Antibodies having the ability to bind to CD27, variants thereof and uses thereof

Abstract

The present invention relates to an antibody having the ability to bind to human CD27 and variants thereof comprising an altered Fc region comprising one or more mutations that enhance the Fc-Fc interaction of the antibody. The present invention further provides pharmaceutical compositions comprising the antibody and uses of the antibody for therapeutic and diagnostic procedures, particularly in cancer treatment. TIFF2024533234000017.tif66170

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibodies having the ability of binding to CD27, and antibody variants thereof comprising one or more mutations in the Fc region, and uses of such antibodies and Fc variants. [Background technology]

[0002] 2. Background of the Invention CD27 (TNFRSF7) is a 55 kDa type I transmembrane protein member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) that costimulates T cell activation after binding to its ligand CD70. In humans, it is expressed on the cell membrane of T, B, and NK cells and their immediate precursors, all of which are part of the lymphoid lineage. On human T cells, CD27 is expressed on the plasma membrane of resting αβ CD4 + (Treg and conventional T cells), CD8 + It is expressed on T cells, stem cell memory cells, and central memory-like cells. On human B cells, CD27 is a memory B cell marker, and CD27 signaling promotes B cell differentiation into plasma cells.

[0003] The only known ligand for CD27 is the type II transmembrane protein CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L), which is expressed quite restrictively and only transiently on activated immune cells, including T, B, NK, and dendritic cells (DCs).

[0004] Upon binding of CD27 to CD70, a truncated 32 kDa form of CD27 can be released through the action of matrix metalloproteinases (known as soluble CD27, sCD27).

[0005] CD27 plays a role in the early generation of primary immune responses and is required for the generation and long-term maintenance of T cell immunity. CD27-CD70 binding leads to activation of the NF-KB and MAPK8 / JNK pathways. The adaptor proteins TRAF2 and TRAF5 have been shown to mediate signaling resulting from CD27 engagement.

[0006] To release their effector functions, T cells require T cell antigen receptor-mediated recognition of their cognate antigen in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs), and activation of costimulatory receptors. CD27 and CD28 are thought to be the most important costimulatory receptors expressed on T cells.

[0007] CD27 stimulation during the priming phase of T cell activation mediates antigen-specific CD4 T cell proliferation through IL-2-independent survival signaling in mice. + and CD8 + CD27 has also been found to promote clonal expansion of T cells (Carr JM et al, Proc Natl Acad Sci USA 2006 Dec 19;130(51):19454-9). CD27 also opposes apoptosis of activated T cells through successive divisions, and also acts on mouse CD8 + It has been shown to play an important role in memory differentiation of T cells (van de Ven K, Borst J. Immunotherapy 2015;7(6):655-67). As a result, CD27 stimulation promotes the generation of effector T cells in lymphoid organs and expands the repertoire of responder T cells. In human naive T cells, CD27 stimulation promotes the generation of CD4 + It promotes T helper-1 (Th1) differentiation of T cells and supports effector differentiation of cytotoxic T lymphocytes (Oosterwijk et al, Int Immunol. 2007 Jun;19(6):713-8).

[0008] Contrary to its presence on tumor cells in some hematological malignancies, CD27 expression has not been detected on tumor cells in solid malignancies. However, CD27-expressing lymphoid cells have been reported in the tumor microenvironment of both hematological malignancies and solid cancers.

[0009] In the treatment of cancer, the engagement and stimulation of immune response has been shown to induce and / or enhance antitumor immunity, resulting in clinical response, as exemplified by the clinical success of immune checkpoint inhibitors. Active immune response and / or existing antitumor immunity can be increased by providing costimulatory signaling, for example CD27 costimulatory signaling.

[0010] In mouse tumor models, T cell function and therefore antitumor immunity can be enhanced by agonistic CD27 antibodies. In a hCD27 transgenic lymphoma mouse model, CD27 activation using agonistic antibodies resulted in potent antitumor activity, as well as enhanced CD4 + and CD8 + It has been shown to induce protective immunity dependent on T cells (He LZ et al., J Immunol. 2013 Oct 15; 191(8): 4174-83). Furthermore, CD27 activation using monoclonal antibodies prevented tumor growth in mouse xenografts, including models derived from leukemia (Vitale et al, Keler T. Clin Cancer Res. 2012 Jul 15; 18(14): 3812-21), melanoma (Roberts DJ, et al., J Immunother. 2010 Oct; 33(8): 769-79), colon cancer, and thymoma (He LZ, et al., J Immunol. 2013 Oct 15; 191(8): 4174-83), among others.

[0011] Monoclonal IgG1 agonistic antibodies against human CD27 have been disclosed in the prior art.

[0012] WO2008 / 051424 relates to CD27 agonists, preferably agonistic CD27 antibodies, alone or in association with another moiety, such as an immunostimulant or immunomodulator, for the treatment of cancer, infectious diseases, inflammation, allergy and autoimmunity, and for enhancing the effectiveness of vaccines, but does not disclose the sequence of any CD27 antibody.

[0013] WO2012 / 004367 describes a humanized anti-human CD27 agonist antibody (referred to as hCD27.15). It is reported that hCD27.15 does not require cross-linking by FcyR expressing cells to activate CD27-mediated costimulation of immune response. However, this antibody does not bind to the frequently occurring SNP (A59T) in hCD27, and does not bind to cynomolgus monkey CD27.

[0014] WO2011 / 130434 discloses a human agonistic anti-human CD27 antibody called 1F5, which activates CD27 upon crosslinking with FcyR-expressing cells and is ligand (sCD70) blocking. 1F5 has been reported to have CDC and ADCC activity in target cells, as well as enhance immune responses, and have antitumor activity in mouse models.

[0015] WO2018 / 058022 discloses an agonistic mouse anti-human CD27 antibody 131A and its humanized version. It is disclosed that 131A binds to a frequently occurring SNP (A59T) in human CD27 and to cynomolgus monkey CD27. WO2018 / 058022 further discloses that antibody 131A had a stronger anti-tumor response in a mouse tumor model compared to antibody 1F5.

[0016] WO2019 / 195452 discloses a non-ligand blocking agonist anti-human CD27 antibody designated BMS-986215, which is reported to have a higher affinity for human and cynomolgus monkey CD27 than the CD27 antibody 1F5 described above. It is disclosed that CD27 costimulation of T cells by binding to its ligand CD70 occurs in the presence of BMS-986215. BMS-986215 inhibits CD4 activation by regulatory T cells (Tregs). + It is further disclosed that BMS-986215 reduces the suppression of responder T cells, and that BMS-986215 induces moderate ADCC and low levels of ADCP, CDC, and binds to C1q. It is further disclosed that BMS-986215 has only weak agonist activity in the absence of FcyR and in the absence of soluble CD70.

[0017] Anti-CD27 antibodies must induce CD27 clustering on the plasma membrane to induce CD27 agonism. In the case of wild-type IgG1 antibodies, CD27 clustering can be achieved through the interaction of membrane-bound CD27 antibodies with FcyR-bearing cells, such as monocytes, macrophages, B cells and other immune cells. As a consequence, anti-CD27 IgG1 molecules may be less efficient when the number of FcyR-expressing cells is limited. Additionally, FcyR engagement may also result in the activation of undesired effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which may cause undesired depletion of CD27-positive T cells.

[0018] Optimizing effector function by modifying the Fc region of an antibody can improve the effectiveness of therapeutic antibodies for treating cancer or other diseases, for example, improving the ability of the antibody to elicit an immune response against antigen-expressing cells. Such efforts are e.g. 2006,177(2):1129-1138;Moore,Chen et al.MAbs 2010 2(2):181-189;Desjarlais and Lazar,Exp Cell Res 2011,317(9):1278-1285;Kaneko and Niwa,BioDrugs 2011,25(1):1-11;Song,Myojo et al.,Antiviral Res 2014,111:60-68;Brezski and Georgiou, Curr Opin Immunol 2016,40:62-69; Sondermann and Szymkowski, Curr Opin Immunol 2016,40:78-87; Zhang, Armstrong et al. MAbs 2017,9(7):1129-1142.; Wang, Mathieu et al. Protein&Cell 2018,9(1):63-73; Beurskens FJ et al., Science. 2014 Mar 14;343(6176):1260-3).

[0019] However, despite these and other efforts in the art, there is a need for agonistic CD27 therapeutic antibodies that have increased agonism and / or increased potency, and / or are effective even when the number of FcyR-expressing cells is limited.Therefore, it is an object of the present invention to provide anti-CD27 antibodies that have high potency and agonism, and induce higher activation of T cell proliferation independent of the number of FcgR-expressing cells that provide secondary cross-linking for CD27 clustering on the cell membrane.Therefore, it is a further object to provide anti-CD27 antibodies that do not require cross-linking by FcyR-expressing cells to activate CD27-mediated costimulation of immune response.It is a further object to provide anti-CD27 antibodies that bind to human CD27, and further bind to the frequently occurring SNP (A59T) in hCD27, and also bind to cynomolgus CD27. It is a further object of the present invention to provide a CD27 agonist antibody that induces CD27 agonism through enhancing IgG hexamer formation in a manner independent of secondary cross-linking by C1q or independent of FcyR.In the context of cancer, such an antibody can increase anti-tumor immunity.There remains a need for anti-CD27 antibodies that exhibit strong agonist activity to enhance anti-tumor immune response. Summary of the Invention

[0020] The present invention relates to CD27 binding antibodies and Fc variants thereof.

[0021] Thus, in one aspect, the present invention relates to an antibody comprising at least one antigen-binding region capable of binding to human CD27, the antibody comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively.

[0022] In one aspect, the invention relates to an antibody comprising VH and VL regions comprising the sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively.

[0023] In one aspect, the invention relates to an antibody comprising a VH and VL region comprising the sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively, and further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

[0024] In one aspect, the present invention relates to an antibody comprising a VH and VL region comprising the sequences set forth in SEQ ID NO:4 and SEQ ID NO:8, respectively, and further comprising a light chain constant region (CL) and a heavy chain constant region (CH), wherein the antibody is of the human IgG1 isotype.

[0025] In one aspect, the invention relates to an antibody as described above having a modified Fc region, wherein the amino acid residue at a position corresponding to position E345 or E430 in a human IgG1 heavy chain according to Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0026] In one aspect, the invention relates to any of the aforementioned antibodies, further comprising an altered Fc region in which the amino acid residue at the position corresponding to position P329 in a human IgG1 heavy chain according to Eu numbering is R.

[0027] In one aspect, the invention relates to an antibody comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively, and further comprising a modified Fc region in which the amino acid residues at positions corresponding to positions E345 and P329 in a human IgG1 heavy chain according to Eu numbering are both R.

[0028] In one aspect, the invention relates to human or humanized antibodies.

[0029] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary artery disease comprising: a. a VH region comprising the amino acid sequence set forth in SEQ ID No:4; b. a VL region comprising the amino acid sequence set forth in SEQ ID No:8; c. a CH region comprising the amino acid sequence set forth in SEQ ID No:15; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. The present invention relates to an antibody comprising the compound

[0030] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary artery disease comprising: a. a VH region comprising the amino acid sequence set forth in SEQ ID No:4; b. a VL region comprising the amino acid sequence set forth in SEQ ID No:8; c. a CH region comprising the amino acid sequence set forth in SEQ ID No:15; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. The present invention relates to an antibody comprising the compound

[0031] In one aspect, the invention relates to an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:35 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0032] In one aspect, the invention relates to an isolated nucleic acid encoding an antibody according to any aspect or embodiment herein.

[0033] In one aspect, the invention relates to an expression vector comprising such a nucleic acid.

[0034] In one aspect, the invention relates to a recombinant host cell producing an antibody according to any aspect or embodiment herein.

[0035] In one aspect, the invention relates to a method for producing an antibody according to any aspect or embodiment herein, comprising culturing such a recombinant host cell in a culture medium and under conditions suitable for producing the antibody.

[0036] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody as defined in any aspect or embodiment herein, and a pharma- ceutically acceptable carrier.

[0037] In one aspect, the invention relates to an antibody according to any aspect or embodiment herein for use as a medicament.

[0038] In one aspect, the invention relates to an antibody according to any aspect or embodiment herein for use in the treatment or prevention of cancer.

[0039] In one aspect, the present invention relates to a method of treating a disease, comprising administering to a subject in need thereof an antibody according to any aspect or embodiment herein, a composition according to any aspect or embodiment herein, or a pharmaceutical composition according to any aspect or embodiment herein.

[0040] In one aspect, the invention relates to a kit-of-parts, e.g., a kit for use as a companion diagnostic / for use in identifying patients within a patient population who have a propensity to respond to treatment with an antibody according to any aspect or embodiment herein.

[0041] In one aspect, the present invention relates to an anti-idiotypic antibody that binds to an antigen-binding region having the ability to bind to CD27 as defined in any one of the aspects or embodiments herein. [Brief description of the drawings]

[0042] [Figure 1]Figure 1 shows the CD27 agonist activity of anti-CD27 antibodies and their hexamerization-enhanced Fc variants as determined in a CD27 Jurkat reporter bioassay. GloResponse NFκB-luc2 / CD27 Jurkat reporter cells used as thawed were incubated for 6 h with the indicated antibody concentration series (0.04 μg / mL, 0.30 μg / mL, 2.50 μg / mL, and 20 μg / mL from left to right). Luciferase activity was quantified by determining luminescence (RLU: relative light units) as a readout for CD27 intracellular signaling. As indicated, the following antibodies were included as WT IgG1 and / or variants with the E430G or E345R mutations: a non-binding anti-HIV-gp120 control antibody containing the E345R mutation (IgG1-b12-E345R, control), the anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, and IgG1-CD27-F, and the prior art anti-CD27 benchmark antibodies IgG1-CD27-131A and IgG1-CD27-15. [Diagram 2] Figure 2 shows binding of anti-CD27 antibodies to (A,B) human CD27 and (C,D) cynomolgus CD27 expressed on (A,C) T cells in PBMCs or (B,D) on CD27-transfected HEK293F cells as determined by flow cytometry. Antibody binding is presented as median fluorescence intensity (MFI). Anti-HIV-gp120 antibody IgG1-b12-FEAR (control) was included as a non-binding negative control antibody. [Diagram 3] Figure 3 shows the binding of anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, and IgG1-CD27-C to the human CD27-A59T variant expressed on HEK293F cells as determined by flow cytometry. Antibody binding is presented as median MFI. Anti-HIV-gp120 antibody IgG1-b12-FEAL (control) was included as a non-binding negative control antibody. [Figure 4A-1]Figure 4 shows heat maps of proliferation of TCR-stimulated (A) CD8+ and (B) CD4+ T cells in the presence of 1 μg / mL of CD27-specific antibody variants IgG1-CD27-A, -B, or -C harboring Fc mutations E430R or E345R in combination with Fc mutations P329R, G237A, or K326A-E33A, as determined by flow cytometry in a CSFE dilution assay. PMBCs from four human healthy donors were used as a source of T cells. T cell proliferation was expressed as a percentage of T cell mitotic index or proliferation of T cells, calculated by gating on cells that underwent CFSE dilution (CFSE low peaks) by using FlowJo software. [Figure 4A-2] See description of Figure 4-1. [Figure 4B-1] See description of Figure 4-1. [Figure 4B-2] See description of Figure 4-1. [Diagram 5] FIG. 5 shows (A-D) percentage of expanded T cells, (E,F) expansion proliferation index of (A,B) unstimulated or (C-F) TCR stimulated, (A,C,E) CD4+ or (B,D,F) CD8+ T cells after incubation of human healthy donor PBMCs with IgG1-CD27-A, IgG1-CD27-A-P329R-E345R, or prior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 as determined by flow cytometry. Anti-HIV-gp120 antibody variant IgG1-b12-E345R-P329R (control) was included as a non-binding negative control antibody. Cell proliferation % was calculated by gating on cells that underwent CFSE dilution (CFSElow peaks). The expansion proliferation index identified the fold increase in cells in a well and was calculated using the Proliferation Modeling tool in FlowJo version 10. Manual adjustments to the peaks were made when necessary to more consistently define the number of peaks present. [Figure 6]Figure 6 shows the binding of C1q to membrane-bound CD27 antibodies of the invention as determined by FACS. IgG1-CD27-A variants containing E430G or E345R hexamerization enhancing mutations (IgG1-CD27-A-E430G and IgG1-CD27-A-E345R) as well as P329R mutation (IgG1-CD27-A-P329R-E345R) were tested for their ability to bind C1q. Anti-HIV-gp120 antibody IgG1-b12-F405L (control) was included as a non-binding negative control antibody. [Figure 7] Figure 7 shows the binding of IgG1-CD27-A-P329R-E345R to human Fc receptors as determined by surface plasmon resonance (SPR). Biacore surface chips were covalently linked to anti-His antibodies and coated with recombinant His-tagged Fc receptors (A) FcγRIa, (B) FcγRIIa-H, (C) FcγRIIa-R, (D) FcγRIIb, (E) FcγRIIIa-F, or (F) FcγRIIIa-V. Anti-HIV-gp120 antibody IgG1-b12 (control) was included as a reference. Shown are absolute resonance units as determined by Biacore SPR after background subtraction (no Fc receptor flow cell). [Figure 8] Figure 8 shows binding of IgG1-CD27-A-P329R-E345R to human (A) CD4+ and (B) CD8+ T cell subsets in human healthy donor PBMC samples as determined by flow cytometry. Negative control antibody IgG1-b12-P329R-E345R (control) is an anti-HIV gp120 non-binding isotype control antibody containing the P329R and E345R mutations. Data presented are the mean MFI+ / -SD of duplicate samples. [Figure 9-1]FIG. 9 shows the CD27 agonist activity of anti-CD27 antibodies in the presence and absence of FcγR-mediated cross-linking as determined in a reporter assay. Fixed numbers of NFκB-luc2 / CD27 Jurkat reporter cells were cultured with (A–E) IgG1-CD27-A-P329R-E345R or IgG1-CD27-A, (F–J) IgG1-CD27-131A, IgG1-CD27-CDX1127 or IgG1-CD27-BMS986215 in the (A,F) absence or (B–J) presence of FcγRIIb-CHO-K1 cells at NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratios of (B,G) 1:1, (C,H) 1:1 / 3, (D,I) 1:1 / 9, or (E,J) 1:1 / 27. IgG1-b12-P329R-E345R and IgG1-b12 are anti-HIV gp120 non-binding control antibodies (control). Luminescence was measured as a readout for CD27 activation and presented as relative luminescence units (RLU). [Figure 9-2] See description of Figure 9-1. [Figure 10] Figure 10 shows human IgG levels in plasma of SCID mice after intravenous injection of 25 mg / kg of IgG-CD27-A or IgG-CD27-A-P329R-E345R antibodies. Total human IgG plasma concentrations were determined by sandwich ELISA and plotted against time after injection. Data shown are the mean plasma concentrations + / - SEM of blood samples per group (n=3 mice). [Figure 11] Figure 11 shows the percentage of viable CD27+ Daudi cells after 4h of co-culture (E:T=2:1) ​​with hMDM in the presence of IgG1-CD27-A-P329R-E345R or wild-type CD20 antibody IgG1-CD20. Daudi cells were labeled with CellTrace™ Violet and cell viability was measured by flow cytometry. Data shown are the mean ± SD of duplicates of the percentage of viable Daudi cells (TO-PRO-3-CTV+CD11b-) normalized to the no-antibody control for one of four donors tested in two experiments. [Figure 12]Figure 12 shows C4d deposition upon incubation of IgG1-CD27-A-P329R-E345R in NHS as determined by ELISA. IgG1-b12-P329R-E345R is an isotype control antibody, IgG1-b12 is a control antibody with WT Fc domain; IgG1-b12-RGY is a positive control antibody for C4d deposition (hexameric antibody in solution). Shown are the mean ± SD of triplicates of one representative experiment out of three performed. [Figure 13] Figure 13 shows the inhibition of CD70 binding on Daudi cells by anti-CD27 antibodies. CD27+ Daudi cells were incubated with 6 μg / mL of biotinylated recombinant human CD70 ECD in the presence or absence of 50 μg / mL of non-binding control antibodies (IgG1-b12-P329E-E345R or IgG1-b12) or CD27 antibodies (IgG1-CD27-A, IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127, IgG1-CD27-BMS986215, or IgG1-CD27-131A). Binding of biotinylated CD70 fragments to Daudi cells was detected by flow cytometry using BV421-labeled streptavidin. Data shown are gMFI ± SD from duplicate wells of one representative experiment out of three performed. [Figure 14A-1]FIG. 14 shows the expression levels of T cell activation markers in polyclonally activated CD4+ and CD8+ T cells upon treatment with anti-CD27 antibody. Human healthy donor PBMCs were incubated with 0.1 μg / mL of CD3 antibody and 30 μg / mL of IgG1-CD27-A-P329R-E345R, CD27 antibody benchmark or non-binding control antibody IgG1-b12-P329R-E345R for 2 or 5 days. Expression levels of T cell activation markers HLA-DR, CD69, GITR, CD25, CD107a, and 4-1BB on the surface of (A) CD4+ and (B) CD8+ T cells in antibody-treated samples were quantified by flow cytometry and presented as the mean fold change in MFI (±SD) compared to non-binding control samples of the same donor. The dotted line indicates the fold change for cells treated with IgG1-b12-P329R-E345R, which was used as a non-binding control and set to 1. Data shown are from three donors tested in duplicate in one experiment. [Figure 14A-2] See description of Figure 14A-1. [Figure 14B-1] See description of Figure 14A-1. [Figure 14B-2] See description of Figure 14A-1. [Figure 15] Figure 15 shows the percentage of OVA-specific CD8+ T cells in the spleens of hCD27-KI mice after immunization with OVA and treatment with anti-CD27 antibodies. hCD27-KI mice were injected with 5 mg OVA sc on days 0, 12 and 21 and simultaneously treated with 30 mg / kg IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127 or non-binding control antibody IgG1-b12-P329R-E345R iv. On day 28, mice were euthanized and spleens were excised and processed as single cell suspensions. Expansion of OVA-specific CD8+ T cells was assessed by flow cytometry. Data shown are the mean ± SD of OVA+% of CD8+ cells per treatment group (5 mice / group) from one experiment performed. [Figure 16]Figure 16 shows the number of IFNγ-producing splenocytes at day 28 after immunization with OVA and treatment with anti-CD27 antibody, measured by IFNγ-ELISpot. hCD27-KI mice were injected sc with 5 mg OVA on days 0, 12 and 21 and simultaneously treated iv with 30 mg / kg IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127, or the non-binding control antibody IgG1-b12-P329R-E345R. On day 28, spleens were excised, processed as single cell suspensions, and IFNγ-ELISpot was used to detect IFNγ-producing splenocytes. Data shown are the mean ± SEM of the number of spots per well for each treatment group (5 mice / group) from one experiment performed. [Figure 17] Figure 17 shows the percentage of activated CD8+ T cells in the spleens of hCD27-KI mice after immunization with OVA and treatment with anti-CD27 antibodies. hCD27-KI mice were injected with 5 mg OVA sc on days 0, 12 and 21 and simultaneously treated with 30 mg / kg IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127 or non-binding control antibody IgG1-b12-P329R-E345R iv. On day 28, mice were euthanized and spleens were excised and processed as single cell suspensions. CD8+ T cell activation was assessed in spleen samples by measuring the PD-1+ percentage of CD8+ cells in the spleen by flow cytometry. Data shown are the mean ± SD per treatment group (5 mice / group) from one experiment performed. [Figure 18]Figure 18 shows the percentage of effector CD8+ T cells in the spleens of hCD27-KI mice after immunization with OVA and treatment with anti-CD27 antibodies. hCD27-KI mice were injected with 5 mg OVA sc on days 0, 12 and 21 and simultaneously treated with 30 mg / kg IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127 or non-binding control antibody IgG1-b12-P329R-E345R iv. On day 28, mice were euthanized and spleens were excised and processed as single cell suspensions. Memory T cell expansion was assessed by CD44 and CD62L expression by flow cytometry. Data shown are the mean ± SD per treatment group (5 mice / group) from one experiment performed. (A) Percentage of CD8+CD44+CD62L- effector memory among CD45+ cells. (B) Percentage of CD44+CD62L- effector memory among CD8+ T cells. (C) Percentage of CD8+CD44-CD62L- pre-effector among CD45+ cells. (D) Percentage of CD44-CD62L- pre-effector among CD8+ T cells. [Figure 19] Figure 19 shows the percentage of T cells in the spleens of hCD27-KI mice after immunization with OVA and treatment with anti-CD27 antibodies. hCD27-KI mice were injected with 5 mg OVA sc on days 0, 12 and 21 and simultaneously treated with 30 mg / kg IgG1-CD27-A-P329R-E345R, IgG1-CD27-CDX1127 or non-binding control antibody IgG1-b12-P329R-E345R iv. On day 28, mice were euthanized and spleens were excised and processed as single cell suspensions. CD3+ cells in blood and spleens were assessed by flow cytometry. Data shown are the mean ± SD per treatment group (5 mice / group) from one experiment performed. [Figure 20]FIG. 20 shows the effect of IgG1-CD27-A-P329R-E345R on T cell cytokine production in an antigen-specific study. Co-cultures of CLDN6-TCR-expressing CD8+ T cells and autologous CLDN6-expressing iDCs expressing (A) endogenous PD-1 or overexpressing (B) PD-1 were incubated with 10 μg / mL of IgG1-CD27-A-P329R-E345R, CD27 benchmark antibody IgG1-CD27-131A, or non-binding control antibody IgG1-b12-P329R-E345R for 2 days. Cytokine levels in the co-culture supernatants were analyzed by multiplex ECLIA. Data shown are the mean ± SD of concentrations of triplicate wells from one representative donor out of seven donors tested in two experiments performed. Abbreviations: CLDN6 = claudin 6; ECLIA = electrochemiluminescence assay; iDC = immature dendritic cells; PD-1 = programmed cell death protein 1; SD = standard deviation; TCR = T cell receptor. [Figure 21] Figure 21 shows the expression of cytotoxicity-related molecules in antigen-specific CD8+ T cells incubated with IgG1-CD27-A-P329R-E345R. CLDN6-TCR electroporated CD8+ T cells were co-cultured with hCLDN6-MDA-MB-231 cells for 2 days in the presence of IgG1-CD27-A-P329R-E345R, CD27 benchmark IgG1-CD27-131A, or non-binding control antibody IgG1-b12-P329R-E345R. Intracellular expression of GzmB and CD107a was determined by flow cytometry. The expression levels of GzmB and CD107a in CD8+ T cells (MFI normalized to IgG1-b12-P329R-E345R) are shown in addition to the percentage of CD8+ T cells expressing both GzmB and CD107a. Data shown are the mean ± SD of six donors tested in a single replicate of two experimental runs. **, P<0.01; *, P<0.05; Friedman test with Dunn's multiple comparison test. Abbreviations: CLDN6 = claudin 6; GzmB = granzyme B; MFI = mean fluorescence intensity; SD = standard deviation; TCR = T cell receptor. [Figure 22]FIG. 22 shows antigen-specific CD8+ T cell-mediated tumor cell killing in the presence of IgG1-CD27-A-P329R-E345R. CD8+ T cell-mediated killing of hCLDN6-MDA-MB-231 cells was assessed by real-time cell analysis. CLDN6 TCR electroporated CD8+ T cells were co-cultured with hCLDN6-MDA-MB-231 cells for 5 days in the presence of IgG1-CD27-A-P329R-E345R, CD27 benchmark IgG1-CD27-131A, or non-binding control antibody IgG1-b12-P329R-E345R. Cell index values ​​were derived from impedance measurements performed at 2-hour intervals. AUC was obtained from cell index data over the 5-day co-culture. The AUC of each treatment condition was normalized to IgG1-b12-P329R-E345R-treated cultures from the same donor. Data shown are the mean ± SD from six donors tested in duplicate in two experiments. **, P<0.01; Friedman test with Dunn's multiple comparison test. Abbreviations: AUC = area under the curve; CLDN6 = claudin 6; SD = standard deviation; TCR = T cell receptor. [Figure 23] Figure 23 shows absolute cell counts of CD4+ and CD8+ T cells as well as NK cells in primary tumor cultures after treatment with IgG1-CD27-A-P329R-E345R. Human NSCLC tumor tissues were cultured with low dose IL-2 (45-50 U / mL) in the presence or absence of 10 μg / mL of IgG1-CD27-A-P329R-E345R. Absolute cell counts of TIL subsets were determined by flow cytometry 14 days after treatment. Data shown are the mean ± SD of four replicate wells from one of five tumor tissues tested in one of four experiments performed. Abbreviations: IL = interleukin; NK = natural killer; NSCLC = non-small cell lung cancer; SD = standard deviation; U / mL = units / mL. [Figure 24]Figure 24 shows molecular proximity determined by bioluminescence resonance energy transfer (BRET) analysis between IgG1-CD27-A-P329R-E345R antibodies on the cell surface of Daudi and huCD27-K562 cells. Cells were incubated with a mixture of NanoLuc (donor) and HaloTag (acceptor) tagged antibodies (5 μg / mL each) as indicated: IgG1-CD27-A-P329R-E345R, WT IgG1-CD27-A or non-binding control IgG1-b12-P329R-E345R. The antibody pairs IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo were used as positive controls. BRET was calculated in milliBRET units (mBU) = (618 nmem / 460 nmem) x 1000 and corrected for donor bleed-through by subtracting the value of the no-ligand control. Data shown are corrected BRET from duplicate wells of one representative experiment out of three performed. [Diagram 25] Figure 25 shows the binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages compared to a WT IgG1 antibody with an unrelated antigen-binding region (IgG1-b12) as a positive control for FcγRIa binding, and a variant of the same antibody with P329R and E345R mutations (IgG1-b12-P329R-E345R). Binding of the antibody to macrophages was detected by flow cytometry using a PE-labeled goat anti-human secondary antibody. Data shown are the mean + SD of two donors tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description of the Invention definition The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, having the ability to specifically bind to an antigen. The antibody of the present invention comprises an Fc domain and an antigen-binding region of an immunoglobulin. An antibody generally contains two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least an Fc domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant or "Fc" region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, e.g., C1q, the first component in the classical pathway of complement activation. As used herein, unless inconsistent with the context, the Fc region of an immunoglobulin typically contains at least the CH2 and CH3 domains of an immunoglobulin CH, and may include a connecting region, e.g., a hinge region. The Fc region is typically in a dimerized form, for example, via a disulfide bridge connecting the two hinge regions and / or a non-covalent interaction between the two CH3 regions. The dimer may be a homodimer (the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (the amino acid sequences of the two Fc region monomers differ in one or more amino acids). As is well known in the art, Fc region fragments of full-length antibodies can be generated, for example, by digestion of full-length antibodies with papain. In addition to the Fc region and the antigen-binding region, an antibody as defined herein may further comprise one or both of the immunoglobulin CH1 and CL regions. The antibody may also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term "bispecific antibody" refers to an antibody that has specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. As indicated above, not otherwise stated,Or, unless the context clearly contradicts, the term antibody in this specification includes fragments of antibodies that contain at least a portion of the Fc region and retain the ability to specifically bind to an antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "Ab" or "antibody" are monovalent antibodies (described by Genmab in WO2007059782); heavy chain antibodies, which consist of only two heavy chains and occur naturally, for example in camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab, Roche, WO2011069104); strand-exchange engineered domain (SEED or Seed-body), which is an asymmetric and bispecific antibody-like molecule (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792); Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768); mAb-Fv (Xencor, WO2011 / 028952); Xmab (Xencor); Dual variable domain immunoglobulins (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain dual head antibodies (Unilever; Sanofi Aventis, WO20100226923); Di-diabody (ImClone / Eli Lilly); Knob-into-hole antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011 / 131746); bispecific IgG1 and IgG2 (Pfizer / Rinat, WO11143545); DuetMab (MedImmune, US2014 / 0348839); electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai,US201000155133;Oncomed, WO2010129304A2;Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545);CrossMAb (Roche, WO2011117329);LUZ-Y (Genentech);Biclonic (Merus, WO2013157953);Dual targeting domain antibodies (GSK / Domantis);Two-in-one antibodies or dual acting Fabs recognizing two targets (Genentech, NovImmune, Adimab);Crosslinked Mabs (Karmanos Cancer Center);Covalently fused mAbs (AIMM);CovX-body (CovX / Pfizer);FynomAb (Covagen / Janssen ilag);DutaMab(Dutalys / Roche);iMab(MedImmune);IgG-like bispecific(ImClone / Eli Lilly, Shen, J., et al.J Immunol Methods, 2007.318(1-2):p.65-74); TIG-body, DIG-body and PIG-body (Pharmabcine); dual affinity retargeting molecules (Fc-DART or Ig-DART, Macrogenics, WO / 2008 / 157379, WO / 2010 / 080538); BEAT (Glenmark); Zybodies (Zyngenia); approaches using common light chains (Crucell / Merus, US7262028) or common heavy chains (κλBodies by NovImmune, WO2012023053), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc region-like scFv fusion, e.g. BsAb by ZymoGenetics / BMS,HERCULES by Biogen Idec (US007951918); SCORPIONS (Emergent BioSolutions / Trubion and Zymogenetics / BMS); Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol,2009.393(3):p.672-92);scFv fusion (Genentech / Roche);scFv fusion (Novartis);scFv fusion (Immunomedics);scFv fusion (Changzhou Adam Biotech Inc, CN 102250246);TvAb (Roche, WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008 / 003116); and dual scFv fusions. The term antibody, unless otherwise specified, should be understood to include monoclonal antibodies (e.g., human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (e.g., bivalent monospecific antibodies), bispecific antibodies, antibodies of any isotype and / or allotype; antibody mixtures (recombinant polyclonal), such as those produced by the technology utilized by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, and fusion proteins as described in WO2014 / 031646. Although these different antibody fragments and formats are generally included within the meaning of antibody, they are unique features of the present invention collectively and each independently and exhibit different biological properties and utility.

