-15 numbering) / N174 (RLI numbering). Although it has been described that N-glycosylation of N79 partially prevents N77 deamidation (Thaysen-Andersen, Chertova et al. 2016), we have indeed seen in mass spectrometry that N77 is deamidated in CHO-expressed RLI2, identifying deamidation as a real issue for potential heterogeneity of RLI2 and RLI-based products. Therefore, deamidation should be avoided.

[0139] Figure 1A shows that RLI2 wt (no mutations) is indeed a heterogeneous product with two major bands of approximately 20 kDa and 25 kDa and a few smaller bands, all immunoreactive with anti-RLI2 antibodies and thus representing different modifications of the RLI2 protein.

[0140] We wanted to avoid mutating N77 as an obvious way to eliminate the deamidation of N77 and thereby remove the polar amide, since a conservative substitution to glutamine did not resolve the risk of deamidation. To eliminate the potential deamidation at position N77, a single substitution G78A (IL-15 numbering) / G175A (RLI numbering) in RLI2 was instead introduced (RLI2A). Although the loss of deamidation was not seen by Coomassie staining or Western blot, the major acidic peak (pI 6.0) in RP-UPLC was significantly reduced in cIEF, as expected for the loss of deamidation, confirming that the deamidation hotspot N174 was indeed deamidated (data not shown). Mass spectrometry of the PEM-RLI AQ construct also showed the absence of deamidation (data not shown).

[0141] Surprisingly, the G78A mutation resulted in a slight increase in glycosylation (see FIG. 1A, better seen in FIG. 1B), with larger / more glycosylated species compared to RLI2 wt. Additional bands appear, indicating this new glycosylation pattern (see dashed box 3 in FIG. 1B). The RP-UPLC peaks were also slightly shifted (data not shown). This altered glycosylation pattern was unexpected, as the effect of the deamination mutation G78A on glycosylation was unpredictable.

[0142] Q (RLI2AQ, RLI2 AQA significant reduction in the larger species of RLI2 was observed with the additional substitution of N79 (IL-15 numbering) / N176 (RLI numbering) with N79 (IL-15 numbering) / N168 (RLI numbering) (see dashed box 1 in FIG. 1B). The remaining larger band (see solid box 2 in FIG. 1B) likely represents glycosylation at N71 (IL-15 numbering) / N168 (RLI numbering) of approximately 20% of the RLI molecules, which appears to be slightly increased compared to RLI2 wt and RLI2. The box 1 band may represent RLI2 glycosylated at N176, whereas the box 3 band may represent RLI2 glycosylated at N176 and N168. However, the box 3 band may also be RLI2 glycosylated with an unfavorable sialic acid glycan structure at N176. Without wishing to be bound by any theory, a possible explanation for this surprising increase in glycosylation at N71 is that glycosylation at the key site N79 sterically interferes with glycosylation at N71 in RLI2 wt, and such interference is alleviated when N79 is mutated.

[0143] In summary, RLI2 with AQ substitutions AQ , and thus IL-15 AQ also represent RLI2, or IL-15, variants with greatly improved homogeneity and reduced risk of deamidation.

[0144] To compare the effect / impact of glycosylation on the biological activity of RLI variants, we specifically inactivated three potential glycosylation sites N71 / N79 / N160 (N168 / N176 / N209 for RLI) of IL-15 by site-directed mutagenesis (Stratagene Site Directed Mutagenesis XL Kit). N168S / N176Q / N209S and RLI1 N168S / N176Q / N209S To confirm the predominant N-glycosylation occupancy at N79 (=N176 in RLI), RLI2 N176QA mutant was generated and transient expression in CHO cells results in a unique 25 kDa band (see FIG. 2, right panel).

[0145] The RLI protein mutated only at the major glycosylation site (RLI2 N176Q ) also showed a unique 25 kDa band, thus confirming the major glycosylation occupancy on the N176 residue of RLI expressed in CHO (transiently expressed). The secretion yields of the deglycosylated mutants transiently expressed in CHO cells were similar to their glycosylated / original counterparts. Thus, there was no significant effect of deglycosylation on the expression levels. The same was observed in a Pichia Pastoris expression system (data not shown).

[0146] Moreover, these mutations on the N-glycosylation sites do not appear to induce any significant effect on the in vitro proliferation activity of RLI on kit225 or 32Dβ cells. As usual, all RLI versions (RLI1 or RLI2, glycosylated or non-glycosylated, CHO or baculo or pichia) stimulated proliferation of kit225 cell lines similarly.

[0147] RLI2 AQ The Potency of Varieties The activity of both IL-2 and IL-15 can be determined by induction of proliferation of kit225 cells as described by Hori et al. (1987). kit225 cells (Hori, Uchiyama et al. 1987) were passaged in kit225 basal medium and used for potency assays at passages 4 to 7. Prior to potency assays, kit225 cells were cultured for 24 hours in kit225 basal medium without IL-2 (starvation period). 1 × 10 4Kit225 cells were plated in 96-well plates and serial dilutions of RLI-15 and the respective molecule PEM-RLI-15 were added to the cells. The cells were incubated at 37° C., 5% CO2 for 72±3 hours. After incubation, 10 μl (10% of the volume in the well) of Alamar Blue was added to each well and after 6 hours, absorbance was measured at 560 nm with a 620 nm reference (mixing was set for 15 seconds before detection) using a Tecan Spark absorbance microplate reader. In some cases, when lower potency RLI2 variants were tested, incubation with kit225 cells was extended from 3 days (72 hours±3 hours) to 5 days.

[0148] Preferably, methods such as colorimetry or fluorescence are used to determine proliferation activation by IL-2 or IL-15 stimulation, for example as described by Soman et al. (Soman, Yang et al. 2009) using CTLL-2 cells. As an alternative to cell lines such as kit225 cells, human peripheral blood mononuclear cells (PBMCs) or buffy coats can be used. A preferred bioassay for determining IL-2 or IL-15 activity is the IL-2 / IL-15 Bioassay Kit (Promega catalog number CS2018B03 / B07 / B05) using STAT5-RE CTLL-2 cells.

[0149] The concentrations of the RLI variants analyzed were as follows: RLI2 supernatant: 0.133mg / ml (ELISA, average from two experiments) RLI2 AQ Supernatant: 0.0297mg / ml (ELISA, average from two experiments)

[0150] [Table 10]

[0151] [Table 11]

[0152] Therefore, the glycosylated mutant RLI2 as a supernatant AQ showed very similar potency in stimulating kit225 and / or 32Db cells when compared to RLI2 from the supernatant, which was surprising since for many glycoproteins loss of glycosylation results in lower activity.

[0153] In SPR (Biacore) binding experiments to the IL-2 / IL-15βγ receptor, the k between RLI2 and RLI AQ was also on speed, k off Rate and equilibrium constant K d No relevant differences in were observed (data not shown).

[0154] In summary, RLI2 with AQ substitutions AQ , and thus IL-15 AQ represents an RLI2, or IL-15, variant with greatly improved homogeneity, reduced risk of deamidation, and equivalent potency in activating immune cells.

[0155] Cynomolgus monkey PK / PD study of hyperglycosylated and hypoglycosylated RLI2 To compare hyperglycosylated and hypoglycosylated RLI2 with respect to their PK and PD properties, a 200 L scale production campaign was performed, harvested on S0SP and X0SP depth filters, and the protein was captured on a PPA column. Virus was inactivated by solvent detergent treatment and purification continued through a Capto Adhere column and a hydroxyapatite type II column (flow-through mode) followed by a second virus removal step by nanofiltration. The RLI preparation was polished on a Capto Impres Phenyl column (CPI Phenyl HIC) and selected fractions for hyperglycosylated RLI2 were pooled (RLI-15-HG) and selected fractions for hypoglycosylated RLI2 were pooled (RLI-15-LG) (see Figure 5A-C). Finally, UFDF filtration was performed onto a 10 kDa cutoff UF membrane into final formulation buffer (20 mM histidine, 6% sorbitol, pH 6.5). RLI-15-HG shows the majority of the RLI in the upper band of glycosylated RLI isomers, whereas RLI-15-LG contains only a smaller fraction of glycosylated RLI isomers (Figures 5B and C).

[0156] A total of 3 male and 3 female cynomolgus monkeys were included in the PK / PD study. Animals were assigned to two groups receiving RLI2 at 15 μg / kg (nominal dose) as RLI-15-HG and RL1-15-LG by daily subcutaneous administration according to a crossover dosing design. Dosing was over two 4-day periods (2×4) separated by a 10-day washout period (Days 1-4: RLI-125-LG for males and RLI-15-HG for females, Days 15-18: RLI-15-HG for males and RLI-15-LG for females). Pharmacodynamic parameters (NK, CD4 + and CD8 +Expression of IgG4-associated leukemia cells (including Ki67 expression in cells) was analyzed from blood samples collected in the pretreatment period, on days 5, 12 and 19. Blood samples for pharmacokinetic studies were taken from all animals on days 1 and 15 after the first dose in each treatment period at the following time points: pre-dose and 0.5, 1, 2, 6, 12 and 24 hours after dosing. Bioanalysis was performed. In addition, back-up serum samples (D1 (pre-dose)). D15 (pre-dose) and D16 (24 hours) were partially used for immunogenicity evaluation (ADA determination).

[0157] Pharmacokinetic (PK) analysis was performed using noncompartmental analysis with Phoenix™ WinNonlin® software (version 6.4, Certara LP (Satara)).

[0158] Pharmacokinetic Profile: All treated animals were exposed to the test article such that quantifiable amounts of RLI2 were measured over the majority of the post-dose sampling periods on Days 1 and 15. Key pharmacokinetic parameters are summarized in Table 10.

[0159] [Table 12]

[0160] C max and AUC 0-t Exposure to C differed between male and female animals. max and AUC 0-t was approximately 2-fold higher in females than in males. Independent of this gender difference, differences in the pharmacokinetics of RLI-15-HG and RLI-15-LG were also observed. Surprisingly, the exposure with RLI-15-HG was lower than that with RLI-15-LG. The ratio between RLI-15-HG and RLI-15-LG was C, independent of the sex of the animals. max and AUC 0-t The values ​​were 0.606 and 0.453, respectively.

[0161] DC-T cell-based assays to determine immunogenicity Buffy coats were obtained from healthy donors. The blood was diluted with PBS-EDTA (to obtain 175 mL of diluted blood) and PBMCs were isolated by Ficoll Paque gradient (15 mL of Ficoll + 35 mL of diluted blood). CD14 + Monocytes were isolated using the EasySep™ Human CD14 Positive Selection Kit II (17858, StemCell) according to the manufacturer's instructions. - The fraction was pipetted into a new Falcon tube and the remainder was centrifuged at 1200 rpm for 10 min, then resuspended in CryoStore medium, frozen and temporarily stored at -80 °C. + Monocytes were resuspended in DC medium (CellGro supplemented with IL-4 and GM-CSF). Cells were incubated at 37°C with 5% CO2 for 5 days, harvested and seeded in 48-well plates. iDCs were loaded with protein for 4 hours and matured overnight with a cytokine cocktail (TNF-α, IL-1β + IL-4 and GM-CSF). They were then washed 4 times with PBS and T cell medium. Cells were stained with autologous CFSE stained CD4 + They were co-cultured with T cells at a ratio of 1:10 (negative magnetic separation) and cultured for 7 days. CFSE dilution was detected by flow cytometry.

[0162] [Table 13]

[0163] Example 2: Rituximab (RTX)-Based Anti-CD20 Immunocytokines This immunocytokine was based on rituximab (VH: SEQ ID NO: 96, VL: SEQ ID NO: 99). The immunocytokines listed in Table 11 were generated and tested for their provided assays.

[0164] [Table 14]

[0165] RTX-RLI immunocytokines showed increased efficacy in vivo at low doses compared to RLI2, and different RTX-RLI immunocytokines were found to contain RLI2 or RLI2. AQ were equivalent to each other, whether they contained x2 or x1, or whether they were with or without a linker.

[0166] Example 3: Reduced production yield of homodimeric RLI2 immunocytokine Immunocytokines were transiently expressed in CHO cells and purified using standard antibody purification protocols using Protein A. Briefly, Mab select sure (GE) was used to capture the immunocytokine product due to the presence of Fc. Nuvia HR-S (CEX) was used in bind / elute mode to separate oligomerized immunocytokine material and partially uncoupled antibodies RTX or PEM, as well as endotoxin and DNA contaminants. Preparative gel filtration (Superdex 200) was used to remove remaining oligomerized ICK uncoupled antibodies. Immunocytokines were concentrated to 2mg / ml using Vivaspin 30kDa. Upstream production of RTX immunocytokines (RTX-ICK) resulted in the presence of contaminants in the supernatant representing 25 kDa and 50 kDa proteins, naked RTX or RTX-RLI x2 (RTX KiH-RLI x1), oligomerized RTX-RLI, with differences in production between homodimeric constructs with two RLI2 molecules and heterodimeric constructs with one RLI2 molecule using KiH technology. Although heterodimeric antibody formats are generally expected to have lower expression due to mispairing of the heavy chains, it was surprisingly observed that for heterodimeric immunocytokines, heterodimeric constructs using KiH technology had higher expression compared to the respective homodimeric constructs. Thus, although the unoptimized production yields were approximately 3-fold higher for RTX-RLI2 x1 (220-300 mg / l) compared to RTX-RLI2 x2 (70-100 mg / l) and 6-fold higher for PEM-RLI2 x1 (90-120 mg / l) compared to PEM-RLI2 x2 (10-20 mg / l), in these unoptimized expressions, the IgG4 PEM constructs generally had worse expression compared to the IgG1 RTX constructs. Without being bound by any theory, the inventors speculate that the significant loss of expression of correctly folded homodimeric immunocytokines is related to the two RLI molecules linked to each heavy chain of the antibody to be folded interfering with proper antibody folding. This is because the RLI molecules have a tendency to interact with each other, thereby limiting the freedom of the heavy chain C-termini to form proper homodimers.

