Antibodies capable of binding to OX40, their variants, and their uses

Abstract

The present invention relates to an antibody capable of binding to human OX40, and variants thereof comprising a modified Fc region including at least one mutation that enhances the Fc-Fc interaction of the antibody and at least one mutation that reduces the Fc effector function of the antibody. The present invention further provides a pharmaceutical composition comprising the antibody, and the use of the antibody for therapeutic and diagnostic procedures, particularly in cancer treatment.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to an antibody capable of binding to OX40, an antibody variant thereof containing one or more mutations in the Fc region, and the use of such an antibody and Fc variant. [Background technology]

[0002] Background of the Invention OX40 (CD134, TNFRSF4), a 277-amino acid type I transmembrane protein, is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and, after binding to its ligand, OX40 ligand (OX40L), co-stimulates T cell activation. In humans, OX40 is expressed on the cell membranes of activated CD4+ and CD8+ T cells, as well as on regulatory T cells (Tregs), but not on resting naive T cells.

[0003] The only known ligand for OX40 is the type II transmembrane glycoprotein OX40L (TNFSF4;CD252). Although OX40L is not constitutively expressed, its expression can be induced on antigen-presenting cells (APCs), including dendritic cells (DCs), macrophages, and B cells. In addition to APCs, OX40L is also expressed on other hematopoietic cells such as activated natural killer (NK) cells or mast cells, as well as on non-hematopoietic cells such as endothelial cells and smooth muscle cells (Croft et al. Immunol Rev. 2009 May;229(1):173-91 (Non-patent Literature 1)).

[0004] In humans, binding of OX40L to OX40 leads to downstream signaling, ultimately resulting in the expansion, proliferation, and survival of effector T cells. In addition, OX40 signaling promotes the generation of memory T cells and inhibits Treg function (Croft et al. Immunol Rev. 2009 May;229(1):173-91 (Non-patent Literature 1)). Furthermore, agonist OX40 antibodies, after interacting with Tregs, help deplete tumor-infiltrating OX40-expressing Tregs via antibody-dependent cell-mediated cytotoxicity (ADCC) induced by myeloid cells and NK cells (Choi et al. J Immunother Cancer. 2020 Oct;8(2):e000966 (Non-patent Literature 2)). However, it has been reported that under certain conditions, such as the absence of IFN-γ and IL-4, OX40 signaling can induce Treg proliferation (Ruby et al. J Immunol. 2009 Oct 15;183(8):4853-7 (Non-Patent Literature 3)).

[0005] Small retrospective studies using melanoma cell lines and patient-derived samples suggest that lower levels of OX40 in the tumor microenvironment (TME) are associated with a poorer prognosis after anti-PD-1 treatment, particularly in patients with low tumor-infiltrating lymphocyte (TIL) counts. Activation of OX40 signaling by treatment with anti-OX40 agonist monoclonal antibodies (mAbs) in combination with adoptive T-cell therapy helps restore or enhance the T-cell-mediated antitumor response, resulting in extended survival in prostate tumor-bearing mice. The antitumor activity of OX40 mAbs is associated with T-cell infiltration into tumors and intratumoral proliferation of effector T cells (He et al. Int Immunopharmacol. 2020 Dec;89(Pt B):107097 (Non-patent Literature 4)).

[0006] However, despite numerous efforts, there is still a need for agonist OX40 therapeutic antibodies that exhibit potent agonist activity to enhance the anti-tumor immune response. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Croft et al. Immunol Rev. 2009 May;229(1):173-91 [Non-Patent Document 2] Choi et al. J Immunother Cancer. 2020 Oct;8(2):e000966 [Non-Patent Document 3] Ruby et al. J Immunol. 2009 Oct 15;183(8):4853-7 [Non-Patent Document 4] He et al. Int Immunopharmacol. 2020 Dec;89(Pt B):107097 [Overview of the project]

[0008] This invention relates to an OX40-conjugated antibody and its Fc variant.

[0009] Accordingly, in one aspect, the present invention relates to an antibody capable of binding to human OX40, comprising an antigen-binding region comprising a heavy chain variable (VH) region in which CDR1, CDR2, and CDR3 each contain the sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable (VL) region in which CDR1, CDR2, and CDR3 each contain the sequences shown in SEQ ID NO: 16, DAS, and SEQ ID NO: 17, respectively, and a human IgG1 Fc region comprising a P329R mutation and an E345R mutation, wherein the amino acid positions are numbered according to Eu numbering.

[0010] In one aspect, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO: 20 and a VL region having the sequence shown in SEQ ID NO: 21.

[0011] In one aspect, the present invention relates to an antibody comprising a VH region having the sequence shown in SEQ ID NO: 20, a VL region having the sequence shown in SEQ ID NO: 21, and further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

[0012] In one aspect, the present invention relates to an antibody that is a human IgG1 isotype, comprising a VH region and a VL region having sequences as shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and further comprising a light chain constant region (CL) and a heavy chain constant region (CH).

[0013] In one aspect, the present invention relates to 3.4 × 10 -9 This relates to an antibody of type M that has binding affinity KD to human OX40.

[0014] In one aspect, the present invention relates to humanized antibodies.

[0015] In one aspect, the present invention relates to an antibody in which the Fc region comprises the sequence shown in SEQ ID NO: 3.

[0016] In one aspect, the present invention relates to an antibody having a heavy chain constant region comprising the sequence shown in SEQ ID NO: 58.

[0017] In one aspect, the present invention relates to an antibody comprising a heavy chain (HC) as shown in SEQ ID NO: 18 and a light chain (LC) as shown in SEQ ID NO: 19.

[0018] In one aspect, the present invention relates to a composition comprising an antibody according to any aspect or embodiment described herein.

[0019] In one aspect, the present invention relates to a composition comprising an antibody according to any aspect or embodiment described herein and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present invention relates to an antibody for pharmaceutical use, according to any aspect or embodiment described herein.

[0021] In one aspect, the present invention relates to an antibody for use as a pharmaceutical, according to any aspect or embodiment described herein, wherein the disease is cancer.

[0022] In one aspect, the present invention relates to a method for treating a disease, comprising the step of administering an antibody according to any aspect or embodiment of this specification to a subject in need thereof.

[0023] In one aspect, the present invention relates to a method for treating a disease, comprising the step of administering a composition or pharmaceutical composition as defined herein to a subject in need thereof.

[0024] In one aspect, the present invention relates to isolated nucleic acids encoding antibodies according to any aspect or embodiment described herein.

[0025] In one aspect, the present invention relates to an expression vector containing such nucleic acids.

[0026] In one aspect, the present invention relates to recombinant host cells that produce antibodies according to any aspect or embodiment described herein.

[0027] In one aspect, the present invention relates to a kit of parts, such as a kit for identifying patients within a patient population who are likely to respond to treatment with an antibody as described herein or in any aspect or manner herein, for use as a companion diagnostic.

[0028] In one aspect, the present invention relates to an anti-idiotype antibody that binds to an antigen-binding region capable of binding to OX40 as defined in any one aspect or embodiment herein. [Brief explanation of the drawing]