[0044] An "agonist antibody" for a natural receptor is a compound that binds to the receptor to form a receptor-antibody complex and activates the receptor, thereby initiating pathway signaling and further biological processes.

[0045] The terms "agonism" and "agonistic" are used interchangeably herein to refer to or describe an antibody that has the ability to substantially induce, promote, or enhance, directly or indirectly, the biological activity or activation of CD27. Optionally, an "agonistic CD27 antibody" is an antibody that has the ability to activate the CD27 receptor by a mechanism similar to that of the ligand of CD27, known as CD70 (tumor necrosis factor superfamily member 7, TNFSF7; CD27 ligand, CD27L), resulting in the activation of one or more intracellular signaling pathways, which may include activation of the NF-KB and MAPK8 / JNK pathways. "Agonism" as defined herein may be determined according to Example 2 herein.

[0046] A "CD27 antibody" or "anti-CD27 antibody" as described herein is an antibody that specifically binds to the protein CD27, in particular human CD27.

[0047] "Variant" as used herein refers to a protein or polypeptide sequence that differs from a parent or reference sequence in one or more amino acid residues. A variant may, for example, have at least 80%, for example 90%, or 95%, or 97%, or 98%, or 99% sequence identity to a parent or reference sequence. Additionally or alternatively, a variant may differ from a parent or reference sequence by 12 or less, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, for example substitution, insertion, or deletion of amino acid residues. Thus, as used interchangeably herein, "variant antibody" or "antibody variant" refers to an antibody that differs in one or more amino acid residues compared to a parent or reference antibody, for example in the antigen binding region, Fc region, or both. Similarly, a "variant Fc region" or "Fc region variant" refers to an Fc region that differs in one or more amino acid residues compared to a parent or reference Fc region, optionally differing from the parent or reference Fc region amino acid sequence by 12 or fewer, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, e.g., substitution, insertion, or deletion of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody, which may be of a particular isotype depending on the context. The variant Fc region may be in a dimerized form, homodimer or heterodimer, e.g., one of the amino acid sequences of the dimerized Fc region contains a mutation and the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference sequence) IgG CH and variant IgG constant region amino acid sequences, including Fc region amino acid sequences, are shown in Table 3.

[0048] The term "immunoglobulin heavy chain" or "heavy chain of immunoglobulin" as used herein is intended to refer to one of the heavy chains of immunoglobulins. Heavy chains typically include a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the immunoglobulin isotype. The heavy chain constant region typically includes three domains, CH1, CH2, and CH3. The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins that consists of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, for example, Fundamental Immunology Ch. 7 Paul, W., 2nd ed. Raven Press, NY 1989). Within the structure of an immunoglobulin, two heavy chains are interconnected through disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain typically comprises several regions; a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically comprises one domain, CL. Furthermore, the VH and VL regions can be further divided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or shape of structurally defined loops), also referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences herein are defined according to IMGT (see Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508 (2008)).

[0049] As used herein, the terms "half molecule", "Fab arm" and "arm" refer to one heavy-light chain pair. When a bispecific antibody is described as comprising a half molecule antibody "derived from" a first antibody and a half molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was generated by recombining the half molecules from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, "recombining" is not intended to be limited by any particular method of recombination, and thus includes all of the methods of producing bispecific antibodies described herein below, including recombination at the nucleic acid level and / or through co-expression of two half molecules in the same cell, in addition to recombination by "half molecule exchange", also described in the art as "Fab arm exchange" and the DuoBody® method.

[0050] The term "antigen-binding region" or "binding region" or antigen-binding domain, as used herein, refers to the region of an antibody that has the ability to bind to an antigen. This binding region is typically defined by the VH and VL domains of an antibody, which can be further divided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or shape of structurally defined loops), also referred to as complementarity determining regions (CDRs), interspersed with more conserved regions, referred to as framework regions (FRs). The antigen can be any molecule, e.g., a polypeptide, present, for example, on a cell, bacteria, or virion. The terms "antigen-binding region" and "antigen-binding site" and "antigen-binding domain" may be used interchangeably in the context of the present invention, unless the context is inconsistent.

[0051] The terms "antigen" and "target" may be used interchangeably in the context of the present invention, unless the context is contradictory.

[0052] The term "binding" as used herein refers to a binding that is typically greater than 1E when determined by biolayer interferometry using an antibody as the ligand and an antigen as the analyte. 6 M or less, e.g. 5E 7 M or less, 1E 7 M or less, e.g. 5E 8 M or less, e.g. 1E 8 M or less, e.g. 5E 9 M or less, or for example 1E 9 M or less K D and which is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower, e.g., at least 100,000-fold lower, than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D The antibody binds to a predetermined antigen with an affinity corresponding to

[0053] The term “K D " (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, k d k a It is obtained by dividing by

[0054] The term "k" d " (sec -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also referred to as k off This is referred to as the off-value or off-rate.

[0055] The term "k" a " " -1 ×sec -1 ) as used herein refers to the association rate constant of a particular antibody-antigen interaction. The value is also referred to as k on This is referred to as the on-rate or on-value.

[0056] The term "CD27" as used herein refers to a human protein designated CD27, also known as tumor necrosis factor receptor superfamily member 7 (TNFRSF7). In the amino acid sequence shown in SEQ ID NO:1 (Uniprot ID P26842), amino acid residues 1-19 are the signal peptide and amino acid residues 20-240 are the mature polypeptide. Unless contradicted by the context, CD27 may also refer to variants of CD27, its isoforms and orthologs. A naturally occurring variant of human CD27 containing the A59T mutation is shown in SEQ ID NO:2.

[0057] In the cynomolgus monkey (Macaca fascicularis), the CD27 protein has the amino acid sequence shown in SEQ ID NO:3 (Genbank XP_005569963). No signal peptide is defined in the 240 amino acid sequence shown in SEQ ID NO:3.

[0058] The term "antibody binding region" refers to the region of an antigen that contains the epitope to which the antibody binds. The antibody binding region may be determined by epitope binding using biolayer interferometry, by alanine scanning, or by shuffle assays (using an antigen construct in which a region of the antigen has been exchanged with that of another species and determining whether the antibody still binds to the antigen). Amino acids within the antibody binding region involved in interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and by crystallography of the antibody bound to the antigen.

[0059] The term "epitope" refers to an antigenic determinant that is specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains or combinations thereof and usually have specific charge characteristics as well as specific three-dimensional structural features. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents while the binding to the latter is not. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by an antibody when it is bound to an antigen (in other words, amino acid residues that are within or closely adjacent to the footprint of a unique antibody).

[0060] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", or "mAb", as used herein, refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse or rat, whose genome includes a human heavy chain transgene and a light chain transgene, fused to an immortalized cell. Monoclonal antibodies may also be produced from recombinantly engineered host cells or from systems using cell extracts that support in vitro transcription and / or translation of nucleic acid sequences encoding the antibody.

[0061] The term "isotype" as used herein refers to an immunoglobulin class (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotypes thereof, such as IgG1m(za) and IgG1m(f), encoded by heavy chain constant region genes. Furthermore, each heavy chain isotype can be paired with either a kappa (κ) or lambda (λ) light chain.

[0062] The term "full-length antibody" as used herein indicates that the antibody contains all of the domains of a particular isotype that are normally found for that isotype in nature, not fragments, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody. In a full-length variant antibody, the heavy and light chain constant and variable domains may contain amino acid substitutions that improve the functional properties of the antibody when compared to the full-length parent or wild-type antibody. A full-length antibody according to the invention may be produced by a method comprising (i) cloning the CDR sequences into a suitable vector containing the complete heavy and light chain sequences, and (ii) expressing the complete heavy and light chain sequences in a suitable expression system. It is within the knowledge of the skilled artisan to produce a full-length antibody when starting from either the CDR sequences or the entire variable region sequences. Thus, the skilled artisan knows how to generate a full-length antibody according to the invention.

[0063] The term "human antibody" as used herein is intended to include antibodies that contain variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibody of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as mice, have been grafted onto human framework sequences.

[0064] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen-binding site onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region may be required. Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain a humanized antibody with favorable characteristics, such as affinity and biochemical properties.

[0065] The terms "Fc region" or "Fc domain" as used herein may be used interchangeably and refer to a region of the heavy chain constant region that includes, from the N-terminus to the C-terminus of an antibody, at least the hinge, CH2 and CH3 regions. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0066] The term "parent polypeptide" or "parent antibody" should be understood as a polypeptide or antibody that is identical to the polypeptide or antibody of the present invention, except that the parent polypeptide or antibody does not have the mutation, unless otherwise stated or clearly contradicted by the context. For example, the antibody IgG1-CD27-A of the present invention is a parent antibody of IgG1-CD27-A-P329R-E345R.

[0067] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering (Eu index) as set forth in Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689 (1991). However, the hinge region may also be of any of the other subtypes described herein.

[0068] The term "CH1 region" or "CH1 domain" as used herein refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering set forth in Kabat, supra. However, the CH1 region may also be of any of the other subtypes described herein.

[0069] The term "CH2 region" or "CH2 domain" as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering set forth in Kabat, supra. However, the CH2 region may also be of any of the other subtypes described herein.

[0070] The term "CH3 region" or "CH3 domain" as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering set forth in Kabat, supra. However, the CH3 region may also be of any of the other subtypes described herein.

[0071] The terms "Fc-mediated effector function" or "Fc effector function", as used herein, are used interchangeably and are intended to refer to a function that is a consequence of binding of a polypeptide or antibody to a target or antigen on a cell membrane, where the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) complement dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc gamma receptor (FcγR) binding, (vi) antibody-dependent, FcγR-mediated antigen cross-linking, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement dependent cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x) antibody-mediated binding of opsonized antibodies to complement receptors, (xi) opsonization, and (xii) any combination of (i)-(xi).

[0072] The terms "reduced Fc effector function" or "reduced Fc-mediated effector function," as used herein, are used interchangeably and are intended to refer to an Fc effector function that is reduced for an antibody when directly compared to the Fc effector function of a parent polypeptide or antibody in the same assay.

[0073] The term "inertness", "inert" or "non-activating" as used herein refers to an Fc region that at least cannot bind to any FcγR, cannot induce Fc-mediated cross-linking of FcγR, cannot induce FcγR-mediated cross-linking of target antigens through two Fc regions of individual antibodies, or cannot bind to C1q.Therefore, in certain embodiments of the present invention, the Fc region is inactive.Therefore, in certain embodiments, some or all of the Fc-mediated effector functions are attenuated or completely absent.

[0074] The term "oligomerization" as used herein is intended to refer to the process of converting monomers to a finite degree of polymerization. The antibodies according to the invention can form oligomers, e.g., hexamers, via non-covalent association of Fc regions after target binding, e.g., at the cell surface. Oligomerization of anti-CD27 antibodies upon cell surface binding through Fc:Fc interactions may increase CD27 clustering, which results in activation of CD27 intracellular signaling. The ability of antibodies comprising E345R or E430G mutations to form oligomers, e.g., hexamers, upon cell surface binding can be assessed as described in de Jong RN et al, PLoS Biol. 2016 Jan 6; 14(1): e1002344. Fc-Fc-mediated oligomerization of antibodies occurs after target binding on the (cell) surface through intermolecular association of Fc regions between adjacent antibodies, and is increased by the introduction of E345R or E430G mutations (numbering according to the Eu index).

[0075] The term "clustering," as used herein, refers to the oligomerization of antibodies through non-covalent interactions.

[0076] The term "Fc-Fc enhancing" as used herein is intended to refer to increasing the binding strength between the Fc regions of two Fc region-containing antibodies or stabilizing the interaction between the Fc regions such that the antibodies form oligomers, e.g., hexamers, on the cell surface. This enhancement can be obtained by certain amino acid mutations in the Fc region of the antibody, e.g., E345R or E430G. The term "monovalent antibody" in the context of the present invention refers to an antibody molecule that can interact with a specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm). In the context of a bispecific antibody, "monovalent antibody binding" refers to the binding of the bispecific antibody to one specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

[0077] The term "monospecific antibody" in the context of the present invention refers to an antibody that has binding specificity for only one epitope. The antibody may be a monospecific, monovalent antibody (i.e., having only one antigen-binding region) or a monospecific, bivalent antibody (i.e., having two identical antigen-binding regions).

[0078] The term "bispecific antibody" refers to an antibody that comprises two non-identical antigen-binding domains, such as two non-identical Fab arms or two Fab arms with non-identical CDR regions. In the context of the present invention, a bispecific antibody has specificity for at least two different epitopes. Such epitopes may be on the same or different antigens or targets. When the epitopes are on different antigens, such antigens may be on the same cell or different cells, cell types or structures, such as extracellular matrix or vesicles and soluble proteins. A bispecific antibody may therefore have the ability to crosslink multiple antigens, such as two different cells. A particular bispecific antibody of the present invention has the ability to bind to CD27 and a second target.

[0079] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions that bind to epitopes on one or two targets or antigens, or that bind to one or two epitopes on the same antigen. Thus, a bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0080] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds that contain an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) that is specific to each amino acid. In the context of the present invention, amino acids may be classified based on structure and chemical characteristics. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0081] Table 1. Major classifications based on the structure and general chemical characterization of the R group TIFF2024533234000002.tif49128

[0082] Table 2. Alternative physical and functional classifications of amino acid residues. TIFF2024533234000003.tif83128

[0083] The substitution of one amino acid for another amino acid may be classified as conservative or non-conservative substitution.In the context of the present invention, "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, for example, the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the above two tables: for example, leucine may be substituted with isoleucine, because they are both aliphatic, branched, hydrophobic substances.Similarly, aspartic acid may be substituted with glutamic acid, because they are both small, negatively charged residues.

[0084] In the context of the present invention, substitutions in an antibody include Original amino acid - position - substituted amino acid Pointed out as; Reference is made to the well-recognized notation, three-letter code, or one-letter code for amino acids, including the code "Xaa" or "X" to indicate any amino acid residue. Thus, Xaa or X can typically represent any of the 20 naturally occurring amino acids. The term "naturally occurring" as used herein refers to any one of the following amino acid residues: glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the notation "K409R" or "Lys409Arg" means that the antibody comprises a substitution of lysine with arginine at amino acid position 409.

[0085] Substitution of an amino acid at a given position with any other amino acid is the original amino acid-position; or for example "K409" It is called.

[0086] For modifications in which the original and / or substituted amino acids may include more than one, but not all, amino acids, the more than one amino acids may be separated by "," or " / ". For example, substitution of lysine at position 409 with arginine, alanine, or phenylalanine is "Lys409Arg,Ala,Phe" or "Lys409Arg / Ala / Phe" or "K409R,A,F" or "K409R / A / F" or "K409→R, A, or F".

[0087] Such designations may be used interchangeably in the context of the present invention and may have the same meaning and purpose.

[0088] Moreover, the term "substitution" encompasses substitutions to any one or the other of the 19 natural amino acids or to other amino acids, e.g., unnatural amino acids. For example, substitution of the amino acid K at position 409 includes each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. Note that this is equivalent to the designation 409X, where X designates any amino acid other than the original amino acid. These substitutions may also be designated K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. The same applies to each and every position mentioned herein, and any such substitutions are specifically included herein.

[0089] Antibodies according to the invention may also include deletions of amino acid residues. Such deletions may be designated as "del", and include, for example, the designation K409del. Thus, in such embodiments, the lysine at position 409 has been deleted from the amino acid sequence.

[0090] The term "host cell" as used herein is intended to refer to a cell into which an expression vector is introduced. It should be understood that such a term is intended to refer not only to a specific target cell, but also to the descendants of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such descendants may not actually be identical to the parent cell, but still fall within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytic cells, as well as prokaryotic cells, such as E. coli, and other eukaryotic hosts, such as plant cells and fungi.

[0091] The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing an antibody or a target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293 cells, Expi293F cells, plant cells, or fungi, including yeast cells.

[0092] For the purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled as "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (identical residues × 100) / (length of alignment − total number of gaps in alignment) It is calculated as follows:

[0093] Retention of similar residues may additionally or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through NCBI using standard settings BLOSUM62, open gap = 11 and extension gap = 1). Suitable variants typically exhibit at least about 45%, e.g., at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or higher (e.g., about 99%) similarity to the parent sequence.