[0167] Both the KiH and L235E mutations did not significantly affect the production yield of the PEM-RLI2 construct, whereas the YTE mutation, alone or in combination with L235E, reduced expression levels by 2-fold.

[0168] Example 4: IL-15 Muteins for Reduced In Vitro Potency To decrease the binding of the RLI conjugate to the IL-2Rβ and / or γ receptors and thus the in vitro potency, and to reduce the heterogeneity of RLI2-containing products, mutations were introduced into the IL-15 portion of the RLI2 conjugate. The indicated amino acid substitutions were made in the mature human IL-15 sequence (see Table 12).

[0169] [Table 15]

[0170] [Table 16]

[0171] Tested IL-15 substitutions that affect binding to IL-2Rβ and / or γ significantly reduced the potency of the RLI molecule on kit225 cells. The single mutant N65A results in a similar reduction in potency as the NQD triple mutant (see Table 13). Other substitutions had only minor effects on potency.

[0172] [Table 17]

[0173] Even for RLI-15 mutant proteins tested without antibody binding, the NA mutation results in an approximately 2 log decrease in activity, measured here as EC50 in kit225 cells.

[0174] Example 5: Comparison of homodimeric and heterodimeric CD20-targeted immune cytokines Immunocytokines based on the anti-CD20 antibody rituximab were generated by using KiH variants of rituximab by fusing the RLI2 wt conjugate to the C-terminus of both antibody heavy chains ("x2") or by fusing one RLI2 mutant protein to the C-terminus of one of the heavy chains ("x1"). Rituximab ("RTX")-based immunocytokines were tested for their in vitro potency on kit225 cells (see Example 1 and Table 15).

[0175] [Table 18]

[0176] Homodimeric RTX-RLI 2x immunocytokines, with two RLI2 conjugates fused to the C-terminus, had a slight reduction in potency, whereas heterodimeric immunocytokines, with only one RLI2 conjugate, showed an approximately 10-fold reduction in potency against kit225 cells.

[0177] Similar activity was observed in hNK cells CD8 after 7 days of stimulation in vitro. + T cell activation (Ki67 + This was observed for cells.

[0178] [Table 19]

[0179] The efficacy of immune cytokines against kit225 was confirmed by activating human NK cells, CD8 + T cells and CD8 + This can be correlated with the potency of the immune cytokines on memory T cells. Only RTX-RLI2 x1 had an approximately 3-fold reduction.

[0180] Example 6: PD activity of rituximab-based anti-CD20 immunocytokines Immune cytokines were tested for PD activity on spleen-derived immune cells following administration of equimolar doses of RTX-ICK IV on day 1 to healthy Balb / c mice (2 mice / group). RLI2 was injected SC daily at 20 μg / mouse for 4 consecutive days (days 1-4). Activation of immune cell populations was detected on day 5 by flow cytometry. The following antibodies (Table 17) were used for PD studies (mice):

[0181] [Table 20]

[0182] There was no difference in PD activity between RTX-RLI2 x2, RTX-RLI AQ x2 and RTX-L40-RLI2 x2 in vivo (Figure 15). The PD activity of equimolar amounts of RTX-RLI2 x1 was lower due to only one RLI2 molecule / antibody, but only about 20-30% less in relative numbers of immune cells compared to double RTX-RLI x2 molecules.

[0183] Example 7: Anti-metastatic activity of rituximab-based anti-CD20 immunocytokines in the Renca mouse metastasis model in vivo The anti-metastatic activity of anti-CD20 immunocytokines at equimolar doses was tested in a Renca renal cell carcinoma metastasis model in Balb / c mice. 3 μg / dose of ICK was injected IV on day 1 (D1), lungs were harvested on day 16, and lung wet weights were measured.

[0184] In vivo, RTX-RLI2 AQ x2, RTX-RLI2 AQ x2, RTX-L40-RLI2 AQ x2 and RTX-RLI2 AQ RLI2 between x1 AQ No significant differences were observed in the anti-metastatic activity of the moieties (Figure 16). A single injection of 3 μg / dose resulted in a 30-40% reduction in metastasis compared to controls.

[0185] Example 8: Anti-CD20 RTX-RLI2 in A20-hCD20 / Balb / c mice AQ Antitumor efficacy of immune cytokines RTX-RLI2 AQ The antitumor efficacy of x2 immunocytokines was tested in Balb / c mice implanted sc with the A20-hCD20 tumor cell line (CrownBiosciences, USA). Mice were randomized into treatment groups based on tumor volume using matched distribution functions provided by the StudyDirector animal management software package (v3.0, StudyLog Systems, USA) to achieve minimal inter- and intragroup variability on day 1. AQ RLIx2 was administered at 0.15 mg / kg on days 1 and 8, and RLI2 was administered at 1 mg / kg on 4 consecutive days on days 1-4. Tumor volumes were measured twice weekly for the duration of the study; measurements were made in two dimensions using calipers and volumes were expressed in mm using the formula "V=(L×W×W) / 2," where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). 3 This is expressed as:

[0186] RTX-RLI2 AQ Two iv injections of x2 showed significant antitumor efficacy in the A20-hCD20 / balbc mouse tumor model when compared to controls. Similar efficacy was shown for RLI2 when administered four times at a nearly 10-fold higher dose (or even higher comparable equimolar doses due to the larger molecular weight of this immunocytokine) compared to the RTX-RLI immunocytokine (Figure 17).

[0187] Example 9: ADCC activity of rituximab-based anti-CD20 immunocytokines compared to rituximab alone Daudi cell line was incubated with the indicated concentrations of RTX and RTX-RLI2 molecules with or without NK92-CD16 cells. Daudi cell death was assessed as the percentage of DAPI positive cells and detected by flow cytometry.

[0188] 4 x 10 per well 4 Daudi tumor cells (CD20-expressing B-cell lymphoma) were seeded in a 96-well plate. NK92-CD16 cells were transfected with RTX-RLI2 AQ Serial dilutions of the molecules (concentrations 0.001, 0.01, 0.1, 1, 10 and 100 nM) were added at a ratio of 1:5. Cells were incubated for 4 hours at 37°C in humidified 5% CO2. After incubation, cells were stained with CD56-Alexa Fluor700 for differentiation of NK cells and tumor cells, CD19-PE antibody, and DAPI to identify dead tumor cells (CD19+DAPI+ cells) and analyzed by flow cytometry.

[0189] RTX-RLI2 AQ The ADCC activity of the molecules was slightly lower than the Rituximab control, however only 60% of the cells were killed by ADCC activity of Rituximab alone compared to 70% or 80% for RTX-RLI AQ and RTX-RLI 1x, respectively (Figure 18).

[0190] Example 10: IL-15 N65A mutation in PD-1 targeted immune cytokines shows reduced potency against kit225 cells Immunocytokines based on the anti-PD-1 antibody pembrolizumab were produced in various formats. Pembrolizumab is a humanized IgG4-κ antibody with a stabilizing S228P mutation in the Fc portion of the antibody. To improve this construct for use in immunocytokines, variations of pembrolizumab ("PEM") were tested. Although it is known that the IgG4 antibody class has relatively low ADCC activity, a L235E mutation (Alegre, Collins et al., 1992) ("LE") was introduced to further reduce ADCC (SEQ ID NO: 43). To limit the potential for immunogenic / anti-drug antibodies, more complex ADCC-inactivating mutations were avoided. One or two RLI2 molecules were genetically fused to the C-terminus of the PEM antibody. For homodimeric PEM variants ("x2"), one RLI2 molecule was fused to each heavy chain, whereas heterodimeric PEM variants ("x1") were generated using knob-in-hole (KiH) technology (Elliott, Ultsch et al. 2014), where one RLI2 molecule was fused to the knob heavy chain (SEQ ID NO: 41) with a T336W substitution, whereas the hole heavy chain (without RLI2 fusion) contained T366S / L368A / Y407V substitutions (SEQ ID NO: 42). When RLI2 was fused to the heavy chain, the terminal lysine (K) was deleted ("dK") to reduce product heterogeneity. Additionally, different RLI2 mutant proteins were used to fuse to the heavy chain of the antibody. All RLI2 molecules have AQ (G78A / N79Q) substitutions to reduce product heterogeneity, and the following substitutions that reduce RLI2 binding to IL-2 / IL-15Rβγ were tested in the PEM-RLI immunocytokines: DA, NA, ND, AD (K10A Q101D) and NQD. The PEM-RLI immunocytokines generated are listed in Table 18, left column.

[0191] Several homodimeric and heterodimeric forms of PEM-RLI2 with different IL-15 substitutions were prepared. AQThe potency of the immunocytokines was compared by measuring the in vitro EC50 in kit225 cells using RLI2 as a standard and set at 100% for relative potency (Table 18). The aim was to identify the least potent mutant protein of RLI2 in kit225 cells. Results shown are the average of 2-5 experiments.

[0192] [Table 21] RLI:RLI2 AQ ND: not detected (limit of assay sensitivity)

[0193] RLI2 in 1x PEM-RLI-NA AQ NA was identified as the least potent RLI mutant protein with a single mutation that reduces IL-2 / IL-15Rβγ, which is still approximately 10-fold more active than the NQD mutant with three amino acid substitutions, thereby carrying a relatively high risk of immunogenicity.

[0194] Heterodimeric PEM-RLI immunocytokines were analyzed by capillary electrophoresis under reducing and non-reducing conditions (Figure 1). All immunocytokines showed high purity with clear separation of antibody heavy chain, heavy chain + RLI (HC-RLI) and light chain. The faint band just above the HC-RLI band represents glycosylated RLI on the heavy chain. Surprisingly, glycosylation appeared to be reduced for the NA mutant.

[0195] Example 11: PEM-RLI x1 or PEM-RLI NA x1 molecules with Fc variants (LE, YTE or LE-YTE) show no difference in their potency against kit225 cells in vitro The potency of several heterodimers PEM-RLI x1 and PEM-RLI NA x1 with Fc variants designed to reduce ADCC (LE substitution) or increase in vivo half-life via increased FcRn binding (YTE substitution), or a combination of both (LE-YTE) was compared in vitro by determining EC50 using kit225 cells (Table 19), with RLI2 used as a standard and set to 100% for relative potency. Data represents the average of two experiments.

[0196] [Table 22] RLI:RLI2 AQ ;

[0197] RLI2 without IL-15 inactivating NA mutations in the RLI conjugate AQ Fc variants of PEM in heterodimeric fusions with (PEM-LE, YTE, or LE / YTE-RLI x1) demonstrated similar potency as PEM-RLI x1 against kit225. Similarly, all compared constructs carrying inactivating IL-15 NA mutations in the RLI conjugate showed similar (reduced) potency against kit225 cells regardless of the Fc variant tested. Thus, the tested mutations in the antibody Fc region did not affect the potency of the PEM-RLI constructs.

[0198] Example 12: Evaluation of PEM LE-RLI NA x1, PEM LE / YTE-RLI NA x1 and PEM-RLI NQD x1 molecules for potency against kit225 in vitro Selected PEM-RLI immunocytokine constructs from two batches were compared for potency in vitro with RLI2 using kit225 cells. The potency of the molecules was evaluated as EC50 and also calculated as relative potency relative to the RLI2 molecule. Data represents one experiment. As shown in Table 20, high batch-to-batch consistency was observed for the potency of the tested immunocytokines measured by determining EC50 on kit225 cells.

[0199] [Table 23]

[0200] Example 13: Comparison of PEM LE-RLI NAx1, PEM LE / YTE-RLI NAx1 and PEM-RLI NQD x1 molecules in potency against kit225 and hPBMC in vitro Selected PEM-RLI immunocytokine constructs (PEM-RLI) carrying one or two RLI2 molecules with or without LE / YTE Fc modification. The indicated PEM-RLI immunocytokines were used at various concentrations for stimulation of human PBMCs from six healthy donors for 7 days in vitro. Human NK cells and CD8 + Potency on T cells was compared to potency on kit225 cells (see Table 21). Surprisingly, the N65A IL-15 variant ("NA") did not inhibit human NK cells or CD8 + No difference in potency of molecules carrying one or two RLI2 molecules (comparing x1 and x2 immunocytokines) was observed with respect to T cell activation. As known from other experiments, LE / YTE in the Fc part of the antibody does not affect the potency of fusion RLI molecules. Immunocytokines containing IL-15 NQD mutant proteins had a further reduced potency of about 10-fold. Human PBMC potency data are the average of 6 donors. kit225 data are the average of 2-3 experiments.