[0029] [Figure 1-1] This shows the binding of anti-human OX40 antibodies to (A) human OX40 and (B) cynomolgus monkey OX40 expressed on OX40-transfected HEK293F cells, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 1-2] Refer to the explanation in Figure 1-1. [Figure 2] The cross-effect concentration (EC50) of anti-human OX40 antibodies for binding to human OX40 and cynomolgus monkey OX40 expressed on OX40-transfected HEK293F cells, as measured by flow cytometry, is shown. The data shown are mean EC50 + SD from two experiments performed. The black dotted line shows the mean EC50 of IgG1-CD134-003-HC6LC2-RR for binding to human OX40. [Figure 3] This shows the sequence alignment of OX40 shuffle constructs with wild-type human OX40 (TNR4) and mouse OX40. Amino acids in the shuffle constructs that differ from those in the human OX40 sequence are highlighted in black. Shuffle 1 = human OX40 with mouse CRD1; Shuffle 2 = human OX40 with mouse CRD2; Shuffle 3 = human OX40 with mouse CRD3; Shuffle 4 = human OX40 with mouse CRD4. [Figure 4] This shows the sequence alignment of human OX40 (TNR4) and mouse OX40. Amino acids in mouse OX40 that differ from those in human OX40 are highlighted in black. [Figure 5]This shows the binding of the anti-human OX40 antibodies IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R to ExpiCHO-S cells transiently transfected to express mouse OX40(A), human OX40(B), or one of four shuffle constructs (C-F) in which individual CRDs of human OX40 are replaced by mouse analogs, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 6] This shows the binding of anti-human OX40 antibodies IgG1-CD134-h3C8-E345R, IgG1-CD134-RG7888, and IgG2s-CD134-SF2-E345R to ExpiCHO-S cells transiently transfected to express mouse OX40(A), human OX40(B), or one of four shuffle constructs (C-F) in which individual CRDs of human OX40 are replaced by mouse analogs, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 7] This shows the binding of anti-human OX40 antibodies IgG1-CD134-11D4, IgG1-CD134-A4453, and IgG1-CD134-ABBV368 to ExpiCHO-S cells transiently transfected to express mouse OX40(A), human OX40(B), or one of four shuffle constructs (C-F) in which individual CRDs of human OX40 are replaced by mouse analogs, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 8]This shows the binding of the anti-human OX40 antibodies IgG1-CD134-GBR830, IgG1-CD134-Hu106, and IgG1-CD134-IBI101 to ExpiCHO-S cells transiently transfected with mouse OX40(A), human OX40(B), or one of four shuffle constructs (C-F) in which individual CRDs of human OX40 are replaced with mouse analogs, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 9] This shows the binding of the anti-human OX40 antibodies IgG1-CD134-INCAGN1949 and IgG1-CD134-MEDI0562 to ExpiCHO-S cells transiently transfected to express mouse OX40(A), human OX40(B), or one of four shuffle constructs (C-F) in which individual CRDs of human OX40 are replaced by mouse analogs, as measured by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 10A] This shows the relative binding of anti-human OX40 antibodies to ExpiCHO-S cells transiently transfected to express human OX40(A), human OX40 with mouse CRD1(B), human OX40 with mouse CRD2(C), human OX40 with mouse CRD3(D), or human OX40 with mouse CRD4(E), as measured by flow cytometry. The data shown are MFI (Mean Fluorescence Intensity) measured at an antibody concentration of 5 μg / mL, normalized to the mean MFI of the antibody IgG1-CD134-A4453. Black dots indicate individual measurements, and horizontal bars indicate the mean and SD of two experiments performed. [Figure 10B] Refer to the explanation in Figure 10A. [Figure 10C] Refer to the explanation in Figure 10A. [Figure 10D]Refer to the explanation in Figure 10A. [Figure 10E] Refer to the explanation in Figure 10A. [Figure 11A] This shows the activation of OX40-expressing reporter cells by IgG1-CD134-003-HC6LC2-RR, as well as by variants of the antibody IgG1-CD134-h3C8 (A), the antibody IgG1-CD134-RG7888 (B), IgG2s-CD134-SF2-E345R (C), IgG1-CD134-007-RR and IgG1-CD134-012-RR (D), and IgG1-CD134-11D4 (E). The unbound antibody IgG1-b12-RR was included as a negative control. The data shown represent the relative luminescence (RLU) from a single measurement, or the average RLU of two consecutive measurements (for IgG1-CD134-003-HC6LC2-RR in Figures A-D) or five consecutive measurements (for IgG1-CD134-003-HC6LC2-RR in Figure E) from a single experiment performed. [Figure 11B] Refer to the explanation in Figure 11A. [Figure 11C] Refer to the explanation in Figure 11A. [Figure 11D] Refer to the explanation in Figure 11A. [Figure 11E] Refer to the explanation in Figure 11A. [Figure 12] This graph shows the half-effect concentration (EC50) of anti-human OX40 antibodies for activation of reporter cells overexpressing human OX40. Individual EC50 values ​​from 2–5 independent experiments are shown, with the horizontal line representing the average EC50 across all experiments. The black dotted line represents the average EC50 of IgG1-CD134-003-HC6LC2-RR. [Figure 13-1]This study shows the effects of IgG1-CD134-003-HC6LC2-RR, as well as variants of the antibodies IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2s-CD134-SF2, on the proliferation of activated primary human CD4+ T cells (A) and CD8+ T cells (B), as analyzed by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the mean expansion growth index ± SD of (A) CD4+ T cells and (B) CD8+ T cells from dipset measurements of one representative donor out of 4–8 donors tested. The black dotted line represents the expansion growth index of CD4+ T cells or CD8+ T cells cultured in the absence of anti-human OX40 antibody. (C) CD4+ T cell growth index in polyclonally activated healthy human donor CD8- PBMCs, and (D) CD8+ T cell growth index in polyclonally activated healthy human donor CD4- PBMCs, analyzed on day 4. The data are from one representative donor out of four donors tested in the three experiments conducted. [Figure 13-2] See the explanation in Figure 13-1. [Figure 14] The effects of IgG1-CD134-003-HC6LC2-RR, as well as variants of the antibodies IgG1-CD134-h3C8 and IgG1-CD134-RG7888, on the percentage of CD4+ central memory T cells in the CD4+ T cell population, as analyzed by flow cytometry, are shown. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown are the mean percentage ± SD of CCR7+CD45RA- cells in (A) CD4+ T cells and (B) CD8+ T cells, from a representative donor (one of six donors tested). The black dotted line represents the percentage of CCR7+CD45RA- cells in CD4+ T cells cultured in the absence of anti-human OX40 antibody. [Figure 15]This shows the binding of IgG1-CD134-003-HC6LC2-RR, its variant without the Fc inactivation mutation (i.e., IgG1-CD134-003-HC6LC2-E345R), and two variants of the antibody IgG1-CD134-RG7888 to immobilized human recombinant FcγRIa(A), FcγRIIa[H](B), FcγRIIa[R](C), FcγRIIb(D), FcγRIIIa[F](E), and FcγRIIIa[V](F) constructs, as analyzed by SPR. The anti-HIV gp120 antibody IgG1-b12, which has a wild-type Fc domain, was included as a positive control. The data shown are relative binding responses, measured individually in a single experiment performed. [Figure 16] This shows the binding of IgG1-CD134-003-HC6LC2-RR, its variant without the Fc inactivation mutation (i.e., IgG1-CD134-003-HC6LC2-E345R), and two variants of the antibody IgG1-CD134-h3C8 to immobilized human recombinant FcγRIa(A), FcγRIIa[H](B), FcγRIIa[R](C), FcγRIIb(D), FcγRIIIa[F](E), and FcγRIIIa[V](F) constructs, as analyzed by SPR. The anti-HIV gp120 antibody IgG1-b12 with a wild-type Fc domain was included as a positive control. The data shown are relative binding responses, measured individually in a single experiment performed. [Figure 17A]This shows the binding of anti-human OX40 antibody to ExpiCHO-S cells transiently transfected to express FcγRIa. Binding is observed for IgG1-CD134-003-HC6LC2-RR, its variant without the Fc mutation (i.e., IgG1-CD134-003-HC6LC2), and its parental chimeric antibody (i.e., IgG1-CD134-003) (A), as well as for the antibodies (or their variants) IgG2-CD134-SF2 (B), IgG1-CD134-11D4, IgG1-CD134-INCAGN1949, and IgG1-CD134-IBI101 (C), IgG1-CD134-h3C8 (D), and IgG1-CD134-RG7888 (E), as analyzed by flow cytometry. The unbound antibody IgG1-b12-RR was included as a negative control. The data shown represents the geometric mean (gMFI) of fluorescence intensity from one representative experiment out of two experiments performed. [Figure 17B] Refer to the explanation in Figure 17A. [Figure 17C] Refer to the explanation in Figure 17A. [Figure 17D] Refer to the explanation in Figure 17A. [Figure 17E] Refer to the explanation in Figure 17A. [Figure 18] The binding ability of IgG1-CD134-003-HC6LC2-RR on primary human CD4+ T cells and CD8+ T cells activated with anti-CD3 / CD28 beads is shown, analyzed by quantitative flow cytometry 1, 2, or 3 days after T cell activation using saturated concentrations of IgG1-CD134-003-HC6LC2-RR. The presented data represent the mean number of antibody binding sites ± SD from three donors, and the symbols indicate the number from each individual donor. [Figure 19]This study demonstrates dose-dependent binding of IgG1-CD134-003-HC6LC2-RR to activated human CD4+ and CD8+ T cells. Human PBMCs were cultured for 2 days in the presence of anti-CD3 / CD28 antibody, and then incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-IBI101, IgG1-CD134-11D4, IgG1-CD134-RG7888, or the unbound control antibody IgG1-b12-RR. Binding of anti-human OX40 antibody to T cells was evaluated by flow cytometry detection of anti-human Fcγ antibody. The data shown are mean ± SD of double wells from one representative donor out of three donors tested. [Figure 20] This shows the binding of IgG1-CD134-003-HC6LC2-RR or soluble OX40L (sOX40L) to activated human CD4+ T cells and CD8+ T cells. Human CD4+ T cells and CD8+ T cells activated for 2 days with anti-CD3 / CD28 beads were incubated with IgG1-CD134-003-HC6LC2-RR or the control antibody IgG1-b12-RR in the presence and absence of sOX40L at a saturated concentration (2 μg / mL). Binding was analyzed by flow cytometry. (A) Antibody binding to CD4+ T cells and CD8+ T cells. (B) sOX40L binding to CD4+ T cells and CD8+ T cells. The data shown are mean geometric mean fluorescence intensity (mean gMFI) ± SD from double wells of one representative donor out of three. [Figure 21A]This shows the activation of OX40-expressing reporter cells by an anti-human OX40 antibody. (A) Mean ± SD bioluminescence as a surrogate for OX40 agonist activity, presented as RLU ± SD, in OX40+ Jurkat reporter T cells incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-E345R, IgG1-CD134-003, IgG1-CD134-003-FEAL, or the unbound control antibody IgG1-b12-RR, in or in the absence of FcγRIIb-CHO-K1 cells. The data shown are from a single experiment. (B) Mean ± SD bioluminescence in OX40+ Jurkat reporter T cells incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-11D4, IgG1-CD134-RG7888, IgG1-CD134-IBI101, or unbound control antibody IgG1-b12-RR, in the absence or presence of FcγRIIb-CHO-K1 cells. The dotted line represents the mean RLU value of the well without antibody treatment. The data shown are representative from one of two experiments. [Figure 21B] Refer to the explanation in Figure 21A. [Figure 22-1]This shows the expression of cell surface expression markers associated with human T cell activation after 2-day or 5-day incubation of polyclonally activated healthy donor PBMC samples with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR. The percentage ± SD of CD4+ T cells (A, C, E, G) and CD8+ T cells (B, D, F, H) expressing 4-1BB, CD25, HLA-DR, or PD-1 was measured using flow cytometry. The data shown are selected from either day 2 or day 5, depending on which day the maximum effect of IgG1-CD134-003-HC6LC2-RR was observed for each marker. The dotted line represents the percentage of marker-expressing T cells incubated with CD3 antibody alone. All data shown are from one representative donor out of seven donors tested in three experiments. Percentages ± SD of CD4+ T cells (I-K) and CD8+ T cells (L-N) expressing 4-1BB, CD25, or HLA-DR after stimulation with 5 μg / mL of IgG1-CD134-003-HC6LC2-RR, IgG1-b12-RR, or the OX40 agonist reference antibody analogs IgG1-CD134-RG7888, IgG1-CD134-11D4, and IgG1-CD134-IBI101, analyzed by flow cytometry 2 and 5 days post-stimulation. The data shown are mean-normalized ± SD pooled data from 4-7 donors evaluated in 2-3 independent experiments. [Figure 22-2] See the explanation in Figure 22-1. [Figure 22-3] See the explanation in Figure 22-1. [Figure 22-4] See the explanation in Figure 22-1. [Figure 23]This shows the dynamics of cytokine concentrations in the supernatant of polyclonally activated healthy human donor PBMC samples incubated with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR for 1, 2, 3, 4, or 6 days. Mean ± SD calculated concentrations of (A) TNFα, (B) IL-2, (C) IFNγ, and (D) IL-13 are shown, measured by multiplex ECLIA. The data presented are from one representative donor out of a total of three donors tested. [Figure 24] This shows cytokine concentrations in the supernatant of polyclonally activated healthy human donor PBMC samples incubated for 4 days with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR. PBMC samples were either not depleted before incubation or depleted of either CD4+ T cells or CD8+ T cells. Mean ± SD calculated concentrations of (A) TNFα, (B) IL-2, (C) IFNγ, and (D) IL-13 are measured by multiplex ECLIA. The data shown are from one representative donor out of a total of three donors tested. [Figure 25] This shows cytokine concentrations in the supernatant of polyclonally activated healthy human donor PBMC samples incubated for 4 days with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-h3C8-K322A-E345R, IgG1-CD134-h3C8-E345R-LALAPG, or the unbound control antibody IgG1-b12-RR. Mean ± SD calculated concentrations of (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα are measured by multiplex ECLIA. The data shown originates from one donor out of a total of two donors tested. [Figure 26]This shows cytokine concentrations in the supernatant of polyclonally activated healthy human donor PBMC samples incubated for 4 days with IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-RG7888-K322A-E345R, IgG1-CD134-RG7888-E345R-LALAPG, or the unbound control antibody IgG1-b12-RR. Mean ± SD calculated concentrations of (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα are measured by multiplex ECLIA. The data shown originates from one donor out of a total of two donors tested. [Figure 27] This shows cytokine concentrations in the supernatant of polyclonally activated healthy human donor PBMC samples incubated with IgG1-CD134-003-HC6LC2-RR, IgG2sCD134-SF2-E345R, or the unbound control antibody IgG1-b12-RR. (A) IFNγ, (B) IL-13, (C) IL-2, and (D) TNFα mean ± SD calculated concentrations, measured by multiplex ECLIA. The data shown originates from one donor out of a total of two donors tested. [Figure 28]This shows CD8+ T cell proliferation on day 4 in an antigen-specific human T cell proliferation assay after treatment with IgG1-CD134-003-HC6LC2-RR or unbound control antibody IgG1-b12-RR, as evaluated by flow cytometry. (A) Mean ± SD expansion index of OX40-expressing CLDN6-specific CD8+ T cells incubated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR in double-well cells from a representative donor (one of three donors tested in two experiments). The black dotted line shows baseline values ​​determined from iDC:CD8+ T cell co-cultures incubated without antibody (medium only). (B) Mean ± SD expansion index of OX40-expressing CLDN6-specific CD8+ T cells incubated with autologous CLDN6-expressing (CLDN6+) iDCs or mock-transfected (CLDN6-) iDCs in the presence of 2 μg / mL IgG1-CD134-003-HC6LC2-RR in double wells. Data are shown for all three donors tested. [Figure 29] This shows the lack of binding of IgG1-CD134-003-HC6LC2-RR to FcγRIa-expressing human monocyte-derived M2c-like macrophages, as analyzed by flow cytometry. The graph shows the binding of IgG1-CD134-003-HC6LC2-RR, or the unbound control antibody IgG1-b12-RR or IgG1-b12, to FcγRIa-expressing human monocyte-derived M2c-like macrophages 15 minutes (A) and 24 hours (B) after incubation. Binding is shown compared to the background control (indicated by the black dotted line, binding with the secondary antibody only). The dots represent three individual donors measured in two independent experiments, and the bar graph and error bars represent the mean change factor and SD for the three donors, respectively. [Figure 30]This shows the binding of IgG1-CD134-003-HC6LC2-RR to the human neonatal receptor FcRn, as analyzed by SPR. The data shown are sensorgrams of the interaction between FcRn and IgG1-CD134-003-HC6LC2-RR at pH 6.0 (A-E) and pH 7.4 (F), with IgG1-CD134-003-HC6LC2-RR tested at various concentrations as shown in each subpanel. The data shown are representative from one of three experiments for binding at pH 6.0 and from one of two experiments for binding at pH 7.4. [Figure 31-1] Figure C shows the binding of IgG1-CD134-003-HC6LC2-RR and IgG1-CD52-E345R to activated CD4+ T cells and CD8+ T cells, as well as C1q binding to them. (A-C) Primary human T cells were stimulated with anti-CD3 / CD28 beads and then incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E345R, or IgG1-b12-RR, and antibody binding to the cells was evaluated by flow cytometry. Figure C shows antibody binding to CD4+ T cells (A) and CD8+ T cells (B and C). Figure C shows the same data for IgG1-b12-RR as Figure B, but with a different Y-axis range. Data are expressed as the mean gMFI ± SD of two-well cells from one representative donor out of three donors tested in three experiments. The binding of C1q to IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E345R, or IgG1-b12-RR to OX40 on the cell membrane of activated CD4+ T cells (D) and CD8+ T cells (E), as analyzed by flow cytometry. All data shown are mean gMFI ± SD of double wells from one representative donor out of three donors tested in three experiments. [Figure 31-2] See the explanation in Figure 31-1. [Figure 32]This shows the binding of monovalent and bivalent anti-human OX40 antibodies to activated human T cells. Human CD4+ T cells (A) and CD8+ T cells (B), activated for 3 days using anti-CD3 / CD28 beads, were incubated with IgG1-CD134-003-HC6LC2-RR, monovalent OX40 antibody BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, IgG1-CD134-003-RR-K409R, IgG1-CD134-003, IgG1-CD134-003-HC6LC2, or unbound control antibody IgG1-b12-RR, and binding was evaluated by flow cytometry. The data shown are the mean gMFI ± SD from two-well dipsets for one representative donor out of four donors tested. [Figure 33] The functional activity of BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, and IgG1-CD134-003-RR-K409R in OX40+ T cell reporter assays and polyclonal T cell proliferation assays is shown. (A) Bioluminescence as a surrogate for OX40 agonist activity in OX40+ Jurkat reporter T cells incubated with BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-RR-K409R, or IgG1-b12-RR is shown as RLU. The data shown are from one representative experiment out of a total of four experiments. (B) Enhancement of CD4+ T cell proliferation in polyclonal T cell proliferation assays after incubation with concentration ranges of BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, IgG1-CD134-003-HC6LC2-RR, or IgG1-b12-RR, analyzed by flow cytometry and presented as mean ± SD of two-well clusters. The data shown are CD4+ T cell expansion and proliferation indices from one donor out of three donors tested in a single experiment. [Figure 34]This shows membrane OX40 expression and soluble OX40 (sOX40) levels in polyclonal activated healthy human donor PBMC cultures after treatment with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR. (A) Mean ± SD of double-well cell surface expression of OX40 on CD4+ T cells and CD8+ T cells, as assessed by flow cytometry. (B) Mean ± SD of double-well sOX40 concentration in the supernatant, as measured by ECLIA. The data shown are from one representative donor out of three donors tested in a similar experimental setup. [Figure 35] This report demonstrates the activation of OX40-expressing reporter cells by IgG1-CD134-003-HC6LC2-RR in the presence of soluble OX40 (sOX40). OX40+ Jurkat reporter cells were cultured for 5 hours in the presence of various concentrations of IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR, and sOX40. Bioluminescence, as a surrogate for OX40 agonist activity, was measured as the mean RLU ± SD of a double-well cell. The data shown are from one of two experiments. [Figure 36] This shows the proliferation of polyclonally activated CD4+ and CD8+ T cells in a polyclonal T cell proliferation assay upon treatment with IgG1-CD134-003-HC6LC2-RR or the unbound control antibody IgG1-b12-RR, in the presence or absence of sOX40. The data shown are the mean ± SD of two-well (A) CD4+ T cell expansion index and (B) CD8+ T cell expansion index, determined by flow cytometry on day 4. The data are from one representative donor out of four donors tested in a single experiment. [Figure 37]The data shows the total huIgG levels in SCID mouse plasma after intravenous administration of anti-human OX40 antibody, as determined by ECLIA in sequentially acquired plasma samples. Data are expressed as the mean huIgG concentration ± SD of 3 mice per treatment group, excluding the groups of mice treated with 0.125 mg / kg or 1.25 mg / kg of IgG1-CD134-003-RR-K409R (N=2), 0.125 mg / kg of IgG1-CD134-003-FEAL (N=1), or 12.5 mg / kg of IgG1-CD134-003-HC6LC2-RR (N=2). Values ​​below the lower limit of quantification (LLOQ) are not shown (N=3 for mice that received 0.125 mg / kg of IgG1-CD134-003-RR-K409R after 8 days, and N=1 for mice that received 0.125 mg / kg of IgG1-CD134-003-FEAL after 14 days). The dashed line shows the predicted plasma concentration of WT huIgG using a two-compartment model. The horizontal dotted line shows the LLOQ and upper limit of quantification (ULOQ) for the ECLIA assay. The data shown are from a single experiment. [Figure 38] The antibody pharmacokinetic parameters in individual mice are shown. SCID mice received a single intravenous injection of IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-RR-K409R, or IgG1-CD134-003-FEAL at t=0. Total huIgG levels were determined by ECLIA in sequentially acquired plasma samples, and CL(A), t1 / 2(B), and Cmax(C) were calculated. The values ​​for individual mice are plotted, with the horizontal bars representing the mean ± SD per treatment group. [Figure 39A]This figure shows tumor volume measured in human OX40 knock-in (hOX40 KI) mice carrying MC38 tumors. Mice were intraperitoneally treated on day 0 with various concentrations of IgG1-CD134-003-HC6LC2-RR or 20 mg / kg of IgG1-b12-FEAL. (A) Tumor volume measured 10 days after the start of treatment. Values ​​for individual mice are plotted, and the horizontal bar shows the mean ± SEM per treatment group. Statistical significance between treatment groups (**P<0.01) was assessed using Mann-Whitney analysis. (B) Mean tumor volume (± SEM) measured over time in hOX40 KI mice carrying MC38 tumors treated as shown in the figure. (C) Kaplan-Meier curve showing progression-free survival (defined as tumor volume < 500 mm3) of MC38 tumor-carrying mice treated as shown. Significance was calculated using Mantel-Cox analysis (*P<0.05 and ***P<0.001 compared to the IgG1-b12-FEAL control group). [Figure 39B] Refer to the explanation in Figure 39A. [Figure 39C] Refer to the explanation in Figure 39A. [Figure 40] This graph shows the percentage and absolute number of T cells in peripheral blood samples from MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL, analyzed by flow cytometry. It also shows the percentage (A, C) and absolute number (B, D) of CD4+ T cells (A, B) and CD8+ T cells (C, D) within the CD3+ T cell population. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups (*P<0.05; **P<0.01) was assessed using Mann-Whitney analysis. [Figure 41]This graph shows the percentage of CD4+ T cells expressing the proliferation markers Ki67 (A), CD25 (B), IA / IE (C), PD-1 (D), and 4-1BB (E), analyzed by flow cytometry in peripheral blood samples collected on day 5 (after two treatments) from MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups (*P<0.05; **P<0.01) was assessed using Mann-Whitney analysis. [Figure 42] The percentages and absolute numbers of proliferating CD8+ T cells and tumor-specific (ADPGK-tetramer-specific) CD8+ T cells are shown. The data shown are (A) the percentage of CD8+ T cells expressing the proliferation marker Ki67, (B) the absolute number of CD8+Ki67+ cells, (C) the percentage of tumor-specific CD8+ T cells, and (D) the absolute number of tumor-specific CD8+ T cells, analyzed by flow cytometry in peripheral blood samples collected on day 5 (after two treatments) from MC38 tumor-carrying hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups (**P<0.01) was assessed using Mann-Whitney analysis. [Figure 43]This graph shows the percentage of CD8+ T cells expressing the proliferation markers CD25 (A), IA / IE (B), PD-1 (C), and 4-1BB (D), analyzed by flow cytometry in peripheral blood samples collected on day 5 (after two treatments) from MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups (*P<0.05; **P<0.01) was assessed using Mann-Whitney analysis. [Figure 44-1] This shows plasma cytokine concentrations in MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR. Plasma samples were collected on day 0 (pre-treatment) and on days 2 and 5 after one or two treatments with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. Cytokine analysis of plasma samples was performed by ECLIA. The data shown are the concentrations and mean concentrations of four individual mice: (A) IFNγ, (B) IP-10, (C) IL-2, (D) IL-4, (E) MCP-1, (F) IL-10, (G) IL-27p28, and (H) TNFα. The data shown are from a single experiment. [Figure 44-2] See the explanation in Figure 44-1. [Figure 45-1] This graph shows the number of intratumor immune cells per mm² of tumor tissue collected from MC38-carrying hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL. The numbers per mm² of tumor tissue are determined by quantitative immunohistochemical analysis of the tumor tissue: (A) CD3+ cells, (B) CD3+Ki67+ cells, (C) CD4+ cells, (D) CD8+ cells, (E) Granzyme B+ cells, and (F) human OX40+ cells. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD. *p<0.05 was determined by the Mann-Whitney test. [Figure 45-2]See the explanation in Figure 45-1. [Figure 46-1] This shows the tumor volume measured in hOX40 KI mice carrying MC38 tumors. Mice were intraperitoneally treated on day 0 with various concentrations of IgG1-CD134-003-HC6LC2-RR or 20 mg / kg of IgG1-b12-FEAL. (A) Tumor volume measured 15 days after the start of treatment. Values ​​for individual mice are plotted, and the horizontal bars show the mean ± SEM per treatment group included from a single experiment. Statistical significance between treatment groups (*P<0.05; **P<0.01) was assessed using Mann-Whitney analysis. (B) Mean tumor volume measured over time (± SEM) in hOX40 KI mice carrying MC38 tumors treated as shown in the figure. (C) Kaplan-Meier curve showing progression-free survival (defined as tumor volume < 500 mm3) of MC38 tumor-carrying mice treated as shown. Significance calculated using Mantel-Cox analysis (*P<0.05 compared to the IgG1-b12-FEAL control group). [Figure 46-2] See the explanation in Figure 46-1. [Figure 47-1] The graphs show the percentages of (A) CD45+ cells, (B) CD3+ cells, (C) CD4+ T cells, (D) CD8+ T cells, and (J) Treg cells detected in tumor samples collected from MC38 tumor-carrying hOX40 KI on day 5 (after two treatments) after initiating treatment with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR using flow cytometry. The absolute numbers of (F) CD45+ cells, (G) CD3+ cells, (H) CD4+ T cells, (I) CD8+ T cells, and (J) Treg cells detected using flow cytometry are also shown. Points in the graphs represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Significance (*P<0.05) was calculated using Mann-Whitney analysis. [Figure 47-2] See the explanation in Figure 47-1. [Figure 48]The graph shows the percentage of conventional CD4+ T cells expressing (A) CD25, (B) granzyme B (GZMB), (C) human OX40, and (D) PD-1, as analyzed by flow cytometry in tumor samples collected on day 5 (after two treatments) from MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups was assessed using Mann-Whitney analysis (*P<0.05; **P<0.01; ***P<0.001). [Figure 49] The graph shows the percentage of CD8+ T cells expressing (A) CD25, (B) granzyme B (GZMB), (C) human OX40, and (D) PD-1, analyzed by flow cytometry in tumor samples collected on day 5 (after two treatments) from MC38 tumor-bearing hOX40 KI mice treated with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR. Points in the graph represent individual mice, and columns and error bars represent the mean ± SD of all animals included from a single experiment. Statistical significance between treatment groups (**P<0.01; ***P<0.001) was assessed using Mann-Whitney analysis. [Modes for carrying out the invention]