[0094] The term "internalized" or "internalization," as used herein, refers to the biological process by which a molecule, such as an antibody according to the present invention, is engulfed by the cell membrane and directed to the interior of the cell. Internalization may also be referred to as "endocytosis."

[0095] As used herein, the term "effector cell" refers to immune cells that participate in the effector phase of immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcgR) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, have the ability to induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells that express FcgR are involved in the specific killing of target cells and / or the presentation of antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments, ADCC can be further enhanced by antibody-driven classical complement activation, resulting in the deposition of activated C3 fragments on target cells. C3 cleavage products are ligands for complement receptors (CRs), such as CR3, expressed on myeloid cells. Recognition of complement fragments by CRs on effector cells can promote the enhancement of Fc receptor-mediated ADCC. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells. These C3 cleavage products can promote direct complement-dependent cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells, which depends on antibody binding and can be mediated by FcγRs expressed by effector cells. The expression of certain FcRs or complement receptors on effector cells can be regulated by humoral factors, such as cytokines. For example, expression of FcγRI has been found to be upregulated by interferon gamma (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-driven classical complement activation leads to C3 fragments on target cells.These C3 cleavage products can promote direct phagocytosis by effector cells or indirectly by enhancing antibody-mediated phagocytosis. In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce Fc-mediated effector function.

[0096] "Effector T cells" or "Teffs" or "Teff" as used herein refers to T lymphocytes that carry out functions of an immune response, such as activating an anti-tumor immune response that can result in the killing of tumor cells and / or the clearance of tumor cells from the body. An example of a Teff phenotype is the CD3 + CD4 + and CD3 + CD8 + Teffs may secrete, contain, or express markers such as IFNγ, Granzyme B, and ICOS. It is understood that Teffs may not be completely restricted to these phenotypes.

[0097] "Memory T cells", as used herein, refer to T lymphocytes that remain in the body for a long period of time after infection has been cleared. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It is understood that memory T cells may not be completely restricted to these phenotypes.

[0098] "Regulatory T cells" or "Tregs" or "Treg" as used herein refers to T lymphocytes that regulate the activity of other T cells and / or other immune cells, usually by suppressing their activity. An example of a Treg phenotype is the CD3 + CD4 + CD25 + CD127dim. Tregs may further express Foxp3. It is understood that Tregs may not be completely restricted to this phenotype.

[0099] As used herein, the term "complement activation" refers to the activation of the classical complement pathway, which is initiated by a large macromolecular complex called C1, which binds to an antibody-antigen complex on a surface. C1 is a complex consisting of six recognition proteins C1q and a heterotetramer of serine protease, C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, with a series of cleavage reactions beginning with the cleavage of C4 into C4a and C4b, and C2 into C2a and C2b. C4b is deposited and forms an enzymatically active convertase called C3 convertase with C2a, which cleaves the complement component C3 into C3b and C3a, which allows the formation of C5 convertase. This C5 convertase splits C5 into C5a and C5b, the last component being deposited on the membrane, which then triggers the late events of complement activation, in which the terminal complement components C5b, C6, C7, C8 and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). In certain embodiments herein where the antibody has an inactive Fc region, the antibody does not induce complement activation.

[0100] Complement activation can be assessed by using C1q binding efficiency, CDC kinetics, CDC assays (described in WO2013 / 004842, WO2014 / 108198), or by the method of cellular deposition of C3b and C4b described in Beurskens et al., J Immunol April 1, 2012 vol. 188 no. 7, 3532-3541.

[0101] The term "C1q binding" as used herein is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to its antigen. Antibody bound to its antigen should be understood to occur both in vivo and in vitro in the context described herein. As described in Example 8 herein, C1q binding can be assessed, for example, by using an antibody immobilized on an artificial surface, or by using an antibody bound to a predetermined antigen on a cell or virion surface. The binding of C1q to an antibody oligomer should be understood herein as a multivalent interaction that results in high avidity binding. The reduction in C1q binding, for example resulting from the introduction of a mutation in an antibody of the present invention, may be measured by comparing the C1q binding of the mutated antibody with the C1q binding of its parent antibody (the antibody of the present invention without the mutation in the same assay).

[0102] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody of the invention with the goal of alleviating, ameliorating, arresting, or eradicating (curing) the symptoms or disease state.

[0103] The term "effective amount" or "therapeutically effective amount" refers to an amount effective for the dosage and time period necessary to achieve the desired therapeutic result. The therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to induce a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody variant are outweighed by the therapeutically beneficial effects.

[0104] The term "pharmacokinetic profile" as used herein may be determined as plasma IgG levels over time as described in Example 12 herein.

[0105] Specific Aspects of the Invention In a first aspect, the present invention provides an antibody comprising at least one antigen binding region capable of binding to human CD27, the antibody comprising a heavy chain variable (VH) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and a light chain variable (VL) region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively. In a further aspect, the present invention provides an antibody comprising two of said antigen binding regions comprising a VH region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and a VL region CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively. The present invention provides an anti-CD27 antibody capable of binding to human CD27 and further binding to a variant of human CD27 comprising the A59T mutation. In one embodiment of the present invention, the antibody binds to CD27, for example, on T cells, and is agonistic in binding to its target. The present invention provides an antibody that stimulates T cell activation and proliferation. The antibody may further stimulate T cell memory formation and survival. Such an antibody is useful, for example, in the treatment of cancer. The antibody further has the ability to bind to cynomolgus monkey CD27, which is useful for antibody toxicology studies.

[0106] It is well known in the art that mutations in the VH and VL of an antibody can be made, for example, to increase the affinity of the antibody for its target antigen, to reduce its potential immunogenicity, and / or to increase the yield of the antibody expressed by a host cell. Thus, in some embodiments, variants of the CDR, VH and / or VL sequences of the antibody according to the invention are also envisaged, in particular antibodies comprising functional variants of the VH and / or VL regions shown in SEQ ID NO:4 and SEQ ID NO:8, respectively. Functional variants may, for example, differ in one or more amino acids in one or more CDRs compared to the parent VH and / or VL sequence, but still allow the antigen-binding region to retain at least a substantial proportion (at least about 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent or higher) or all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include variants that differ from the respective parent VH or VL regions by 12 or less, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, e.g., substitutions, insertions, or deletions of amino acid residues. Exemplary variants include variants that differ from the VH and / or VL and / or CDR regions of the parent sequence mainly by conservative amino acid substitutions; e.g., 12, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further aspect of the invention, the antibody may contain up to 1, 2 or 3 mutations in the VH CDR region and / or the VL CDR region, respectively. Such mutations may be substitutions. It is preferred that such substitutions do not significantly alter the binding affinity and / or binding specificity of the anti-CD27 antibody of the invention.Thus, the present invention encompasses variants of the anti-CD27 antibodies of the present invention that have the same functional characteristics as antibodies comprising the VH region CDR sequences shown in SEQ ID NOs:5, 6, and 7, and the VL region CDR sequences shown in SEQ ID NOs:9, 10, and 11.

[0107] In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 80% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 85% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 90% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 95% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 96% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 97% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 98% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 99% identical to the VH region shown in SEQ ID NO:4. In another aspect of the invention, the antibody comprises a VH region comprising a sequence shown in SEQ ID NO:4.

[0108] In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 80% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 85% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 90% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 95% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 96% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 97% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 98% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence at least 99% identical to the VH region shown in SEQ ID NO:8. In another aspect of the invention, the antibody comprises a VH region comprising a sequence shown in SEQ ID NO:8.

[0109] In another aspect of the invention, the antibody comprises VH and VL regions comprising the sequences shown in SEQ ID NO:4 and SEQ ID NO:8, respectively.

[0110] In one aspect, an antibody of the invention is an isolated antibody.

[0111] In one embodiment, the antibody is a human antibody. In another embodiment, the antibody is a humanized antibody. In another aspect, the antibody is a chimeric antibody.

[0112] In a preferred embodiment, the antibody of the present invention is a full-length antibody.Therefore, the antibody of the present invention may further comprise a light chain constant region (CL) and a heavy chain constant region (CH).The CH preferably comprises a CH1 region, a hinge region, a CH2 region and a CH3 region.

[0113] The antibody of the present invention may comprise a light chain constant region that is a human kappa light chain. In another aspect, it may comprise a human lambda light chain constant region.

[0114] The antibody according to the present invention may preferably further comprise a heavy chain constant region that is a heavy chain constant region of the human IgG isotype. It may optionally comprise a constant region of a modified human IgG. Such a human IgG comprises an Fc region that includes CH2 and CH3 regions. By modifying the IgG constant region in the Fc region, it is possible, for example, to modulate the Fc effector function of the antibody or to increase the Fc-Fc interaction and thus the tendency of the antibody to form a cluster, for example a hexamer. In one aspect of the present invention, the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4. In one embodiment, it is IgG1. In another aspect, it is IgG2. In yet another aspect, it is IgG3. In a further aspect, it is IgG4. In one particular aspect, the IgG is a modified human IgG that comprises one or more amino acid substitutions in the Fc region. In one embodiment, it may be a modified human IgG1 that comprises one or more amino acid substitutions in the Fc region. In a further aspect of the invention, the IgG1 comprises two or more amino acid substitutions in the Fc region. In one embodiment, the IgG1 Fc region has two amino acid substitutions.

[0115] In a further aspect of the invention, the modified human IgG heavy chain constant region comprises up to 10 amino acid substitutions in the Fc region. In another aspect, it comprises up to 9 amino acid substitutions. In another aspect, it comprises up to 8 amino acid substitutions. In another aspect, it comprises up to 7 amino acid substitutions. In another aspect, it comprises up to 6 amino acid substitutions. In another aspect, it comprises up to 5 amino acid substitutions. In another aspect, it comprises up to 4 amino acid substitutions. In another aspect, it comprises up to 3 amino acid substitutions. In another aspect, it comprises up to 2 amino acid substitutions in the Fc region.

[0116] Mutations in amino acid residues at positions corresponding to E430, E345 and S440 in the human IgG1 heavy chain, numbered according to the EU index, can improve the ability of an antibody to induce CDC. Without being bound by theory, it is believed that substituting one or more amino acids at these positions can stimulate oligomerization of the antibody, thereby modulating Fc-mediated effector functions, for example increasing C1q binding, complement activation, CDC, ADCP, internalization, or other related functions that may provide in vivo efficacy.

[0117] The present invention relates in one aspect to a variant antibody comprising an antigen-binding region and a variant Fc region.

[0118] In certain embodiments, the antibody variant that binds to human CD27 is (a) a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region comprising a mutation at one or more of E430, E345 and S440 (amino acid residues are numbered according to the EU index); (b) a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11. Includes.

[0119] In certain other embodiments, the antibody variant that binds to human CD27 is (a) a heavy chain comprising a VH region comprising SEQ ID NO:4 and a human IgG1 CH region comprising a mutation at one or more of E430, E345 and S440 (amino acid residues are numbered according to the EU index); and (b) a light chain comprising a VL region comprising SEQ ID NO:8 Includes.

[0120] The variant antibodies of the invention comprise a variant Fc region or a variant human IgG1 CH region comprising mutations at one or more of P329, E430 and E345. Hereinafter, references to mutations in the Fc region may equally apply to mutations in the human IgG1 CH region, and vice versa.

[0121] As described herein, the position of the amino acid to be mutated in the Fc region can be given with respect to (i.e., "corresponding to") its position in a naturally occurring (wild-type) human IgG1 heavy chain when numbered according to the Eu index. Thus, if the parent Fc region already contains one or more mutations and / or is, for example, an IgG2, IgG3 or IgG4 Fc region, the position of the amino acid corresponding to an amino acid residue, such as E430, in the human IgG1 heavy chain numbered according to the Eu index can be determined by alignment. In particular, the parent Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue at the position corresponding to E430 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence can be useful for this purpose, including any one of the different human IgG1 allotypes shown in Table 3.

[0122] In one aspect of the invention, the modification in the IgG Fc region induces increased CD27 agonism compared to an identical antibody, except that it comprises a wild-type IgG Fc region of the same isotype, e.g. IgG1. This may be obtained, for example, by introducing an amino acid other than E at the amino acid positions corresponding to positions E345 and / or E430 in the human IgG1 heavy chain according to the Eu numbering. In one embodiment of the invention, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to the Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W and Y. In another aspect of the invention, the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to the Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W.

[0123] In a preferred embodiment, the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to Eu numbering is R. Thus, the antibody of the invention may comprise an E345R substitution in the Fc region. In another aspect of the invention, the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to Eu numbering is G. Thus, the antibody of the invention may comprise an E430G substitution in the Fc region. In another embodiment, the antibody comprises an amino acid substitution selected from the group comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y.

[0124] The present invention provides antibodies that have enhanced Fc-Fc interactions that can lead to antibody-dependent clustering of CD27 on the cell surface upon antibody binding, thereby increasing the agonism of the antibodies of the invention.

[0125] In another embodiment of the antibody of the invention, the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering is substituted with an amino acid selected from the group comprising A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y. Thus, the antibody of the invention may further comprise a mutation at position 329.

[0126] In a further aspect of the invention, the antibody has an amino acid residue R at a position corresponding to position P329 in a human IgG1 heavy chain according to Eu numbering. Thus, the antibody of the invention may have a P329R substitution in the Fc region. Without being bound by theory, it is believed that the antibody of the invention comprising an E345R mutation in the Fc region (e.g. as shown in SEQ ID NO:13) has increased serum clearance. The inventors have found that further introducing a mutation at position 329, e.g. P329R (e.g. as shown in SEQ ID NO:15), restores the clearance of the antibody of the invention to the level of an antibody comprising wt IgG1, e.g. as shown in SEQ ID NO:12.

[0127] In another preferred embodiment, the amino acid residues at positions corresponding to positions P329 and E345 in a human IgG1 heavy chain according to Eu numbering are both R. The present invention provides antibodies that have increased CD27 receptor agonism and comparable pharmacokinetic properties, such as serum clearance, when compared to an antibody that comprises the same VH and VL regions and that comprises the same IgG1 heavy chain constant region with the exception of the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345.

[0128] Thus, in one aspect, the invention provides CD27 binding antibodies that have increased receptor agonism in binding to CD27 and further have pharmacokinetic properties that are comparable, e.g., similar or identical, when compared to the pharmacokinetic properties of an antibody comprising the same VH and VL regions but comprising a wild-type IgG1 heavy chain constant region, such as that shown in SEQ ID NO: 12. In other words, the invention provides CD27 binding antibodies that have pharmacokinetic properties that do not differ materially from the pharmacokinetic properties of an identical CD27 binding antibody except that it comprises a wild-type IgG1 heavy chain constant region.

[0129] In another embodiment of the invention, the antibody comprises a variant Fc region according to any one of the preceding sections, wherein the variant Fc region is a variant of a human IgG Fc region selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 Fc regions. That is, the mutations at one or more of the amino acid residues corresponding to E430 and E345 and P329 are made in a parent Fc region which is a human IgG Fc region selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 Fc regions. Preferably, the parent Fc region is a naturally occurring (wild type) human IgG Fc region, such as a human wild type IgG1, IgG2, IgG3 or IgG4 Fc region, or a mixed isotype thereof. Thus, the variant Fc region may be of human IgG1, IgG2, IgG3 or IgG4 isotype, or a mixed isotype thereof, except for the mutations described (at one or more of the amino acid residues selected from E430 and E345 and P329).

[0130] In one embodiment, the parent Fc region and / or human IgG1 CH region is of the wild-type human IgG1 isotype.

[0131] Thus, the variant Fc region may be a human IgG1 Fc region, except for the mutations described (at E430 or E345 or P329).

[0132] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(f) isotype.

[0133] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(z) isotype.

[0134] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(a) isotype.

[0135] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(x) isotype.

[0136] In specific embodiments, the parent Fc region and / or human IgG1 CH region is a human wild-type IgG1 of mixed allotypes, such as IgG1m(za), IgG1m(zax), or IgG1m(fa).

[0137] Thus, the variant Fc region and / or human IgG1 CH region may be of the human IgG1m(f), IgG1m(a), IgG1m(x), IgG1m(z) allotype or a mixed allotype of any two or more thereof, except for the mutations described (at E430 or E345 or P329).

[0138] In a specific embodiment, the parent Fc region and / or human IgG1 CH region is of the human wild-type IgG1m(za) isotype.

[0139] In a specific embodiment, the parent Fc region is a human wild-type IgG2 isotype.

[0140] In a specific embodiment, the parent Fc region is a human wild-type IgG3 isotype.

[0141] In a specific embodiment, the parent Fc region is a human wild-type IgG4 isotype.

[0142] The CH region amino acid sequences of specific examples of wild-type human IgG isotypes and IgG1 allotypes are shown in Table 3.

[0143] In another aspect, the invention provides an antibody comprising a heavy chain constant region comprising an amino acid sequence selected from the group comprising SEQ ID Nos 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:12. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:13. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:14. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:15. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:18. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:19. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:20. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:21. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:22. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:23. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:27. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:28. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:29. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:30. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:31. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO:32. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 33. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 34. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 36.

[0144] In one embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 15; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0145] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 12; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0146] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 13; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0147] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 14; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0148] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 18; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0149] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 19; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0150] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No:20; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0151] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No:21; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0152] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No:22; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0153] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No:23; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0154] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No:27; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0155] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No:28; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0156] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No:29; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0157] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 30; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0158] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence shown in SEQ ID No: 31; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0159] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 32; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0160] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 33; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0161] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No:34; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0162] In another embodiment, the antibody of the present invention comprises a. A VH region comprising the amino acid sequence shown in SEQ ID No:4 b. A VL region comprising the amino acid sequence shown in SEQ ID No:8 c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 36; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 16. Includes.

[0163] In an alternative embodiment of the above antibody, the CL region may be the amino acid sequence shown in SEQ ID No:17.

[0164] In one embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 15; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0165] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 12; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0166] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 13; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0167] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 14; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0168] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 18; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0169] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 19; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0170] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No:20; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0171] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No:21; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0172] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No:22; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0173] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 23; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0174] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 27; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0175] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 28; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0176] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No:29; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0177] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 30; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0178] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 31; h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0179] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 32; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0180] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 33; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0181] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 34; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0182] In another embodiment, the antibody of the present invention comprises e. A VH region comprising the amino acid sequence shown in SEQ ID No:4 f. A VL region comprising the amino acid sequence shown in SEQ ID No:8 g. a CH region comprising the amino acid sequence set forth in SEQ ID No: 36; and h. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17. Includes.

[0183] In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:24 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0184] In another embodiment, an antibody of the invention comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:35 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:25.

[0185] In yet another aspect, the present invention provides an antibody comprising a heavy chain constant region modified such that the antibody induces Fc-mediated effector functions to a lesser extent than the same antibody except for the modification. An example is a CD27-binding antibody of the present invention comprising P329R and E345R substitutions. Such an antibody induces one or more Fc-mediated effector functions to a lesser extent than an antibody comprising the same sequence except for the P329R substitution, and also compared to the same antibody comprising the same sequence except for the P329R and E345R substitutions, e.g., wild-type IgG1 heavy chain. In one embodiment, the Fc-mediated effector function is reduced by at least 20%. In another aspect, the Fc-mediated effector function is reduced by at least 30%. In another aspect, the Fc-mediated effector function is reduced by at least 40%. In another aspect, the Fc-mediated effector function is reduced by at least 50%. In another aspect, the Fc-mediated effector function is reduced by at least 60%. In another aspect, the Fc-mediated effector function is reduced by at least 70%. In another aspect, the Fc-mediated effector function is reduced by at least 80%. In another aspect, the Fc-mediated effector function is reduced by at least 90%. In another aspect, the antibody does not induce one or more Fc-mediated effector functions. The one or more Fc effector functions that are reduced or not induced at all may be selected from the following group: complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated cytotoxicity (CDCC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding and FcγR binding. Thus, in one embodiment, the antibody of the present invention induces CDC to an extent that is reduced by at least 20%, for example at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, compared to the same antibody except for the wild-type IgG1 HC constant region. In another embodiment, the antibodies of the invention do not induce CDC.

[0186] In another aspect, an antibody of the invention induces CDCC to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical antibody except having a wild-type IgG1 HC constant region. In another embodiment, an antibody of the invention does not induce CDCC.

[0187] In another aspect, an antibody of the invention induces ADCC to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical antibody except having a wild-type IgG1 HC constant region. In another embodiment, an antibody of the invention does not induce ADCC.

[0188] In another aspect, the antibodies of the invention induce ADCP to a degree that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced compared to an identical antibody except having a wild-type IgG1 HC constant region. In another embodiment, the antibodies of the invention do not induce ADCP.

[0189] In another aspect, the antibody of the invention induces C1q binding to an extent that is at least 20%, such as at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced, compared to an identical antibody except that it has a wild-type IgG1 HC constant region. In another embodiment, the antibody of the invention does not induce C1q binding. Preferably, C1q binding is determined as in Example 8.

[0190] In another aspect, the antibody of the present invention induces FcγR binding to a degree that is at least 20%, for example at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% reduced, compared to the same antibody except that it has a wild-type IgG1 HC constant region. In another embodiment, the antibody of the present invention does not induce FcγR binding. Preferably, FcγR binding is determined as in Example 9.

[0191] In one embodiment, an antibody of the invention has reduced C1q binding and reduced FcγR binding compared to an antibody containing the same amino acid sequence except that it does not contain the P329R substitution.

[0192] In one embodiment, the antibody according to any aspect or embodiment herein is a human antibody, except for the mutations described.

[0193] In one embodiment of the invention, the antibody is a monovalent antibody.

[0194] In another embodiment, the antibody is a bivalent antibody.

[0195] Furthermore, the antibodies of the present invention may be monospecific antibodies.

[0196] In one embodiment, the antibody according to any aspect or embodiment herein is a monoclonal antibody, such as a human monoclonal antibody, such as a human bivalent monoclonal antibody, such as a human bivalent full length monoclonal antibody.

[0197] In a preferred embodiment, the antibody according to any aspect or embodiment herein is an IgG1 antibody, such as a full length IgG1 antibody, such as a human full length IgG1 antibody, optionally a human monoclonal full length bivalent IgG1,κ antibody, such as a human monoclonal full length bivalent IgG1m(f),κ antibody, except for any described mutations in the Fc region.

[0198] The antibody according to the present invention is advantageously a bivalent monospecific format, comprising two antigen-binding regions that bind to the same epitope. However, a bispecific format is also envisaged, in which one of the antigen-binding regions binds to a different epitope. Thus, the antibody according to any aspect or embodiment herein can be either a monospecific antibody or a bispecific antibody, unless the context indicates otherwise.

[0199] Thus, in another embodiment, the antibody of the invention is a bispecific antibody comprising a first antigen-binding region capable of binding to human CD27 as described herein, and a second antigen-binding region capable of binding to a different epitope on human CD27. In another embodiment, the antibody of the invention is a bispecific antibody comprising a first antigen-binding region capable of binding to human CD27 as described herein, and a second antigen-binding region capable of binding to a different target. Such a target may be on a different cell or on the same cell as CD27.

[0200] In one aspect of the invention, the antibody has the ability to bind to human CD27 having the sequence shown in SEQ ID NO:1. However, human CD27 may be expressed as variants thereof in some individuals. Thus, in another aspect, the antibody of the invention further has the ability to bind to a human CD27 variant, such as the human CD27 variant shown in SEQ ID NO:2. In another embodiment, the antibody of the invention further has the ability to bind to cynomolgus CD27, such as that shown in SEQ ID NO:3.

[0201] In a further embodiment of the invention, the antibody has the capacity of binding to CD27-expressing human T cells.

[0202] In another embodiment of the invention, the antibody is capable of binding to CD27-expressing cynomolgus monkey T cells.

[0203] In one embodiment of the invention, the full length IgG1 antibody has the C-terminal lysine of the HC truncated. Such an antibody is also considered a "full length antibody."

[0204] In another embodiment of the invention, the antibody is directed to a human T cell, e.g., CD4 + and CD8 + It has the ability to induce proliferation of T cells, such as helper T cells and cytotoxic T cells. Such activity may be assayed as described in Examples 6 or 7 herein.

[0205] In another embodiment of the invention, the antibody has the ability to induce activation of human CD27-expressing Jurkat reporter T cells, such as those described in Example 2 herein.

[0206] In another embodiment of the invention, the antibody has the ability to induce activation of human CD27-expressing Jurkat reporter T cells in the absence of Fcγ receptor IIb cross-linking, such as that described in Example 11 herein.

[0207] In another embodiment of the invention, the antibody is a CD4+ and CD8 T cell with a central memory T cell phenotype. + It has the ability to induce T cell proliferation.

[0208] In another embodiment of the invention, the antibody is capable of inducing the production of IFN gamma.

[0209] Antibodies are well known as therapeutic agents that can be used in the treatment of various diseases. Another method for administration of an antibody to a subject in need thereof includes administration of a nucleic acid or a combination of nucleic acids encoding said antibody for in vivo expression of said antibody.