[0201] [Table 24]

[0202] Example 14: Evaluation of low potency PEM-RLI mutants bound to HC or LC in kit225 cells in vitro Several low potency IL-15 muteins in PEM-RLI immunocytokine with or without mutated Fc antibody portion (LE-YTE) were compared for their potency compared to PEM LE / YTE-RLI2 NA x1 as a reference. In the "Lc" immunocytokine, the RLI conjugate was fused to the C-terminus of the light chain of the antibody (while all other constructs have the RLI conjugate fused to the C-terminus of one of both heavy chains). In vitro potency testing was accomplished using kit225 cell line with a modified protocol (extended cell incubation). The potency of the molecules was evaluated as EC50 and was also calculated as relative potency to the PEM LE / YTE-RLI NA x1 molecule. Data in Table 22 represent the average of 2-4 experiments.

[0203] [Table 25]

[0204] Substitution combinations QDQA (Q101D / Q108A), NQD (D30N / E64Q / N65D), DANA (D61A / N65A) and DANAQD (D61A / N65A / Q101D) further reduced the potency of the PEM-RLI immunocytokine construct until it was not measurable for the DANAQD construct. Immunocytokines with an RLI conjugate fused to the light chain of the antibody showed similar potency compared to constructs with only one RLI conjugate with the same IL-15 mutations on one heavy chain of the antibody.

[0205] Example 15: Comparison of low potency mutants with and without mutated Fc portions The aim was to evaluate and compare the potency of several muteins less potent than PEM-RLI NA x1 with or without a mutated Fc antibody portion (LE-YTE). These molecules are a fusion protein of pembrolizumab (IgG4) and RLI-15 (PEM-RLI-15). RLI2 was used as a standard. In vitro potency testing was accomplished using the kit225 cell line. The potency of the molecules was evaluated as EC50 and was also calculated as relative potency related to the naked RLI-15 molecule. Data represent the average of several experiments after 3.5 or 7 days of kit225 growth.

[0206] [Table 26]

[0207] Example 16: The functionality of pembrolizumab is not affected by the fusion of RLI2 or RLI2 mutant proteins or by Fc modification of the antibody The functionality of the anti-PD-1 antibody derivatives of pembrolizumab was determined by measuring the blocking of PD-1 / PD-L1 interaction using a bioluminescent cell-based assay "PD-1 / PD-L1 Blockade Bioassay" (J1250, Promega) according to the manufacturer's instructions. The indicated PEM RLI immunocytokines were tested to evaluate their potency in terms of their activity in blocking PD-1 / PD-L1 interaction (see Table 24). No significant differences were observed between the immunocytokines tested. Therefore, the functionality of the PEM part of the immunocytokines (PD-1 blocking) is preserved regardless of the number of RLI2 molecules bound, the mutations in the RLI2 conjugate or the mutations in the Fc part of the antibody.

[0208] [Table 27]

[0209] Example 17: PEM-RLI NA x1 immunocytokine shows anti-tumor efficacy in mouse tumor models The in vivo therapeutic efficacy of PEM-RLI NA x1 immunocytokine was compared to pembrolizumab as monotherapy in the treatment of HuCell MC38-hPD-L1 tumors in female human PD-1 single KI HuGEMM mice (n=8 mice / group). The mean tumor size was 108 mm on randomization day 0. 3 Treatment was initiated when ≥ 100% CI, 0.01-0.05 and 0.1-0.20, respectively. PEM-RLI NA x1 was administered IV at 20 mg / kg on day 0 and pembrolizumab was administered IP at 5 mg / kg on days 0, 3, 6, and 9.

[0210] PEM-RLI NA x1 strongly reduced tumor volume in this model compared to the control untreated group (p value was <0.05) and similar to the pembrolizumab treated group (see Figure 7). It should be noted that for immunocytokines, no significant difference with pembrolizumab was observed, but a single injection of immunocytokines achieved similar results as four doses of pembrolizumab. Furthermore, since mice are known to be about 10 times less sensitive to RLI, the full functionality of PEM-RLI NA x1 could not be tested in this mouse model, and therefore, the treatment effect in humans is expected to be better.

[0211] Example 18: PEM LE / YTE-RLI NA x1 molecule enhances IFN-γ production in mixed lymphocyte reactions more than pembrolizumab and RLI-15 monotherapy To evaluate the potential of PEM-RLI constructs to enhance T cell activation and IFNγ production, a mixed lymphocyte reaction (MLR) was employed. The MLR is an in vitro assay in which leukocytes from two genetically distinct individuals of the same species are co-cultured, resulting in cell blast transformation, DNA synthesis, and proliferation. The generation of the MLR occurs as a result of incompatibility of allogeneic determinants expressed on the surface of cell populations and encoded by the major histocompatibility complex (MHC).

[0212] T cell activation via IFNγ production was evaluated for PEM RLI-NA x2, PEM RLI-NQD x1, PEM LE / YTE-RLI NA x1 and PEM YTE-RLI x1 molecules in an in vitro MLR assay. PEM-RLI constructs were compared to RLI2 and pembrolizumab. The respective RLI-15 muteins not fused to an antibody, which would be a much more suitable matched control, were not available at the time of this study. When mismatched human PBMC donor pairs were incubated with PEM LE / YTE-RLI NA x1 (1000 nM), IFNγ production was increased compared to equimolar pembrolizumab, and the adjusted RLI2 concentration was reduced 300-fold, equaling the potency of the RLI2 NA mutant protein. Data represent the mean ± SE of six donor pairs for pembrolizumab and PEM LE / YTE-RLI NA x1, and three donor pairs for RLI2 (see Figure 8).

[0213] Example 19: PEM LE / YTE-RLI NA x1 molecules exhibit longer in vivo half-life than PEM-RLI wt x1 molecules, which correlates with higher PD activity The pharmacokinetics of PEM-RLI x1 and PEM LE / YTE-RLI NA x1 were studied in cynomolgus monkeys (n=2) following administration of 10 or 30 μg / kg (PEM-RLI x1) and 30 or 90 μg / kg (PEM LE / YTE-RLI NA x1) on days 1 and 15, respectively. Blood for serum isolation was collected at 1, 4, 8, 12, 24, 48, 60, 72, 96, 120, and 168 hours. Concentrations of PEM-RLI x1 and PEM LE / YTE-RLI NA x1 in serum were determined by ELISA. Selected immune cell populations (NK cells and CD8 + T cell proliferation – Ki67 + Blood for flow cytometric assessment of lymphocyte counts (IL-1, lymphocyte counts) was collected before dosing and on days 5, 8, 12, 15, 19, 22, and 26. The PEM LE / YTE-RL1 NA x1 molecule, which has reduced RLI-15 affinity for IL-2 / 15Rβγ, showed a significantly extended half-life relative to PEM-RL1 x1 after IV administration in cynomolgus monkeys (Figure 9A). There was an increase in the number of lymphocytes (Figure 9B) and NK cells as determined by Ki67 positivity (Figure 9C) and CD8 + PEM LE / YTE-RLI NA x1 molecules, which have an RLI mutant protein with significantly reduced affinity for IL-2Rβγ and potency, retained high PD activity in fusions with PEM antibodies (Figure 9 ), as shown by increased proliferation of T cells (Figure 9 D).

[0214] Example 20: PEM-RLI NA x2 molecule shows no benefit over PEM-RLI NA x1 molecule (PK and PD profiles) The pharmacokinetics of PEM-RLI NA x1 and PEM-RLI NA x2 were studied in cynomolgus monkeys (n=2) after dosing with 30 μg / kg on day 1 to evaluate the benefit of two RLI2 molecules over one RLI2 molecule bound to an antibody. Blood for serum isolation was collected at 1, 4, 8, 12, 24, 48, 72, 96 and 168 hours. Concentrations of PEM-RLI NA x1 and PEM-RLI NA x2 in serum were determined by ELISA. Selected immune cell populations (NK cells and CD8 + T cell proliferation – Ki67 + Blood for flow cytometric assessment of lymphocyte counts (IL-1, lymphocyte counts) was collected before dosing and on days 5, 8, 12, 15, 19, 22, and 26. Pharmacokinetics as determined by serum concentrations (Figure 10A) or pharmacodynamics as determined by lymphocyte counts (fold change, Figure 10B) after IV administration in cynomolgus monkeys. + NK cells (Fig. 10C) and %Ki67 + CD8 + For T cells (FIG. 10D), there was no benefit of the PEM-RLI immunocytokine, which has two RLI2 molecules with reduced affinity for antibody-bound IL-2 / 15Rβγ, over a single molecule of RLI2.

[0215] Example 21: PEM-RLI NA x1 molecules with LE mutations exhibit longer half-lives than molecules with LE / YTE mutations The pharmacokinetics of PEM LE / YTE-RLI NA x1 and PEM LE-RLI NA x1 were studied in cynomolgus monkeys (n=3) after IV dosing of 600 μg / kg on day 1. Blood for serum isolation was collected at 1, 8, 24, 48, 60, 72, 84, 96, and 120 hours. Serum concentrations of PEM LE / YTE-RLI NA x1 and PEM LE-RLI NA x1 were determined by ELISA. Although YTE mutations have been reported to increase FcRn binding and thus plasma half-life, in an immunocytokine format with one RLI NA mutant protein, the half-life was surprisingly decreased compared to constructs with only LE mutations (FIG. 11).

[0216] Example 22: The less potent mutant PEM-RLI NQD x1 exhibits a longer half-life than the PEM-RLI NAx1 molecule The pharmacokinetics of PEM LE-RLI NA x1 and PEM-RLI NQD x1 (NQD refers to D30N / E64Q / N65D) were studied in cynomolgus monkeys (n=3) after IV dosing of 600 μg / kg on day 1. Blood for serum separation was collected at 1, 8, 24, 48, 60, 72, 84, 96, 120, and 144 hours. Concentrations of PEM L-RLI NA x1 and PEM-RLI NQD x1 in serum were determined by ELISA. PEM-RLI constructs with triple mutant NQDs (see Table 13), which showed reduced potency compared to the NA mutant, showed increased half-life in vivo compared to PEM-RLI constructs with N65A substitutions (FIG. 12).

[0217] Example 23: ADCC activity of immunocytokines based on anti-Claudin18.2 hCl1a antibodies with altered effector functions cell line Human cell lines PA-TU-8988S (Creative Bioarray, Cat. No. CSC-C0326) and A549 (ATCC CCL-185) overexpressing Claudin18.2 (A549-Cldn18.2) were grown in DMEM medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM glutamine (GlutaMAX, Gibco), 100 U / ml penicillin, 0.1 mg / ml streptomycin (Invitrogen), and 2 μg / ml puromycin (Gibco). A549 cells were co-transfected by electroporation with a transposase expression construct (pcDNA3.1-hy-mPB), a construct carrying transposable full-length huCLDN18.2 (pPB-Puro-huCLDN18.2) together with a puromycin resistance cassette and a construct carrying EGFP as a transfection control (pEGFP-N3) (Waldmeier, Hellmann et al., 2016). After electroporation, cells were allowed to recover for 2 days in growth medium at 37 °C in a humidified incubator with a 5% CO2 atmosphere. Transfection was verified by FC analysis of EGFP expression. Cells expressing CLDN18.2 were then selected by adding puromycin at 1 μg / ml to the culture and further expanded to generate frozen stocks in FCS containing 10% DMSO. Expression of CLDN18.2 in transfected cells was analyzed by FC. To have a more homogenous PA-TU-8988S cell population, cells were sorted by FACS to select only cells with higher CLDN18.2 expression. Briefly, PA-TU-8988S cells suspended in FACS buffer (PBS, 2% FCS) were incubated with 2 μg / ml zolbetuximab for 30 min on ice. After washing in FACS buffer, cells were incubated with PE-labeled Fcγ-specific IgG goat anti-human secondary antibody (eBioscience) for 30 min on ice. After washing, stained cells were resuspended in FACS buffer and analyzed and sorted by FACSAria™ instrument to separate moderately expressing cells from high expressing cells. After sorting, harvested PA-TU-8988S-High cells (PaTu) were resuspended in growth medium, expanded, and frozen aliquots were stored in liquid N2. The human NK cell line NK92 (ATCC CRL-2407) exogenously expressing human CD16 (NK92-hCD16, referred to herein as NK92) was generated as described in Clemenceau et al., 2013 (Clemenceau, Vivien et al., 2013). The cells were grown in RPMI1640 medium (Gibco) supplemented with 10% AB human serum (One Lambda), 2 mM glutamine (GlutaMAX, Gibco) and 5 ng / ml IL-2 (Peprotech). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0218] Cell-based ADCC assay A549-Cldn18.2 or PaTu cells were seeded in 96-well plates at the appropriate concentration (A549-Cldn18.2 - 20000 cells, PaTu - 30000 cells) and incubated for 24 hours. NK92 cells or isolated human NK cells were harvested by centrifugation, washed, and resuspended in ADCC assay medium (RPMI1640 (without phenol red) supplemented with 2 mM glutamine and 10% heat-inactivated (20 min at 56 °C) pooled complement human serum (Innovative Research)). Medium was removed from the 96-well plates containing adherent cells (target cells T) and NK92 cells in suspension in ADCC assay medium (effector cells E) were added to the adherent target cells at an E:T ratio of 10 for A549-Cldn18.2 and 5 for PA-TU-8988S cells. Antibodies or immunocytokines (ICKs) to be tested were added at concentrations ranging from 0.001 to 100 nM or 0.0001 to 10 μg / ml. A human IgG1 isotype antibody (Ultra-LEAF™ Purified Human IgG1 Isotype Control Recombinant Antibody, Biolegend, Cat. No. 403502) was included as a non-specific control. The mixture was incubated overnight at 37°C. After 24 hours, cytotoxicity, expressed as the activity of lactate dehydrogenase enzyme released from dead cells, was measured using the LDH Cytotoxicity Assay (Abcam, ab65393) according to the manufacturer's instructions. 10 μl of the supernatant was transferred to a new 96-well plate, mixed with LDH substrate, and the color change developed was measured at OD of 450 nm using a spectrophotometer.Cytotoxicity was calculated according to the following formula: Cytotoxicity (%) = ((Test sample - Effector cell control - Low control) / (High control - Low control)) x 100; "Test sample": effector / target mixture; "Effector cell control": one well with NK92 cells only (determines LDH activity released from effector cells); "Low control": one well with target cells only (determines spontaneous release of LDH activity from untreated target cells); "High control": one well with target cells permeabilized with lysis buffer (determines maximum releasable LDH activity).