[0030] Detailed description of the invention definition In the context of this invention, the term “antibody” (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen. The antibodies of this invention include the Fc domain and antigen-binding region of the immunoglobulin. Antibodies generally contain two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g., at least the Fc domain. Thus, the antibodies of this invention may include the Fc region and the antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region or “Fc” region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, which is the first component in the classical pathway of complement activation. Where used herein, unless inconsistent with the context, the Fc region of an immunoglobulin typically contains at least the CH2 and CH3 domains of the 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 two hinge regions and / or non-covalent interactions between two CH3 regions. The dimer may be a homodimer (where the amino acid sequences of the two Fc region monomers are identical) or a heterodimer (where the amino acid sequences of the two Fc region monomers differ by one or more amino acids). Fc region fragments of full-length antibodies can be produced, for example, by digestion of a full-length antibody with papain, as is well known in the art. Antibodies as defined herein may further include, in addition to the Fc region and antigen-binding region, one or both of the immunoglobulin CH1 region and CL region. Antibodies may also be multispecific antibodies, e.g., bispecific antibodies, or similar molecules. 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 target or on different targets. If the epitopes are located on different targets, those targets may be located on the same cell.Alternatively, they may be on different cells or cell types. As shown above, unless otherwise specified or clearly inconsistent with the context, the term antibody as used herein includes fragments of an antibody that include 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 may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" include, non-limitingly, monovalent antibodies (Genmab, described in WO2007059782); heavy chain antibodies consisting of only two heavy chains, naturally occurring in, for example, camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMab (Roche, WO2011069104); asymmetric and bispecific antibody-like molecules, such as chain exchange domains (SEED or Seed-body) (Merck, WO2007110205); Triomab (Pharma / Fresenius Biotech, Lindhofer et al. 1995 J Immunol 155:219; WO2002020039); FcΔAdp (Regeneron, WO2010151792); and Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768); mAb-Fv (Xencor, WO2011 / 028952); Xmab (Xencor); Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Patent No. 7,612,181); Dual domain double head antibody (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 format (Amgen,EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat Neurosciences Corporation, WO11143545); CrossMAb (Roche, WO2011117329); LUZ-Y (Genentech); Biclonic (Merus, WO2013157953); Dual-targeting domain antibody (GSK / Domantis); Two-in-one antibody or dual-action Fab recognizing two targets (Genentech, NovImmune, Adimab); Cross-linked Mab (Karmanos Cancer Center); Covalent fusion mAb (AIMM); CovX-body (CovX / Pfizer); FynomAb (Covagen / Janssen) ilag); DutaMab (Dutalys / Roche); iMab (MedImmune); IgG-like bispecificity (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); Common light chain approach (Crucell / Approaches such as Merus (US7262028) or common heavy chain approaches (κλBodies by NovImmune, WO2012023053), as well as scFv fusion-like approaches such as BsAb by ZymoGenetics / BMS and HERCULES by Biogen Idec (US007951918),Fusion proteins containing polypeptide sequences fused to antibody fragments containing an Fc region; these include SCORPIONS (Emergent BioSolutions / Trubion and Zymogenetics / BMS); Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92)); scFv fusions (Genentech / Roche); scFv fusions (Novartis); scFv fusions (Immunomedics); scFv fusions (Changzhou Adam Biotech Inc, CN 102250246); TvAb (Roche, WO 2012025525, WO 2012025530); mAb2 (f-Star, WO2008 / 003116); and dual scFv fusions. It should be understood that the term antibody, unless otherwise specified, includes monoclonal antibodies (such as human monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, monospecific antibodies (such as bivalent monospecific antibodies), bispecific antibodies, antibodies of any isotype and / or allotype; for example, antibody mixtures (recombinant polyclonals) produced by techniques developed by Symphogen and Merus (Oligoclonics), multimeric Fc proteins as described in WO2015 / 158867, and fusion proteins as described in WO2014 / 031646. While these various antibody fragments and forms are generally included in the meaning of antibody, they, collectively and individually, are distinctive features of the present invention, exhibiting a variety of biological properties and utility.