[0210] Therefore, in one aspect, the present invention also relates to a nucleic acid encoding a heavy chain of an antibody according to the present invention, said heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO:5, a VH CDR2 comprising the sequence shown in SEQ ID NO:6, a VH CDR3 comprising the sequence shown in SEQ ID NO:7, and a human IgG1 CH region.

[0211] In another aspect, the present invention also relates to a nucleic acid encoding the heavy chain of an antibody according to the present invention, said heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence as shown in SEQ ID NO:5, a VH CDR2 comprising the sequence as shown in SEQ ID NO:6, a VH CDR3 comprising the sequence as shown in SEQ ID NO:7, and a human IgG1 CH region, and having a mutation at one or both of E430 and E345 (amino acid residues numbered according to the Eu index).

[0212] In another aspect, the present invention also relates to a nucleic acid encoding a heavy chain of an antibody according to the present invention, said heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence as shown in SEQ ID NO:5, a VH CDR2 comprising the sequence as shown in SEQ ID NO:6, a VH CDR3 comprising the sequence as shown in SEQ ID NO:7, and a human IgG1 CH region having a mutation at one or both of P329 and E345 (amino acid residues are numbered according to the Eu index).

[0213] In one aspect, the present invention also relates to a nucleic acid or a combination of nucleic acids encoding an antibody according to the present invention.

[0214] In some aspects, the present invention provides a method for producing a) an antigen-binding region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11, and b) a variant Fc region comprising a mutation at one or both amino acids corresponding to P329 and E345 in a human IgG1 heavy chain, said amino acid residues being numbered according to the Eu index. The present invention relates to a nucleic acid or combination of nucleic acids encoding an antibody comprising:

[0215] In one embodiment, an antibody of the invention is encoded by a single nucleic acid, such that nucleotide sequences encoding an antibody of the invention are present in a single nucleic acid sequence or in the same nucleic acid molecule.

[0216] In another embodiment, the antibody of the present invention is encoded by a combination of nucleic acid sequences, typically by two nucleic acid sequences, in one embodiment, said combination of nucleic acid sequences comprises a nucleic acid sequence encoding the heavy chain of said antibody and a nucleic acid sequence encoding the light chain of said antibody.

[0217] In some aspects, the present invention provides a method for producing a) a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region comprising a mutation at one or both of P329 and E345 (amino acid residues are numbered according to the Eu index); b) a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11. The present invention relates to a nucleic acid sequence or combination of nucleic acid sequences encoding an antibody comprising:

[0218] In one embodiment, the antibodies of the invention are encoded by one nucleic acid, such that nucleotide sequences encoding the antibodies of the invention are present in one nucleic acid or in the same nucleic acid molecule.

[0219] In another embodiment, the antibody of the present invention is encoded by a combination of nucleic acid sequences, typically by two nucleic acid sequences, in one embodiment, said combination of nucleic acid sequences comprises a nucleic acid sequence encoding the heavy chain of said antibody and a nucleic acid sequence encoding the light chain of said antibody.

[0220] As noted above, the nucleic acid sequences may be used as a means to deliver a therapeutic protein, such as an antibody, to a subject in need thereof.

[0221] In some embodiments, the nucleic acid may be deoxyribonucleic acid (DNA). Suitable DNA and methods for preparing DNA for in vivo expression of therapeutic proteins, such as antibodies, are well known to those skilled in the art, including but not limited to those described in Patel A et al., 2018, Cell Reports 25, 1982-1993.

[0222] In some embodiments, the nucleic acid may be a ribonucleic acid (RNA), such as a messenger RNA (mRNA). In some embodiments, the mRNA may contain only naturally occurring nucleotides. In some embodiments, the mRNA may contain modified nucleotides, where modified refers to the nucleotides being chemically different from naturally occurring nucleotides. In some embodiments, the mRNA may contain both naturally occurring and modified nucleotides.

[0223] Different nucleic acid sequences suitable for the in vivo expression of therapeutic proteins, such as antibodies, in a subject are well known to those skilled in the art.For example, mRNA suitable for the expression of therapeutic antibodies in a subject often comprises an open reading frame (ORF), which is flanked by untranslated regions (UTRs) containing unique sequences, and the 5' and 3' ends are formed by cap structures and poly(A) tails (see, for example, Schlake et al., 2019, Molecular Therapy Vol.27 No 4 April).

[0224] Examples of RNA and methods for optimizing RNA molecules, such as mRNA, suitable for in vivo expression include, but are not limited to, those described in US9254311; US9221891; US20160185840 and EP3118224.

[0225] Naked nucleic acid sequences administered to a subject for in vivo expression tend to degrade and / or cause an immunogenic response in the subject. Furthermore, for in vivo expression of an antibody encoded by a nucleic acid sequence, the nucleic acid sequence is typically administered in a form suitable for the nucleic acid sequence to enter the cells of the subject. There are different methods of delivering nucleic acid sequences for in vivo expression, including both methods involving mechanical means and methods involving chemical means. For example, such methods may involve electroporation or tattooing the nucleic acid into the skin (Patel et al., 2018, Cell Reports 25, 1982-1993). Other methods suitable for administration of nucleic acid sequences to a subject involve administration of the nucleic acid in a suitable formulation. Therefore, the present invention also relates to a delivery vehicle comprising the nucleic acid of the present invention.

[0226] In some embodiments, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain of an antibody according to the invention. Thus, in one embodiment, the nucleic acid sequence may encode a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region having a mutation at one or both of P329 and E345 (amino acid residues numbered according to the Eu index).

[0227] In some embodiments, the present invention also relates to a delivery vehicle comprising a nucleic acid sequence encoding a light chain of an antibody according to the present invention. Thus, in one embodiment, said nucleic acid sequence may encode a light chain comprising a VL region comprising a VL CDR1 comprising the sequence shown in SEQ ID NO:9, a VL CDR2 comprising the sequence shown in SEQ ID NO:10, and a VL CDR3 comprising the sequence shown in SEQ ID NO:11.

[0228] The present invention also relates to a mixture of delivery vehicles comprising a nucleic acid sequence encoding the heavy chain of the antibody of the present invention and a nucleic acid sequence encoding the light chain of the antibody of the present invention. Thus, in one embodiment, said mixture of delivery vehicles comprises a delivery vehicle comprising a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO:5, a VH CDR2 comprising the sequence shown in SEQ ID NO:6, a VH CDR3 comprising the sequence shown in SEQ ID NO:7, and a human IgG1 CH region having a mutation at one or both of E430 and E345 (amino acid residues are numbered according to the Eu index); and a delivery vehicle comprising a nucleic acid sequence encoding a light chain comprising a VL region comprising a VL CDR1 comprising the sequence shown in SEQ ID NO:9, a VL CDR2 comprising the sequence shown in SEQ ID NO:10, and a VL CDR3 comprising the sequence shown in SEQ ID NO:11.

[0229] In some embodiments, the delivery vehicle comprises a nucleic acid sequence or a combination of nucleic acid sequences encoding the heavy and light chains of an antibody of the present invention.

[0230] Thus, in one embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region having a mutation at one or both of E430 and E345 (amino acid residues are numbered according to the Eu index); and a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0231] In yet another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region with the mutations P329R and E345R (amino acid residues are numbered according to the Eu index); and a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0232] In another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a WT human IgG1 CH region; and a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0233] Thus, the nucleic acid sequences encoding the heavy and light chains of the antibody according to the invention are present in one (and the same) nucleic acid molecule.

[0234] In another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a WT human IgG1 CH region; and a nucleic acid encoding a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0235] In another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region having a mutation at one or both of E430 and E345 (amino acid residues are numbered according to the Eu index); and a nucleic acid sequence encoding a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0236] In another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, and a human IgG1 CH region having the P329R and E345R mutations (amino acid residues are numbered according to the Eu index); and a nucleic acid sequence encoding a light chain comprising a VL region comprising a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0237] Thus, the nucleic acid sequences encoding the heavy and light chains of the antibody variants according to the invention are present on separate or different nucleic acid molecules.

[0238] In some embodiments, the delivery vehicle may be a lipid formulation. The lipid of the formulation may be a particle, such as a lipid nanoparticle (LNP). The nucleic acid sequence or combination of nucleic acid sequences of the present invention may be encapsulated within the particle, such as within the LNP.

[0239] Different lipid formulations suitable for administration of nucleic acids to a subject for in vivo expression are well known to those skilled in the art. For example, the lipid formulation may typically comprise a lipid, an ionizable amino lipid, a PEG-lipid, cholesterol, or any combination thereof.

[0240] Various forms and preparation methods of lipid formulations suitable for administering nucleic acid sequences to subjects for expressing therapeutic antibodies are well known in the art.Examples of such lipid formulations include but are not limited to those described in US20180170866 (Arcturus), EP 2391343 (Arbutus), WO 2018 / 006052 (Protiva), WO2014152774 (Shire Human Genetics), EP 2 972 360 (Translate Bio), US10195156 (Moderna) and US20190022247 (Acuitas).

[0241] The present invention also provides isolated nucleic acid sequences and vectors encoding the antibody variants according to any one of the aspects and embodiments described herein, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors and expression systems for antibodies and their variants are known in the art, including but not limited to those described in the Examples. In embodiments where the variant antibody comprises HC and LC that are separate polypeptides rather than being contained in a single polypeptide (e.g., as in scFv-Fc fusion proteins), the nucleotide sequences encoding the heavy and light chains may be present in the same or different nucleic acids or vectors.

[0242] Thus, in one aspect, the invention provides an isolated nucleic acid sequence or combination of nucleic acid sequences encoding an antibody according to any aspect or embodiment herein. The invention also provides a nucleic acid sequence encoding a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO:5, a VH CDR2 comprising the sequence shown in SEQ ID NO:6, and a VH CDR3 comprising the sequence shown in SEQ ID NO:7.

[0243] The present invention further provides a nucleic acid sequence encoding a VL region comprising a VL CDR1 comprising the sequence shown in SEQ ID NO:9, a VL CDR2 comprising the sequence shown in SEQ ID NO:10, and a VL CDR3 comprising the sequence shown in SEQ ID NO:11.

[0244] Furthermore, the present invention provides a nucleic acid sequence encoding a VH region comprising the amino acid sequence shown in SEQ ID NO: 4. The present invention also relates to a nucleic acid sequence encoding a VL region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0245] In a further aspect, the invention provides a nucleic acid sequence encoding a heavy chain of an antibody according to any aspect or embodiment described herein. In a further aspect, the invention provides a nucleic acid sequence encoding a light chain of an antibody according to any aspect or embodiment described herein. In another aspect, the invention relates to a nucleic acid sequence encoding a heavy chain comprising a VH region comprising the sequence shown in SEQ ID NO:4 and a human IgG1 CH region comprising a mutation at P329 and / or E345 (amino acid residues numbered according to the Eu index). In yet another aspect, the invention provides a nucleic acid sequence encoding a light chain comprising a VL region comprising the sequence shown in SEQ ID NO:8 and a human kappa constant region comprising the sequence shown in SEQ ID NO:16. In yet another aspect, the invention provides a nucleic acid sequence encoding a light chain comprising a VL region comprising the sequence shown in SEQ ID NO:8 and a human lambda constant region comprising the sequence shown in SEQ ID NO:17.

[0246] In one embodiment of the invention, the nucleic acid sequence or combination of nucleic acid sequences is RNA or DNA. In one embodiment of the invention, the nucleic acid sequence or combination of nucleic acid sequences is mRNA.

[0247] The present invention further provides an expression vector comprising a nucleic acid sequence according to any aspect or embodiment described herein, or a combination thereof.

[0248] In another aspect, the invention relates to a nucleic acid sequence or combination of nucleic acid sequences described herein for use in expression in a mammalian cell.

[0249] In a further aspect, the present invention relates to a recombinant host cell producing an antibody as defined herein, optionally comprising an expression vector as described above. In certain particular aspects, the recombinant host cell is a eukaryotic or prokaryotic cell.

[0250] In another aspect, the invention relates to a method of producing an antibody according to any aspect or embodiment herein, comprising culturing a recombinant host cell as described above in a culture medium and under conditions suitable for producing the antibody, and optionally purifying or isolating the antibody from the culture medium.

[0251] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary artery disease comprising: (i) a nucleotide sequence encoding the heavy chain sequence of the antibody according to any one of the embodiments disclosed herein; (ii) a nucleotide sequence encoding the light chain sequence of the antibody according to any one of the embodiments disclosed herein; or (iii) Both (i) and (ii) The present invention relates to a nucleic acid or expression vector comprising:

[0252] In one aspect, the invention relates to a nucleic acid or expression vector comprising a nucleotide sequence encoding the heavy chain sequence of an antibody variant according to any one of the embodiments disclosed herein.

[0253] In one aspect, the invention relates to a nucleic acid sequence or an expression vector comprising a nucleotide sequence encoding the heavy and light chain sequences of an antibody according to any one of the embodiments disclosed herein.

[0254] In one aspect, the present invention relates to a combination of a first and a second nucleic acid or a first and a second expression vector, optionally in the same host cell, the first comprising a nucleotide sequence according to (i) and the second comprising a nucleotide sequence according to (ii).

[0255] An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences that include a suitable set of expression control elements). Examples of such vectors include SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and derivatives of viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid is, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid vector (e.g., as described in US 6,077, 835 and / or WO 00 / 70087), a plasmid vector, such as pBR322, pUC 19 / 18, or pUC 118 / 119, a "midge" minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793 800 (2001)), or a precipitated nucleic acid vector construct, such as the CaP04 precipitated construct (e.g., as described in WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0256] In one embodiment, the vector is suitable for expressing the antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989)), pET vectors (Novagen, Madison WI), and the like.

[0257] The expression vector may additionally or alternatively be a vector suitable for expression in yeast system. Any vector suitable for expression in yeast system may be used. Suitable vectors include, for example, vectors that contain constitutive or inducible promoters, such as alpha factor, alcohol oxidase and PGH (reviewed in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153,516 544 (1987)).

[0258] The expression vector may additionally or alternatively be a vector suitable for expression in mammalian cells, such as a vector containing glutamine synthetase as a selectable marker, such as those described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0259] The nucleic acid and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of targeting a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides.

[0260] The expression vector may contain or be associated with any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include a strong expression promoter (e.g., RSV, SV40, SL33, MMTV, and HIV LTR promoters, in addition to the human CMV IE promoter / enhancer), an effective poly(A) termination sequence, an origin of replication for plasmid production in E. coli, an antibiotic resistance gene as a selection marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter as opposed to a constitutive promoter, e.g., CMV IE.

[0261] In one embodiment, an expression vector encoding the antibody may be placed into and / or delivered to a host cell or animal via a viral vector.

[0262] The present invention also provides a recombinant host cell that produces the antibody disclosed herein, optionally comprising an isolated nucleic acid or vector according to the present invention. Typically, the host cell is transformed or transfected with the nucleic acid or vector. The claimed recombinant host cell can be, for example, a eukaryotic cell, a prokaryotic cell, or a microorganism cell, such as a transfectoma. In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is a prokaryotic cell. In some embodiments, the antibody is a heavy chain antibody. In most embodiments, however, the antibody contains both a heavy chain and a light chain, so that the host cell expresses both a construct encoding a heavy chain and a construct encoding a light chain, on the same or different vectors.

[0263] Examples of host cells include yeast, bacteria, plants and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, NS0 cells, Sp2 / 0 cells or lymphocytic cells. In one embodiment, the host cell is a CHO (Chinese Hamster Ovary) cell. For example, in one embodiment, the host cell may comprise a first and a second nucleic acid construct stably integrated into the cell genome, the first encoding the heavy chain and the second encoding the light chain of the antibody variant disclosed herein. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising the first and second nucleic acid constructs identified above.

[0264] In one embodiment, the host cell is a cell capable of Asn-linked glycosylation of proteins, eg, a eukaryotic cell, eg, a mammalian cell, eg, a human cell.

[0265] In one embodiment, the host cell is a host cell that does not have the ability to efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97:2426 (hereby incorporated by reference) lists a number of such antibody production systems, such as Sp2 / 0, NS / 0 or transgenic mammary gland (goat), in which only partial removal of the C-terminal lysine is obtained. In one embodiment, the host cell is a host cell with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells to express the variants of the present invention and thereby produce antibodies with altered glycosylation. See, e.g., Shields, R Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03 / 035835; and WO99 / 54342. Additional methods for generating engineered glycoforms are known in the art and include those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO 02 / 30954A1; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49, as well as those described in WO2018 / 114877, WO2018 / 114878 and WO2018 / 114879, but are not limited to these.

[0266] In an even further aspect, the present invention relates to transgenic non-human animals or plants comprising nucleic acids encoding one or two sets of human heavy and human light chains that produce the antibodies disclosed herein.

[0267] In one embodiment there is provided an antibody obtained or obtainable by the method described above.

[0268] In another aspect, the present invention also relates to a method for increasing or decreasing at least one effector function of an antibody of the present invention, comprising the step of introducing mutations into the antibody at one or more amino acid residues corresponding to E430, E345, and P329 in the Fc region of human IgG1 heavy chain, as numbered according to the Eu index.

[0269] Thus, in certain embodiments, there is provided a method of increasing an effector function, such as an Fc-mediated effector function, of a parent antibody, or increasing, for example, a biological activity of an antibody, such as CD27 agonism, wherein said parent antibody comprises an Fc region and an antigen binding region that binds to CD27, said method comprising introducing mutations in the Fc region at one or both amino acid residues corresponding to E430 and E345 in the Fc region of human IgG1 heavy chain, where the amino acid residues are numbered according to the Eu index; and wherein the antigen binding region comprises a VH CDR1 comprising the sequence set forth in SEQ ID NO:5, a VH CDR2 comprising the sequence set forth in SEQ ID NO:6, a VH CDR3 comprising the sequence set forth in SEQ ID NO:7, a VL CDR1 comprising the sequence set forth in SEQ ID NO:9, a VL CDR2 comprising the sequence set forth in SEQ ID NO:10, and a VL CDR3 comprising the sequence set forth in SEQ ID NO:11.

[0270] In certain other embodiments, a method is provided for reducing effector functions, such as C1q binding or FcgR binding, of a parent antibody comprising a VH CDR1 comprising the sequence shown in SEQ ID NO:5, a VH CDR2 comprising the sequence shown in SEQ ID NO:6, a VH CDR3 comprising the sequence shown in SEQ ID NO:7, a VL CDR1 comprising the sequence shown in SEQ ID NO:9, a VL CDR2 comprising the sequence shown in SEQ ID NO:10, and a VL CDR3 comprising the sequence shown in SEQ ID NO:11, and further comprising an amino acid substitution of E345R in the Fc region of the human IgG1 heavy chain, the amino acid residues being numbered according to the Eu index, comprising introducing a further amino acid substitution in the Fc region at an amino acid position corresponding to P329 of the human IgG1 heavy chain, numbered according to the Eu index. In a preferred embodiment of the invention, the method comprises the substitution of P329R. According to the invention, the effector functions, such as C1q binding or FcgR binding, of the parent antibody can be reduced or completely eliminated.

[0271] In one embodiment of any one of the aforementioned methods, the effector function that is increased comprises CD27 agonism.

[0272] In one embodiment of any one of the aforementioned methods, the effector function is C1q binding.

[0273] In one embodiment of any one of the aforementioned methods, the effector function is FcgR binding.

[0274] In one embodiment of any one of the aforementioned methods, the effector function that is decreased includes both C1q binding and FcgR binding.

[0275] In one embodiment of any of the above-mentioned methods, the mutation at one or more amino acid residues is selected from the group including E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and P329K. For example, the mutation at one or more amino acid residues may include or consist of E430G or E345R.

[0276] In one embodiment of any of the above methods, the Fc region of the antibody is a human IgG1, IgG2, IgG3 or IgG4 Fc region, or a mixture of these isotypes, except for the mutations described, and optionally includes an Fc region of one of the sequences set forth as SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:36. In a particular embodiment, the Fc region of the antibody is a human IgG1 Fc region. For example, the antibody can be a human full length IgG1 antibody, optionally a human monoclonal full length bivalent IgG1,κ antibody. Additionally, the antibody can be a monospecific or bispecific antibody, e.g., a monospecific antibody.

[0277] The Fc region of the antibody may be the naturally occurring (wild-type) sequence, but in some embodiments, the Fc region of the antibody comprises one or more additional mutations, as described elsewhere herein.

[0278] The present invention also relates to an antibody obtained or obtainable according to any of the above methods.

[0279] The present invention also relates to a composition comprising an antibody according to the invention, a nucleic acid according to the invention, an expression vector according to the invention or a host cell according to the invention.

[0280] In a further embodiment, the composition according to the invention is a pharmaceutical composition, typically comprising a pharma- ceutically acceptable carrier.In one embodiment, the pharmaceutical composition contains an antibody as defined in any aspect or embodiment disclosed herein, or an expression vector as defined in any aspect or embodiment disclosed herein.

[0281] In yet a further aspect, the present invention provides a method for producing a composition comprising the steps of: - an antibody as defined in any of the aspects and embodiments disclosed herein, and - A pharma- ceutically acceptable carrier The present invention relates to a pharmaceutical composition comprising:

[0282] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0283] The present invention also relates to a kit-of-parts, e.g. a kit for use as a companion diagnostic for identifying patients within a patient population who have a propensity to respond to treatment with an antibody defined herein, said kit comprising an antibody as defined in any aspect or embodiment disclosed herein and instructions for use of said kit.

[0284] The present invention also relates to a kit-of-parts for use in therapy comprising an antibody according to the invention or a composition comprising an antibody according to the invention, optionally comprising more than one dosage of the antibody.

[0285] In one embodiment, the kit-of-parts comprises such an antibody or composition in one or more containers, e.g., vials.

[0286] In one embodiment, the kit-of-parts comprises such antibodies or compositions for simultaneous, separate or sequential use in therapy.

[0287] The antibodies of the present invention have numerous therapeutic uses involving the treatment of diseases and disorders that can be treated by activating immune cells expressing CD27. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat or prevent various disorders and diseases. As used herein, the term "subject" is intended to include humans and non-human animals that can benefit or respond to the antibody. Subjects can be, for example, human or non-human animals that can benefit from or respond to the antibody. + and / or CD8 + This may include human patients having a disease or disorder that may be corrected or ameliorated by modulating CD27 function such that T cell populations are expanded. Thus, the antibodies may be used to induce in vivo or in vitro expansion of T cell populations, such as helper T cells and cytotoxic T cells.

[0288] Therefore, in one aspect the present invention relates to an antibody according to the present invention, a nucleic acid or a combination of nucleic acids according to the present invention, a delivery vehicle according to the present invention, an expression vector according to the present invention, a host cell according to the present invention, a composition according to the present invention or a pharmaceutical composition according to the present invention for use as a medicament.

[0289] In one aspect, the present invention relates to the use of an antibody according to the invention, a nucleic acid or a combination of nucleic acids according to the invention, a delivery vehicle according to the invention, an expression vector according to the invention, a host cell according to the invention, a composition according to the invention, or a pharmaceutical composition according to the invention in the preparation of a medicament for treating or preventing a disease or disorder.

[0290] In one aspect, the present invention relates to a method of treating a disease or disorder, comprising administering to a subject in need thereof an antibody according to the present invention, a nucleic acid or a combination of nucleic acids according to the present invention, a delivery vehicle according to the present invention, an expression vector according to the present invention, a host cell according to the claims of the present invention, a composition according to the present invention, or a pharmaceutical composition according to the present invention.

[0291] In one aspect, the invention relates to an antibody according to any aspect or embodiment for use as a medicament.

[0292] In one aspect, the invention relates to the use of an antibody according to any aspect or embodiment in the preparation of a medicament for treating or preventing a disease or disorder.

[0293] In one aspect, the invention relates to an antibody according to any aspect or embodiment for use in the treatment or prevention of a disease or disorder.

[0294] In one aspect, the invention relates to an antibody according to any aspect or embodiment for use in a diagnostic or for use in a diagnostic method.

[0295] In one aspect, the invention relates to a method of treating a disease or disorder comprising administering an antibody according to any aspect or embodiment to a subject in need thereof, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0296] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody according to any aspect or embodiment for use as a medicament.

[0297] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody according to any aspect or embodiment for use in the treatment or prevention of a disease or disorder.

[0298] In one aspect, the invention relates to a method of treating a disease or disorder comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody according to any aspect or embodiment, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder.

[0299] In one aspect, the present invention provides a method of treating a disease or disorder, comprising: selecting a subject suffering from a disease or disorder, and administering to a subject an antibody according to any aspect or embodiment, or a pharmaceutical composition comprising said antibody, typically in a therapeutically effective amount and / or for a time sufficient to treat the disease or disorder. The present invention relates to a method comprising the steps of:

[0300] In one embodiment, the disease or disorder is cancer, i.e., a tumorigenic disorder, such as a hematological cancer or a solid malignant tumor, etc. In another embodiment, the disease or disorder is an inflammatory and / or autoimmune disease or disorder.

[0301] In a further aspect, the present invention relates to an antibody comprising at least one antigen-binding region capable of binding to CD27, i.e. an anti-idiotypic antibody that binds to an antibody according to the invention described herein. In a particular embodiment, the anti-idiotypic antibody binds to an antigen-binding region capable of binding to CD27 described herein.