[0219] Figure 13 shows the ADCC activity of immunocytokines based on the hCl1a antibody with altered effector functions. All of the immunocytokines tested have a heterodimeric Fc domain and one RLI2 AQ The conjugate was fused to the C-terminus of one of the heavy chains. When immunocytokines with mutations in the effector domain that reduce ADCC were tested, the immunocytokine hCl1a LALAPG-RLI DANA showed nearly abolished ADCC activity when tested against A549-CLDN18.2 cells (upper panel) or PA-TU-8988S (lower panel) in the presence of NK92 cells when compared to the hCl1a-DANA immunocytokine with the hCl1a antibody alone. The hCl1a-LALA antibody also showed reduced ADCC activity compared to the hCl1a antibody, however, ADCC activity was not completely abolished. The addition of the conjugate did not affect the ADCC activity of the immunocytokines when the ADCC activity was reduced when compared to the ADCC activity of the antibody alone. Table 25 summarizes the ADCC EC50 values ​​measured for each immunocytokine or antibody tested. EC 50 Values ​​were determined using Graphpad Prism Software with the built-in "log(AGONIST) vs. response - variable slope (four parameters)" EC50 determination. When immunocytokines with effector domain mutations that enhance ADCC were tested, all tested immunocytokines based on hCl1a antibodies with DLE, DE, AAA, TE or IE mutations in the Fc domain showed enhanced ADCC activity compared to the same immunocytokines or antibodies alone without those mutations (Figure 14). To enhance ADCC activity, afucosylation was also tested. Figure 14F shows that in A549-Cldn18.2 and PA-TU-8988S cells, the defucosylated immunocytokine hCl1a-DANA afuc has enhanced ADCC activity compared to hCl1a-DANA, and ADCC activity comparable to that of the immunocytokines with the DE and DLE mutations mentioned above. However, when defucosylation was combined with effector domain-enhancing mutations, defucosylation surprisingly had a negative effect on the ADCC enhancement induced by the DE or DLE mutations (see Figures 14B and A). Nevertheless, when defucosylation was combined with AAA mutations, the enhanced ADCC activity was maintained (Figure 14C).

[0220] [Table 28]

[0221] Example 24: Assessment of antibody Fc binding to the ADCC activating receptors FcγRIIIa V158 and FcγRIIIa F158 and the ADCC inhibitory receptor FcγRIIb by surface plasmon resonance (SPR) The human FcγRIIIa receptor (hFcγRIIIa; CD16a) exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158. FcγRIIIa activates ADCC activity, whereas FcγRIIb inhibits ADCC. The ADCC activity of immunocytokines can be expressed as the ratio of the EC50 binding affinity to FcγRIIIa to the EC50 binding affinity to FcγRIIb when their affinity to the receptor is measured by SPR. SPR experiments were performed on a Biacore 8K (Cytiva, Chicago, IL, USA) using a CM5 sensor chip (Cytiva) immobilized with THE His-tag antibody (Genscript). FcγRIIIa V158, FcγRIIIa F158 or FcγRIIb protein was used for capture in 1×HBS-EP+ running buffer at a flow rate of 10 μl / min with a contact time of 30 s. The association / dissociation rate was measured for each immunocytokine tested in the appropriate range of concentration serial dilutions using association / dissociation times of 300 s / 300 s at a flow rate of 30 μl / min, except for constructs with DLE and DE with or without defucosylation. For constructs with DLE and DE, association / dissociation times of 120 s / 1200 s were applied. Table 26 below summarizes the results of the SPR measurements.

[0222] [Table 29] A / I ratio=(affinity for FcgRIIIa) / (affinity for FcgRIIb). Affinity=1 / Kd. "afuc" refers to defucosylation.

[0223] The A / I ratio allows one to assess the strength of binding to an ADCC activating receptor ("A"; FcγRIII) compared to an ADCC inhibitory receptor ("B"; FcγRIIb). The higher this ratio, the stronger the binding of the antibody or immunocytokine to the activating receptor.

[0224] The SPR data confirm that overall, all immunocytokines with ADCC-enhancing mutations exhibit higher A / I ratios than immunocytokines that do not have ADCC-enhancing mutations that are part of the TL mutations. The relatively low A / I ratios of the TL mutations may be due to increased glycosylation of such mutations (see Example 25).

[0225] Example 25: Stability / developability of hCl1a-based immunocytokines with enhanced ADCC activity Immunocytokines based on hCl1a that have ADCC-enhancing DLE, DE, AAA, TL or IE mutations or are defucosylated are administered to patients with ADCC. H The stability and developability of the two domains were assessed by evaluating their melting temperature, sequence liability and glycosylation (N-glycan) profile.

[0226] C H The melting temperatures of the two domains were measured by differential scanning calorimetry (DSC) using a MicroCal PEAQ-DSC automated system (Malvern Panalytical). Briefly, the immunocytokine samples were diluted to 1 mg / ml in their storage buffer. Heating was performed from 20°C to 100°C at a rate of 1°C / min. The protein solution was then cooled in situ and the same thermal scan was performed to obtain a baseline for subtraction from the first scan.

[0227] For N-glycan analysis, proteins were first reduced with DTT and then transferred to an HPLC column equipped with a glass insert vial for injection. Proteins were separated by reversed-phase chromatography and detected by a Waters / XEVO G2XS-QTOF online LC-MS coupled with a UV detector. The molecular weights of the detected glycan chains were matched with known N-glycan types, and the relative abundance of N-glycans was calculated and expressed by the intensity of the detected peaks.

[0228] The amino acid sequences of the immunocytokine constructs carrying ADCC enhancing mutations were analyzed for the presence of the following additional sequence instabilities (not present in constructs without ADCC enhancing mutations), as listed in Table 27.

[0229] [Table 30]

[0230] The TL mutation introduced N-glycosylation sequence instability (mutation K392T adjacent to N390 in the IgG1 sequence). No sequence instability was introduced by the other mutations (see Table 28).

[0231] [Table 31] Score 4: the parameter is within the expected range for a mAb-based drug product; Score 3: careful monitoring / evaluation of required quality attributes during development; Score 2: Likely to have a significant impact on timelines and / or costs; Score 1: High risk of not being adequately controlled.

[0232] Overall, defucosylation does not affect the stability and developability and may therefore be used to enhance the ADCC activity of immunocytokines. HThe TL mutations caused a significant decrease in the melting temperature of the 2 domains (see Table 29), with the potential to affect the stability of the immunocytokines in solution. However, these mutations did not affect the glycosylation of the immunocytokines. The sequence instability introduced by the TL mutations resulted in the introduction of undesirable sialylated and high mannose glycan species (see Table 30). These species could adversely affect the pK of the immunocytokines. Similarly, the immunocytokines with IE mutations had a high proportion of mannose species, which had the potential to affect the properties of the immunocytokines. The immunocytokines with AAA mutations resulted in an increase in mannose species (see Table 30). However, the generation of defucosylated immunocytokines partially restored glycosylation to an acceptable level for developability. Therefore, if enhancement of an hCl1a-based immunocytokine is desired, the AAA mutation, optionally in combination with defucosylation, may be the recommended mutation with the least impact on its stability and developability. Defucosylation did not affect the properties evaluated. The DLE and DE mutations caused a significant decrease in Tm and had the potential to destabilize the molecule. The TL mutation introduced an additional glycosylation site into the Fc. The construct with the IE mutation had a high proportion of mannose species.

[0233] [Table 32]

[0234] [Table 33]

[0235] Example 26: Mouse in vivo efficacy study The aim of this study is to test the in vivo therapeutic efficacy of hCl1a-RLI immunocytokine in a mouse model. Female NMRI nude mice are implanted with pancreatic human cell line-derived xenografts BXPC3 (ATCC CRL-1687™) exogenously expressing Claudin18.2 (BXPC3-CLDN18.2) at 5-7 weeks of age. Tumors are implanted by unilateral subcutaneous injection. The animals are cultured at approximately 100 mm 3 Mice are randomized based on tumor volume in mm. Mice are assigned to different groups (n=7 per group) and treated according to Table 31 on day 1. Animals are checked twice weekly for weight loss and tumor volume. Tumor volumes are measured by caliper and expressed in mm using the formula: V=(L×W×W) / 2, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). 3 It is expressed as 2000mm. 3 Mice are euthanized when they reach a tumor burden of 0.05 mg / kg or experience significant weight loss (>30% overall or >20% on two consecutive days).

[0236] [Table 34]

[0237] Example 27: Anti-PD-1 antibody and SOT201 bind to CD8 + Synergize in T cell activation SOT201 binds RLI-15 at the C-terminus of the knob heavy chain. AQASOT201 is a heterodimeric immunocytokine having an antibody derived from humanized IgG4 pembrolizumab with heavy chain T366W-knob / T366S, L368A, Y407V-hole, L235E substitutions and terminal K deleted (see SEQ ID NO:21, SEQ ID NO:101, SEQ ID NO:23) fused to SOT201. SOT201 and Keytruda® (pembrolizumab) were compared in a PD-1 / PD-L1 blocking assay according to Example 1. Figure 19A shows that SOT201 effectively blocks PD-1 / PD-L1 interaction similar to the anti-PD-1 antibody Keytruda. The K determined for SOT201 and pembrolizumab D The values ​​are shown in Table 32.

[0238] [Table 35]

[0239] Human PBMCs from 11 healthy donors were cultured using RLI2AQ N65A (RLI-15 AQA ) variant, or RLI2, which has the same antibody heavy and light chains as SOT201 but does not have reduced binding of the IL-15 moiety to IL-2 / IL-15Rβγ. AQ The control molecule with the variant ("SOT201 wt") was stimulated in vitro for 7 days. Cell proliferation was assessed by flow cytometry analysis using Ki-67 and + NK cells and CD8 + SOT201 was determined to activate NK cells and CD8 T cells at a higher EC50 concentration than a comparable immunocytokine molecule (SOT201 wt) with unreduced receptor binding to the RLI-15 molecule. + Activates T cell proliferation (Figure 19B).

[0240] RLI-15 AQAThe murine surrogate SOT201 (mSOT201: see SEQ ID NO: 102, SEQ ID NO: 103 and SEQ ID NO: 104) containing the anti-mouse PD-1 antibody RMP1-14 (BioXCell, Lebanon, NH, USA) with similar substitutions for heterodimerization (E356K, N399K / K409E, K439D), ADCC silencing (D265A) and stabilization (dK) fused to the monoclonal anti-mouse PD-1 antibody RMP1-14 itself (mPD1) and RLI-15, which has a similar in vivo half-life as mSOT201. AQA Anti-human PD1 mouse IgG1-RLI-15 as a control AQA The results were compared to a single activity control represented by (hPD1-mSOT201), which does not exert any PD-1 blocking activity in C57BL / 6 mice. Cell proliferation (Ki67) was detected in the spleen by flow cytometry 5 days after IV injection of the compound equimolar to 5 mg / kg mSOT201 in healthy C57BL / 6 mice (n=2 / group). Anti-PD-1 antibody and RLI-15 in mouse surrogate mSOT201 AQA The mutant protein portion is CD8 + It showed a synergistic effect on T cell proliferation (Figure 19C).

[0241] Example 28: Tumor regression in the MC38 mouse model C57BL / 6 mice (hPD1 transgenic) were implanted with the syngeneic MC38 cell line. The test drugs mSOT201, hPD1-mSOT201, and mPD1 were administered on day 1 (randomization day, tumor volume 80-100 mm 3) were injected IV at equimolar doses with 5 mg / kg mSOT201 (n=10 / group) and compared with control (NaCl). mSOT201 induced tumor regression in 9 out of 10 mice after a single IV administration, whereas in comparison, monoclonal anti-mouse PD-1 antibody (mPD1) and anti-human PD-1 mouse IgG1-RLI-15 mutant protein immunocytokine (hPD1-mSOT201), which exerts no anti-PD-1 effect in mice, only showed a marginal effect on tumor growth compared with control mice (Figure 20A). Similarly, anti-mouse PD-1 antibody alone (mPD1) or RLI-15 AQA Anti-mouse PD-1 antibody and RLI-15 in a fusion protein (mSOT201) compared to anti-human PD1 mouse IgG1-RLI-15 mutant protein immunocytokine (hPD1-mSOT201) as a control for the mutant protein alone AQA The synergistic activity of the mutant proteins is demonstrated in surviving mice over a time course up to 100 days after treatment (FIG. 20B).