[0031] An "agonist antibody" against a natural receptor is a compound that binds to the receptor, forms a receptor-antibody complex, and activates the receptor, thereby initiating pathway signaling and further biological processes.

[0032] The terms “agonism” and “agonist” are used interchangeably herein and refer to or describe an antibody that can substantially induce, promote, or enhance the biological activity or activation of OX40, either directly or indirectly. Optionally, an "agonist OX40 antibody" is an antibody that can activate the OX40 receptor by a mechanism similar to that of the ligand for OX40, known as OX40L (CD134L, OX-40L, TNLG2B, OX4OL, GP34, CD252 antigen, CD134 ligand, TAX transcription-activated glycoprotein 1, CD252, OX40 ligand, OX40L, TNFSF4, tumor necrosis factor ligand superfamily member 4, glycoprotein Gp34, TNF superfamily member 4, TXGP1, Tax transcription-activated glycoprotein 1 (34kD), tumor necrosis factor (ligand) superfamily member 4, tumor necrosis factor (ligand) superfamily member 4, tumor necrosis factor superfamily member 4), resulting in the activation of one or more intracellular signaling pathways, which may include activation of the NF-KB and MAPK8 / JNK pathways.

[0033] The terms "OX40 antibody" or "anti-OX40 antibody" as used herein refer to antibodies that specifically bind to the protein OX40, particularly human OX40.

[0034] As used herein, “variant” refers to a protein sequence or polypeptide sequence that differs from a parent sequence or reference sequence by one or more amino acid residues. A variant may have, for example, at least 80%, for example 90%, 95%, 97%, 98%, or 99% sequence identity with respect to the parent sequence or reference sequence. Similarly or alternatively, a variant may differ from the parent sequence or reference sequence by 12 or fewer mutations, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, such as substitutions, insertions, or deletions of amino acid residues. Accordingly, as used interchangeably herein, “variant antibody” or “antibody variant” refers to an antibody that differs from a parent antibody or reference antibody by one or more amino acid residues, for example, in the antigen-binding region, the Fc region, or both. Similarly, a “variant Fc region” or “Fc region variant” refers to an Fc region that differs from a parent or reference Fc region by one or more amino acid residues, optionally by 12 or fewer mutations, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, such as substitutions, insertions, or deletions of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody and, depending on the context, may be a specific isotype. In dimerized form, the variant Fc region may be homodimer or heterodimer, for example, where one amino acid sequence of the dimerized Fc region contains mutations, while the other is identical to the parent or reference wild-type amino acid sequence. Examples of wild-type (typically parent or reference) IgG CH and variant IgG constant region amino acid sequences, including the Fc region amino acid sequence, are shown in Table 3.

[0035] As used herein, the terms “immunoglobulin heavy chain” or “immunoglobulin heavy chain” are intended to refer to one of the immunoglobulin heavy chains. A heavy chain typically consists of 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 consists of three domains: CH1, CH2, and CH3. As used herein, the term “immunoglobulin” is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four of which are potentially interconnected by disulfide bonds. The structures of immunoglobulins are well-characterized (see, for example, Fundamental Immunology Ch.7 Paul, W., 2nd ed. Raven Press, NY 1989). Within the structure of immunoglobulins, two heavy chains are interconnected via disulfide bonds in a so-called "hinge region." Equal to the heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of a single domain, the CL. Furthermore, the VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs) (or hypervariable regions whose sequence may be hypervariable and / or in the form of structurally defined loops). Each VH and VL typically consists of three CDRs and four FRs, aligned from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, CDR sequences 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)).

[0036] As used herein, the terms “halving,” “Fab arm,” and “arm” refer to a single heavy-light chain pair. Where a bispecific antibody is described as comprising a hapmon antibody “derived” from a first antibody and a hapmon antibody “derived” from a second antibody, the term “derived” indicates that the bispecific antibody is produced by recombining the hapmones from each of the first and second antibodies by any known method, resulting in a bispecific antibody. In this context, “recombination” is not intended to be limited to any particular recombination method, and therefore includes all methods for producing bispecific antibodies described herein below, including, for example, “hapmon exchange” as also described in the Art as “Fab arm exchange,” recombination by the DuoBody® method, and recombination at the nucleic acid level and / or through co-expression of two hapmones in the same cell.

[0037] As used herein, the terms “antigen-binding region,” “binding region,” or “antigen-binding domain” refer to a region of an antibody capable of binding to an antigen. This binding region is typically defined by the VH and VL domains of the antibody, which can be further subdivided into a hypervariable region, also called a complementarity-determining region (CDR), which is dotted with more conserved regions called a framework region (FR) (or a hypervariable region whose sequence may be hypervariable and / or may be in the form of structurally defined loops). The antigen can be any molecule, such as a polypeptide, present on a cell, bacterium, or virion. The terms “antigen-binding region,” “antigen-binding site,” and “antigen-binding domain” may be used interchangeably in the context of this invention, as long as they do not contradict the context.

[0038] The terms "antigen" and "target" may be used interchangeably in the context of this invention, provided that they do not contradict the context.

[0039] As used herein, the term "binding" refers to the binding of an antibody to a given antigen or target, typically determined by biolayer interferometry using the antibody as a ligand and the antigen as an analyte, with a binding affinity corresponding to 1E 6 M or less, for example, 5E 7 M or less, 1E 7 M or less, for example 5E 8 M or less, for example 1E 8 M or less, for example 5E 9 M or less, or for example 1E 9 M or less of K D and refers to binding with a binding affinity at least 10-fold lower, for example at least 100-fold lower, by way of example at least 1,000-fold lower, for example at least 10,000-fold lower, by way of example at least 100,000-fold lower than its affinity for binding to the given antigen with respect to binding to a non-specific antigen (e.g., BSA, casein) other than the given antigen or an antigen closely related thereto, and binds with an affinity corresponding to K D .

[0040] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is obtained by dividing k d by k a .

[0041] As used herein, the term "k d " (sec -1 ) refers to the dissociation rate constant of a specific antibody-antigen interaction. The said value is also referred to as the k off value or off-rate.

[0042] As used herein, the term "k a " (M -1 ×sec -1 ) refers to the association rate constant of a specific antibody-antigen interaction. The said value is also referred to as the k on value or on-rate.

[0043] As used herein, the term "OX40" refers to a human protein named OX40, also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4). In the amino acid sequence shown in SEQ ID NO: 52, amino acid residues 1-28 are the signal peptide, and amino acid residues 29-277 are the mature polypeptide.

[0044] In cynomolgus monkeys (Macaca fascicularis), the OX40 protein has the amino acid sequence shown in SEQ ID NO: 51.

[0045] An agonist IgG1 antibody against human OX40 for cancer treatment has been disclosed.