[0302] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen-binding site of an antibody. Anti-Id antibodies may be prepared by immunizing an animal of the same species and genotype as the source of the anti-CD27 monoclonal antibody with the monoclonal antibody from which the anti-Id is prepared. The immunized animal is typically capable of recognizing the idiotypic determinants of the immunizing antibody and, in response, producing antibodies against these idiotypic determinants (anti-Id antibodies). Methods for producing such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0303] The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody may be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies against the idiotypic determinants of the monoclonal antibody, it is possible to identify other clones expressing antibodies of the same specificity. The anti-Id antibody may be modified (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, for example by the techniques described elsewhere herein for the CD27-specific antibodies of the invention. For example, the monoclonal anti-Id antibody may be coupled to a carrier, such as keyhole limpet hemocyanin (KLH), and used to immunize BALB / c mice. Serum from these mice typically contains anti-anti-Id antibodies with similar, if not identical, binding properties to the original / parent anti-CD27 antibody.

[0304] Fc regions may have a lysine at their C-terminus. The source of this lysine is the naturally occurring sequence found in the human from which these Fc regions are derived. During cell culture production of recombinant antibodies, this terminal lysine can be cleaved off by proteolytic hydrolysis by endogenous carboxypeptidases, resulting in a constant region with the same sequence but lacking the C-terminal lysine. For the purpose of antibody production, the DNA encoding this terminal lysine can be omitted from the sequence such that the antibody is produced without the lysine. Antibodies produced from either a nucleic acid sequence that encodes or does not encode a terminal lysine are substantially identical in sequence and function, since the degree of processing of the terminal lysine is typically high, for example, when using antibodies produced in a CHO-based production system (Dick, LWet al. Biotechnol. Bioeng. 2008;100:1132-1143). It is therefore understood that the proteins, e.g., antibodies, of the present invention can be produced with or without encoding or carrying a terminal lysine. It is also understood in accordance with the present invention that a sequence having a terminal lysine, e.g., a constant region sequence having a terminal lysine, can be understood as the corresponding sequence without the terminal lysine, and that a sequence without a terminal lysine can also be understood as the corresponding sequence with a terminal lysine. EXAMPLES

[0305] Example 1: Generation of anti-human CD27 antibodies and their Fc variants The generation of anti-human CD27 antibodies through immunization and hybridoma generation was performed at Aldevron GmbH (Freiburg, Germany). cDNA encoding human CD27 (full length and ECD) was cloned into Aldevron's proprietary expression plasmids. Anti-CD27 antibodies were generated by immunization of OmniRat animals (transgenic rats expressing a diversified repertoire of antibodies with fully human idiotypes; Ligand Pharmaceuticals Inc.) using intradermal application of human CD27 cDNA-coated gold particles using a handheld device for particle bombardment ("gene gun"). Serum samples were collected after the immunization series and tested by flow cytometry in HEK cells transiently transfected with the above-mentioned expression plasmid for full-length human CD27 expression. Antibody-producing cells were isolated from rat spleens and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from hybridomas producing CD27-specific antibodies was extracted for sequencing.

[0306] From the panel of 71 CD27 antibodies, six antibodies were selected for further characterization based on diversity in binding to primary T cells and in in vitro CD27 binding competition assays. These six antibodies are designated herein as IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E and IgG1-CD27-F.

[0307] The variable regions, which may have single point mutations to remove amino acid residues that were deemed disadvantageous for production of the heavy and light chains of interest (e.g., free cysteines or glycosylation sites), were gene synthesized and cloned into expression vectors containing framework sequences for a human antibody light chain and a human IgG1 heavy chain.

[0308] Six different antibody Fc variants were generated by introduction of one or more of the following amino acid mutations according to Eu numbering: E345R, E430G, P329R, G237A, K326A, E333A (see Tables 3 and 5 below). After in vitro functional characterization as described below, the CD27-specific IgG1-CD27-A appeared to have the most optimal biological properties. The sequences of CD27-targeting antibodies of the prior art used herein as benchmarks were obtained as follows: IgG1-CD27-15 (WO2012004367; SEQ ID Nos 3 and 4), IgG1-CD27-131A (WO2018 / 058022; SEQ ID Nos 10 and 15), IgG1-CD27-CDX1127 (WO2016145085; SEQ ID Nos: 1 and 2), and IgG1-CD27-BMS986215 (WO2019195452A1; SEQ ID Nos 8 and 9). The VH and VL sequences of type I anti-human CD20 antibodies have been previously described in WO2019 / 145455A1 (SEQ ID Nos 35 and 39).

[0309] Table 3: List of amino acid sequences TIFF2024533234000004.tif248156TIFF2024533234000005.tif238156TIFF2024533234000006.tif238156TIFF20245332340 00007.tif238156TIFF2024533234000008.tif238156TIFF2024533234000009.tif238156TIFF2024533234000010.tif202156

[0310] Example 2: Agonistic activity of anti-CD27 antibodies in a CD27 activation reporter cell assay The CD27 agonist activity of different anti-CD27 antibodies with and without the E345R or E430G hexamerization-enhancing Fc mutations was measured using the CD27 Thaw and Use Bioassay kit (Promega, Custom Assay Services, CAS#CS1979A25). The kit contains NF-κB reporter-Jurkat recombinant cells expressing the firefly luciferase gene under the control of NF-κB response elements along with constitutive expression of human CD27, which was used essentially according to the manufacturer's instructions. Briefly, Thaw-and-Use GloResponse NFκB-luc2 / CD27 cells were thawed and incubated with a dilution series of antibodies (final concentration range 0.04–20 μg / mL) in Bio-Glo Luciferase Assay Buffer in 96-well flat-bottom culture plates (PerkinElmer, catalog #6005680) for 6 h at 37 °C, 5% CO2. The anti-CD27 antibody was wild-type (WT *) IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, IgG1-CD27-D, IgG1-CD27-E, IgG1-CD27-F, and their respective variants harboring E430G or E345R mutations. The anti-CD27 benchmark antibodies were IgG1-CD27-131A (WT and E430G variants) and non-hexamerizing IgG1-CD27-15 (IgG1-CD27-15-P329R-E345R-K439E; harboring a combination of Fc mutations that prevent hexamerization, so the mutations are not functionally relevant in the context of this experiment and are therefore referred to as WT in the figures) as well as the hexamerizing variant of IgG1-CD27-15 containing the E345R mutation. Anti-HIV gp120 human antibody, IgG1-b12-E345R, was used as a non-binding negative control antibody (control). After antibody incubation, Bio-Glo Luciferase Assay Reagent (equilibrated to RT) was added to each well and incubated at RT for 5-10 min. Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer) and presented as relative luminescence units (RLU) in a bar graph generated using GraphPad Prism software.

[0311] Introduction of hexamerization-enhancing Fc mutations (E345R or E430G) resulted in enhanced CD27 agonism for antibody clones IgG1-CD27-A to -E as well as the benchmark antibodies IgG1-CD27-131A (tested with E430G) and IgG1-CD27-15 (tested with E345R) compared to the corresponding WT antibodies (Figure 1).

[0312] IgG-CD27-A, B and C demonstrated enhanced CD27 agonist activity after introduction of E430G or E345R at all concentrations tested, whereas IgG1-CD27-D and E variants containing hexamerization enhancing mutations showed no increase in agonism at the lowest antibody concentrations. IgG1-CD27-F variants with E430G or E345R mutations showed enhanced CD27 agonism only at the highest antibody concentrations tested. For variants IgG1-CD27-A to -E, introduction of the E345R mutation resulted in stronger CD27 activation than the E430G mutation. Antibodies IgG1-CD27-A to -E with the E345R mutation showed higher or similar CD27 activation levels compared to IgG1-CD27-131A with the E430G mutation or CD27-15 with the E345R mutation, respectively. * The WT antibodies for IgG1-CD27-B and IgG1-CD27-F had the F405L mutation in the IgG Fc domain, which is not functionally relevant in the context of this experiment.

[0313] Example 3: Binding affinity of anti-human CD27 antibodies for recombinant human, mouse and cynomolgus CD27 The binding affinity of five anti-human CD27 IgG1 antibodies (IgG1-CD27-A, -B, -C, -D and -E) for recombinant human, cynomolgus and mouse CD27 proteins was determined using label-free biolayer interferometry on an Octet HTX instrument (ForteBio, Portsmouth, UK). Experiments were performed using bispecific antibodies that contain one CD27-specific Fab arm and a non-binding Fab arm such that the antibody is monovalent for CD27. These bispecific antibodies were generated by controlled Fab arm exchange between CD27 and non-binding antibodies (as described in Labrijn AF et al., Nat Protoc. 2014 Oct;9(10):2450-63).

[0314] To determine the affinity of CD27 antibodies for human and mouse CD27, 100 nM of recombinant His-tagged mouse or human CD27 protein (Sino Biological, Catalog #10039-H08B1 [human], Catalog #50110-M08H [mouse]) was loaded onto a preconditioned anti-Penta-HIS (HIS1K) biosensor (ForteBio, Catalog #18-5120) for 600 seconds.

[0315] To evaluate the affinity of CD27 antibodies for cynomolgus CD27, 5 μg / mL of recombinant cynomolgus CD27-Fc fusion protein (R&D systems, catalog #9904-CD-100) was loaded onto an activated Amine Reactive 2nd Generation (AR2G) biosensor (ForteBio, catalog #18-5092).

[0316] After a 300 second baseline measurement in Sample Diluent (ForteBio, Cat #18-1104), CD27 antibody association (200 seconds) and dissociation (1,000 seconds) were determined for an antibody concentration series from 0.78 to 800 nM with 2-fold dilution steps in Sample Diluent. An antibody molecular mass of 150 kDa was used for calculations. The reference sensor was incubated with Sample Diluent.

[0317] Data were acquired using Data Acquisition Software v11.1.1.19 (ForteBio) and analyzed using Data Analysis Software v9.0.0.14 (ForteBio). Data traces were corrected for each antibody by subtraction of the reference sensor. The Y-axis was aligned to the last 10 seconds of baseline, and Interstep Correction alignment for dissociation and Savitzky-Golay filtering were applied. Data traces were excluded from analysis if the response was <0.05 nM and the calculated equilibrium was close to saturation (Req / Rmax>95% using a dissociation time of 50 seconds). Data were fitted with a 1:1 model with the window of interest for the association set at 200 seconds and the dissociation time set at 50 seconds. Dissociation times were calculated using the coefficient of determination (R 2 ) (preferentially >0.98), were selected based on visual inspection of the curves and a signal decay of at least 5% during the association step.

[0318] The affinity for human CD27 was determined using a K in the nanomolar range. D The K values ​​could be accurately determined for the three CD27 antibodies (IgG1-CD27-A, B, C) along with their K values ​​(Table 4). For IgG1-CD27-D and -E, biolayer interferometry experiments confirmed binding to human CD27 with similar ranges of affinities, although the exact K values ​​could not be determined due to suboptimal curve fitting. D It was not possible to calculate values ​​(as indicated in Table 4).

[0319] IgG1-CD27-A and -B also have the same K range as human CD27. D Results with IgG1-CD27-C, -D and -E also confirmed binding to cynomolgus CD27 with a similar range of affinities, although the exact K values ​​were unclear due to suboptimal curve fitting. D It was not possible to calculate values ​​(as indicated in Table 4).

[0320] Binding to recombinant mouse CD27 was only observed for the antibody IgG1-CD27-C.

[0321] Table 4. Binding affinities of IgG1-CD27-A to -E antibodies to CD27 from the indicated species TIFF2024533234000011.tif69156 * : Binding was observed, but KD, k on and k dis is a low confidence value due to suboptimal curve fitting resulting in unreliable interpretation using the 1:1 model. Note: No binding was observed.

[0322] Example 4: Binding of anti-CD27 antibodies to cell surface expressed human and cynomolgus CD27 Anti-CD27 antibodies IgG1-CD27-A to -E against human and cynomolgus monkey CD27 expressed on the cell surface * and IgG1-CD27-131A of the prior art * Binding of was analyzed by flow cytometry using transiently transfected HEK293F cells and primary T cells that endogenously express CD27. The non-binding control antibody IgG1-b12-FEAR was used as a negative control antibody.

[0323] FreeStyle 293-F suspension cells (HEK293F; ThermoFisher, catalog #R79007) were transiently transfected with the mammalian expression vector pSB encoding full-length human or cynomolgus CD27 using 293fectin Transfection Reagent (ThermoFisher, catalog #12347019) according to the manufacturer's instructions.

[0324] Human and cynomolgus monkey PBMCs were purified from buffy coats obtained from healthy human donors (Sanquin Blood Bank, the Netherlands) or cynomolgus monkeys (BPRC, the Netherlands, catalog #S-1135) by low-density gradient centrifugation using Lymphocyte Separation Medium (LSM; Corning, catalog #25-072CV) according to the manufacturer's instructions.

[0325] Cells were seeded in 96-well plates (100,000 cells / well; Greiner Bio-one, catalog #650180) for sequential incubations with intervening washing steps using FACS buffer consisting of PBS (Lonza, catalog #BE17-517Q) + 1% BSA (Roche, catalog #10735086001) + 0.02% sodium azide (Bio-World, catalog #41920044-3). The following incubations were applied: antibody concentration series (final concentrations of 0.0001-10 μg / mL) for 30 min at 4 °C; live / dead marker FVS510 (BD, Cat #564406, dilution of 1:1,000 in PBS) for 20 min at RT; PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, Cat #109-116-098, dilution of 1:500) for 30 min at 4 °C; and anti-CD3 antibodies for T cell identification (anti-human CD3: BD, Cat #555335, dilution of 1:10; anti-cynomolgus CD3: Miltenyi, Cat #130-091-998, dilution of 1:10) for 30 min at 4 °C. All samples were analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism.

[0326] All tested antibodies showed dose-dependent binding to human CD27 in both human T cells and transfected HEK293F cells (Figure 2A, Figure 2B). The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-C, compared to intermediate binding for IgG1-CD27-A and IgG1-CD27-131A, and low binding for IgG1-CD27-D and IgG1-CD27-E, with the difference being most pronounced using human T cells. For binding to cynomolgus monkey CD27 T cells, the highest binding was observed for IgG1-CD27-B, followed by Ig1-CD27-131A and IgG1-CD27-A. Lower binding was observed for IgG1-CD27-D and -E, while IgG1-CD27-C showed the least binding to cynomolgus monkey T cells. All CD27 antibodies showed dose-dependent binding to cynomolgus CD27-transfected HEK cells. The highest maximal binding was observed for IgG1-CD27-B and IgG1-CD27-131-A, and somewhat lower binding was observed for IgG1-CD27-A, -D, and -E. IgG1-CD27-C showed the lowest binding to cynomolgus CD27-transfected HEK cells (Figure 2C, Figure 2D).

[0327] In conclusion, IgG1-CD27-A and IgG1-CD27-B showed dose-dependent binding to human and cynomolgus CD27 expressed endogenously on human or cynomolgus T cells and transiently expressed in transfected HEK cells. IgG1-CD27-A and IgG-CD27-131A showed comparable binding to human T cells, while IgG1-CD27-B showed higher maximal binding. *Note: IgG1-CD27-A, -B, -C, -D and -E had the mutations F405L-L234F-L235E-D265A in the IgG Fc domain that are not functionally relevant in the context of this experiment. IgG1-CD27-131A had the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0328] Example 5: Binding of anti-CD27 antibodies to the native human CD27-A59T variant Approximately 19% of the human population expresses a naturally occurring CD27 variant harboring the A59T mutation in the extracellular domain (SEQ ID NO. 2). Binding to human CD27-A59T was confirmed by the anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, and IgG1-CD27-D. * and benchmark IgG1-CD27-131A were tested by flow cytometry. The non-binding antibody IgG1-b12-FEAL was used as a negative control antibody. Transiently transfected HEK293F cells (15,000 cells / well) expressing human CD27-A59T were incubated with the primary test antibodies IgG1-CD27-A to -C, the non-binding control antibody IgG1-b12 (control), and a concentration series (0.0001 to 10 μg / mL using 10-fold dilution steps) of the prior art benchmark IgG-CD27-131A, which has previously been described (WO2018 / 058022) to bind to CD27-A59T. After incubation, the antibodies were PE-labeled using a polyclonal goat anti-human IgG. Binding was analyzed on a FACSCelesta flow cytometer (BD) and FlowJo software. Data were processed and visualized using GraphPad Prism v.8.

[0329] The tested anti-CD27 antibodies IgG1-CD27-A, IgG1-CD27-B, IgG1-CD27-C, and IgG1-CD27-131A showed dose-dependent binding to CD27-A59T transfected HEK293F cells with similar binding curves between the different antibodies (Figure Example 5). *Note: IgG1-CD27-A, -B and -C had the mutations F405L-L234F-L235E-D265A in the IgG Fc domain that are not functionally relevant in the context of this experiment. IgG1-CD27-131A had the functionally irrelevant F405L mutation in the IgG1 Fc domain.

[0330] Example 6: Induction of human T cell proliferation by anti-CD27 antibodies Because enhancing IgG hexamerization through Fc-Fc interactions with the introduction of the E345R or E430G mutation enhanced the CD27 agonist activity of anti-CD27 antibodies (Example 2), the ability of IgG1-CD27-A, IgG1-CD27-B, and IgG1-CD27-C antibody variants carrying the E430G or E345R mutation to increase proliferation of TCR-activated T cells was tested in vitro.

[0331] Additionally, Fc mutations reported to reduce binding to C1q and FcgR (G237A or P329R) or enhance binding to C1q (K326A / E333A double mutation) were introduced to test their potential effect on the CD27 agonist activity of CD27 antibodies with E345R or E430G mutations. The K326A / E333A double mutation was previously shown to contribute to enhancing C1q binding and enhancing the agonist activity of a DR5-specific humanized IgG1 antibody containing Fc-Fc interaction enhancing mutations (WO2018 / 146317A1). In addition to E430G or E345R, the mutations G237A, P329R, or K326A / E333A were introduced into IgG1-CD27-A, IgG1-CD27-B and IgG1-C (Table 5) and their effects on T cell proliferation were determined using human PBMCs obtained from healthy donors (Sanquin Blood Bank, the Netherlands).

[0332] Table 5. Mutations in the Fc domain of antibodies IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C and their biological effects TIFF2024533234000012.tif68156 * X in IgG1-CD27-X refers to the IgG1-CD27 clones IgG1-CD27-A, IgG1-CD27-B, or IgG1-CD27-C.

[0333] PBMCs, 5 x 10 in PBS 6 The PBMCs were resuspended at a density of 100,000 cells / mL and labeled with CFSE using the CellTrace CFSE Cell Proliferation Kit (Invitrogen, Cat#C34564; 1:10,000) according to the manufacturer's instructions. CFSE-labeled PBMCs (100,000 cells / well) were incubated in 96-well round-bottom plates (Greiner Bio-one, Cat#650180) in T-cell Activation Medium (ATCC, Cat#80528190) supplemented with 5% Normal Human Serum (NHS; Sanquin, Product#B0625) with 0.1 μg / mL of anti-CD3 antibody clone UCHT1 (Stemcell Technologies, Cat#60011) to activate T cells, and CD27 antibody (final concentration of 1 μg / mL) for 96 h at 37°C / 5% CO2. CD4 by flow cytometry was analyzed. + and CD8 +For identification of viable cells in T cell subsets, cells were sequentially incubated with the live / dead marker FVS510 (1:1,000) for 20 min at RT and with a staining mix for lymphocyte markers containing APC-eFluor780-labeled anti-human CD4 antibody (Invitrogen, Cat. #47-0048-42, 1:50), AlexaFluor700-labeled anti-human CD8a antibody (BioLegend, Cat. #301028; 1:100), PE-Cy7-labeled mouse anti-human CD14 antibody (BD Biosciences, Cat. #557742; 1:50), and BV785-labeled anti-human CD19 antibody (BioLegend, Cat. #363028; 1:50) for 30 min in the dark at 4 °C. Samples were measured on a FACSCelesta (BD Biosciences) flow cytometer and viable CD4 + and CD8 + T cell subsets (FVS510 - CD14 - CD19 - CD4 + and FVS510 - CD14 - CD19 - CD8 + ) were analyzed as a readout for T cell proliferation using FlowJo 10 software. T cell proliferation was expressed as a percentage of cell proliferation or mitotic index, both calculated by using FlowJo software (version 10). The percentage of cell proliferation (division) was calculated based on the percentage of cells that underwent CFSE dilution (CFSE low peaks ) was determined by gating on the mean number of divisions a cell underwent. Mitotic index is the average number of divisions a cell underwent. Heat maps were generated using GraphPad Prism version 8. Proliferation assays were performed using PBMCs from four different healthy donors.

[0334] IgG1-CD27-A, -B and -C variants carrying the E430G or E345R mutations upregulated CD8 +Introduction of additional mutations (P329R, G237A or K326A / E333A) into the IgG1-CD27-A, -B or -C variants carrying the E430G mutation induced a small increase in CD8 T cell proliferation across four PBMC donors. + In contrast, introduction of the P329R mutation into IgG1-CD27-A and IgG1-CD27-C variants carrying the E345R mutation suppressed activation of CD8 + Consistently, they increased the ability of IgG1-CD27-A to enhance T cell proliferation, which was particularly true for IgG1-CD27-A, which increased the measured CD8 + T cell proliferation was comparable for IgG-CD27-A-E345R, IgG1-CD27-B-E345R and IgG1-CD27-C-E345R in each of the donors, while introduction of the additional P329R mutation significantly increased CD8 proliferation for clone IgG1-CD27-A-E345R compared to IgG1-CD27-B-E345R or IgG1-CD27-C-E345R. + This consistently led to higher increases in T cell proliferation. + The effect of the E345R mutation in combination with the P329R mutation on T cell proliferation was consistently greater for clone IgG1-CD27-A than for IgG1-CD27-B and IgG1-CD27-C. Across all antibody variants tested, IgG1-CD27-A-E345R-P329R significantly increased CD8 T cell proliferation in all donors. + induced the greatest increase in T cell proliferation (Figure 4A).

[0335] The addition of the mutations G237A or K326A-E333A to CD27 antibody variants with the E345R mutation significantly increased CD8 expression in any of the clones tested compared to antibodies containing the single mutation E345R. + It did not or only minimally increased T cell proliferation (Figure 4A).

[0336] CD4 +In T cells, the highest and most consistent increase in T cell proliferation was observed in the presence of IgG1-CD27-A-E345R-P329R. + T cell proliferation was generally comparable between IgG1-CD27-A, -B and -C variants carrying only the E430G or E345R mutations, whereas introduction of the additional P329R mutation significantly increased CD4 T cell proliferation for IgG1-CD27-A variants carrying the E345R variant compared to IgG1-CD27-A-E430G or IgG1-CD27-B or -C variants carrying either the E430G or E345R mutations. + This led to a greater increase in T cell proliferation. This effect was observed in three of the four donors tested. In donor 1, CD4 + The effects of additional mutations besides E430G or E345R on T cell proliferation were generally small, and the effects observed in this donor were not reproduced in the other three donors.

[0337] The combination of E345R with the P329R mutation also inhibited CD4 + Although it consistently increased T cell proliferation, the difference between the E345R mutation alone and the combination of E345R and P329R was smaller for clone IgG1-CD27-C than for clone-A. + A modest increase in T cell proliferation was observed for IgG1-CD27-B-E345R-P329R compared to IgG1-CD27-B-E345R in two of four donors.

[0338] Introduction of P329R, G327A or K326A / E333A mutations into IgG1-CD27-A, -B or -C variants carrying the E430G mutation enhances CD4 +Similarly, no or inconsistent effects were observed following introduction of G327A or K326A / E333A in IgG1-CD27-A, -B or -C variants carrying the E345R mutation.

[0339] In summary, IgG1-CD27-A-E345R-P329R inhibits activated CD8 + and CD4 + We demonstrated that IgG1-CD27-A-E345R-P329R consistently induced the highest increase in T cell proliferation and induced the most efficient CD27 agonism. DR5-specific, hexamerization-enhanced antibodies with the P329R mutation have previously shown a reduced ability to induce DR5 agonism compared to DR5-specific, hexamerization-enhanced antibodies without the P329R mutation (Overdijk et al, Mol Canc Ther 2020). Therefore, it was considered surprising that introducing the P329R mutation in addition to the E345R mutation in IgG1-CD27-A would enhance CD27 agonist activity. Furthermore, it is not known why the combined effect of the E345R+P329R mutations was consistently greater for IgG1-CD27-A than for IgG1-CD27-B or IgG1-CD27-C.

[0340] Example 7: Induction of human T cell proliferation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R TCR stimulated human CD4 + and CD8 + The ability of IgG1-CD27-A-P329R-E345R to increase T cell proliferation was analyzed in a CSFE dilution assay using human healthy donor PBMCs and compared with the prior art anti-CD27 clone IgG1-CD27-131A. * , IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 *The T cell proliferation assay was performed as described in Example 6 with minor deviations (75,000 cells / well; concentration range 0.002-10 μg / mL). Samples using T cells without anti-CD3 stimulation were included to test potential CD27 agonist activity of the antibodies in the absence of T cell receptor activation (Figure 5A and Figure 5B). Such activity is undesirable as it poses a safety risk if the antibody is able to induce proliferation of resting T cells.