[0242] Example 29: Induction of pathways and genes related to antitumor immunity in MC38 tumors and activation of immune cells in the spleen and lymph nodes RNA isolation: RNA samples were isolated from tumors in syngeneic MC38 tumor-bearing C57BL / 6 mice 7 days after a single IV dose of mSOT201 (5 mg / kg). Day 1 (randomization day, tumor volume 80–100 mm 3 Three mice were treated IV with mSOT201 (5 mg / kg) and four control mice were left untreated. RNA was isolated from tumor tissues using an RNeasy MicroKit. The quality of the RNA samples was checked using an Agilent Bioanalyzer RNA Nano Chip and Qubit HS RNA assay.

[0243] RNA sequence analysis: Sequencing libraries were prepared from RNA samples by SMARTer® Stranded Total RNA-Seq Kit v3-Pico Input Mammalian Kit (Takara Bio USA, Inc.), library quality control was performed using a capillary gel electrophoresis system (Agilent Bioanalyzer with HS DNA chip) and Qubit HS DNA assay, and sequencing was performed on a NovaSeq 6000 with a 2x151bp run using the NovaSeq 6000 300 cycles Reagent Kit.

[0244] Data analysis: Raw data were processed according to a standard RNA-seq pipeline including the following steps: quality control (by FastQC and FastqScreen), adapter trimming (8bp trimming in Read2 by using seqtk), mapping to the reference genome GRCm39 (using HISAT2) and transcript counting (using ht-seq). The resulting output, a quantification file containing the number of transcripts for each sample, was further processed via R packages and ggplot2, tydiverse, dplyr. Raw counts were normalized to median ratio normalization by DESeq2. Differential gene expression analysis was performed using DESeq2 (version 1.24.0) in R (abs(log2FC)=1, FDR<0.05). Heatmaps were generated using the ComplexHeatmap package in R. Analysis of the function and enrichment of DEGs was performed using ClusterProfiler and the web-based tool Gene ontology (GO). To calculate TPM values ​​for cell population analysis, the salmon tool was used on the trimmed fastq files. Cell population analysis was performed with TIMER 2.0 and xCell tools.

[0245] Results: Differential expression analysis (abs(log2FC)=1, FDR<0.05) resulted in the upregulation of 800 mouse genes and the downregulation of 1910 mouse genes in mSOT201-treated tumors compared to control samples. Gene Ontology (GO) term enrichment analysis identified upregulated DEGs mainly related to αβ T cells, γδ T cells, B cells, NK cell activation, cytotoxicity, cell killing, cytokine production, cell chemotaxis and cell adhesion, whereas downregulated genes were related to tumor development and tumor signaling. These data indicate that mSOT201 activates both innate and adaptive immunity in the tumor microenvironment. Next, we employed "metagene" markers to estimate the relative abundance of different immune cell populations in the tumor microenvironment. In accordance with the whole transcriptome findings, mSOT201-treated samples were analyzed using a 3-fold analysis of the CD8 + T cells (p<0.001), CD8 + Naive T cells (p<0.0005), CD8 + Effector memory T cells (p=0.001), CD8 + Central memory T cells (p<0.001), γδ T cells (p=0.0002), NK cells (p<0.001), CD4 + T cells (p=0.0157), CD4 + Naive T cells (p=0.1176), CD4 + Gene sets associated with effector memory T cells (p=0.003), B cells (p=0.0602), and myeloid dendritic cells (p=0.0120) were enriched, whereas gene sets associated with cancer-associated fibroblasts were significantly depleted (p=0.0254) (Figure 21A).

[0246] mSOT201 induced the proliferation of selected immune cell populations in the spleen and lymph nodes of MC38 tumor-bearing mice (Figure 21B). 3 ) On day 7 after mSOT201 treatment, cell proliferation (Ki67) was detected by flow cytometry (n=2).

[0247] Example 30: EC50 values ​​of different IL2 / IL-15Rβγ agonists in kit225 cells RLI-15(SOT101), SOT201(PEM-RLI-15 AQA ), hPD-1-IL-2v and αhPD1-IL-15m M1 EC50 values ​​were determined as described in Example 1. In hPD-1-IL-2v, one IL-2 mutein IL-2v (SEQ ID NO: 106) is fused to the C-terminus of one heavy chain of an anti-human PD-1 antibody as described in WO 2018 / 184964 A1 (sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 25 in WO 2018 / 184964 A1). In αhPD1-IL-15m M1, one IL-15 mutein with mutations N1A-D30N-E46G-V49R (SEQ ID NO: 107) is fused to the C-terminus of one heavy chain of an anti-human PD-1 antibody as described in WO 2019 / 166946 A1 (see FIG. 1D therein, SEQ ID NO: 89, SEQ ID NO: 74 and SEQ ID NO: 65 therein). The EC50 values ​​are shown in Table 33.

[0248] [Table 36]

[0249] A further interesting candidate to be tested is αhPD1-IL-15m M2 in which one IL-15 mutein with the mutations N1G-D30N-E46G-V49R-E64Q (SEQ ID NO: 108) is fused to the C-terminus of one heavy chain of the anti-human PD-1 antibody described in WO 2019 / 166946 A1 (see FIG. 1C therein, SEQ ID NO: 90, SEQ ID NO: 74 and SEQ ID NO: 65 therein).

[0250] Thus, SOT201 has a substantially lower EC50 than PD1-IL-2v and αhPD1-IL-15m M1 in kit225 cells, allowing for higher doses and a longer in vivo half-life, and is also expected to exert a stronger and longer lasting effect with respect to activity in disrupting the anti-PD-1 / PD-L1 interaction.

[0251] Example 31: Comparison of mSOT201 and mPD1-IL-2Rβγ agonist in MC38 tumor model mSOT201 (a mouse SOT201 surrogate) was compared with control (NaCl), anti-mouse PD-1 antibody fused to IL-2v IL-2 mutant protein RMP1-14 (mPD1-IL-2Rβγ agonist), and RLI-15 AQA The combination of mSOT201 and mPD1 antibody was compared in the MC38 tumor model with a single IV dose as described in Example 28. Administration of mPD1-IL-2Rβγ significantly increased NK cell and CD8+ counts on day 5 after IV administration of 5 mg / kg mSOT201 in healthy C57 / BL6 mice. + The dose was chosen to match T cell proliferation and was equivalent to 0.25 mg / kg mPD1-IL-2Rβγ. + ) was detected by flow cytometry. + It induced activation of T cells and NK cells that, in contrast to the mPD1-IL-2Rβγ agonist, persisted until day 8 (Figure 22B).

[0252] mPD1-IL-2Rβγ is an IL-2 / IL-15Rβγ agonist in which the IL-2 mutein IL-2v (SEQ ID NO: 106) contains substitutions F42A, Y45A and L72G to the IL-2 sequence that reduce affinity for IL-2Rα (see WO 2018 / 184964A1, e.g., the paragraph spanning pages 27 and 28), as well as further substitutions, T3A to eliminate O-glycosylation at position 3 (paragraph spanning pages 28 and 29), and C125A to increase expression or stability (page 30, 3rd paragraph).

[0253] A murine surrogate of SOT201 (mSOT201) induced tumor regression in 9 of 10 MC38 tumor-bearing mice after a single IV administration compared with 5 of 10 for the mPD1-IL-2Rβγ agonist, but not RLI-15. AQA The combination of IFN-γ with mPD1 antibody only resulted in a delay in tumor growth compared to control mice (Figure 22A).

[0254] mSOT201 inhibited NK cells and CD8 + Induced T cell proliferation, which was observed with the mPD1-IL-2Rβγ agonist and equimolar amounts of RLI-15 in combination with mPD1. AQA In contrast to the MC38 tumors, treatment lasted for 7 days after randomization. 3 (n=10 / group).

[0255] Furthermore, mSOT201 still showed persistent CD8+ proliferation at day 8, in contrast to the mPD1-IL-2Rβγ agonist, which showed a marked reduction in proliferating cells at day 8. + It induced a significant and prolonged activation of T cells and NK cells (Figure 22B).

[0256] SOT201 also inhibited NK cells and CD8 + Induce T cell proliferation, which is achieved by combining mPD1-IL-2v and an equimolar amount of RLI-15. AQA and mPD1 antibody combination, persisted for 7 days after administration (Figure 22C).

[0257] Example 32: PK profile of SOT201 in cynomolgus monkeys SOT201 was administered IV at 0.6 mg / kg on day 1 to cynomolgus monkeys, and NK cell and CD8 + T cell proliferation (Ki67 + ) and absolute cell counts were determined over time by flow cytometry and hematology. SOT201 significantly increased NK (approximately 90% at day 5) and CD8 cell counts in the blood of cynomolgus monkeys after IV administration. +It induced high proliferation and expansion of T cells (approximately 80% on day 5) (FIG. 23A). Pharmacokinetic parameters are shown in Table 34.

[0258] [Table 37]

[0259] SOT201 increased NK cells and CD8 cells after repeated IV administration in cynomolgus monkeys. + induced T cell activation (Figure 23B).

[0260] Example 33: PD activity of mouse SOT201 surrogates The primary objective of this study was to determine whether treatment with the murine surrogate molecule mSOT201 (see Example 27) reduced CD8 expression in C57BL / 6 mice compared with treatment with hPD1-mSOT201 or mPD-1. + The aim of the study was to evaluate whether mSOT201 wt murine surrogate molecule has additive / synergistic effects on T cell proliferation. The secondary objective of the study was to compare the pharmacodynamic activity of the murine surrogate molecule mPD1-IL2v in C57BL / 6 mice. A description of the murine surrogate molecules tested is provided in Table 35. PD activity was assessed on days 5 and 8. FACS analysis was performed as described above.

[0261] [Table 38]

[0262] [Table 39]

[0263] Because pembrolizumab does not recognize mouse PD-1, hPD-1-mSOT201 was compared with RLI-15 bound to a non-conjugated antibody with a similar PK profile. AQA and therefore has such a PK profile. AQAThe mPD-1 molecule reflects the PD activity of the anti-PD-1 antibody alone. CD8 + With respect to T cell activation, mSOT201 exhibits more than additive (i.e., synergistic) effects on days 5 and even 8 compared to its single component surrogates hPD1-mSOT201 and mPD-1, administered at equimolar amounts. In comparison, both mPD1-IL2v and mSOT201 wt (both with more active IL-2 / IL-15Rβγ agonists), administered at lower doses to account for the anticipated higher activity on day 5, exhibited greater CD8 T cell activation on day 5. + Although it shows slightly higher activation of T cells, such effects are short-lived and do not affect CD8 + Activation of T cells is much stronger for mSOT201 on day 8. Looking at activated NK cells, the difference is less pronounced. As expected, mPD-1 does not activate NK cells, but hPD1-mSOT201, mPD1-IL2v, mSOT201 and mSOT201 wt strongly activate NK cells on day 5, with mSOT201 being somewhat weaker than the others. On day 8, mSOT201 again shows stronger activation of NK cells compared to mPD1-IL2v and mSOT201 wt (Figure 24A).

[0264] Although mSOT201, hPD1-mSOT201, and mPD-1 were administered at twice the doses as in A, similar behavior was observed when administered at lower doses, likely because maximal activation of cells had already been reached in experiment A (see FIG. 24B). As expected, mSOT201 wt and mPD1-IL2v suppressed the activation of CD8 + We showed a decrease in activation of both T cells and NK cells, which was mediated by CD8 + For T cells, there was again a decline towards control levels by day 8.

[0265] These data suggest that SOT201, which has significantly reduced binding to IL-2 / IL-15Rβγ, together with its anti-PD-1 moiety, inhibits NK and CD8 +It is a potent and long-lasting activator of T cells, whereas molecules with higher IL-2 / IL-15Rβγ agonistic activity are specifically targeted to CD8 + This shows that the activation of T cells is much shorter than that of the control. + T cells) or in trans (i.e., in close proximity to different CD8 + PD-1 expression on CD8 T cells + The avidity effect of simultaneous binding of PD-1 and IL-2 / IL-15Rβγ on T cells is that CD8 + It is hypothesized that this results in such preferential activation of T cells.

[0266] Example 34: Antitumor efficacy activity of mSOT201 in PD-1 sensitive and PD-1 treatment resistant mouse models The purpose of this study was to evaluate the antitumor activity of mSOT201 in anti-PD-1 treatment-sensitive (CT26, MC38) and anti-PD-1 treatment-resistant (B16F10, CT26 STK11 ko) mouse models. Descriptions of the mouse surrogate molecules tested are listed in Table 37.

[0267] [Table 40]

[0268] The murine surrogate molecule of SOT201, mSOT201, showed synergistic effects compared to its single component surrogates mPD-1 and hPD1-mSOT201 in the PD-1-sensitive tumor models tested, CT26 and MC38, showing complete responses in 5 of 10 and 9 of 10 mice, respectively (Figure 25A).