[0046] WO2009 / 079335A1 discloses the human OX40-binding antibody 11D4, which demonstrated agonist activity in multiple mouse tumor models. The antibody clone 11D4 was shown to induce T cell activation during incubation of primary T cells or healthy donor PBMC samples pre-incubated with anti-human CD3. 11D4 was shown to block the binding of the natural ligand OX40L and also bind to cynomolgus monkey T cells. Gutierrez et al. reported that the 11D4-based clinical candidate antibody BMS-986178, when administered as monotherapy, did not induce dose-limiting toxicity or objective response in patients with advanced cancer during a phase 1 / 2a clinical trial (Gutierrez et al. Clin Cancer Res. 2021 Jan 15;27(2):460-472).

[0047] Other OX40 antibodies described as inducing T cell activation via human OX40 involvement include clone A4453 (WO2019 / 223733), Hu106 (WO2020 / 030570A1), MEDI0562 (INN 10420, tavolimab), ABBV368 (INN 11242, revdofilimab), IBI101 (INN 11200; cudarolimab), INCAGN1949 (US10259882B2), GSK-3174998 (US9006399), and 49B4 (WO2019 / 086497A2).

[0048] 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 can be determined by epitope binding using biolayer interferometry, by alanine scanning, or by shuffle assay (which uses an antigen construct in which the antigen region has been replaced with that of another species to determine whether the antibody still binds to the antigen). The amino acids within the antibody-binding region that are involved in the interaction with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and by crystal structure analysis of the antibody bound to that antigen.

[0049] The term "epitope" refers to an antigenic determinant to which an antibody specifically binds. Epitopes typically consist of surface arrangements of molecules, such as amino acids, sugar side chains, or combinations thereof, and usually possess specific three-dimensional structural and specific charge properties. Constructive and non-constructive epitopes are distinguished in that, in the presence of a denaturing solvent, binding to the former is lost, while binding to the latter is not. Epitopes may include amino acid residues directly involved in binding, and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the antibody when the antibody is bound to the antigen (in other words, those amino acid residues are within or immediately adjacent to the footprint of the specific antibody).

[0050] As used herein, terms such as “monoclonal antibody,” “monoclonal Ab,” “monoclonal antibody composition,” and “mAb” refer to preparations of antibody molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. Therefore, 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 can be produced by hybridomas, including B cells obtained from non-human animals with transgenic or transchromosomes having a genome containing human heavy chain and light chain transgenes, fused with immortalized cells, such as transgenic mice or transgenic rats. Monoclonal antibodies can also be produced from recombinantly modified host cells or from systems using cell extracts that assist in vitro transcription and / or translation of nucleic acid sequences encoding antibodies.

[0051] As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, e.g., IgG1m(za) and IgG1m(f), encoded by a heavy chain constant region gene. Furthermore, each heavy chain isotype can be combined with either a kappa (κ) light chain or a lambda (λ) light chain.

[0052] As used herein, the term “full-length antibody” means that the antibody contains, rather than fragments, all of the domains of a particular isotype that are naturally and commonly found for that isotype; for example, for an IgG1 antibody, the VH, CH1, CH2, CH3, hinge, VL, and CL domains. In full-length variant antibodies, the constant and variable domains of the heavy and light chains may contain amino acid substitutions that, in particular, improve the functional properties of the antibody compared to a full-length parental antibody or wild-type antibody. Full-length antibodies according to the present invention can be produced by a method comprising (i) cloning a CDR sequence into a suitable vector containing a complete heavy chain sequence and a complete light chain sequence, and (ii) expressing the complete heavy chain sequence and light chain sequence in a suitable expression system. Producing full-length antibodies starting from either a CDR sequence or a complete variable region sequence is within the knowledge of those skilled in the art. Therefore, those skilled in the art will know how to produce full-length antibodies according to the present invention.

[0053] As used herein, the term “human antibody” is intended to include antibodies comprising variable regions and framework regions derived from human germline immunoglobulin sequences, as well as human immunoglobulin constant domains. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced in vitro by random mutagenesis or site-directed mutagenesis, or in vivo by somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as mouse, have been transplanted onto human framework sequences.

[0054] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to exhibit a high level of sequence homology to the human variable domain. This can be achieved by transplanting six non-human antibody complementarity-determining regions (CDRs) that together form an antigen-binding site onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). Substitution (reverse mutation) of framework residues derived from the parent antibody (i.e., the non-human antibody) into the human framework region may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues in the framework region that are important for the antibody’s binding properties. Therefore, a humanized antibody may contain a non-human CDR sequence, a human framework region optionally containing one or more amino acid reverse mutations, primarily into the non-human amino acid sequence, and a fully human constant domain. Optionally, additional amino acid modifications, not necessarily reverse mutations, may be applied to obtain humanized antibodies with desirable characteristics such as affinity and biochemical properties.

[0055] As used herein, the terms “Fc region” or “Fc domain” may be used interchangeably and refer to the heavy chain constant region of an antibody, including at least the hinge region, CH2 region, and CH3 region, from the N-terminus to the C-terminus. The Fc region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system.

[0056] The terms "parent polypeptide" or "parent antibody" should be understood, unless otherwise specified or clearly contradicted by the context, as being identical to the polypeptide or antibody according to the present invention, but without mutations. For example, antibody IgG1-CD134-003 is the parent antibody of IgG1-CD134-003-P329R-E345R.

[0057] As used herein, the term “hinge region” refers to the hinge region of an immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216–230, following the EU numbering (Eu-index) as shown 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 any of the other subtypes described herein.

[0058] As used herein, the terms “CH1 region” or “CH1 domain” refer to the CH1 region of an immunoglobulin heavy chain. For example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118–215, following the Eu numbering as shown in Kabat (ibid.). However, the CH1 region may also be any of the other subtypes described herein.

[0059] As used herein, the terms “CH2 region” or “CH2 domain” refer to the CH2 region of an immunoglobulin heavy chain. Therefore, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231–340, following the Eu numbering as shown in Kabat (ibid.). However, the CH2 region may also be any of the other subtypes described herein.

[0060] As used herein, the terms “CH3 region” or “CH3 domain” refer to the CH3 region of an immunoglobulin heavy chain. Therefore, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341–447, following the Eu numbering as shown in Kabat (ibid.). However, the CH3 region may also be any of the other subtypes described herein.

[0061] As used herein, the terms “Fc-mediated effector function” or “Fc effector function” are interchangeable and intended to refer to the function resulting from the binding of a polypeptide or antibody to its target or antigen on the cell membrane, where 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 crosslinking, (vii) antibody-dependent cellular phagocytosis (ADCP), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated binding of opsonized antibodies to complement receptors, (x) opsonization, and (xi) any combination of (i) to (x).

[0062] As used herein, the terms “reduced Fc effector function” or “reduced Fc-mediated effector function” are interchangeable and are intended to refer to the reduced Fc effector function of an antibody when directly compared to the Fc effector function of the parent polypeptide or parent antibody in the same assay.

[0063] As used herein, the terms “inactive,” “inactive,” or “deactivated” refer to an Fc region that is unable to bind to at least one or more FcγRs, unable to induce Fc-mediated crosslinking of FcγRs, unable to induce FcγR-mediated crosslinking of a target antigen via two Fc regions of an individual antibody, or unable to 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.

[0064] As used herein, the term “oligomerization” is intended to refer to the process of converting a monomer into a finite degree of polymerization. Antibodies according to the present invention can form oligomers, such as hexamers, after target binding, for example, on the cell surface via non-covalent association of Fc regions. Oligomerization of anti-OX40 antibodies upon cell surface binding via Fc:Fc interactions can increase OX40 cluster formation and result in activation of OX40 intracellular signaling. The ability of antibodies containing the E345R mutation or E430G mutation to form oligomers, such as hexamers, upon cell surface binding can be evaluated 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 via intermolecular association of Fc regions between adjacent antibodies and is increased by the introduction of the E345R mutation or E430G mutation (numbered according to the Eu-index).

[0065] As used herein, the term "cluster formation" refers to the oligomerization of antibodies through non-covalent interactions.

[0066] As used herein, the term "Fc-Fc enhancement" is intended to refer to increasing the binding strength between the Fc regions of two Fc region-containing antibodies, or stabilizing the interaction between them, so that the antibodies form oligomers such as hexamers on the cell surface. This enhancement can be achieved by certain amino acid mutations in the Fc region of the antibody, such as E345R or E430G.

[0067] In the context of this invention, the term "monovalent antibody" 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 bispecific antibodies, "monovalent antibody binding" refers to the binding of a bispecific antibody to a single specific epitope on an antigen using only one antigen-binding domain (e.g., one Fab arm).

[0068] In the context of this invention, the term "monospecific antibody" refers to an antibody that has binding specificity to only one epitope. The antibody may be a monospecific monovalent antibody (i.e., possessing only one antigen-binding region) or a monospecific bivalent antibody (i.e., possessing two identical antigen-binding regions).

[0069] The term "bispecific antibody" refers to an antibody comprising two non-identical antigen-binding domains, for example, two non-identical Fab arms, or two Fab arms having 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 present on the same or different antigens or targets. If the epitopes are on different antigens, such antigens may be on the same cell, or on different cells, cell types, or structures, such as the extracellular matrix or vesicles, and on soluble proteins. Therefore, a bispecific antibody may crosslink multiple antigens, for example, two different cells. Certain bispecific antibodies of the present invention can bind to OX40 and a second target.

[0070] The term "bivalent antibody" refers to an antibody that has two antigen-binding regions that bind to one or two epitopes on a target or antigen, or that binds to one or two epitopes on the same antigen. Therefore, a bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.

[0071] The terms “amino acid” and “amino acid residue” may be used interchangeably herein and should not be understood as restrictive. Amino acids are organic compounds containing an amine (-NH2) functional group and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of this invention, amino acids can be classified based on their structure and chemical characteristics. Therefore, the classes of amino acids may be reflected in one or both of the following tables.

[0072] (Table 1) Main classifications based on the structure of the R group and general chemical characterization. TIFF2026516142000001.tif52128

[0073] (Table 2) Different physical and functional classifications of amino acid residues TIFF2026516142000002.tif88128

[0074] Substitution of one amino acid with another can be classified as either a conservative or non-conservative substitution. In the context of this invention, a “conservative substitution” is the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, and such substitution of one amino acid residue with another amino acid residue of the same class is as defined in either of the two tables above: for example, leucine can be substituted with isoleucine because they are both aliphatic branched hydrophobic substances. Similarly, aspartic acid can be substituted with glutamic acid because they are both small negatively charged residues.

[0075] In the context of the present invention, substitution in an antibody is Original amino acid - Position - Substituted amino acid It is shown as follows.

[0076] Referring to well-recognized amino acid nomenclature, a three-letter code or a one-letter code is used, which includes 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. As used herein, the term "naturally occurring" 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. Therefore, the notation "K409R" or "Lys409Arg" means that the antibody contains a substitution of lysine with arginine at amino acid position 409.

[0077] Substitution of an amino acid at a given position with any other amino acid is: Original amino acid - position; or, for example, "K409" It is called that.

[0078] For modifications where the original amino acid and / or substituted amino acids may contain more than one but not all amino acids, more than one amino acid may be separated by a comma or a slash. For example, the 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 to R, A, or F" That is the case.

[0079] Such designations may be used interchangeably in the context of the present invention, but they have the same meaning and purpose.

[0080] Furthermore, the term “substitution” encompasses any substitution of any one or any other 19 native amino acids, or substitution of other amino acids, such as non-native amino acids. For example, substitutions of amino acid K at position 409 include 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. This is, by the way, equivalent to the designation 409X, where X specifies any amino acid other than the original amino acid. These substitutions may also be referred to as K409A, K409C, etc., or K409A,C, etc., or K409A / C / , etc. The same applies by analogy to each and every position referred to herein, so that any one of such substitutions is specifically included herein.