[0341] Using FlowJo software, the percentage of proliferated T cells (Figure 5A, Figure 5B, Figure 5C, Figure 5D) was calculated as the percentage of cells with a reduction in CFSE fluorescence, indicative of cell division. The expansion proliferation index (Figure 5E and Figure 5F) identified the fold increase in cells in a well and was calculated using the Proliferation Modeling tool in FlowJo version 10. Manual adjustments to the peaks were made when necessary to more consistently define the number of peaks present.

[0342] Neither the CD27 antibodies of the invention nor the prior art antibodies tested here induced proliferation of unstimulated T cells, i.e., in the absence of CD3 cross-linking (FIGS. 5A and 5B).

[0343] Most CD27 antibodies were not associated with activating CD4 at the highest antibody concentrations tested. + and CD8 + The antibody IgG1-CD27-A-P329R-E345R of the present invention induced a certain degree of proliferation of T cells (FIGS. 5C and 5D). Based on this, the expansion proliferation index was calculated (FIGS. 5E and 5F). The antibody IgG1-CD27-A-P329R-E345R of the present invention showed a significantly higher in vitro CD4 T cell proliferation rate than the prior art anti-CD27 clones IgG1-CD27-131A, IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215. + and CD8 + It significantly enhanced T cell proliferation. *For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation, which are not functionally relevant in the context of this experiment, were used.

[0344] Example 8: Binding of C1q to membrane-bound CD27 antibodies The P329R mutation was previously described to reduce the interaction of IgG1 antibodies with C1q and FcgR (Overdijk et al, Molecular Cancer Therapeutics 2020). The effect of the P329R mutation on C1q binding of IgG1-CD27-A containing the E345R mutation was tested in an in vitro cellular C1q binding assay using human healthy donor T cells. The anti-HIV gp120 antibody IgG1-b12-F405L was used as a non-binding isotype control antibody (control). T cells were enriched from human healthy donor PBMCs using RosetteSep Human T cell Enrichment cocktail (Stemcell, catalog #15061) and resuspended in culture medium (RPMI 1640 [Gibco, catalog #A10491-01] supplemented with 0.1% BSA and 1% Pen / Strep [Lonza, catalog #DE17-603E]). T cells (2 × 10 6 T cells (1000 cells / well) were pre-incubated for 15 min at 37 °C in polystyrene 96-well round-bottom plates containing an antibody dilution series (eight 5-fold dilutions starting at a final assay concentration of 15 μg / mL) to allow antibody binding to T cells. Cells were then chilled on ice, supplemented with NHS as a source of human C1q (final assay concentration of 20% NHS) and incubated on ice for 45 min. Cells were subsequently incubated with FITC-labeled rabbit anti-human C1q antibody (DAKO, catalog #F0254; 20 μg / mL) for 30 min on ice and resuspended in FACS buffer containing TO-PRO-3 (ThermoFisher, catalog #T3605; 1:5,000 dilution). C1q binding was determined by flow cytometry measuring the FITC signal in live cells.

[0345] Membrane-bound WT IgG1-CD27-A antibody did not exhibit C1q binding (Figure 6). Introduction of the hexamerization-enhancing mutations E430G or E345R (IgG1-CD27-A-E430G and IgG1-CD27-A-E345R) resulted in binding of C1q to CD27 antibodies on the T cell surface (Figure 6), consistent with increased binding avidity of hexameric C1q protein to hexameric antibody ring structures on the cell surface. Introduction of the P329R mutation in IgG1-CD27-A-E345R (IgG1-CD27-A-P329R-E345R) resulted in loss of C1q binding (Figure 6), demonstrating that IgG1-CD27-A-P329R-E345R cannot bind C1q.

[0346] These data indicate that IgG1-CD27-A-P329R-E345R is unable to bind C1q upon binding to CD27 on the cell surface of T cells. This indicates that C1q binding does not contribute to the antibody-induced CD27 agonist activity of IgG1-CD27-A-P329R-E345R. This is in contrast to what has been previously described for other hexamerization-enhancing agonist antibodies. Furthermore, the lack of C1q binding indicates that IgG1-CD27-A-P329R-E345R is unable to activate the classical pathway of complement activation. Therefore, it is expected that IgG1-CD27-A-P329R-E345R will not induce complement activation and CDC in T cells, where these activities are undesirable.

[0347] Example 9: Binding of anti-CD27 antibodies to human Fc receptors Binding of IgG1-CD27-A-P329R-E345R to human FcγR variants was analyzed using a Biacore surface plasmon resonance (SPR) system and compared to anti-HIV gp120 antibody IgG1-b12 (control). Biacore Series S Sensor Chips CM5 (Cytiva, Cat#29104988) were covalently coated with anti-His antibody using an Amine-Coupling and His Capture Kit (Cytiva, Cat#BR100050 and Cat#29234602) according to the manufacturer's instructions. Next, 125 nM of Fcγ receptors FcγRIa, FcγRIIa (167-His[H] and 167-Arg[R]), FcγRIIb, or FcγRIIIa (176-Phe[F] and 176-Val[V]) (Sino Biological, Catalog #10256-H08S-B, Catalog #10374-H27H, Catalog #10374-H27H1-B, Catalog #10259-H27H-B, Catalog #10389-H27H-B, and Catalog #10389-H27H1-B) in HBS-P+ (Cytiva, Catalog #BR100827) were captured onto the surface. After three cycles of buffer, antibody samples were injected over 36 cycles to generate binding curves using an antibody range of 0-3,000 nM for FcγRI and 0-10,000 nM for other FcγRs. Each sample analyzed on the FcR-coated surface (active surface) was also analyzed on a parallel flow cell without FcR (reference surface) that was used for background correction. Dissociation from the anti-His-coated surface was performed by regeneration of the surface using 10 mM glycine-HCl (pH 1.5) (Cytiva, catalog #BR100354). Sensorgrams were generated using Biacore Insight Evaluation software (Cytiva) and a four-parameter logistic (4PL) fit was applied to calculate the relative binding of IgG1-CD27-A-P329R-E345R to the reference sample (control).

[0348] Binding of IgG1-CD27-A-P329R-E345R to the high affinity receptor FcγRIa was strongly reduced compared to the control antibody, although some binding was observed at higher antibody concentrations (Figure 7A). IgG1-CD27-A-P329R-E345R did not bind to the human low affinity receptors FcγRIIa (Figures 7B and 7C), FcγRIIb (Figure 7D) and FcγRIIIa (Figures 7E and 7F).

[0349] In conclusion, IgG1-CD27A-P329R-E345R shows minimal or no binding to human IgG Fc receptors (FcγRIa) or (FcγRIIa, FcγRIIb, and FcγRIIIa).

[0350] Example 10: Binding of anti-CD27 antibody IgG1-CD27-A-E345R-P329R to human T cells Flow cytometry was used to characterize the binding of IgG1-CD27-A-P329R-E345R to CD27 on human healthy donor T cells in more detail. The anti-HIV gp120 antibody variant IgG1-b12-P329R-E345R was used as a non-binding control antibody (control). Human PBMCs were isolated from buffy coats obtained from human healthy donors. PBMCs (1 × 10 5Cells were pelleted by centrifugation at 300 x g for 3 min at 4 °C. Cells were resuspended in 50 μL / well of serial antibody dilutions in FACS buffer (ranging from 0.0015 to 10 μg / mL in 3-fold dilution steps) and incubated for 30 min at 4 °C. Cells were pelleted, washed twice with FACS buffer, and incubated with FITC-conjugated secondary antibody (FITC AffiniPure F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific, Jackson ImmunoResearch, catalog #109-096-097, diluted 1:100) in 50 μL / well for 30 min in the dark at 4 °C. Cells were pelleted again, washed twice with FACS buffer, and stained with BV711-conjugated anti-human CD19 antibody (BioLegend, catalog #302246, 1:50), AlexaFluor700-conjugated anti-human CD8a antibody (BioLegend, catalog #301028, 1:100), APC-eFluor780-conjugated anti-human CD4 antibody (Invitrogen, catalog #47-0048-42, 1:50), PE-CF594-conjugated mouse anti-human CD56 antibody (BD Biosciences, catalog #564849, 1:100), and PE-Cy7-conjugated mouse anti-human CD14 antibody (BD The cells were incubated in 50 μL / well of staining mix for lymphocyte markers containing 50 μL / well of 50-μL ...Binding curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 8 software.

[0351] The anti-CD27 antibody IgG1-CD27-A-P329R-E345R inhibits CD4 + and CD8 + It showed dose-dependent binding to healthy donor T cells with similar binding characteristics for T cells (Figure 8).

[0352] Example 11: FcγR-independent induction of CD27 cell signaling by anti-CD27 antibody IgG1-CD27-A-P329R-E345R CD27-specific monoclonal antibodies that can induce CD27 signaling independently of secondary FcγR-mediated cross-linking may be immunostimulatory in the absence of FcγR-positive cells, which is an advantage in tumors with a low frequency of FcγR-bearing cells.

[0353] The CD27 agonist activity of IgG1-CD27-A-P329R-E345R was tested in the presence or absence of FcγR-bearing cells and compared with the corresponding WT antibody IgG1-CD27-A as well as the prior art antibody IgG1-CD27-131A. * , IgG1-CD27-CDX1127, and IgG1-CD27-BMS986215 * The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control (control). CD27 reporter assays were performed essentially as described in Example 2, with the exception that in this example, Thaw-and-Use GloResponse NFκB-luc2 / CD27 Jurkat cells were cultured in the presence of human FcyRIIb expressing cells that can facilitate FcgR-mediated cross-linking of membrane-bound antibodies.

[0354] Thaw-and-Use effector FcγRIIb CHO-K1 cells (Promega, Cat#JA2251) were plated undiluted or in three increasing dilutions (1 / 3, 1 / 9, 1 / 27) in 96-well flat-bottom culture plates (PerkinElmer, Cat#0815) and incubated overnight at 37°C / 5% CO2. Supernatants of adherent FcyRIIb-expressing cells were replaced by Thaw-and-Use NFκB-luc2 / CD27 Jurkat cell suspensions at fixed cell concentrations (starting with a 1:1 NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio for undiluted FcγRIIb CHO-K1 cells) in Bio-Glo Luciferase Assay Buffer containing serial dilutions of antibody (final concentration range 0.0002-10 μg / mL). After 6 h incubation at 37° C. / 5% CO 2 , plates were equilibrated to RT and bioluminescence was measured and presented as RLU as described in Example 2.

[0355] IgG1-CD27-A-P329R-E345R induced dose-dependent CD27 activation independent of FcγRIIb-expressing cells (Figure 9A). In contrast, the corresponding WT antibody IgG1-CD27-A, which does not have the E345R hexamerization-enhancing mutation and the P329R mutation, showed CD27 agonism only in the presence of FcγRIIb-expressing cells (Figures 9A-E). Similarly, CD27 activation by the prior art antibodies IgG1-CD27-131A, IgG1-CD27-CDX1127 and IgG1-CD27-BMS986215 was also dependent on the presence of FcγRIIb-expressing cells and gradually decreased with decreasing NFκB-luc2 / CD27 Jurkat:FcγRIIb CHO-K1 ratio (Figures 9F-J).

[0356] In conclusion, these data indicate that IgG1-CD27-A-P329R-E345R can induce CD27 agonism independent of secondary FcγR-mediated cross-linking, in contrast to prior art anti-CD27 antibodies that were dependent on the presence of FcγR-bearing cells to induce CD27 agonism. * For IgG1-CD27-131A and IgG1-CD27-BMS986215, variants with the F405L mutation, which are not functionally relevant in the context of this experiment, were used.

[0357] Example 12: Pharmacokinetic (PK) analysis of anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding studied in mice Anti-CD27 antibody IgG1-CD27-A-P329R-E345R in the absence of target binding * The pharmacokinetic characteristics of the corresponding WT antibody IgG1-CD27-A were analyzed in mice. *IgG1-CD27-A does not bind to mouse CD27 (Example 3, Table 4), therefore, experiments were designed to test the pharmacokinetic behavior of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R in vivo in the absence of target binding. The study was carried out by Crown Bioscience (China) by qualified personnel in accordance with the approved IACUC protocol and Crown Bioscience, Inc. Standard Operating Procedures. Eleven to 12 week old female SCID mice (CB-17, Vital River Laboratory Animal Technology Co., Ltd. (VR, Beijing, China; 3 mice / group) were intravenously injected with 500 μg of antibody (25 mg / kg) in an injection volume of 200 μL. 40 μL blood samples were collected at 10 min, 4 h, 1 day, 2 days, 7 days, 14 days, and 21 days after antibody administration, and plasma was collected from the blood samples and stored at -80 °C until determination of total human IgG concentrations by ELISA. 96-well ELISA plates (Greiner, Cat #655092) were filled with 2 μg / mL of anti-human IgG (Sanquin, The The anti-human IgG-coated plates were then coated overnight at 4° C. with ELISA kit (Roche, Netherlands, product #M9105, lot #8000260395) and subsequently blocked for 1 h with PBSA (PBS supplemented with 0.2% bovine serum albumin [BSA, Roche, catalog #10735086001]). The anti-human IgG-coated plates were then incubated at RT for 1 h on a plate shaker with an intermediate washing step for 1 h. Plasma samples serially diluted in Buffer (PBSA supplemented with 0.05% Tween 20 [Sigma-Aldrich, Cat. #P1379]) were sequentially incubated with a polyclonal peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson, Cat. #109-035-098) for 1 h at RT, and finally with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Cat. #11112422001). The reaction was stopped by adding 2% oxalic acid (Riedel de Haen, Cat. #33506).A reference curve was generated using a dilution series of each material used for injection. The absorbance was measured at 405 nm in an EL808 microtiter plate reader (BioSPX) and the total human IgG concentration (μg / mL) was plotted.

[0358] There were no substantial differences between the PK profiles of IgG1-CD27-A-P329R-E345R and the counterpart WT antibody IgG1-CD27-A, as determined by measuring plasma IgG levels at different time points after intravenous injection in mice (Figure 10).

[0359] A steeper decline in the early (distribution) phase was observed for IgG1-CD27-A-P329R-E345R and its WT counterpart (IgG1-CD27-A) compared to that predicted for human IgG1 in mice, but the terminal clearance of both antibodies was consistent with the predicted kinetics for human wild-type IgG1 based on a two-compartment model (Bleeker WK, Teeling JL, Hack CE. Blood. 2001 Nov 15;98(10):3136-42).

[0360] Together, this demonstrates that the introduction of the P329R and E345R mutations did not affect the pharmacokinetic properties of IgG1-CD27-A in the absence of target binding. Note: The experiments described in this example used variants of IgG1-CD27-A and IgG1-CD27-A-P329R-E345R with the F405L mutation, which is not functionally relevant in the context of this experiment.

[0361] Example 13: Induction of antibody-dependent cellular phagocytosis by anti-CD27 antibody IgG1-CD27-A-P329R-E345R Antibody-dependent cellular cytotoxicity (ADCC) is mediated primarily through FcγRIIIa expressed on NK cells, whereas antibody-dependent cellular phagocytosis (ADCP) can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRI, FcγRIIa, and FcγRIII (Hayes, JM et al. 2016). To understand the effect of the residual binding of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to FcγRIa (Example 9) on the effector function of FcγRIa-expressing immune cells, we used CTV-labeled CD27 + The ability of IgG1-CD27-A-P329R-E345R to induce ADCP was analyzed in vitro using Burkitt's lymphoma Daudi cells as target cells and human monocyte-derived macrophages (hMDMs) as effector cells (E:T=2:1).

[0362] hMDMs were isolated from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotec, Cat. No. 130-050-201) according to the manufacturer's instructions. PBMCs were centrifuged (1,200 RPM, 5 min, RT) and diluted to 1.25 × 10 in ice-cold monocyte isolation buffer (PBS, 0.5% BSA, 2 mM EDTA). 7 PBMCs were resuspended at a density of 1000 ng / mL. 20 μL of CD14 microbeads were added per 80 μL of PBMC suspension and incubated with agitation for 15 min at 4°C on a roller bank. 30 mL of ice-cold monocyte isolation buffer was added and the PBMC / CD14 microbead mixture was centrifuged (300×g, 10 min, 4°C) and resuspended in 6 mL of ice-cold monocyte isolation buffer. LS columns (Miltenyi Biotec, Cat. No. 130-042-401) were rinsed with 3 mL of ice-cold monocyte isolation buffer and 3 mL of the PBMC / CD14 microbead mixture was loaded onto each column. CD14 - After draining the cells and washing the column three times with ice-cold monocyte isolation buffer, CD14 +Monocytes were harvested in 3 mL of ice-cold monocyte isolation buffer. CD14 cells were detected using ViaStain™ Viability Dye Acridine Orange / Propidium Iodide (AOPI; Nexcelom Bioscience, Cat. No. CS2-0106) on a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience). + 100 mm 200 µm plate with UpCell™ Surface to count cells and allow cell harvesting by placing plates at room temperature 2 Macrophage colony-stimulating factor (M-CSF; Gibco, catalog no. PH9501; final concentration of 50 ng / mL) and 3 mL of monocyte suspension (i.e., 2.4 × 10 6 monocytes) in Celgene® GMP DC medium (CellGenix, catalog no. 20801-0500) supplemented with 0.8 × 10 6 Macrophages were resuspended at a density of 1 × 10 cells / mL. After 3 days of incubation, 2 mL of fresh medium containing 5× M-CSF was added to the plate. After 7 days of incubation (37 °C, 5% CO2), macrophages were detached from the surface by leaving the plate at RT for 1–1.5 h. Detached macrophages were pelleted by centrifugation, counted using an AOPI, and resuspended in culture medium (RPMI 1640 with 10% DBSI) at a density of 1 × 10 6 The cells were resuspended at a density of 1000 cells / mL.

[0363] Human Burkitt's lymphoma Daudi cells (ATCC® CCL-213™) were labeled using the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific, Cat. No. C34557) according to the manufacturer's instructions. Briefly, Cell Trace Violet (CTV) was added at 1 × 10 per mL in PBS to a final concentration of 0.2 μM. 6Daudi cells and incubated for 20 min at 37°C in the dark (15 mL incubation volume). 10 mL of DBSI was added to inactivate unbound dye. Cells were pelleted by centrifugation (300 × g, 5 min), washed in PBS, and counted in an AOPI. CTV-labeled Daudi cells were added to culture medium at 0.5 × 10 6 The cells were resuspended at a density of 1000 cells / mL.

[0364] For the ADCP assay, hMDMs (50,000 cells / well) and CTV-labeled Daudi cells (25,000 cells / well) were seeded together (E:T=2:1) ​​in a final volume of 150 μL of culture medium in 96-well plates on ice and incubated for 4 h (37 °C, 5% CO2) with anti-CD27 antibody IgG1-CD27-A-P329R-E345R or anti-CD20 antibody IgG1-CD20 (concentration range of 0.000001-10 μg / mL in 10-fold dilutions). After incubation, 100 μL of Human BD Fc Block™ (BD Biosciences, catalog no. 564220; 1:100 in FACS buffer) was added and incubated for 10 min at 4 °C. Cells were pelleted by centrifugation (300×g, 5 min), resuspended in FACS buffer containing PE-Cy7 conjugated anti-human CD11b antibody (BioLegend, Cat. No. 301322; 1:80) and TO-PRO-3 (Thermo Fisher Scientific, Cat. No. T3605; 1:25,000) and incubated at 4° C. for 30 min. Cells were washed, resuspended in FACS buffer, collected and analyzed on a FACSymphony™ A3 Cell Analyzer (BD Biosciences). Data was analyzed using FlowJo software to measure viable target cell numbers and phagocytic hMDMs, and processed and visualized using GraphPad Prism software.

[0365] The percentage of viable Daudi cells for each condition was calculated by the following formula: TIFF2024533234000013.tif16128

[0366] The amount of phagocytic hMDM for each condition was determined by TO-PRO-3 - CD11b + CTV + Determined as % of cells.

[0367] IgG1-CD27-A-P329R-E345R did not increase the percentage of phagocytic hMDMs or reduce the percentage of viable Daudi cells in phagocytosis assays using hMDMs from four different human healthy donors, demonstrating that residual FcγRIa binding did not result in FcγRIa-mediated effector function for IgG1-CD27-A-P329R-E345R (data from a representative human healthy donor is shown in FIG. 11). The positive control antibody IgG1-CD20 efficiently induced phagocytosis of Daudi cells expressing high levels of CD20, as demonstrated by an increase in the percentage of phagocytic hMDMs and a decrease in the percentage of viable Daudi cells.

[0368] In conclusion, residual binding to FcγRIa is consistent with CD27 + was not sufficient to induce IgG1-CD27-A-P329R-E345R-dependent ADCP of cells.

[0369] Example 14: Target-independent fluid-phase complement activation by anti-CD27 antibody IgG1-CD27-A-P329R-E345R as determined by measuring C4d deposition Antibodies with enhanced Fc-Fc interactions generally exist as monomeric IgG1 molecules in solution, which upon target binding hexamerize on the cell surface to form a C1q docking site in the case of the active Fc region (Diebolder, CA et al. 2014; de Jong, RN et al., 2016). The IgG Fc domain of the anti-CD27 antibody IgG1-CD27-A-P329R-E345R was silenced by the introduction of the P329R mutation, which results in the lack of C1q binding for membrane-bound IgG1-CD27-A-P329R-E345R (Figure 6). To confirm that IgG1-CD27-A-P329R-E345R is unable to activate complement in solution in the absence of target binding, target-independent complement activation was examined by the determination of C4d deposition, which is considered an indicator for activation of the classical complement pathway. Fluid-phase C4d fragment deposition by IgG1-CD27-A-P329R-E345R was analyzed by enzyme-linked immunosorbent assay (ELISA) using MicroVue™ C4d Enzyme Immunoassay (EIA; Quidel, Cat. No. A008) and was performed according to the manufacturer's protocol. Heat Aggregated Gamma Globulin (HAGG; complement activator; Quidel, Cat. No. A114) was used as a positive control for the assay. IgG1-b12 and IgG1-b12-RGY (WO2014006217A1) were included as control antibodies. Introduction of E345R / E430G / S440Y (RGY) Fc mutations in IgG1 antibodies has been described to induce the formation of hexamers in solution, resulting in fluid-phase complement activation (Diebolder, CA et al., 2014; Wang, G., RN et al., 2016; de Jong, RN et al., 2016). IgG1-b12-P329R-E345R was included as an isotype control antibody.

[0370] Antibody dilutions were prepared in phosphate-buffered saline (PBS) to a concentration of 1 mg / mL, except for HAGG, which was diluted to a concentration of 10 mg / mL. Test samples were then further diluted in 90% (final concentration) normal human serum (NHS) (CompTech, Lot No. 42a) to a concentration of 100 μg / mL (monoclonal IgG) or 1,000 μg / mL (HAGG) and incubated for 1 h at 37 °C. In parallel, a "No Antibody" sample (no antibody, 90% NHS) and a "PBS Alone" sample (no antibody, no NHS) were included as negative controls. Samples were then diluted 1:250 in chilled Complement Specimen Diluent provided in the kit. Meanwhile, mouse anti-human C4d antibody-coated strips were placed into a 96-well plate and the assay wells were washed three times with 250–300 μL of wash buffer with a 1 min waiting step after the first wash. Test samples were added to wells (100 μL / well) and, as a negative control, only complement sample diluent (blank) was used in the ELISA. In parallel, 100 μL of standard (standard AE) and internal control provided by the kit were added to separate wells. The plate was incubated for 30 min at RT. Then, the plate was washed 5 times with the washing buffer described above. 50 μL of C4d conjugate (peroxidase-conjugated goat anti-human C4d) was added to the wells and the plate was incubated for 30 min at RT. After 5 washing steps with the washing buffer described above, 100 μL of C4d substrate [0.7% 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid diammonium salt] was added and the plate was again incubated for 30 min at RT. Finally, 50 μL of stop solution provided by the kit was added and within 1 h, the optical density was measured at 405 nm using an ELISA plate reader (EL808 BioSPX, BioTek).

[0371] IgG1-CD27-A-P329R-E345R and the control antibody IgG1-b12-P329R-E345R (with the same Fc backbone as IgG1-CD27-A-P329R-E345R) did not induce fluid-phase C4d deposition at the tested concentration of 100 μg / mL; the measured C4d levels were similar to the background levels of a control antibody with a wild-type Fc domain (IgG1-b12) and a no-antibody control (FIG. 12). In contrast, the positive control antibody IgG1-b12-RGY, known to form hexamers in solution, induced C4d deposition to the same level as HAGG.

[0372] These data indicate that IgG1-CD27-A-P329R-E345R did not induce target-independent, fluid-phase complement activation in vitro.