[0269] Even in tumor models known to be resistant to anti-PD-1 therapy, mSOT201 showed synergistic effects compared with its single components, but the therapeutic effect was not as strong as in the sensitive models, with only 1 out of 10 mice showing a complete response in the B16F10 model.

[0270] Example 35: mSOT201 vs. RLI-15AQA Antitumor efficacy of mutant protein + anti-PD-1 antibody The objective of this study was to evaluate the antitumor activity of mSOT201 versus RLI-15 AQA mutant protein + anti-PD-1 treatment in the MC38 mouse model. A description of the mouse surrogate molecules tested is provided in Table 38.

[0271] [Table 41]

[0272] Anti-PD-1 moiety and IL-2 / IL-15βγ agonist RLI-15 AQA (2 doses, G2 and G3) were fused with a combination of equimolar amounts of each component (G4: RLI-15 AQA The results showed a strong synergistic effect compared to G11:hPD1-mSOT201+mPD1, or G11:hPD1-mSOT201+mPD1. See Figure 26. It is hypothesized that the temporal and spatial linkage of activation of PD-1 positive immune cells is mechanistically stronger than the activation of immune cells by individual components.

[0273] Example 36: Antitumor efficacy activity of mSOT201 vs. SOT101 + anti-PD-1 antibody The objective of this study was to evaluate the antitumor activity of mSOT201 versus SOT101 + anti-PD-1 treatment in the MC38 mouse model. Descriptions of the mouse surrogate molecules tested are listed in Table 39.

[0274] [Table 42]

[0275] A single dose of 2 mg / kg mSOT201 (G3) was associated with an RLI of 1 mg / kg AQ + 4 doses of mPD1 at 5 mg / kg (G8) or 1 mg / kg of RLI2 AQThe combination therapy with 4 doses of mPD1 at 5 mg / kg (G9) showed similar therapeutic efficacy. However, a single dose of mSOT201 at 5 mg / kg (G2) outperformed multiple doses of the individual components (G8 and G9).

[0276] Example 37: Mechanistic studies on the differences in immune cell activation under mSOT201 vs. SOT101 + anti-PD-1 antibody treatment The purpose of this study was to evaluate the antitumor activity of similar effective doses of mSOT201 versus SOT101 + anti-PD-1 treatment in the MC38 mouse model. Descriptions of the mouse surrogate molecules tested are listed in Table 39.

[0277] Differences in the relative numbers of various immune cell populations in both treatments were detected in the tumor, spleen and lymph nodes. + T cells and αβTCR-bearing CD3 + The relative proliferation of cells did not change between both treatments in the spleen and lymph nodes. However, in the tumor, mSOT201 significantly increased the proliferation of CD8 + Induced a higher relative increase in T cells, but RLI2 AQ + anti-PD-1 treatment increased more NK cells. Interestingly, mSOT201 increased the percentage of γδTCR-bearing CD3 + Cells were induced but not RLI2 AQ + Anti-PD-1 combination treatment primarily stimulated a higher proportion of γδTCR-bearing CD3 + The cells were induced (see FIG. 28).

[0278] Example 38: DC-T cell-based assay and Fluorospot assay to determine immunogenicity The aim of this study was to evaluate in vitro the immunogenic risk of a pembrolizumab-based immunocytokine carrying one RLI-15 mutant protein (PEM-RLI-15 candidate molecule). A DC-T cell assay was used for this purpose, in which the test product was first incubated with immature dendritic cells (iDCs) and later presented to autologous T cells as processed peptides of the candidate molecule loaded on MHC molecules of mature DCs (mDCs). After a 7-day co-incubation period, T cell proliferation was measured as a surrogate marker of anti-drug antibody formation. The detection of DC-induced T cell proliferation was used to mitigate the stimulatory activity of the RLI-15 components in the test system, which could have a strong influence on the results, but this strong influence is not due to immunogenicity. Keyhole limpet hemocyanin (KLH) was used as a positive control, since KLH is known to induce a strong immune response induction. Pembrolizumab was used as a negative control. Control DCs that were not loaded with protein were used as a control for the assessment of non-specific T cell proliferation.

[0279] [Table 43]

[0280] PEM-RLI-15 candidate molecules from Table 40 were used at two concentrations each for stimulation of iDCs. Maturation of DCs was induced by proinflammatory cytokines. After 24 hours, mDCs were washed and incubated with autologous CD4+ pre-stained with CFSE. + T cells were incubated with 100% T cells, and T cell proliferation was assessed after 7 days based on CFSE detection by flow cytometry.

[0281] This assay could not be performed with SOT201 (PEM L-RLI N65A x1) because the still too high activity of the RLI N65A mutant protein would result in direct T cell activation and spill over to RLI-15 activity.

[0282] Human CD14 +DCs generated from monocytes (11 healthy donors from three separate experiments) were incubated with 10 μg / ml (not shown) or 50 μg / ml of PEM-RLI-15 candidate molecules, pembrolizumab or KLH for 24 h in the presence of maturation signals (proinflammatory cytokines TNFα and IL-1β). Protein-loaded washed mDCs were subsequently cultured with autologous CFSE-stained CD4 + T cells were cultured together. T cell proliferation was measured by flow cytometry after 7 days. CFSE low Considering cells as going through the cell cycle, proliferating CD4 + The percentage of T cells was evaluated based on the CFSE signal. KLH was used as a positive control and pembrolizumab was used as a negative control (see FIG. 29). The PEM-RLI-15 candidate molecule PEM L-RLI DANA x1 / PEM LY-RLI DANA x1 did not induce significant proliferation of T cells compared to the negative control, reflecting a low immunogenicity risk (positive response detected in 1 of 11 donors). The candidate molecule PEM LY-RLI DANAQD x1 induced significant proliferation of T cells compared to the negative control (p=0.0208, paired t-test), pointing out a potential immunogenicity risk for this RLI-15 mutant protein with three mutations to reduce binding to IL-2 / IL-15Rβγ (positive response detected in 4 of 11 donors).

[0283] Since the overactive RLI-15 mutant protein stimulates an immune response, DC-T cell assays showed that RLI-15 (wild-type sequence) is more potent than RLI-15. AQA Therefore, pairs of peptides in which substitutions were introduced were generated across the substitutions and tested in the Fluorospot assay.

[0284] [Table 44]

[0285] CD8-depleted PBMCs from 40 donors were plated and incubated with test peptides in RPMI + 10% huAb and IL-7. Media was refreshed for IL-7 on day 1 and IL-7 and IL-2 on day 4. On day 7, CD8-depleted PBMCs were harvested, rested overnight, plated on FluoroSpot plates the next day and restimulated with peptide. INF-γ and TNF-α FluoroSpot plates were developed on day 9. Figure 29B shows that the confidence intervals for all tested conditions overlap 0, meaning that there is no evidence of a shift in the average dSFU when comparing the mutant peptides with the paired wild-type sequences, and therefore no associated increase in immunogenicity is seen for both the N65A and G175A / N176Q substitution pairs.

[0286] Example 39: Efficacy of different anti-PD-1 IL-2 / IL-15Rβγ agonist immunocytokines The following anti-PD-1 IL-2 / IL-15Rβγ agonist immunocytokines (Table 42) were generated and their activities compared.

[0287] [Table 45]

[0288] The efficacy of anti-PD-1 IL-2 / IL-15Rβγ agonist immunocytokines was determined in kit225 cells (see Table 43) and hPBMCs (see Table 44).

[0289] [Table 46]

[0290] [Table 47]

[0291] Example 40: PD-1 / PD-L1 Blocking Activity of Anti-PD-1 IL-2 / IL-15Rβγ Agonist Immunocytokines To evaluate the blocking activity of the PD-1 / PD-L1 axis, the anti-PD-1 IL-2 / IL-15Rβγ agonist immunocytokines were tested using the PD-1 / PD-L1 Blockade Bioassay (Promega, No. J1250) as described above. The results are shown in Table 45.

[0292] [Table 48]

[0293] SOT201 exhibits the highest PD-1 / PD-L1 blocking activity among the three tested anti-PD-1 IL-2 / IL-15Rβγ agonist immunocytokines.

[0294] Example 41: Efficacy of human and mouse surrogate SOT202 molecules with altered effector functions on kit225 cells SOT202 binds RLI-15 at the C-terminus of the knob heavy chain. AQA It is a heterodimeric immunocytokine with an antibody derived from humanized IgG1 hCl1a with T366W-knob / T366S, L368A, Y407V-hole substitutions in the heavy chain and with the terminal K deleted (see SEQ ID NO: 111, SEQ ID NO: 110 and SEQ ID NO: 88) fused to IgG1. In the following examples, the term SOT202-XXX denotes a molecule in which further modifying mutations have been made to SOT202, such as the DANA mutation in RLI2 shown in Table 13. For clarity, SOT202-DANA differs from SOT202 only by the additional DA (D61A) mutation, since SOT202 already contains the NA (N65A) mutation (numbers refer to IL-15 numbering). Mutations in the effector domain of the IgG1 molecule that modify the ADCC properties of the antibody, such as the AAA, DE and DLE mutations shown in Table 2, as shown. The term "afuc" denotes a defucosylated IgG1 molecule. Defucosylated antibodies also have altered ADCC properties.

[0295] The activity of human and mouse surrogate SOT202 ADCC modified molecules for inducing proliferation of kit225 cells was evaluated as described in Example 1, and the EC50 and relative potency compared to SOT101 are shown in Tables 46 and 47. Mouse SOT202 was generated by replacing the human hIgG1 constant domain of SOT202 with its mouse equivalent mIgG2a (mSOT202: SEQ ID NO: 112, SEQ ID NO: 128 and SEQ ID NO: 129; mSOT2020 LALAPG: SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 129; mSOT202 isotype: mSOT202 isotype HC knob, SEQ ID NO: 133 and SEQ ID NO: 134; mSOT202 LALAPG isotype: SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 134).

[0296] [Table 49]

[0297] This potency assay shows that SOT202 shows the same potency as SOT201 on kit225 cells (see Table 36) and that the ADCC modification did not affect the potency of the immunocytokine. Therefore, this toolbox allows to adjust the ADCC activity of the antibody without affecting the potency of the immunocytokine on the activation of kit225 cells.

[0298] [Table 50]

[0299] For human SOT202, ADCC modification (LALAPG mutation) did not affect the potency of mouse SOT202 surrogates in activating kit225 cells. However, mouse SOT202 surrogates were less potent than their human counterparts. This is likely due to kit225 cells not expressing CD16, which is required for co-signaling with IL-15Rβγ in human and mouse NK cells.

[0300] Example 42: Human NK cells and CD8 + Potency of human SOT202 ADCC engineered molecules on T cells Human NK cells and CD8 + The activity of human SOT202 ADCC modified molecules for inducing T cell proliferation was assessed as described in Example 1 (hPBMC potency assay) and the EC50 and relative potency compared to SOT202 are shown in Figure 30 and Table 48.

[0301] [Table 51]

[0302] SOT202-DANA with DLE and DE mutations that enhance ADCC significantly increased human NK cell activity compared to SOT202-DANA without ADCC modification. Defucosylated SOT202 also increased ADCC activity, but to a lesser extent than the DE and DLE mutations. On the other hand, mutations that reduce ADCC, such as the LALAPG mutation, almost abolished NK cell activation. These mutations suppressed the activation of CD8 + It had only a minor effect on T cell activation. Without wishing to be bound by theory, it is hypothesized that the higher binding to the CD16 receptor due to the enhancing mutations synergizes with IL-15Rβγ signaling.

[0303] Example 43: Human NK cells and CD8 compared to SOT201 molecule + Potency of human SOT202 molecules on T cells Human NK cells and CD8 + The activity of the human SOT202 molecule in inducing T cell proliferation was compared to that of SOT201. The EC50 and relative potency compared to SOT202 and SOT201 are shown in FIG. 31 and Table 49.

[0304] [Table 52]

[0305] Human NK cells and CD8 + The activity of the human SOT202 molecule was compared with that of SOT201-DANA for inducing T cell proliferation. The EC50 and the relative potency compared to SOT202 and SOT201 are shown in Figure 32 and Table 49. The reduced stimulatory activity of the molecule with the DANA mutation compared to the molecule with only the NA mutation confirms the lower stimulatory activity of this mutation, as already described in the previous examples. The SOT202 molecule with enhanced ADCC activity via defucosylation (SOT202 with NA mutation) increases NK cell activity, but does not increase CD8 + It does not increase T cell activity, confirming the results shown in Example 42. SOT201 is based on an IgG4 antibody, which therefore has inherently low ADCC activity.

[0306] [Table 53]

[0307] The SOT202 and SOT201 molecules bind to human CD8 + It has the same potency on T cells, but not on NK cells. Defucosylation increased human NK cell activity.

[0308] Example 44: mSOT202 activates immune cells in the spleen of healthy C57BL / 6 mice Murine SOT202 was generated by replacing the human hIgG1 constant domain of SOT202 with its murine equivalent mIgG2a (SEQ ID NO: 127, SEQ ID NO: 128 and SEQ ID NO: 129). Cell proliferation (Ki67) was detected in the spleen by flow cytometry 5 days after IV injection of 5, 10 or 20 mg / kg of mSOT202 compound in healthy C57BL / 6 mice. mSOT202 increased the expression of NK cells and CD8 + Dose-dependent stimulation of T cells was observed (Figures 33(A) and (B)).