[0081] The antibodies described herein may also contain deletions of amino acid residues. Such deletions may be denoted as "del," including, for example, K409del. Thus, in such embodiments, lysine at position 409 is deleted from the amino acid sequence.

[0082] As used herein, the term “host cell” is intended to refer to the cell into which the expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Because certain modifications may occur in the next generation due to either mutation or environmental influence, such offspring may not actually be identical to the parent cell, but they are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK293 cells, Expi293F cells, PER.C6 cells, NS0 cells, and lymphocytes, as well as prokaryotic cells such as Escherichia coli (E. coli), and other eukaryotic hosts such as plant cells and fungi.

[0083] As used herein, the term “transfectoma” includes recombinant eukaryotic host cells expressing antibodies or target antigens, such as CHO cells, PER.C6 cells, NS0 cells, HEK293 cells, Expi293F cells, plant cells, or fungi including yeast cells.

[0084] For the purposes of this invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably 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 start penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) permutation matrix. The Needle output, labeled "longest identity" (obtained using the -nobrief option), is used as the identity percentage and is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment).

[0085] Retention of similar residues can also be measured by a similarity score, similarly or alternatively, by using the BLAST program (e.g., BLAST 2.2.8, available through NCBI, with standard settings BLOSUM62, Open Gap=11, and Extended Gap=1). Suitable variants typically exhibit at least about 45% similarity to the parent sequence, 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%).

[0086] As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, e.g., lymphocytes (including T cells, such as B cells and cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, e.g., neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcγR) or complement receptors and perform specific immune functions. In some embodiments, effector cells, such as natural killer cells, can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells expressing FcγR are involved in the specific killing of target cells and / or the presentation of antigens to other components of the immune system, or their binding to antigen-presenting cells. 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 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 directly promote complement-dependent cell-mediated cytotoxicity (CDCC). In some embodiments, effector cells can phagocytose target antigens, target particles, or target cells, which may be antibody-binding-dependent and mediated by FcγR expressed by the effector cells. Expression of specific FcγRs or complement receptors on effector cells may be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon-γ (IFNγ) and / or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-carrying cells against their targets. Effector cells can phagocytose the target antigen or phagocytose or lyse the target cells.In some embodiments, antibody-driven classical complement activation results in C3 fragments on target cells. These C3 cleavage products may promote direct phagocytosis by effector cells or indirectly promote it 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.

[0087] As used herein, “effector T cells,” “Teffs,” or “Teff” refer to T lymphocytes that perform immune response functions, such as killing tumor cells and / or activating an anti-tumor immune response, which can result in the clearance of tumor cells from the body. An example of a Teff phenotype is CD3 + CD4 + and CD3 + CD8 + This includes Teff, which may secrete, contain, or express markers such as IFNγ, granzyme B, and ICOS. It is recognized that Teff may not be entirely limited to these phenotypes.

[0088] As used herein, "memory T cells" refer to T lymphocytes that remain in the body for an extended period after an infection has been eliminated. Examples of memory T cells include central memory T cells (CD45RA-CCR7+) and effector memory T cells (CD45RA-CCR7-). It is acknowledged that memory T cells may not be entirely limited to these phenotypes.

[0089] As used herein, “regulatory T cells,” “Tregs,” or “Treg” refer 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 CD3 + CD4 + CD25 + This is CD127dim. Tregs may further express Foxp3. It is observed that Tregs may not be entirely limited to this phenotype.

[0090] As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is initiated by the binding of a large macromolecular complex called C1 to an antibody-antigen complex on its surface. C1 is a complex consisting of six recognition proteins C1q and a serine protease heterotetramer C1r2C1s2. C1 is the first protein complex in the early events of the classical complement cascade, involving a series of cleavage reactions beginning with the cleavage of C4 into C4a and C4b, and C2 into C2a and C2b. C4b deposits and, together with C2a, forms an enzymatically active convertase called C3 convertase, which cleaves complement component C3 into C3b and C3a, forming C5 convertase. This C5 convertase splits C5 into C5a and C5b, the last component of which deposits on the membrane, which then triggers a late event of complement activation in which the terminal complement components C5b, C6, C7, C8, and C9 assemble to form the membrane invasion complex (MAC). The complement cascade consequently creates pores in the cell membrane, which causes cell lysis, also known as complement-dependent cytotoxicity (CDC). In certain embodiments herein in which the antibody has an inactive Fc region, the antibody does not induce complement activation.

[0091] Complement activation can be evaluated by C1q binding efficacy, CDC kinetics CDC assay (as described in WO2013 / 004842, WO2014 / 108198), or by the C3b and C4b cell deposition methods described in Beurskens et al., J Immunol April 1, 2012 vol. 188 no. 7, 3532-3541.

[0092] As used herein, the term “C1q binding” is intended to refer to the binding of C1q to an antibody bound to an antigen. Antibody binding to the antigen should be understood to occur both in vivo and in vitro in the context described herein. C1q binding can be evaluated, for example, by using an antibody immobilized on an artificial surface, or by using an antibody bound to a given antigen on a cell surface or virion surface, as described in Example 8 herein. The binding of C1q to an antibody oligomer should be understood herein as a multivalent interaction resulting in binding with high binding strength. For example, a decrease in C1q binding due to the introduction of a mutation in the antibody of the present invention can be measured by comparing the C1q binding of the mutant antibody with the C1q binding of its parent antibody (the antibody of the present invention that does not have the mutation in the same assay).

[0093] The term "treatment" refers to the administration of an effective amount of the therapeutically active antibody of the present invention for the purpose of alleviating, improving, stopping, or eradicating (curing) a symptom or disease condition.

[0094] The term "effective dose" or "therapeutic effective dose" refers to the amount of medication that is effective over the required period of time to achieve the desired therapeutic outcome. The therapeutic effective dose of an antibody may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the antibody's ability to elicit the desired response in the individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effects of any antibody variant outweigh the toxic or adverse effects.

[0095] Specific aspects of the invention In a first aspect, the present invention provides an antibody capable of binding to human OX40, comprising an antigen-binding region comprising a heavy-chain variable (VH) region in which CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and a light-chain variable (VL) region in which CDR1, CDR2, and CDR3 comprise the sequences shown in SEQ ID NO: 16, DAS, and SEQ ID NO: 17, respectively, and a human IgG1 Fc region comprising P329R and E345R mutations, wherein the amino acid positions are numbered according to Eu numbering. This provides an anti-OX40 antibody capable of binding to humans. In one aspect of the present invention, the antibody is agonist when it binds to OX40 on a T cell, for example, and to its target. This provides an antibody that stimulates T cell activation and proliferation. The antibody may further stimulate T cell memory formation and survival. Such antibodies are useful, for example, in the treatment of cancer. Furthermore, the antibodies can bind to cynomolgus monkey OX40, which is useful for toxicological studies of the antibodies. Therefore, in one embodiment of the antibody according to the present invention, the antibody can bind to cynomolgus monkey OX40 having the sequence SEQ ID NO: 51.

[0096] In another aspect of the present invention, an antibody can bind to human OX40 having the sequence SEQ ID NO: 52. Human OX40 contains four extracellular domains, designated CRD1, CRD2, CRD3, and CRD4, respectively. Without being constrained by theory, an antibody that binds to the CRD1 domain is intended to have an enhanced ability to activate T cells. In one aspect of the present invention, an antibody binds to the CRD1 domain of human OX40. In one aspect of the present invention, an antibody binds to an epitope of human OX40, enabling the binding of the antibody to human OX40 in the presence of OX40L.

[0097] It is well known in the art that binding affinity can be important for antibody function. Therefore, the affinity of an antibody to its congener antigen can be either too high or too low for the antibody to activate a desired intracellular pathway. In one embodiment, the antibody is 1 × 10⁻⁶ -9 M~6×10 -9 M's binding affinity K to human OX40 D It has. In one embodiment, the antibody is 2 × 10 -9 M~5×10 -9 M's binding affinity K to human OX40 D It has. In one embodiment, the antibody is 3 × 10 -9 M~4×10 -9 M's binding affinity K to human OX40 D It has. In another embodiment, the antibody is 3.4 × 10 -9 M's binding affinity K to human OX40 D It holds.

[0098] In another aspect of the present invention, the antibody is a humanized antibody or a chimeric antibody.

[0099] The immunogenicity of an antibody can be reduced through humanization, which may thereby reduce the level of the anti-drug antibody (ADA) response. Therefore, in one aspect of the present invention, the antibody is a humanized antibody.

[0100] In one embodiment of the present invention, the antibody comprises a VH region having the sequence shown in SEQ ID NO: 20 and a VL region having the sequence shown in SEQ ID NO: 21. This provides an antibody comprising a humanized VH region as shown in SEQ ID NO: 20 and a humanized VL region as shown in SEQ ID NO: 21. It is well known in the art that mutations can be introduced into proteins having well-characterized sequences and three-dimensional structures, such as antibodies, without loss of function. Accordingly, in some embodiments of the present invention, variants having mutations in the framework regions of the VH and / or VL sequences are also intended, and specific variants of the VH and / or VL regions are as shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. A variant may differ from the parent VH and / or VL sequences in one or more framework regions, i.e., FR1, FR2, FR3, and FR4, by one or more amino acids in one or more framework regions, but the antigen-binding region can still retain at least a significant percentage (at least about 90 percent, 95 percent, or more) of the affinity and / or specificity of the parent antibody, or even all of it. Typically, such a functional variant retains significant sequence identity with respect to the parent sequence. Exemplary variants include those that differ from the respective parent VH or VL region by five or fewer amino acid mutations, e.g., 5, 4, 3, 2, or 1 mutation, e.g., substitutions. Exemplary variants include those that differ from the parent VH and / or VL regions by primarily conservative amino acid substitutions; for example, five of the amino acid substitutions in the variant, e.g., 5, 4, 3, 2, or 1, may be conservative. In a further aspect of the present invention, the antibody may contain at most one, two, or three mutations in the VH framework region and / or the VL framework region, respectively. Such mutations may be substitutions. It is preferable that such substitutions do not significantly alter the binding affinity and / or binding specificity of the anti-OX40 antibody of the present invention.Accordingly, the present invention encompasses variants of the anti-OX40 antibody of the present invention, which have the same functional characteristics as the antibody comprising the VH region CDR sequence as shown in SEQ ID NO: 12, 13, and 14, and the VL region CDR sequence as shown in SEQ ID NO: 16, DAS, and SEQ ID NO: 17.

[0101] In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 80% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 85% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 90% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 95% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 96% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 97% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 98% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing a sequence that is at least 99% identical to the VH region shown in SEQ ID NO: 20. In another aspect of the present invention, the antibody includes a VH region containing the sequence shown in SEQ ID NO: 20.

[0102] In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 80% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 85% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 90% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 95% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 96% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 97% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 98% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing a sequence that is at least 99% identical to the VL region as shown in SEQ ID NO: 21. In another aspect of the present invention, the antibody includes a VL region containing the sequence as shown in SEQ ID NO: 21.

[0103] In another aspect of the present invention, the antibody comprises a VH region and a VL region, each containing the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively.

[0104] In one respect, the antibody of the present invention is an isolated antibody.

[0105] 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.

[0106] The antibody according to the present invention may include a light chain constant region, which is a human kappa light chain. In another context, it may include a human lambda light chain constant region.