[0373] Example 15: Ability of anti-CD27 antibody IgG1-CD27-A-P329R-E345R to compete for ligand binding with CD70 To determine whether the anti-CD27 antibody IgG1-CD27-A-P329R-E345R interferes with the interaction of CD27 with its natural ligand CD70, binding of saturating concentrations of biotinylated recombinant human CD70 extracellular domain (ECD) to CD27 endogenously expressed on the human Burkitt's lymphoma cell line Daudi was studied in the presence and absence of excess IgG1-CD27-A-P329R-E345R.

[0374] Daudi cells (ATCC® CCL-213™) cultured in RPMI 1640 medium (Gibco, Cat. No. A10491-01) supplemented with 10% donor bovine serum with iron (DBSI; Gibco, Cat. No. 20731-030) were seeded at 50,000 cells / well in round-bottom 96-well plates (Greiner Bio One, Cat. No. 650261). Cells were pelleted by centrifugation (300×g, 3 min, 4° C.) and resuspended in FACS buffer (PBS, 1% BSA [Roche, Cat. No. 1073508600]) containing anti-CD27 or control antibodies (final concentration of 50 μg / mL). Biotinylated recombinant human CD70 ECD (Abcam, Cat. No. ab271443) was added at saturating concentration (6 μg / mL) and cells were incubated at 4° C. for 30 min.

[0375] Cells were washed twice and resuspended in FACS buffer containing Brilliant Violet (BV) 421™-labeled streptavidin (BioLegend, Cat. No. 405225; 0.0025 μg / mL final concentration) and R-Phycoerythrin (PE)-labeled polyclonal AffiniPure F(ab')2 fragment goat-anti-human IgG Fc (Jackson ImmunoResearch, Cat. No. 109 116098; 0.0025 μg / mL final concentration) for 30 minutes at 4°C. Cells were washed twice and resuspended in FACS buffer containing TO-PRO-3 iodide (Thermo Fisher Scientific, Cat. No. T3605; 1:25,000) and analyzed. Data were collected on a BD FACSymphony™ A3 flow cytometer (BD Biosciences) and analyzed using FlowJo software. For compensation, one drop of UltraComp eBeads™ Compensation Beads (Life Technologies, Cat. No. 01-2222-42) was added to each well. 2 μL of each antibody was added and the mix was incubated for 20 minutes. The plates were spun down and the beads were resuspended in FACS buffer and measured. For viability compensation, cells were treated at 65° C. for 10 minutes and mixed 1:1 with viable cells. Cells were spun down and resuspended in TO-PRO-3 diluted in FACS buffer. Data was processed and visualized using GraphPad Prism.

[0376] IgG1-CD27-A-P329R-E345R or IgG1-CD27-A inhibits CD27 +It did not block binding of CD70 ECD to Daudi cells, and CD70 binding levels were comparable to Daudi cells incubated with non-binding isotype control antibodies IgG1-b12-P329R-E345R or IgG1-b12, or cells without antibody (Figure 13). Prior art anti-CD27 antibodies IgG1-CD27-BMS986215 and IgG1-CD27-131A also showed a weak blocking effect on CD27 binding to CD70 ECD. In contrast, CD70 was unable to bind to surface CD27 on Daudi cells in the presence of prior art anti-CD27 antibody IgG1-CD27-CDX1127, previously reported to block ligand binding (Vitale et al., 2012) (Figure 13).

[0377] In conclusion, binding of IgG1-CD27-A-P329R-E345R does not block binding of CD27 by its natural ligand CD70 on Daudi cells.

[0378] Example 16: Expression of T cell activation markers upon incubation of polyclonally stimulated human PBMCs with anti-CD27 antibodies The effect of IgG1-CD27-A-P329R-E345R on the expression of T cell activation markers in polyclonally activated T cells was studied using PBMCs obtained from three different healthy human donors. The expression of HLA-DR, CD25, CD107a, and 4-1BB was analyzed after 2 and 5 days of incubation of PBMCs with IgG1-CD27-A-P329R-E345R or a prior art anti-CD27 antibody.

[0379] 75,000 freshly isolated PBMCs were seeded per well in cell culture medium in 96-well U-bottom plates (Greiner Bio-One). Duplicate wells were incubated simultaneously with anti-CD3 antibodies (UCHT1 clone; Stemcell; 0.1 μg / mL); and IgG1-CD27-A-P329R-E345R (0.0005 to 30 μg / mL in 3-fold dilutions); or prior art anti-CD27 antibodies IgG1-CD27-CDX1127, IgG1-CD27-131A, and IgG1-CD27-BMS986215 (30 μg / mL); or non-binding control antibody IgG1-b12-P329R-E345R (10 μg / mL). To determine the expression of each activation marker in the absence of treatment, duplicate control wells containing untreated (no anti-CD3 or anti-CD27 antibodies) cells were supplemented with culture medium alone. A fluorescence minus one (FMO) control was used to set the gates to identify activation marker-positive cells. For the FMO control, all antibodies used in the experiment except those corresponding to the activation markers in duplicate wells were added to 75,000 PBMCs per well from one donor activated with anti-CD3 antibodies. Untreated cells from each donor in a single well without staining antibody were included as negative controls. To detect viable cells, untreated cells from each donor were stained with 4',6-diamidino-2-phenylindole (DAPI) alone in a single well.

[0380] After 2 or 5 days of incubation (37°C, 5% CO2), plates were washed once with FACS buffer and analyzed by flow cytometry with antibodies for T cell activation markers 4-1BB, CD25, CD107a, human leukocyte antigen (HLA)-DR; as well as CD4 + and CD8 +After 30 min incubation at 4°C, all plates were washed twice with FACS buffer and cells were resuspended in FACS buffer. Samples were analyzed on a BD LSRFortessa Cell Analyzer using FlowJo software to identify CD4 + and CD8 + The median fluorescence intensity (MFI) and percentage of positive cells for each T cell activation marker on T cells were determined. Anti-CD27 antibody-induced changes in the expression levels of T cell activation markers were presented as the fold change in MFI of anti-CD27 antibody samples compared to the non-binding control antibody IgG1-b12-P329R-E345R. Samples were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences) using FlowJo software.

[0381] IgG1-CD27-A-P329R-E345R inhibits activated CD4 + Increased expression of CD25, CD107a and 4-1BB on T cells (Figure 14A). These effects were more pronounced after 2 days of incubation than after 5 days of incubation. CD8 + On T cells, incubation with IgG1-CD27-A-P329R-E345R resulted in increased expression of HLA-DR, CD107a and 4-1BB after both 2 and 5 days of incubation (Figure 14B).

[0382] The expression of T cell activation markers was also evaluated after 2 and 5 days of incubation with the three prior art antibodies. IgG1-CD27-131A and IgG1-CD27-BMS986215 inhibited CD4 + and CD8 +Although it induced comparable increases in HLA-DR, 4-1BB, CD25, and CD107a expression on T cells, the effect of incubation with IgG1-CD27-CDX1127 for 2 or 5 days on T cell activation marker expression was weaker.

[0383] In conclusion, incubation of polyclonally activated PBMCs with IgG1-CD27-A-P329R-E345R significantly inhibited CD4 + and CD8 + This resulted in increased expression of activation markers HLA-DR, CD25, CD107a and 4-1BB on T cells.

[0384] Example 17: OVA-specific CD8 in OVA protein-immunized mice after injection of anti-CD27 antibody in human CD27-KI mouse model + T cell percentage The effect of IgG1-CD27-A-P329R-E345R treatment on the expansion of antigen-specific T cells in the hCD27 KI OVA model in splenocytes was analyzed by flow cytometry.

[0385] Homozygous human CD27 (hCD27)-KI mice on a C57BL / 6 background (hCD27 KI mice) were obtained from Beijing Biocytogen Co., Ltd. (strain name C57BL / 6-Cd27tm1(CD27) / Bcgen, stock number 110006). This strain was developed in collaboration with Crown Bioscience's HuGEMM™ platform and featured a humanized drug target (CD27 in this case) in mice with a functional immune system. In the hCD27 KI mice, exons 1-5 of the mouse CD27 gene, which encodes the extracellular domain, were replaced by human CD27 exons 1-5. OVA-specific T cells were induced in vivo in hCD27-KI mice by subcutaneous (sc) injection of the immunogen ovalbumin (OVA) and the agonistic effect of IgG1-CD27-A-P329R-E345R was tested by simultaneously treating the mice with the antibody intravenously (iv).

[0386] On day 0, mice were injected sc with 5 mg OVA (InvivoGen, Cat. No. vac-pova-100, Lot No. EFP-42-04) and treated by iv tail vein injection with IgG1-CD27-A-P329R-E345R (30 mg / kg), IgG1-CD27-CDX1127 (30 mg / kg) or IgG1-b12-P329R-E345R (30 mg / kg). On days 12 and 21, mice were boosted with OVA and treated with the same antibodies as on day 0. On days 10, 19 and 24, blood was collected via the cheek pouch or saphenous vein into BD Microtainer® blood collection tubes containing dipotassium ethylenediaminetetraacetate (K2-EDTA; BD, Cat. No. 365974) and used immediately in further analysis. On day 28, mice were euthanized and spleens were removed under aseptic conditions.

[0387] Excised splenic tissue in RPMI1640 medium (Thermo Fisher Scientific, Cat. No. C22400500BT) was transferred to gentleMACs™ C Tubes (Miltenyi Biotec, Cat. No. 130-093-237) and mechanically dissociated into a single cell suspension using a gentleMACS™ Dissociator (Miltenyi, Cat. No. 130-093-235) according to the manufacturer's instructions. After dissociation, the cell suspension was filtered through a 70 μm cell strainer (Falcon, Cat. No. 352350). Samples were then washed twice by resuspension in 3 mL of wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, Cat. No. 10099 141]). Cells were counted with a Cellometer Auto T4 (Nexcelom Bioscience) and the number of cells was determined to be 2 × 10 per tube. 6 The cells were adjusted to 100 spleen cells.

[0388] 2×10 6Spleen cells were transferred to FACS tubes (Falcon, Cat. No. 352052) and resuspended in wash buffer (sterile PBS [Hyclone, SH0256.01B] supplemented with 4% FBS [Gibco, Cat. No. 10099 141]) supplemented with 1 μg / mL purified rat anti-mouse CD16 / CD32 (Mouse BD Fc Block™, BD Biosciences, Cat. No. 553141). After 10 min preincubation in the dark at 2-8 °C, 10 μL of PE-labeled OVA tetramer (MBL Life science, Cat. No. TS 5001 1C) was added and the samples were gently vortexed before further incubation for 30-60 min in the dark at 2-8 °C. Without washing, labeled antibodies and compounds used for flow cytometry gating of T cell subsets were added. Samples were gently vortexed and incubated for an additional 30 min in the dark at 2–8 °C. Samples were then washed twice by resuspension in 2 mL of wash buffer and centrifuged at 300 × g for 5 min. Finally, cells were resuspended in 250 μL of wash buffer and analyzed on a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences). Data were processed using Kaluza Analysis Software (Beckman Coulter).

[0389] IgG1-CD27-A-P329R-E345R increased OVA-specific CD8 in the blood and spleen of mice co-injected with OVA protein vaccination. + Increased the percentage of OVA-specific CD8 T cells in mice treated with 30 mg / kg IgG1-CD27-CDX1127 + The percentage of T cells was lower than in the IgG1-CD27-A-P329R-E345R-treated group and similar to the IgG1-b12-P329R-E345R-treated group (Figure 15).

[0390] Example 18: OVA-specific CD8 from spleens of OVA-immunized mice injected with anti-CD27 antibody + IFNγ secretion by T cells Excised spleen tissue (see Example 17) in RPMI1640 medium was gently mashed through a 70 μm cell strainer (Falcon, Cat. No. 352350), pelleted by centrifugation (1,500 rpm, 5 min), and resuspended in 10 mL of Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Invitrogen, Cat. No. A1049201). After 3-5 min of incubation at RT, samples were washed twice with 10-20 mL of PBS and resuspended in 5 mL of Cellular Technology Limited (CTL) Test™ Medium (ImmunoSpot, Cat. No. CTLT-005) supplemented with 50 U / mL penicillin and 50 μg / mL streptomycin (pen / strep, Gibco, Cat. No. 15070-063). The harvested spleen cells were again filtered through a 70 μm cell strainer and counted on a Vi-CELL™ XR Cell Viability Analyzer (Beckman Coulter) to obtain a total of 3.125 × 10 6 The concentration was adjusted to cells / mL.

[0391] IFNγ production by splenocytes was analyzed using the Mouse IFN-γ ELISpotPLUS kit (Mabtech, Cat. No. 3321-4HPW-2) essentially as described by the manufacturer. Pre-coated MultiScreenHTS IP Filter (MSIP) white plates (mAb AN18) were washed 4 times with 200 μL of sterile PBS per well and conditioned with 200 μL of CTL-Test Medium containing pen / strep (RT, 30 min). The medium was removed and 5 × 10 cells were plated per well. 5 10 spleen cells were inoculated with 2 μg / mL OVA in duplicate. 257-264Plates were incubated with peptide SIINFEKL (Invivogen, Cat. No. vac-sin), or scrambled control peptide FILKSINE (SB-PEPTIDE, Cat. No. SB073-1MG) in a total volume of 180 μL / well for 20 h in a humidified incubator (37 °C, 5% CO2). As a positive control for IFNγ production, spleen cells were incubated in parallel with a cell stimulation cocktail consisting of 500 ng / mL phorbol myristate acetate (PMA) and 10 μg / mL ionomycin (PMA + ionomycin, Dakewe Biotech, Cat. No. DKW ST PI). Cultures of spleen cells without peptide were included as negative controls. After incubation, cells were removed and plates were washed 5 times with PBS. Plates were then sequentially incubated with biotinylated detection mAb (R4-6A2; RT, 2 h), streptavidin-horseradish peroxidase (HRP; RT, 1 h), and finally 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution (all provided by the kit), with five washing steps with PBS in between. When distinct spots appeared, the reaction was stopped by extensive washing in deionized water. Spots were counted on an AID iSpot ELISpot Reader (Autoimmun Diagnostika [AID] GMBH, ELR08IFL) using spotAID V8 software (AID). ELISpot data were analyzed using GraphPad Prism software and presented in bar graphs and as the mean number of spots per well ± SEM from all mice (n = 5) per treatment group.

[0392] As demonstrated by ELISpot analysis, spleen cells from all IgG1-CD27-A-P329R-E345R-treated animal groups showed increased IFNγ production in response to treatment with OVA peptide (Figure 16). Stimulation of spleen cells with a scrambled control peptide did not induce IFNγ production or induced minimal IFNγ production, suggesting that IFNγ was produced by OVA-specific T cells. In contrast, IFNγ production was not observed in spleen cells from mice treated with 30 mg / kg IgG1-CD27-CDX1127.

[0393] Example 19: Effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation in OVA-immunized mice in vivo CD8 + The effect of IgG1-CD27-A-P329R-E345R treatment on T cell activation was measured using CD8 + In vivo studies were performed by analyzing the expression of PD-1 on T cells. Mice were treated as described in Example 17. Methods for obtaining splenocytes and analyzing them by FACS are also described in Example 17.

[0394] IgG1-CD27-A-P329R-E345R inhibited CD8 + Induced an increase in the percentage of CD8 T cells. + PD-1 + The percentage of T cells was lower in animals treated with IgG1-CD27-CDX1127 or the control antibody IgG1-b12-P329R-E345R (Figure 17).

[0395] Example 20: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo induction of T cell subsets in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on the expansion of T cell subsets was studied by analyzing the expression of CD44 and CD62L in splenocyte samples from OVA-treated hCD27-KI mice. Memory CD8 T cells derived from the spleens of IgG1-CD27-A-P329R-E345R-treated, OVA-immunized, hCD27-KI mice were + T cells were quantified by flow cytometry. Memory T cells were classified as effector memory (CD44 + CD62L - ) and pre-effector T cells (CD44 - CD62L - ;Nakajima,Y.,K et al 2018). Mice were treated as described in Example 17. Methods for obtaining spleen cells and analyzing them by FACS are also described in Example 17.

[0396] IgG1-CD27-A-P329R-E345R (30 mg / kg) significantly increased pre-effector T cells and effector memory CD8 T cells in the spleen at day 28 compared to spleen cells from mice treated with IgG1-b12-P329R-E345R. + Induced an increase in the percentage of CD45 T cells (Figure 18). + Within the population, IgG1-CD27-A-P329R-E345R induced a higher percentage of pre-effector and effector memory T cells than IgG1-CD27-CDX1127 (30 mg / kg), but similar mean percentages of these T cell populations were downregulated by CD8 + In the fractions, both anti-CD27 antibodies induced

[0397] Example 21: Effect of IgG1-CD27-A-P329R-E345R treatment on in vivo expansion of T cells in OVA-immunized mice The effect of IgG1-CD27-A-P329R-E345R on T cell expansion was studied by analyzing the expression of CD3 in spleen cells and blood samples from OVA-treated hCD27-KI mice. Mice were treated as described in Example 17. Methods for obtaining spleen cells and blood samples and analyzing them by flow cytometry are also described in Example 17.

[0398] Treatment of OVA-immunized hCD27-KI mice with 30 mg / kg IgG1-CD27-A-P329R-E345R significantly increased CD3+ expression in the spleen compared to treatment with the non-binding control antibody IgG1-b12-P329R-E345R. + In contrast, treatment with the benchmark antibody IgG1-CD27-CDX1127 (30 mg / kg) did not increase the percentage of CD3 T cells in the spleen. + This resulted in a depletion of T cells. Similar observations were made in peripheral blood samples.

[0399] Example 22: Effect of IgG1-CD27-A-P329R-E345R on T cell cytokine production in antigen-specific studies The ability of IgG1-CD27-A-P329R-E345R to increase cytokine production was studied using T cells stimulated with cognate antigen.PBMCs were isolated from buffy coats obtained from healthy human donors by Ficoll-Paque density gradient separation (GE Healthcare, Cat. No. 17 1440 03) according to the manufacturer's instructions.

[0400] Human magnetic CD14 and CD8 microbeads (Miltenyi Biotec, catalog numbers 130 050 201 and 130 045 201, respectively) were used to isolate CD14 from human PBMCs. + Positive selection of monocytes and CD14 - Negative selection of PBLs and CD8 from frozen PBLs +The cell suspension was centrifuged and sorted into magnetic activated cell sorting (MACS) buffer (Dulbecco's phosphate-buffered saline [DPBS] containing 5 μM EDTA and 0.2% human albumin) at 1 × 10 per 80 μL of MACS buffer. 7 Viable cells were resuspended at 1 x 10 7 Twelve μL of CD14 or CD8 microbeads were added per cell. Subsequent MACS separation was performed using an automated magnetic cell separation instrument or by manual separation. Automated MACS separation was performed using an autoMACS® Pro Separator (Miltenyi Biotec) according to the manufacturer's instructions. Eluted CD14 + Monocytes and CD8 + T cells were centrifuged (8 min, 300 × g, RT), resuspended in X-VIVO 15 medium (Lonza) and used for further use with Erythrosin B solution; i.e., monocyte differentiation into iDCs or CD8+ cells with PD-1 and / or CLDN6 specific T cell receptor (TCR) mRNA. + T cells were counted for electroporation.

[0401] For generation of monocyte-derived iDCs, up to 40 x 10 6 PBMC-derived CD14 +Monocytes were cultured in T175 flasks for 5 days (37°C, 5% CO2) in DC medium (RPMI 1640, 5% pooled human serum [PHS; One Lambda, catalog number A25761], 1x minimum essential medium non-essential amino acid solution [MEM NEAA, Life Technologies, catalog number 11140 035], 1 mM sodium pyruvate [Life Technologies, catalog number 11360 039]) supplemented with 100 ng / mL human granulocyte / macrophage colony-stimulating factor (GM-CSF; Miltenyi Biotec, catalog number 130-093-868) and 50 ng / mL human IL-4 (Miltenyi Biotec, catalog number 130093 924). After 3 days of culture, half of the medium per flask was replaced. Non-adherent monocytes in the medium obtained from the flask were pelleted (8 min, 300×g, RT), resuspended in fresh DC medium supplemented with 200 ng / mL GM-CSF and 100 ng / mL IL-4, and then transferred back to the original flask. After 5 days of incubation, iDCs that had adhered to the culture flask were detached using 10 mL of DPBS containing 2 mM EDTA (37° C., 10 min). The isolated iDCs were washed, pelleted (8 min, 300×g, RT), and used for electroporation with CLDN6 mRNA.

[0402] Human CD8 + T cells were electroporated with RNA encoding the alpha and beta chains of the mouse TCR specific for human CLDN6, either alone or together with RNA encoding PD-1, and human monocyte-derived iDCs were electroporated with RNA encoding human CLDN6. 6 pcs iDC or 15 x 10 6 CD8 +T cells were electroporated in 250 μL of X-VIVO 15 medium at RT using an ECM 830 Square Wave Electroporation System (BTX®). Cells were mixed with RNA, pulsed (500V, 3ms for T cells or 300V, 12ms for iDCs) and immediately diluted in 750 μL of pre-warmed assay medium (IMDM GlutaMAX with 5% PHS [Life technologies, Cat. No. 31980030]). Electroporated iDCs were transferred to 6- or 12-well plates and cultured O / N (37°C, 5% CO2). After O / N incubation, electroporated CD8 + T cells and iDCs were assessed by flow cytometry to determine cell purity, expression of transfected RNA (CD8 + PD-1 and CLDN6 on T cells (TCR and CLDN6 on iDCs), and CD8 + Baseline expression of CD27 and PD-1 on T cells and PD-L1 on iDCs was assessed. + Approximately 78%–93%, 78%–92%, and 36%–98% of T cells expressed CLDN6-TCR, PD-1, and endogenous CD27, respectively. Approximately 47%–91% and 94%–99% of electroporated iDCs expressed CLDN6 and endogenous PD-L1, respectively (not shown).

[0403] CD8 + T cells and iDCs were cultured at a ratio of 10:1 (7.5 × 10 per well). 4 7.5 x 10 T cells and 3iDCs were seeded in 96-well round-bottom plates with 100 μL of each antibody. IgG1-CD27-A-P329R-E345R was diluted in assay medium and 25 μL of diluted IgG1-CD27-A-P329R-E345R was added to the wells to reach a final concentration of 10 μg / mL. Similarly, control antibodies IgG1-CD27-131A and IgG1-b12-P329R-E345R were added to reach a final concentration of 10 μg / mL. Antigen-specific T cell activity upon antibody treatment was analyzed in vitro by measuring cytokines in the supernatants of T cells transduced to express CLDN6-TCR co-cultured with iDCs transduced to express and present CLDN6. Supernatants were collected 2 days later, and concentrations of multiple proinflammatory cytokines and chemokines were determined by multiplex electrochemiluminescence assay (ECLIA) using the 10-spot U-PLEX ImmunoOncology Group 1 (human) kit (MSD; catalog number K151AEL 2) according to the manufacturer's instructions.

[0404] For the 10-spot U-PLEX Immuno-Oncology Group 1 kit, biotinylated capture antibodies were pre-incubated with the assigned linker with a biotin-binding domain for 30 min at RT, followed by 30 min incubation with stop solution. The plate was coated with a mix of linker-linked capture antibodies by incubating for 1 hr at RT with shaking. The plate was washed 3 times with 1x MSD wash buffer. Supernatant samples or kit standards were diluted 1:2 in assay diluent, added to the wells and incubated for 2 h at RT with constant shaking. The plate was washed 3 times with wash buffer and incubated with SULFO-TAG conjugated detection antibody from the kit for 1 h at RT with constant shaking. After washing the plate 3 times with wash buffer, lead buffer B was added to catalyze the electrochemiluminescence reaction. The plate was analyzed immediately by measuring the light intensity on a MESO QuickPlex SQ 120 imager (MSD).

[0405] CD8 after 2 days of incubation+ IgG1-CD27-A-P329R-E345R-induced changes in cytokine production were assessed by multiplex ECLIA in supernatants from T cell / iDC co-cultures (n=4 different donors). IgG1-CD27-A-P329R-E345R inhibited CD8 + CD8 using T cells + induced a significant increase in the production of GM-CSF and IFN-γ in T cell / iDC cocultures (Figure 20A), while increases in IL-13 and TNFα production were also observed. A considerable increase for the same cytokines was observed in cultures containing PD-1-overexpressing T cells (Figure 20B). Although cytokine levels generally decreased when T cells overexpressed PD-1, the relative increase (fold increase) in cytokine production in the presence of IgG1-CD27-A-P329R-E345R was highest in this setting (Figures 20A and 20B). In contrast, the prior art anti-CD27 antibody IgG1-CD27-131A showed minimal effects on cytokine production compared to the non-binding control antibody IgG1-b12-P329R-E345R (Figures 20A and 20B).

[0406] Example 23: Antigen-specific CD8 incubated with IgG1-CD27-A-P329R-E345R + Expression of cytotoxicity-associated molecules by T cells The induction of T cell-mediated cytotoxicity upon antibody treatment was studied by analyzing the expression of cytotoxicity-associated molecules on antigen-specific T cells by flow cytometry in cocultures of human healthy donor T cells transduced to express the CLDN6-TCR and MDA-MB-231_hCLDN6 target cells.