[0309] Example 45: mSOT202 induces synergy between ADCC activity and RLI2 stimulation of NK cell proliferation Cell proliferation (Ki67) was detected in the spleen by flow cytometry 5 and 10 days after IV injection of 5 mg / kg of mSOT202 molecule in healthy C57BL / 6 mice. The proliferation activity of mSOT202 (hCl1a-mIgG2a-NA 1x) on NK cells was higher than the effect of hCl1a-mIgG2a (molecule without RLI2) in addition to the effect of mSOT202-LALAPG (hCl1a-mIgG2a-LALAPG-NA 1x without ADCC activity), indicating synergy between the ADCC activity of the antibody in mSOT202 and the proliferation activity of RLI2, which is likely due to CD16 signaling (Figure 34(A)). Therefore, ADCC may contribute to the increase in the activity of NK cells. CD8 + For T cell stimulation, no synergy could be measured in this experimental model (Figure 34(B)).

[0310] The present invention is illustrated by the following embodiments.

[0311] 1. An immunocytokine comprising: a. a conjugate comprising interleukin 15 (IL-15) or a derivative thereof and a polypeptide comprising the sushi domain of interleukin 15 receptor alpha (IL-15Rα) or a derivative thereof; b. An antibody or a functional variant thereof, i. heterodimeric Fc domain, ii. altered effector functions, and / or iii. Increased in vivo half-life and an antibody or a functional variant thereof, characterized by Including, The conjugate is fused directly or indirectly to the C-terminus of both antibody heavy or light chains, or in the case of i. to the C-terminus of one antibody heavy chain. Immune cytokines.

[0312] 2. The altered effector function is a reduced antibody-dependent cellular cytotoxicity, and the antibody or a functional variant thereof is a. an IgG1 antibody or a functional variant thereof, comprising mutations selected from L234A / L235A, P329G, L234A / L235A / P329G, G236R / L328R, D265A, N297A, N297Q, N297G or L234A / L235A / G237A / P238S / H268A / A330S / P331S; b. an IgG4 antibody or a functional variant thereof, comprising mutations selected from L235E, F234A / L235A, F234A / L235A / P329G, P329G, S228P / L235E, S228P / F234A / L235A or E233P / F234V / L235A / D265A / R409K; c. a hybrid of IgG2 (IgG2a or IgG2b) and IgG4 or a functional variant thereof, comprising the CH1 and hinge regions from IgG2, and the CH2 and CH3 regions from IgG4 (IgG2 amino acids 118-260 and IgG4 amino acids 261-447); or d. an IgG2 antibody or a functional variant thereof, comprising mutations selected from H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S; Numbering follows EU numbering The immunocytokine according to embodiment 1.

[0313] 3. The above antibody or a functional variant thereof (a) an IgG4 antibody or a functional variant thereof, which comprises the L235E mutation; or (b) an IgG1 antibody or a functional variant thereof, comprising the L234A / L235A mutation; The immunocytokine according to embodiment 2.

[0314] 4. The modified effector function is an enhanced antibody-dependent cellular cytotoxicity, and the antibody or a functional variant thereof is a. an IgG1 antibody or a functional variant thereof, comprising in one heavy chain: F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; Preferably, it comprises mutations selected from S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E and, in the other heavy chain, further comprises mutations D270E / K326D / A330M / K334E, and / or b. a defucosylated IgG1, IgG2, or IgG4 antibody or a functional variant thereof; Numbering follows EU numbering The immunocytokine according to embodiment 1.

[0315] 5. The heterodimeric Fc domain is KiH, KiH S-S , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT S-S 5. The immunocytokine according to any one of embodiments 1 to 4, selected from the group consisting of A107, SEED and A107, preferably KiH.

[0316] 6. An immunocytokine described in any one of embodiments 1 to 5, wherein the heterodimeric Fc domain results in a higher yield of the immunocytokine upon expression in cell culture compared to an immunocytokine having a homodimeric Fc domain.

[0317] 7. The half-life of said immune cytokine is increased, and said antibody or functional variant thereof is an IgG1 or IgG4 antibody or functional variant thereof and comprises mutations selected from M252Y / S254T / T256E, M428L / N434S or T250Q / M428L, Numbering follows EU numbering An immunocytokine according to any one of embodiments 1 to 6.

[0318] 8. The immunocytokine according to any one of embodiments 1 to 3, wherein the antibody or functional variant thereof has reduced antibody-dependent cellular cytotoxicity, and wherein the antibody or functional variant thereof is an IgG4 antibody or functional variant thereof and comprises an L235E mutation and a KiH heterodimerized Fc domain.

[0319] 9. The conjugate is a fusion protein comprising, in the order of N-terminus to C-terminus, an IL-15Rαsushi domain or a derivative thereof, a linker, and the IL-15 or a derivative thereof, preferably, the IL-15Rαsushi domain comprises the sequence of SEQ ID NO:5; The linker has a length of 18 to 22 amino acids, is preferably composed of glycine or serine and glycine, and more preferably has the sequence of SEQ ID NO: 7, and the IL-15 has the sequence of SEQ ID NO: 2. An immunocytokine according to any one of embodiments 1 to 8.

[0320] 10. The above IL-15 variants are a. at least one mutation that increases the homogeneity of said IL-15 variant with respect to post-translational modification, Preferably, the mutation reduces deamidation at N77 and / or glycosylation at N79 of IL-15 (SEQ ID NO:2); More preferably, said mutations are selected from the mutations G78A, G78V, G78L or G78I, and N79Q, N79S or N79T, Most preferably, said mutation is G78A / N79Q, and / or b. IL-2 / IL-15Rβ and / or γ c at least one mutation that reduces binding to the receptor, Preferably, the mutated amino acid is selected from N1, N4, S7, D8, K10, K11, D30, D61, E64, N65, L69, N72, E92, Q101, Q108, I111 of IL-15 (SEQ ID NO: 2), more preferably, the mutated amino acid is selected from D61, N65 and Q101, and most preferably, the mutated amino acid is N65. 10. The immunocytokine of any one of embodiments 1 to 9, comprising:

[0321] 11. The IL-2 / IL-15Rβ and / or the γ c The at least one mutation that reduces binding to the receptor is N1D, N1A, N1G, N4D, S7Y, S7A, D8A, D8N, K10A, K11A, D30N, D61A, D61N, E64Q, N65D, N65A, N65E, N65R, N65K, L69R, N72R, Q101D, Q101E, Q108D, Q108A, Q108E and Q108R, preferably D8A, D8N, D61 11. An immunocytokine according to any one of embodiments 1 to 10, wherein the substitution is selected from: D8N / N65A, D61A / N65A or D61A / N65A / Q101D.

[0322] 12. The above antibody or a functional variant thereof a. Binds to a tumor antigen, preferably selected from EGFR, HER2, FGFR2, FOLR1, CLDN18.2, CEA, GD2, O-acetyl-GD-2, GM1, CAIX, EPCAM, MUC1, PSMA, c-MET, ROR1, GPC3, CD19, CD20, CD38; b. binds to a tumor extracellular matrix antigen, preferably selected from FAP, the EDA domain of fibronectin, the EDB domain of fibronectin and LRRC15, preferably FAP and the EDB domain of fibronectin; c. binds to an angiogenic antigen, preferably VEGF or endoglin; d. An immunomodulatory antibody or a functional variant thereof; said immunomodulatory antibody stimulates a costimulatory receptor and is preferably selected from a CD40 agonist, a CD137 / 4-1BB agonist, a CD134 / OX40 agonist and a TNFRSF18 / GITR agonist; or Said immunomodulatory antibody inhibits an immunosuppressive receptor and is preferably selected from PD-1 antagonists, CTLA-4 antagonists, LAG3 antagonists, TIGIT antagonists, inhibitory KIR antagonists, BTLA antagonists, HAVCR2 antagonists and ADORA2A antagonists, more preferably PD-1 antagonists. An immunocytokine according to any one of embodiments 1 to 11.

[0323] 13. The cytokine domain comprises the sequence of SEQ ID NO: 10; The antibody is i. the heavy chain knob sequence of SEQ ID NO:20; ii. the heavy chain hole sequence of SEQ ID NO:22, and iii. Light chain sequence of SEQ ID NO: 16 wherein the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker. An immunocytokine according to any one of embodiments 1.

[0324] 14. The cytokine domain comprises the sequence of SEQ ID NO: 10, and the antibody comprises i. the heavy chain knob sequence of SEQ ID NO:84; ii. the heavy chain hole sequence of SEQ ID NO: 87, and iii. Light chain sequence of SEQ ID NO: 88 wherein the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker. An immunocytokine according to any one of embodiments 1.

[0325] 15. The cytokine domain comprises the sequence of SEQ ID NO: 10, and the antibody comprises i. the heavy chain knob sequence of SEQ ID NO:93; ii. the heavy chain hole sequence of SEQ ID NO: 95, and iii. Light chain sequence of SEQ ID NO: 92 wherein the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker. An immunocytokine according to any one of embodiments 1.

[0326] 16. A nucleic acid encoding an immunocytokine described in any one of embodiments 1 to 15.

[0327] 17. A vector comprising the nucleic acid described in embodiment 16.

[0328] 18. A host cell comprising the nucleic acid of embodiment 16 or the vector of embodiment 17.

[0329] 19. An immunocytokine according to any one of embodiments 1 to 15, a nucleic acid according to embodiment 16 or a vector according to embodiment 17 for use in therapy.

[0330] 20. A pharmaceutical composition comprising an immunocytokine according to any one of embodiments 1 to 15, a nucleic acid according to embodiment 16 or a vector according to embodiment 17, and a pharma- ceutically acceptable carrier.

[0331] 21. An immunocytokine according to any one of embodiments 1 to 15, a nucleic acid according to embodiment 16 or a vector according to embodiment 17 for use in treating a subject suffering from, at risk of developing and / or diagnosed with a neoplastic or infectious disease.

[0332] 22. A method for treating a patient suffering from, at risk of developing, and / or diagnosed with a neoplastic or infectious disease, comprising administering an immunocytokine described in any one of embodiments 1 to 15, a nucleic acid described in embodiment 16, or a vector described in embodiment 17.

[0333] The present invention is also described by the following embodiments.

[0334] 1. An immunocytokine comprising: a. a conjugate comprising interleukin 15 (IL-15) or a derivative thereof and a polypeptide comprising the sushi domain of interleukin 15 receptor alpha (IL-15Rα) or a derivative thereof; b. A PD-1 antibody or a functional variant thereof, i. heterodimeric Fc domain, ii. altered effector functions, and / or iii. Increased in vivo half-life and a PD-1 antibody or a functional variant thereof, characterized by Including, The conjugate is fused directly or indirectly to the C-terminus of both antibody heavy or light chains, or in the case of i. to the C-terminus of one antibody heavy chain. Immune cytokines.

[0335] 2. The altered effector function is a reduced antibody-dependent cellular cytotoxicity, and the antibody or a functional variant thereof is a. an IgG1 antibody or a functional variant thereof, comprising mutations selected from L234A / L235A, P329G, L234A / L235A / P329G, G236R / L328R, D265A, N297A, N297Q, N297G or L234A / L235A / G237A / P238S / H268A / A330S / P331S; b. an IgG4 antibody or a functional variant thereof, comprising mutations selected from L235E, F234A / L235A, F234A / L235A / P329G, P329G, S228P / L235E, S228P / F234A / L235A or E233P / F234V / L235A / D265A / R409K; c. a hybrid of IgG2 (IgG2a or IgG2b) and IgG4 or a functional variant thereof, comprising the CH1 and hinge regions from IgG2, and the CH2 and CH3 regions from IgG4 (IgG2 amino acids 118-260 and IgG4 amino acids 261-447); or d. an IgG2 antibody or a functional variant thereof, comprising mutations selected from H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S; Numbering follows EU numbering The immunocytokine according to embodiment 1.

[0336] 3. The above antibody or a functional variant thereof (a) an IgG4 antibody or a functional variant thereof, which comprises the L235E mutation; or (b) an IgG1 antibody or a functional variant thereof, comprising the L234A / L235A mutation; The immunocytokine according to embodiment 2.

[0337] 4. The heterodimeric Fc domain is KiH, KiH S-S , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT S-S 4. The immunocytokine according to any one of embodiments 1 to 3, selected from the group consisting of A107, SEED and A107, preferably KiH.

[0338] 5. An immunocytokine according to any one of embodiments 1 to 4, wherein the heterodimeric Fc domain results in a higher yield of the immunocytokine upon expression in cell culture compared to an immunocytokine having a homodimeric Fc domain.

[0339] 6. The immunocytokine according to any one of embodiments 1 to 5, wherein the half-life of said immunocytokine is increased, and wherein said antibody or functional variant thereof is an IgG1 or IgG4 antibody or a functional variant thereof and comprises mutations selected from M252Y / S254T / T256E, M428L / N434S or T250Q / M428L, wherein the numbering is according to EU numbering.

[0340] 7. The immunocytokine according to any one of embodiments 1 to 6, wherein the antibody or functional variant thereof has reduced antibody-dependent cellular cytotoxicity, and wherein the antibody or functional variant thereof is an IgG4 antibody or functional variant thereof and comprises an L235E mutation and a KiH heterodimerized Fc domain.