[0107] The antibody according to the present invention may further include a heavy chain constant region, preferably a human IgG1 isotype. Thus, in one embodiment, the antibody includes a human IgG1 Fc region. Human IgG1 isotypes exist in various allotypes such as IgG1m(f), IgG1m(a), IgG1m(x), and IgG1m(z), and the allotypes may also be written as IgG1mf, IgG1ma, IgG1mx, and IgG1mz, respectively.

[0108] In one embodiment of the present invention, the antibody comprises an Fc region selected from the human IgG1mf, human IgG1ma, human IgG1mx, or human IgG1mz allotypes. In one embodiment of the present invention, the Fc region is of the human IgG1mf allotype. In one embodiment of the present invention, the antibody comprises an Fc region having the sequence shown in SEQ ID NO: 1.

[0109] For example, optimizing effector function by modifying the Fc region of an antibody to improve its ability to elicit an immune response against antigen-expressing cells can improve the efficacy of therapeutic antibodies for treating cancer or other diseases. Such efforts are described, for example, in WO2013 / 004842 A2;WO2014 / 108198 A1;WO2018 / 146317;WO2018 / 083126;WO2018 / 031258 A1.

[0110] US2014 / 0377284A1 discloses human OX40-binding antibody clone 12H3 (also known as SF2) that does not block the binding of OX40L to OX40. The SF2 antibody has been shown to induce T cell activation and reduce the inhibitory effect of Tregs in vitro. Zhang et al. demonstrated that the agonist effect and effector function of antibody clone SF2 could be enhanced by various engineering approaches, including the introduction of the hexamerization-enhancing mutation E345R (Zhang et al. J Biol Chem. 2016 Dec 30;291(53):27134-27146).

[0111] WO2016 / 164480A1 discloses human OX40-conjugated antibody clones containing clone h3C8 that have been molecularly engineered to confer an enhanced agonist effect. Engineering strategies included the introduction of hexamerization-enhancing mutants E345R, E430G, S440Y, or various combinations thereof. In addition, Fc-inactivating mutants such as L234A-L235A-P329G were introduced.

[0112] US20190276549A1 describes a method for engineering antibodies with enhanced Fc-Fc interactions, while further incorporating mutations that reduce the binding of complement factor C1q and the Fcγ receptor, in order to generate non-depleting immune cell targeting antibodies that possess agonist properties independently of Fcγ receptor involvement.

[0113] Further engineering approaches for developing human OX40 agonist constructs are described, including a fusion protein (US10449233) comprising Fc domains (WO2017 / 019805A1) covalently linked to six single-domain antibodies to create a hexavalent construct, and the extracellular domain (ECD) of PD1 linked to the ECD of the OX40L trimer via an IgG4 Fc-linked protein.

[0114] The antibody according to the present invention preferably comprises a modified human IgG1 constant region. Such human IgG1 comprises an Fc region including a CH2 region and a CH3 region. By modifying the IgG1 constant region in the Fc region, it is possible, for example, to control the Fc effector function of the antibody or to increase Fc-Fc interactions, thereby increasing the tendency of the antibody to form clusters such as hexamers. In one aspect of the present invention, human IgG1 or modified human IgG1 is selected from IgG1mf, IgG1ma, IgG1mx, or IgG1mz. In one aspect, it is a modified IgG1 having at least two mutations. In another aspect, it is IgG1mf having at least two mutations. In yet another aspect, it is IgG1ma having at least two mutations. In yet another aspect, it is IgG1mx having at least two mutations. In yet another aspect of the present invention, it is IgG1mz having at least two mutations. In one particular aspect, IgG is a modified human IgG containing two or more amino acid substitutions in the Fc region. In one embodiment, it may be human IgG1 containing two or more amino acid substitutions in the Fc region. In a further aspect of the present invention, IgG1mf contains two or more amino acid substitutions in the Fc region. In a further aspect of the present invention, IgG1mf contains three amino acid substitutions in the Fc region. In a further aspect of the present invention, IgG1mf contains four amino acid substitutions in the Fc region. In a further aspect of the present invention, IgG1mf contains five amino acid substitutions in the Fc region. In one embodiment, the IgG1 Fc region has at most three amino acid substitutions. In one embodiment, the IgG1mf Fc region has at most three amino acid substitutions. In one embodiment, the IgG1mf Fc region has at most four amino acid substitutions. In one embodiment, the IgG1mf Fc region has at most five amino acid substitutions.

[0115] In a further aspect of the present invention, the modified human IgG1 heavy chain constant region contains at most five amino acid substitutions in the Fc region. In another aspect, it contains at most four amino acid substitutions. In yet another aspect, it contains at most three amino acid substitutions. In yet another aspect, it contains at most two amino acid substitutions.

[0116] Mutations in the amino acid residue at the position corresponding to E345 in the human IgG1 heavy chain (amino acid residues are numbered according to Eu numbering) can improve the antibody's ability to induce CDC and other effector functions. Without being constrained by theory, it is thought that introducing an E345R substitution can stimulate antibody oligomerization, thereby modulating Fc-mediated effector functions to enhance, for example, C1q binding, complement activation, CDC, ADCP, internal translocation, or other relevant functions that may provide in vivo efficacy. In one embodiment, the antibody according to the present invention comprises an Fc region containing the sequence shown in SEQ ID NO: 2.

[0117] In one aspect, the present invention relates to a variant antibody comprising an antigen-binding region and a variant Fc region, wherein the antigen-binding region is capable of binding to OX40.

[0118] This provides an antibody having enhanced Fc-Fc interaction, which can induce antibody-dependent cluster formation of OX40 on the cell surface upon antibody binding, thereby increasing the agonism of the antibody of the present invention.

[0119] In one aspect of the present invention, the antibody comprises a variant human IgG1 Fc region or a variant human IgG1 CH region containing the E345R mutation and the P329R mutation. Hereafter, references to mutations in the Fc region may be applied similarly to mutations in the human IgG1 CH region, and vice versa.

[0120] As described herein, the positions of amino acids to be mutated in the Fc region can be given in relation to (i.e., "correspondingly") their positions in the naturally occurring (wild-type) human IgG1 heavy chain when numbered according to Eu numbering. Therefore, if the parent Fc region already contains one or more mutations, and / or if the parent Fc region is, for example, an IgG1, IgG1mf, IgG1ma, IgG1mz, or IgG1mz Fc region, the positions of amino acids corresponding to amino acid residues such as E345 in the human IgG1 heavy chain, numbered according to Eu numbering, can be determined by alignment. Specifically, the parent Fc region is aligned with the wild-type human IgG1 heavy chain sequence to identify the residue at the position corresponding to E345 in the human IgG1 heavy chain sequence. Any wild-type human IgG1 constant region amino acid sequence containing any one of the various human IgG1 allotypes shown in Table 3 may be useful for this purpose.

[0121] In one aspect of the present invention, modification in the IgG1 Fc region induces increased OX40 agonism compared to antibodies containing the wild type of the IgG Fc region of the same isotype or allotype, such as IgG1, even though the antibody is the same antibody. This can be achieved, for example, by introducing an R amino acid at the amino acid position corresponding to position E345, and thus introducing the E345R mutation into the human IgG1 heavy chain according to Eu numbering.

[0122] 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. Therefore, the antibody of the present invention may contain an E345R substitution in the Fc region.

[0123] This provides an antibody having enhanced Fc-Fc interaction, which can induce antibody-dependent cluster formation of OX40 on the cell surface upon antibody binding, thereby increasing the agonism of the antibody of the present invention.

[0124] In another aspect of the antibody of the present invention, the amino acid residue at the position corresponding to position P329 in the human IgG1 heavy chain according to the EU numbering is substituted with an R amino acid, and thus, a P329R mutation is introduced into the human IgG1 heavy chain according to the EU numbering.

[0125] In a further aspect of the present invention, the antibody has an amino acid residue R at the position corresponding to position P329 in the human IgG1 heavy chain according to the EU numbering. Thus, the antibody of the present invention contains a P329R substitution in the Fc region. Without being bound by theory, the antibody of the present invention containing an E345R mutation in the Fc region is thought to have increased serum clearance. The inventors have found that by further introducing a mutation at position 329, such as P329R, the clearance of the antibody of the present invention has been restored to the level of an antibody containing the wild-type sequence of the human IgG1 Fc region (i.e., having no mutations in the human IgG1 Fc region). Furthermore, the P329R mutation reduces the ability of the antibody to bind to FcγR receptors such as FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa.

[0126] In a preferred aspect of the present invention, the Fc region contains a P329R mutation and an E345R mutation at the positions corresponding to positions P329 and E345 in the human IgG1 heavy chain according to the EU numbering. Thereby, an antibody is provided that has increased OX40 receptor agonism and comparable pharmacokinetic properties, such as serum clearance, when compared to an antibody containing the same IgG1 heavy chain constant region except that it contains the wild-type amino acid P at position 329 and the wild-type amino acid E at position 345 and contains the same VH and VL regions.

[0127] Accordingly, one aspect of the present invention provides an OX40-conjugating antibody that, when compared to the pharmacokinetic properties of an antibody containing the same VH and VL regions but also containing the wild-type IgG1 heavy chain constant region, such as shown in SEQ ID NO: 1, exhibits increased receptor agonism upon binding to OX40, and further possesses comparable pharmacokinetic properties, such as similar or even identical pharmacokinetic properties. In other words, the present invention provides an OX40-conjugating antibody having pharmacokinetic properties that are not significantly different from those of an OX40-conjugating antibody that is identical except for containing the wild-type IgG1 heavy chain constant region.

[0128] In one embodiment, the parent Fc region and / or the human IgG1 CH region is the wild-type human IgG1 isotype.

[0129] In one embodiment of the present invention, the parent human IgG1 Fc region and / or human IgG1 CH region is the wild-type human IgG1mf allotype. In one embodiment of the present invention, the parent human IgG1 Fc region and / or human IgG1 CH region is the wild-type human IgG1ma allotype. In one embodiment of the present invention, the parent human IgG1 Fc region and / or human IgG1 CH region is the wild-type human IgG1mx allotype. In one embodiment of the present invention, the parent human IgG1 Fc region and / or human IgG1 CH region is the wild-type human IgG1mz allotype.

[0130] Therefore, the variant Fc region can be the human IgG1 Fc region, with the exception of the listed mutations E345R and P329R.

[0131] In another aspect, the present invention provides an antibody comprising a heavy chain constant region having an amino acid sequence selected from the group including SEQ ID NO: 58, 59, 60, and 61. In one aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 58. In another aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 59. In another aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 60. In another aspect, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 61.

[0132] In one embodiment, the antibody according to the present invention is a. VH region containing the amino acid sequence shown in SEQ ID NO: 20, b. The VL region containing the amino acid sequence shown in SEQ ID NO: 21, and c. Fc region containing the amino acid sequence shown in SEQ ID NO: 3 Includes.

[0133] In another embodiment, the antibody according to the present invention is a. VH region containing the amino acid sequence shown in SEQ ID NO: 20, b. The VL region containing the amino acid sequence shown in SEQ ID NO: 21, and c. CH region containing the amino acid sequence shown in SEQ ID NO: 58 Includes.

[0134] In another embodiment, the antibody according to the present invention comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 18 and a light chain containing the amino acid sequence shown in SEQ ID NO: 19.

[0135] In another aspect, the present invention provides an antibody comprising a heavy chain constant region that is modified to induce Fc-mediated effector function to a lesser extent than an antibody that is identical except for the modification. An example of this is the OX40-conjugated antibody of the present invention comprising P329R substitution and E345R substitution. 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 absence of the P329R substitution, such as a wild-type IgG1 heavy chain, and also compared to an antibody comprising the same sequence except for the absence of the P329R substitution and E345R substitution. 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, Fc-mediated effector function is reduced by at least 70%. In another aspect, Fc-mediated effector function is reduced by at least 80%. In another aspect, 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. Therefore, in one embodiment, the antibody of the present invention induces CDC by reducing it by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the constant region of the same antibody but wild-type IgG1 HC. In another embodiment, the antibody of the present invention does not induce CDC.