[0407] MDA-MB-231_hCLDN6 cells were generated by lentiviral transduction. For this purpose, 2 × 10 cells were cultured in 250 µL of Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, catalog number 31966-047) supplemented with 10% FBS (non-heat inactivated). 5MDA-MB-231 cells were seeded per well in a 12-well tissue culture plate. The cells were incubated at 37 °C (7.5% CO2) for 1–2 h. The supernatant containing the lentiviral vector encoding human CLDN6 (pL64b42E(EF1a-hClaudin6)Hygro-T2A-GFP) was thawed on ice and diluted in a total volume of 750 µL of DMEM / 10% FBS to give 2 × 10 5 , 8×10 4 , and 3.2 × 10 4 Titers in TU / mL were obtained. These titers corresponded to MOIs of 1, 0.4, and 0.16, respectively. The supernatants were then added to MDA-MB-231 cells and the cells were incubated for 72 h at 37° C. (5% CO2) without disturbance. For the experiments described in this example, MDA-MB-231-hCLDN6 cells were cultured in DMEM / 10% FBS. Cells were passaged and harvested for experiments at 70%-90% confluence. Cells were detached by treatment with Accutase (Thermo Fisher Scientific, Cat. No. A11105010) for 5 min (37° C., 7.5% CO2) and resuspended by addition of culture medium. Cells were centrifuged (300×g, 4 min, RT) and counted. MDA-MB-231_hCLDN6 cells were not cultured for more than 20 passages.

[0408] 1.2–1.5 × 10 MDA-MB-231_hCLDN6 cells 4 Cells / well were seeded into 96-well flat-bottom plates (for flow cytometry analysis) and xCELLigence E-plates (Agilent, Cat. No. 05232368001; for impedance measurements) and left at RT for 30 min. Plates were then incubated for 1 day in an incubator and xCELLigence real-time cell analysis (RTCA) instrument (ACEA Biosciences), respectively (37°C, 5% CO2).

[0409] Isolated CD8 +T cells (see Example 22) were electroporated with CLDN6-specific TCR mRNA and incubated O / N. CD8 + Following T cell isolation and electroporation, T cell cultures were 49%-99% CD8 + These electroporated CD8 T cells were + Among T cells, approximately 78% to 93% express CLDN6-TCR. + CD8 + 59%-98% of cells are CD27 + The cells were centrifuged (8 min, 300 × g, RT), resuspended in DMEM / 10% FBS, and counted. The cells were centrifuged again and 3 × 10 6 The cells were resuspended in DMEM / 10% FBS at 1000 cells / mL and added to wells containing previously seeded MDA-MB-231_hCLDN6 cells (1.5 × 10 5 CD8 + T cells / well; T cell:tumor cell ratio, effector:target ratio was 10:1). IgG1-CD27-A-P329R-E345R, IgG1-CD27-131A, and the non-binding control antibody IgG1-b12-P329R-E345R were added to the co-cultures at 10 μg / mL. Expression of CD107a and GzmB was determined by flow cytometry.

[0410] After 2 days of incubation in the presence of 10 μg / mL IgG1-CD27-A-P329R-E345R, GzmB + CD107a + CD8 + The percentage of T cells was significantly enhanced compared to treatment with a non-binding control antibody or the prior art anti-CD27 antibody IgG1-CD27-131A (Figure 21).

[0411] In conclusion, these data show that IgG1-CD27-A-P329R-E345R was able to induce cytotoxicity-associated molecules on activated antigen-specific T cells.

[0412] Example 24: Ability of IgG1-CD27-A-P329R-E345R to induce T cell-mediated tumor cytotoxicity To assess T cell-mediated cytotoxicity, electroporate CD8 CLDN6-TCR + T cells were co-cultured with MDA-MB-231_hCLDN6 cells in the presence of IgG1-CD27-A-P329R-E345R, the prior art anti-CD27 antibody IgG1-CD27-131A, or the non-binding control antibody IgG1-b12-P329R-E345R for 5 days with impedance measurements at 2-hour intervals as described in Example 23 in an xCELLigence real-time cell analysis instrument (Acea Biosciences). Cell index values ​​were derived from impedance measurements performed at 2-hour intervals. Areas under the curve (AUC) were obtained from cell index data over 5 days of co-culture. AUC was normalized to co-cultures treated with IgG1-b12-P329R-E345R. The magnitude of impedance depends on cell number, cell morphology, and cell size as well as the strength of cell adhesion to the plate, which are both used in this particular case as an indirect readout of tumor cell mass. The decrease in impedance in this experimental setting indicates that CD8 + It is considered a surrogate for tumor cell killing by T cells. It should be noted that impedance may underestimate tumor cell killing due to T cell proliferation.

[0413] IgG1-CD27-A-P329R-E345R induced a decrease in the cell index, an indicator of tumor cell killing. IgG1-CD27-131A had no visible effect on the cell index, indicating a minimal ability to increase tumor cell killing (FIG. 22).

[0414] Example 25: Ability of IgG1-CD27-A-P329R-E345R to induce expansion of tumor-infiltrating lymphocytes Tumor-infiltrating lymphocyte (TIL) subsets (CD4 + and CD8 +The ability of IgG1-CD27-A-P329R-E345R to induce expansion of T cells, NK cells, and regulatory T cells (Tregs) was evaluated ex vivo using cryopreserved tumors surgically resected from NSCLC patients.

[0415] Surgically resected human NSCLC tissue was received in transport medium (HypoThermosol® FRS Preservation Solution [BioLife Solutions, Catalog No. 101104], 7.5 μg / mL amphotericin B [Thermo Fisher Scientific, Catalog No. 15290026], and 300 units / mL (U / mL) pen / strep [Thermo Fisher Scientific, Catalog No. 15140-122]). Samples were washed three times with wash medium (5 mL of X-VIVO 15 [Lonza], 2.5 μg / mL amphotericin B [Thermo Fisher Scientific], and 100 U / mL pen / strep [Thermo Fisher Scientific]) and transferred to a cell culture dish. Adipose tissue and necrotic compartments were removed with a scalpel, and tissue was cut into approximately 5 mm 3 The tissue was cut into 100 x 100 pieces. Each piece was placed into an individual cryovial and 1 mL of freezing medium (FBS, 10% DMSO) was added to each vial. The vials were transferred to a controlled freezing chamber (Mr. Frosty freezing container) placed in a -80°C freezer. After at least 16 h at -80°C, the vials were transferred to liquid nitrogen for long-term storage.

[0416] Approximately 5 mm from one tumor specimen 3 Four to six cryopreserved vials containing tumor fragments were thawed for each experiment in a 37 °C water bath for approximately 2 min, washed five times with washing medium, and transferred to a cell culture dish. The tumor fragments were cut into pieces with a scalpel to approximately 1 mm 3 The tissue was further dissected into 100x fragments. Most of the fragments were used for TIL expansion in culture with IL-2 and treatment antibodies, and the remaining fragments were used to determine the expression of specific cell surface markers at baseline without any treatment.

[0417] Two tumor fragments (on average) per well were seeded in 0.1 mL of pre-warmed TIL culture medium (X-VIVO 15 [Lonza] containing 2% human serum albumin [HSA; CSL Behring, catalog number PZN-00504775], 100 U / mL pen / strep [Thermo Fisher Scientific], and 2.5 μg / mL amphotericin B [Thermo Fisher Scientific]) in 24-well plates (a total volume volume of 2 mL / well was used in the assay). IgG1-CD27-A-P329R-E345R was diluted in TIL culture medium containing 45-50 U / mL IL-2 and 900 μL of this dilution was added to the appropriate wells. The final IgG1-CD27-A-P329R-E345R concentration in the wells was 1 or 10 μg / mL. As a control, medium containing 45-50 U / mL IL-2 without antibody was added to tumor fragments in separate wells. A total of 8-16 wells were incubated for each experimental condition (37°C, 5% CO2) per donor.

[0418] After 3 days of culture, fresh TIL culture medium containing 45-50 U / mL IL-2 and IgG1-CD27-A-P329R-E345R was added to the wells (1 mL / well, same antibody concentrations as above). 5-14 / 17 days after the start of the assay, cultures were routinely monitored by microscopy for proliferation of TILs that had migrated from the tissue fragments and for the formation of TIL microclusters. If more than 25 TIL microclusters were observed in one well after 7 or 8 days of culture, cells and tissue fragments from two identically treated original wells were resuspended and pooled in one well of a 6-well plate containing culture medium (a total volumetric volume of 5-6 mL / well was used in the assay) and fresh IL-2-containing TIL culture medium was added (estimated final IL-2 concentration of 33 U / mL).

[0419] Cultures were replenished with fresh IL-2-containing TIL culture medium every 2-3 days. The IL-2 concentration in the medium added to the cultures was either reduced to 10 U / mL or initially to 25 U / mL and then to 10 U / mL, and wells were replenished with medium throughout the assay. On days 14 or 17, cells were harvested for flow cytometry analysis.

[0420] IgG1-CD27-A-P329R-E345R enhanced the expansion of TIL subtypes compared with control cultures treated with IL-2 alone, with the greatest relative increase in cell counts in CD8 + This was observed for T cells and Tregs, followed by CD4 + For all TIL subsets, expansion was more pronounced with IgG1-CD27-A-P329R-E345R at 1 μg / mL than with 10 μg / mL (Table 6 and FIG. 23).

[0421] Table 6. Fold expansion of IgG1-CD27-A-P329R-E345R-treated TILs Tumor tissues derived from human NSCLC specimens were cultured with low dose IL-2 in the presence or absence of IgG1-CD27-A-P329R-E345R. Absolute cell counts of the indicated cell subsets were determined by flow cytometry after 14-17 days of treatment. Fold difference in cell numbers for IgG1-CD27-A-P329R-E345R treated cultures compared to IL-2 treated cultures is shown. Data shown are from five tumor tissues from individual patients tested in five independent experiments. P=0.0236, 1 μg / mL vs. 10 μg / mL IgG1-CD27-A-P329R-E345R (two-way ANOVA). TIFF2024533234000014.tif68156 a Calculation of the mean and SD excluded patient #561 for better comparability between cell populations. Abbreviations: ANOVA = analysis of variance; nd = not determined; NK = natural killer; NSCLC = non-small cell lung cancer; SD = standard deviation; TIL = tumor-infiltrating lymphocytes; Treg = regulatory T cells.

[0422] Example 26: BRET analysis to assess the intermolecular interactions of IgG1-CD27-A-P329R-E345R molecules on the cell surface The ability of a CD27 antibody harboring a hexamerization-enhancing mutation (E345R) that increases intermolecular Fc-Fc interactions after binding to CD27 on the cell surface was determined using bioluminescence resonance energy transfer (BRET) analysis. This molecular proximity-based assay detects protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. Energy transfer occurs only when the donor and acceptor are in close proximity (<10 nm [Wu and Brand, 1994; Dacres et al, 2012]).

[0423] First, cell surface expression of CD20 and CD37 (as positive control molecules), in addition to CD27, was determined on a human chronic myeloid leukemia cell line genetically modified to stably express human CD27, huCD27-K562, and on Daudi cells using an indirect immunofluorescence assay (QIFIKIT, Agilent Technologies, Cat. No. K0078). Cells were seeded at 100,000 cells / well and incubated with 10 μg / mL of primary antibodies (CD27: IgG1-7730-143-C102S-FEAL; CD20: IgG1-11B8-FEAR; CD37: IgG1-3009-010-FEAR). This was followed by incubation with FITC-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, Cat. No. 109-096-097) and in parallel with QIFIKIT beads coated with a defined number of antibody molecules. The number of antibody molecules per cell was determined by interpolating the measured MFI of the test sample in a soft positive curve generated by plotting the mean fluorescence intensity (MFI) of the individual bead populations against the known number of antibody molecules per bead. Samples were measured on an LSRFortessa Cell Analyzer flow cytometer (BD Biosciences) and analyzed using FlowJo software.

[0424] QiFi analysis showed moderate CD27 expression and high CD20 and CD37 expression on Daudi cells, while huCD27-K562 cells expressed high levels of CD27 but no CD20 or CD37 (Table 7).

[0425] Table 7. Cell surface expression of antibody molecules per cell TIFF2024533234000015.tif29156

[0426] BRET assays (NanoBRET™ System, Promega, Cat. No. N1661) were performed essentially according to the manufacturer's instructions. To generate NanoLuc (donor) and HaloTag (acceptor) tagged antibodies, variable light chain sequences (Table 3, sequences 37-44) bearing either NanoLuc or HaloTag were prepared by gene synthesis, cloned into appropriate expression vectors, and full-length antibodies were produced as described in Example 1. For analysis, 0.5×10 5 huCD27-K562 or Daudi cells were seeded in a total volume of 100 μL in 96-well round-bottom plates (Greiner Bio-One, Cat. No. 650101). Cells were pelleted by centrifugation (3 min, 300 × g) and resuspended in 50 μL of assay medium (Opti-MEM I [Gibco, Cat. No. 11058-021] + 4% FBS [ATCC, Cat. No. 30-2020]) containing a mixture of NanoLuc or HaloTag tagged antibody pairs at a concentration of 5 μg / mL each. Then, 50 μL of HaloTag NanoBret 618 ligand (Promega, Cat. No. G980A, 1:1000 dilution in assay medium) was added. For each antibody mixture, no-ligand control samples were prepared in parallel by adding 50 μL of medium without HaloTag NanoBret 618 ligand. Cells were incubated at 37°C in the dark for 30 minutes, washed twice with medium, and resuspended in 100 μL of assay medium without FBS. 25 μL of NanoBRET NanoGLO substrate (Promega, Cat. No. N1571, diluted 1:200 in assay medium without FBS) was added to each well. Plates were shaken for 30 s, and 120 μL of each sample was transferred to an OptiPlate (Perkin Elmer, Cat. No. 6005299). Donor emission was measured at 460 nm and acceptor emission at 618 nm using an EnVision Multilabel Reader (Perkin Elmer).

[0427] BRET is milliBRET unit (mBU) = (618nm em / 460nm em ) × 1000.

[0428] Results are reported as corrected BRET, which is corrected for background or bleed-through of donor contributions, and is calculated as mBU ligand minus mBU no ligand control.

[0429] The proximity of NanoLuc and HaloTag labeled IgG1-CD27-A-P329R-E345R antibodies after binding to CD27 on the cell surface was compared to WT IgG1-CD27-A antibodies with the same tag. IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo antibodies, containing the hexamerization-inducing E430G mutation (WO2019243636A1), were used as positive controls for proximity-induced BRET. IgG1-CD20-11B8-E430G and IgG1-CD37-37.3-E430G have previously been shown to form heterohexamers on binding to cells expressing CD20 and CD37 using a molecular proximity assay (Oostindie, SC et al, Haematologica, 2019). The non-binding antibody IgG1-b12-P329R-E345R was used as a negative control.

[0430] As positive and negative controls for BRET signal induction, Daudi cells (high CD20 and CD37 expression) and huCD27-K562 cells (no CD20 and CD37 expression) were opsonized with the antibody pairs IgG1-CD20-11B8-E430G-LNLuc and IgG1-CD37-37.3-E430G-LHalo. BRET induction was only detected in Daudi cells, but not in huCD27-K562 cells lacking CD20 and CD37 (Figure 24). Similarly, the non-binding control antibody pair (IgG1-b12-P329R-E345R-LNLuc+IgG1-b12-P329R-E345R-LHalo) did not induce BRET in either cell line. When huCD27-K562 cells were opsonized with a mixture of NanoLuc and HaloTag-labeled CD27 antibodies with hexamerization-enhancing mutations (IgG1-CD27-A-P329R-E345R-LNLuc+IgG1-CD27-A-P329R-E345R-LHalo), high BRET was detected, but BRET in Daudi cells did not exceed background levels (Figure 24). The mixture of IgG1-CD27-A-LNLuc and IgG1-CD27-A-LHalo (WT) antibodies induced significantly lower BRET in huCD27-K562 cells and no BRET in Daudi cells compared to CD27 antibodies with P329R and E345R mutations. These results indicate that BRET signals were associated with higher target expression. CD27 expression on huCD27-K562 cells was found to be approximately 26-fold higher than on Daudi cells, and BRET levels for CD27-binding IgG1-CD27-A-P329R-E345R on huCD27-K562 cells were approximately 24-fold higher than in Daudi cells.Mixtures of NanoLuc and HaloTag-labeled non-binding and CD27-binding antibody pairs (IgG1-b12-P329R-E345R-LNLuc+IgG1-CD27-A-P329R-E345R-LHalo, and IgG1-CD27-A-P329R-E345R-LNLuc+IgG1-b12-P329R-E345R-LHalo, respectively) did not induce BRET in either cell line, confirming that the observed BRET was dependent on the simultaneous interaction of donor and acceptor antibodies bound to cell surface targets.

[0431] In summary, IgG1-CD27-A-P329R-E345R induced higher BRET in huCD27-K562 cells compared to its WT variant. This finding supports enhanced proximity between membrane-bound IgG1-CD27-A-P329R-E345R molecules compared to its WT variant, consistent with E345R-enhanced Fc-Fc interactions between cell surface-bound antibodies. Note: The experiments described in this example used a variant of IgG1-CD27-A with the F405L mutation, which is not functionally relevant in the context of this experiment.

[0432] Example 27: FcγRIa + Binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages In Example 9, surface plasmon resonance (SPR) was used to assess the binding of IgG1-CD27-A-P329R-E345R to human FcγR variants and showed minimal binding (FcγRIa) or no binding (FcγRIIa, FcγRIIb, and FcγRIIIa) to recombinant human IgG Fc receptor molecules. This residual FcγRIa binding was observed in CD27 +However, this was not sufficient to induce IgG1-CD27-A-P329R-E345R-dependent ADCP of cells (see Example 13). To further exclude the interaction of IgG1-CD27-A-P329R-E345R with FcγRIa-positive macrophages, the Fc-mediated binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages was determined.

[0433] Human CD14 as described in Example 13 + Monocytes were isolated from PBMCs from two healthy donors and differentiated into monocyte-derived macrophages by culturing the cells in medium (CellGenix, Cat. No. 20801-0500) supplemented with 50 ng / mL M-CSF (Gibco, Cat. No. PHC9501) to obtain M0 macrophages, or 50 ng / mL GM-CSF (Immunotools, Cat. No. 11343125) for differentiation into M1 macrophages. After 6 days of culture, M0 and M1 phenotypes were confirmed by FACS analysis according to the expression of markers defined in Table 8. Additionally, both macrophage subtypes were confirmed to express human Fc receptors FcγRIa, FcγRII and FcγRIIIa (Table 8).

[0434] (Table 8) TIFF2024533234000016.tif62156

[0435] Binding of IgG1-CD27-A-P329R-E345R to M0 and M1 macrophages was compared to that of a WT IgG1 antibody with an irrelevant antigen-binding region (IgG1-b12) as a positive control for FcγRIa binding, and a variant of the same antibody (IgG1-b12-P329R-E345R) that also has a P329R mutation previously described to reduce interaction with FcγR. Since macrophages should not express CD27, it is hypothesized that any binding observed occurs via FcγRIa, the only FcγR that binds monovalent IgG. Differentiated macrophages were incubated for 15 min with IgG1-CD27-A-P329R-E345R or control antibodies (30 μg / mL in DC medium) and PE-labeled polyclonal goat anti-human IgG (Jackson Immuno Research, Cat. No. 109-116-097, dilution 1:200, 30 min, 4°C). After incubation, cells were washed and resuspended in 100 μL of FACS buffer containing the nuclear stain DAPI (BD Pharmingen, Cat. No. 564907, dilution 1:5000). Samples were measured on a FACSymphony flow cytometer (BD Biosciences) and analyzed using FlowJo software.

[0436] No binding above background (secondary antibody only) was observed with either IgG1-CD27-A-P329R-E345R or control IgG1-b12-P329R-E345R to M0 or M1 macrophages isolated from two independent donors (Figure 25). WT IgG1-b12, which contains an active Fc region, bound consistently to both M0 and M1 macrophages.

[0437] In conclusion, IgG1-CD27-A-P329R-E345R and the control IgG1-b12-P329R-E345R do not bind to M0 or M1 macrophages expressing FcγRIa, FcγRII and FcγRIIIa.

Claims

1. An antibody comprising at least one antigen-binding region capable of binding to human CD27, the antibody comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs:9, 10, and 11, respectively.

2. comprising two antigen-binding regions capable of binding to human CD27; heavy chain variable (VH) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NOs: 9, 10, and 11, respectively; The antibody of claim 1.

3. 2. The antibody of claim 1, comprising VH and VL regions comprising the sequences set forth in SEQ ID NO:4 and SEQ ID NO:8, respectively.

4. The antibody of claim 1, which is a human or humanized antibody.

5. The antibody of claim 1, which is a full-length antibody further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

6. The antibody of claim 5, wherein the light chain constant region is human kappa or human lambda.

7. The antibody further comprising a heavy chain constant region, the heavy chain constant region being of a human IgG isotype, optionally a modified human IgG heavy chain constant region; Preferably, the human IgG or modified human IgG is selected from IgG1, IgG2, IgG3 or IgG4, such as human IgG1. The antibody of claim 1.

8. 8. The antibody of claim 7, wherein the amino acid residue at a position corresponding to position E345 or E430 in a human IgG1 heavy chain according to Eu numbering is selected from the group comprising A, C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

9. 8. The antibody of claim 7, wherein the amino acid residue at the position corresponding to position E345 in the human IgG1 heavy chain according to Eu numbering is R.

10. 8. The antibody of claim 7, wherein the amino acid residue at the position corresponding to position E430 in the human IgG1 heavy chain according to Eu numbering is G.

11. 8. The antibody of claim 7, wherein the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to Eu numbering is R.

12. 8. The antibody of claim 7, wherein the amino acid residues at positions corresponding to positions E345 and P329 in a human IgG1 heavy chain according to Eu numbering are both R.

13. 8. The antibody of claim 7, which has a pharmacokinetic profile similar to that of a parent antibody comprising a wild-type IgG1 heavy chain constant region.

14. 2. The antibody of claim 1, comprising a heavy chain constant region comprising a sequence selected from the group consisting of SEQ ID Nos. 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32, 33, 34 and 36.

15. 2. The antibody of claim 1, comprising a heavy chain constant region comprising the sequence shown in SEQ ID No.

15.

16. The antibody of claim 1, which is a monovalent antibody or a bivalent antibody.

17. The antibody of claim 1, which is a monospecific antibody.

18. A bispecific antibody comprising a first antigen-binding region capable of binding to human CD27 according to claim 1, and a second antigen-binding region capable of binding to a different epitope on human CD27 or capable of binding to a different target. The antibody of claim 1, wherein

19. a. a VH region comprising the amino acid sequence set forth in SEQ ID No:4; b. a VL region comprising the amino acid sequence set forth in SEQ ID No:8; c. a CH region comprising the amino acid sequence set forth in SEQ ID No: 15; and d. A CL region comprising the amino acid sequence set forth in SEQ ID No: 17 The antibody of claim 1, comprising:

20. 2. The antibody of claim 1, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:35 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

25.

21. A composition comprising an antibody as defined in any one of claims 1 to 20.

22. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

23. An antibody as defined in any one of claims 1 to 20 for use as a medicament.

24. 24. The antibody for use as a medicament according to claim 23, for use in the treatment of a disease.

25. 25. The antibody for use as a medicament according to claim 24, wherein the disease is cancer.

26. 26. The antibody for use as a medicament according to claim 25, wherein the cancer is a solid tumor or a blood cancer.

27. 21. An isolated nucleic acid sequence or combination of nucleic acid sequences encoding the antibody of any one of claims 1 to 20.

28. 28. The nucleic acid sequence or combination of nucleic acid sequences according to claim 27, wherein the nucleic acid is RNA or DNA, such as mRNA.

29. 28. The nucleic acid sequence of claim 27, which is an isolated nucleic acid sequence.

30. 28. An expression vector comprising the nucleic acid sequence of claim 27 or a combination thereof.

31. 28. A nucleic acid sequence, or a combination of nucleic acid sequences, according to claim 27 for use in expression in mammalian cells.

32. A recombinant host cell producing an antibody as defined in any one of claims 1 to 20, optionally comprising an expression vector as defined in claim 30, and preferably being a eukaryotic or prokaryotic cell.

33. 28. A nucleic acid sequence or a combination of nucleic acid sequences according to claim 27, or an expression vector comprising said nucleic acid sequence or said combination of nucleic acid sequences; a pharmaceutically acceptable carrier; 10. A pharmaceutical composition comprising:

34. 32. A method for producing an antibody according to any one of claims 1 to 20, comprising culturing a recombinant host cell according to claim 32 in a culture medium and under conditions suitable for producing said antibody, and optionally purifying or isolating said antibody from the culture medium.

35. A kit-of-parts comprising an antibody as defined in any one of claims 1 to 20 and instructions for use of the kit, For example, as a companion diagnostic / kit for use in identifying patients in a patient population who have a propensity to respond to treatment with an antibody as defined in any one of claims 1 to 20. Kit of parts.

36. An anti-idiotypic antibody that binds to an antigen-binding region capable of binding to human CD27 as defined in any one of claims 1 to 20.

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