[0341] 8. The immunocytokine according to any one of embodiments 1 to 7, wherein the conjugate is a fusion protein comprising, in the order of N-terminus to C-terminus, an IL-15Rαsushi domain or a derivative thereof, a linker, and the IL-15 or a derivative thereof, preferably wherein the IL-15Rαsushi domain comprises the sequence of SEQ ID NO: 5, the linker has a length of 18 to 22 amino acids and is preferably composed of glycine or serine and glycine, more preferably, has the sequence of SEQ ID NO: 7, and the IL-15 has the sequence of SEQ ID NO: 2.

[0342] 9. The cytokine domain comprises the sequence of SEQ ID NO: 10, and the antibody comprises i. the heavy chain knob sequence of SEQ ID NO:20; ii. the heavy chain hole sequence of SEQ ID NO:22, and iii. Light chain sequence of SEQ ID NO: 23 wherein the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker. An immunocytokine according to any one of embodiments 1 to 8.

[0343] Further aspects of the present invention are described in the following embodiments.

[0344] 1. An immunocytokine comprising: a. a conjugate comprising interleukin 15 (IL-15) or a derivative thereof and a polypeptide comprising the sushi domain of interleukin 15 receptor alpha (IL-15R) or a derivative thereof; b. Claudin18.2 antibody or a functional variant thereof, i. heterodimeric Fc domain, ii. altered effector functions, and / or iii. Increased in vivo half-life Claudin18.2 antibody or a functional variant thereof, Including, The conjugate is fused directly or indirectly to the C-terminus of both antibody heavy or light chains, or in the case of i. to the C-terminus of one antibody heavy chain. Immune cytokines.

[0345] 2. The modified effector function is enhanced antibody-dependent cellular cytotoxicity, and the antibody or a functional variant thereof is a. an IgG1 antibody or a functional variant thereof, comprising in one heavy chain: F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; Preferably comprising mutations selected from S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E, and further comprising D270E / K326D / A330M / K334E mutations in the other heavy chain, and / or b. a defucosylated IgG1, IgG2, or IgG4 antibody or a functional variant thereof; Numbering follows EU numbering The immunocytokine according to embodiment 1.

[0346] 3. The heterodimeric Fc domain is KiH, KiH S-S , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT S-S 3. The immunocytokine according to embodiment 1 or embodiment 2, selected from the group consisting of A107, SEED and A107, preferably KiH.

[0347] 4. An immunocytokine according to any one of embodiments 1 to 3, wherein the heterodimeric Fc domain results in a higher yield of the immunocytokine upon expression in cell culture compared to an immunocytokine having a homodimeric Fc domain.

[0348] 5. The immunocytokine according to any one of embodiments 1 to 4, wherein the half-life of said immunocytokine is increased, and wherein said antibody or functional variant thereof is an IgG1 or IgG4 antibody or functional variant thereof and comprises mutations selected from M252Y / S254T / T256E, M428L / N434S or T250Q / M428L, wherein the numbering is according to EU numbering.

[0349] 6. The immunocytokine according to any one of embodiments 1 to 5, wherein the conjugate is a fusion protein comprising, in the order of N-terminus to C-terminus, an IL-15Rαsushi domain or a derivative thereof, a linker, and the IL-15 or a derivative thereof, preferably, the IL-15Rαsushi domain comprises the sequence of SEQ ID NO: 5, the linker has a length of 18 to 22 amino acids and is preferably composed of glycine or serine and glycine, more preferably, has the sequence of SEQ ID NO: 7, and the IL-15 has the sequence of SEQ ID NO: 2.

[0350] 7. The immunocytokine according to any one of embodiments 1 to 6, wherein said conjugate comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and wherein said antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of Table 4, said IgG1 variant being heterodimeric via a KiH mutation in Table 3 and having enhanced ADCC activity via a DE, DLE, AAA, TL or IE mutation in Table 2, or via defucosylation, or via a combination of the above listed mutations and defucosylation.

[0351] 8. The immunocytokine according to any one of embodiments 1 to 7, wherein said conjugate comprises the sequence of SEQ ID NO: 10, and said antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, and said IgG1 variant is heterodimeric via a KiH mutation in Table 3 and has enhanced ADCC activity via a DE, DLE, AAA, TL or IE mutation in Table 2, or via defucosylation, or via a combination of the above listed mutations and defucosylation.

[0352] 9. The immunocytokine of any one of embodiments 1 to 7, wherein the conjugate comprises the sequence of SEQ ID NO: 10, and the antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, and wherein the IgG1 variant is heterodimeric via a KiH mutation in Table 3 and has enhanced ADCC activity via defucosylation.

[0353] 10. The immunocytokine of any one of embodiments 1 to 7, wherein the conjugate comprises the sequence of SEQ ID NO: 11 and the antibody variant is a heterodimeric IgG1 anti-CLDN18.2 antibody having a heavy chain knob sequence of SEQ ID NO: 84, a heavy chain hole sequence of SEQ ID NO: 87, and a light chain sequence of SEQ ID NO: 88.

[0354] 11. The immunocytokine of any one of embodiments 1 to 7, wherein the conjugate comprises the sequence of SEQ ID NO: 10 and the antibody variant is a heterodimeric IgG1 anti-CLDN18.2 antibody having a heavy chain knob sequence of SEQ ID NO: 84, a heavy chain hole sequence of SEQ ID NO: 87, and a light chain sequence of SEQ ID NO: 88.

[0355] 12. The immunocytokine of any one of embodiments 1 to 7, wherein said conjugate comprises the sequence of SEQ ID NO: 10, and said antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, said IgG1 variant being heterodimeric via KiH mutations in Table 3 and having ADCC enhancing mutations of S239D / I332E(DE) in the IgG1 Fc domain.

[0356] 13. The immunocytokine of any one of embodiments 1 to 7, wherein said conjugate comprises the sequence of SEQ ID NO: 11, and said antibody is an anti-CLDN18.2 heterodimeric IgG1 antibody variant having the VH and VL domain sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, said IgG1 variant being heterodimeric via KiH mutations in Table 3 and having ADCC enhancing mutations of S239D / I332E(DE) in the IgG1 Fc domain.

[0357] 14. Immunocytokines of sequences SEQ ID NO: 85 ("HC knob"), SEQ ID NO: 87 ("HC hole") and SEQ ID NO: 88 (LC).

[0358] 15. Immunocytokines of sequences SEQ ID NO: 86 ("HC knob"), SEQ ID NO: 87 ("HC hole") and SEQ ID NO: 88 (LC).

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Claims

1. An immune cytokine, comprising: a. A conjugate comprising a polypeptide containing the sushi domain of interleukin 15 (IL-15) or a derivative thereof and interleukin 15-receptor α (IL-15Rα) or a derivative thereof; The IL-15 derivative is a conjugate comprising at least one mutation that reduces binding to the IL-2 / IL-15Rβ and / or γ ｃ receptor, and b. An antibody or a functional variant thereof, comprising: i. A heterodimeric Fc domain; ii. Modified effector function, and / or iii. Increased in vivo half-life characterized antibody or functional variant thereof, and wherein the conjugate is fused directly or indirectly to the C-terminus of both antibody heavy chains or antibody light chains, or in the case of i., to the C-terminus of one antibody heavy chain. An immune cytokine.

2. The immune cytokine according to claim 1, wherein the immune cytokine comprises a functional variant of an antibody.

3. The modified effector function is reduced antibody-dependent cell cytotoxicity activity, and the antibody or a functional variant thereof is: a. An IgG1 antibody or a functional variant thereof, comprising a mutation selected from L234A / L235A, P329G, L234A / L235A / P329G, G236R / L328R, D265A, N297A, N297Q, N297G or L234A / L235A / G237A / P238S / H268A / A330S / P331S; b. An IgG4 antibody or a functional variant thereof, comprising a mutation selected from L235E, F234A / L235A, F234A / L235A / P329G, P329G, S228P / L235E, S228P / F234A / L235A or E233P / F234V / L235A / D265A / R409K; c. A hybrid of IgG2 (IgG2a or IgG2b) and IgG4 or a functional variant thereof, comprising the CH1 and hinge regions derived from IgG2, and the CH2 and CH3 regions are derived from IgG4 (IgG2 amino acids 118-260 and IgG4 amino acids 261-447); or d. An IgG2 antibody or a functional variant thereof, comprising a mutation selected from H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S, numbering according to EU numbering. The immune cytokine according to claim 1 or claim 2.

4. The antibody or a functional variant thereof is: ​ (a) An IgG4 antibody or a functional variant thereof, which contains the L235E mutation, or (b) An IgG1 antibody or a functional variant thereof, which contains the L234A / L235A mutation The immune cytokine according to claim 3.

5. The modified effector function is enhanced antibody-dependent cell cytotoxic activity, and the antibody or its functional variant is a. An IgG1 antibody or a functional variant thereof, in one heavy chain, contains a mutation selected from F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, K392T / P396L, V264I / I332E or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and in the other heavy chain, further contains the D270E / K326D / A330M / K334E mutation, and / or b. A defucosylated IgG1, IgG2 or IgG4 antibody or a functional variant thereof, The numbering follows EU numbering The immune cytokine according to claim 1.

6. The heterodimeric Fc domain is KiH, KiH Ｓ－Ｓ , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT Ｓ－Ｓ , the immune cytokine according to claim 1 or claim 2 selected from SEED and A107.

7. The immune cytokine according to claim 1 or claim 2, wherein the heterodimeric Fc domain results in a higher yield of the immune cytokine during expression in cell culture as compared to an immune cytokine having a homodimeric Fc domain.

8. The half-life of the immune cytokine is increased, and the antibody or its functional variant is an IgG1 or IgG4 antibody or a functional variant thereof, and contains a mutation selected from M252Y / S254T / T256E, M428L / N434S or T250Q / M428L. The numbering follows EU numbering The immune cytokine according to claim 1 or claim 2.

9. The antibody or its functional variant has reduced antibody-dependent cell cytotoxic activity, and the antibody or its functional variant is an IgG4 antibody or a functional variant thereof, and contains the L235E mutation and a KiH heterodimeric Fc domain. The immune cytokine according to claim 1 or claim 2.

10. The conjugate is a fusion protein that contains, in order from the N-terminus to the C-terminus, the IL-15Rα sushi domain or a derivative thereof, a linker, and the IL-15 or a derivative thereof. The linker has a length of 18 to 22 amino acids The immune cytokine according to claim 1 or claim 2.

11. The IL-15 derivative is a. at least one mutation that increases the homogeneity of the IL-15 derivative with respect to post-translational modification, and / or b. At least one mutation that reduces binding to said IL-2 / IL-15Rβ and / or said γ ｃ receptor, wherein The mutated amino acid is at least one mutation selected from N1, N4, S7, D8, K10, K11, D30, D61, E64, N65, L69, N72, E92, Q101, Q108, I111 of IL-15 (SEQ ID NO: 2) The immune cytokine according to claim 1 or claim 2, comprising

12. said IL-2 / IL-15Rβ and / or said γ ｃ The at least one mutation that reduces binding to the receptor is a substitution selected from N1D, N1A, N1G, N4D, S7Y, S7A, D8A, D8N, K10A, K11A, D30N, D61A, D61N, E64Q, N65D, N65A, N65E, N65R, N65K, L69R, N72R, Q101D, Q101E, Q108D, Q108A, Q108E and Q108R. The immune cytokine according to claim 11.

13. The antibody or its functional variant is a. binds to a tumor antigen, b. binds to a tumor extracellular matrix antigen, c. binds to an angiogenesis antigen, d. is an immunomodulatory antibody or its functional variant, The immunomodulatory antibody stimulates a costimulatory receptor or The immunomodulatory antibody inhibits an immunosuppressive receptor The immune cytokine according to claim 1 or claim 2.

14. The cytokine domain comprises the sequence of SEQ ID NO: 10, The antibody is i. the heavy chain knob sequence of SEQ ID NO: 20, ii. the heavy chain hole sequence of SEQ ID NO: 22 or SEQ ID NO: 101, and iii. the light chain sequence of SEQ ID NO: 16 comprising, and the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker The immune cytokine according to claim 1.

15. The cytokine domain comprises the sequence of SEQ ID NO: 10, The antibody is i. the heavy chain knob sequence of SEQ ID NO: 84 ii. the heavy chain hole sequence of SEQ ID NO: 87, and iii. the light chain sequence of SEQ ID NO: 88 comprising, and the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker The immune cytokine according to claim 1.

16. The cytokine domain comprises the sequence of SEQ ID NO: 10, The antibody is i. the heavy chain knob sequence of SEQ ID NO: 93, ii. the heavy chain hole sequence of SEQ ID NO: 95, and iii. the light chain sequence of SEQ ID NO: 92 comprising, and the cytokine domain is fused to the C-terminus of the heavy chain knob sequence without a linker The immune cytokine according to claim 1.

17. The cytokine domain comprises the sequence of SEQ ID NO: 10, The antibody is i. the heavy chain knob sequence of SEQ ID NO: 109, ii. the heavy chain hole sequence of SEQ ID NO: 110, and iii. the light chain sequence of SEQ ID NO: 88 comprising, wherein the cytokine domain is fused to the C-terminus of the heavy chain knob array without a linker The immune cytokine according to claim 1.

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