[0136] In another aspect, the antibody of the present invention induces CDCC by reducing it by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the same antibody but with a wild-type IgG1 HC constant region. In another aspect, the antibody of the present invention does not induce CDCC.

[0137] In another aspect, the antibody of the present invention induces ADCC by reducing it by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the same antibody but with a wild-type IgG1 HC constant region. In another aspect, the antibody of the present invention does not induce ADCC.

[0138] In another aspect, the antibody of the present invention induces ADCP by reducing it by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the same antibody having a wild-type IgG1 HC constant region. In another aspect, the antibody of the present invention does not induce ADCP.

[0139] In another aspect, the antibody of the present invention induces C1q binding by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the same antibody having a wild-type IgG1 HC constant region. In another aspect, the antibody of the present invention does not induce C1q binding.

[0140] In one embodiment, the antibody of the present invention does not bind to C1q. This describes an embodiment in which, when the antibody binds to the cell membrane, it is not possible to bind C1q to the Fc region of the antibody. In one embodiment, the antibody does not induce target-independent liquid-phase complement activation.

[0141] In another aspect, the antibody of the present invention induces FcγR binding by at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, or at least 90%, at least 95%, or at least 98%, compared to the same antibody having a wild-type IgG1 HC constant region. In another aspect, the antibody of the present invention does not induce FcγR binding. Preferably, FcγR binding is determined as in Example 8.

[0142] In one embodiment, the antibody of the present invention has reduced C1q binding and reduced FcγR binding compared to an antibody having the same amino acid sequence except that it does not contain the P329R substitution. In one embodiment, the antibody of the present invention has reduced binding to FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa. In one embodiment, the antibody of the present invention has binding to FcγRIa 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%. In one embodiment, the antibody of the present invention has binding to FcγRIIa, FcγRIIb, and FcγRIIa that is reduced by at least 60%, for example, at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%. In one embodiment of the present invention, the antibody does not have binding to FcγRIIa, FcγRIIb, and FcγRIIa.

[0143] In one aspect, an antibody according to any aspect or embodiment herein is a human antibody, except for the recited mutations.

[0144] In one embodiment of the invention, the antibody is a monovalent antibody.

[0145] In another aspect, the antibody is a bivalent antibody.

[0146] Furthermore, the antibodies of the invention can be monospecific antibodies.

[0147] In one aspect, an 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.

[0148] In a preferred aspect, an antibody according to any aspect or embodiment herein is a humanized bivalent full-length monoclonal antibody. In a preferred aspect, an antibody according to the invention is a humanized antibody comprising a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 19. Thereby, an aspect of a humanized antibody with a SI mean score of 2.66 corresponding to an antibody with low clinical immunogenicity is described.

[0149] In a preferred aspect, an 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 recited mutations in the Fc region.

[0150] The antibodies according to the present invention are advantageously in a bivalent, monospecific form, comprising two antigen-binding regions that bind to the same epitope. However, a bispecific form in which one of the antigen-binding regions binds to a different epitope is also intended. Thus, an antibody according to any aspect or embodiment of this specification may be either a monospecific antibody or a bispecific antibody, as long as it is not inconsistent with the context.

[0151] Accordingly, in another embodiment, the antibody of the present invention is a bispecific antibody comprising a first antigen-binding region capable of binding to human OX40 as described herein, and a second antigen-binding region capable of binding to a different epitope on human OX40. In another embodiment, the antibody of the present invention is a bispecific antibody comprising a first antigen-binding region capable of binding to human OX40 as described herein, and a second antigen-binding region capable of binding to a different target. Such targets may be on different cells or on the same cells as OX40.

[0152] In one aspect of the present invention, the antibody can bind to human OX40 having the sequence shown in SEQ ID NO: 52. In another aspect, the antibody of the present invention can further bind to cynomolgus monkey OX40, for example, shown in SEQ ID NO: 51.

[0153] In a further embodiment of the present invention, the antibody can bind to OX40-expressing human T cells.

[0154] In another embodiment of the present invention, the antibody can bind to OX40-expressing cynomolgus monkey T cells.

[0155] In another aspect of the present invention, the antibody has increased agonist activity compared to a wild-type parent antibody that does not have the P329R and E345R mutations.

[0156] In another aspect of the present invention, the antibody induces increased T cell proliferation.

[0157] In another aspect of the present invention, the antibody induces increased T cell proliferation compared to a parental antibody that does not have the P329R and E345R mutations.

[0158] In one embodiment of the present invention, a full-length IgG1 antibody has the C-terminal lysine of HC cleaved. Such an antibody is also considered a "full-length antibody."

[0159] In another embodiment of the present invention, the antibody is used to target human T cells, e.g., CD4, as described in Example 7. + T cells and CD8 + The antibody can induce the proliferation of T cells, such as T helper cells and cytotoxic T cells. In another embodiment of the present invention, the antibody can induce the proliferation of human CD4+ T cells. In another embodiment of the present invention, the antibody can induce the proliferation of human CD8+ T cells.

[0160] In another embodiment of the present invention, the antibody can induce the activation of human OX40-expressing T cells.

[0161] In another embodiment of the present invention, an antibody can induce the expression of OX40 on T cells, such as CD4+ T cells and CD8+ T cells. Thus, the antibody can increase the expression of OX40 on CD4+ T cells and CD8+ T cells.

[0162] In another aspect of the present invention, an antibody can induce activation of human OX40-expressing T cells in the absence of Fcγ receptor IIb crosslinking.

[0163] In another embodiment of the present invention, the antibody can induce the expression of 4-1BB, CD25, and HLA-DR on CD4+ T cells and CD8+ T cells.

[0164] In another aspect of the present invention, the antibody is a CD4+ T cell and CD8 T cell having a central memory T cell phenotype. +It can induce the proliferation of T cells.

[0165] In another aspect of the present invention, the antibody can induce the secretion of TNFα, IFNγ, IL-2, and IL-13. In another aspect of the present invention, the antibody can induce the secretion of TNFα, IFNγ, IL-2, and IL-13 from CD8+ T cells.

[0166] In another embodiment of the present invention, an antibody can induce granzyme B in T cells, for example, CD4+ T cells. In another embodiment of the present invention, an antibody can induce elevated levels of granzyme B in T cells, for example, CD4+ T cells. Granzyme B can be used as a marker for cytotoxic T cells. This discloses an embodiment in which the antibody can generate T cells having elevated levels of cytolytic ability. Antibodies are well known as therapeutic agents that can be used in the treatment of various diseases. Another method for administering an antibody to a target that needs it includes the administration of a nucleic acid or combination of nucleic acids encoding the antibody for in vivo expression of the antibody.

[0167] Accordingly, in one aspect, the present invention also relates to a nucleic acid sequence encoding a heavy chain of an antibody according to the present invention, wherein the heavy chain comprises a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO: 12, a VH CDR2 comprising the sequence shown in SEQ ID NO: 13, and a VH CDR3 comprising the sequence shown in SEQ ID NO: 17.

[0168] In another aspect, the present invention also relates to a nucleic acid sequence encoding a heavy chain of an antibody according to the present invention, the heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO: 12, a VH CDR2 comprising the sequence shown in SEQ ID NO: 13, a VH CDR3 comprising the sequence shown in SEQ ID NO: 17, and a human IgG1 CH region.

[0169] In another aspect, the present invention also relates to a nucleic acid sequence encoding a VH region comprising the amino acid sequence shown in SEQ ID NO: 20.

[0170] In another aspect, the present invention also relates to a nucleic acid sequence encoding a VL region comprising the amino acid sequence shown in SEQ ID NO: 21.

[0171] In another aspect, the present invention also relates to a nucleic acid sequence encoding a heavy chain (HC) as shown in SEQ ID NO: 18.

[0172] In another aspect, the present invention also relates to a nucleic acid sequence encoding a light chain (LC) as shown in SEQ ID NO: 19.

[0173] In another aspect, the present invention also relates to nucleic acid sequences encoding a heavy chain (HC) as shown in SEQ ID NO: 18 and a light chain (LC) as shown in SEQ ID NO: 19.

[0174] In one embodiment, the antibody of the present invention is encoded by a single nucleic acid. Therefore, the nucleotide sequence encoding the antibody of the present invention resides in a single nucleic acid sequence or in the same nucleic acid molecule.

[0175] 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, the combination of nucleic acid sequences includes a nucleic acid sequence encoding the heavy chain of the antibody and a nucleic acid sequence encoding the light chain of the antibody.

[0176] In some embodiments, the present invention relates to a nucleic acid sequence or combination of nucleic acid sequences encoding an antibody comprising the following:

[0177] In one embodiment, the antibody of the present invention is encoded by a single nucleic acid. Therefore, the nucleotide sequence encoding the antibody of the present invention resides in a single nucleic acid or in the same nucleic acid molecule.

[0178] 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, the combination of nucleic acid sequences includes a nucleic acid sequence encoding the heavy chain of the antibody and a nucleic acid sequence encoding the light chain of the antibody.

[0179] As described above, nucleic acid sequences can be used as a means of supplying therapeutic proteins, such as antibodies, to targets that require them.

[0180] In some embodiments, the nucleic acid may be deoxyribonucleic acid (DNA). DNA and methods for preparing DNA suitable 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 by Patel A et al., 2018, Cell Reports 25, 1982-1993.

[0181] In some embodiments, the nucleic acid may be ribonucleic acid (RNA), such as messenger RNA (mRNA). In some embodiments, the mRNA may consist only of naturally occurring nucleotides. In some embodiments, the mRNA may also consist of modified nucleotides, where modified means that the nucleotide is chemically different from a naturally occurring nucleotide. In some embodiments, the mRNA may consist of both naturally occurring nucleotides and modified nucleotides.

[0182] Various nucleic acid sequences suitable for in vivo expression of therapeutic proteins such as antibodies in a target are well known to those skilled in the art. For example, mRNA suitable for the expression of therapeutic antibodies in a target often includes an open reading frame (ORF) with adjacent untranslated regions (UTRs) containing specific sequences, as well as 5' and 3' ends formed by a cap structure and a poly(A) tail (see, for example, Schlake et al., 2019, Molecular Therapy Vol.27 No.4 April).

[0183] Examples of methods for optimizing RNA and RNA molecules suitable for in vivo expression, such as mRNA, include, but are not limited to, those described in US9254311;US9221891;US20160185840 and EP3118224.

[0184] Naked nucleic acid sequences administered to a subject for in vivo expression are readily degradable and / or prone to eliciting an immunogenic response in the subject. Furthermore, for the in vivo expression of antibodies encoded by a nucleic acid sequence, the nucleic acid sequence is typically administered in a form suitable for entry into the target cells. Various methods exist for delivering nucleic acid sequences for in vivo expression, including both mechanical and chemical methods. For example, such methods may include electroporation or tattooing of nucleic acids onto the skin (Patel et al., 2018, Cell Reports 25, 1982-1993). Other methods suitable for administering nucleic acid sequences to a subject include the administration of nucleic acids in a suitable formulation. Thus, the present invention also relates to a delivery vehicle containing the nucleic acid of the present invention.

[0185] In some embodiments, the delivery vehicle may include a nucleic acid sequence encoding the heavy chain of the antibody according to the present invention. Thus, in one embodiment, the nucleic acid sequence may encode a heavy chain comprising a VH region including VH CDR1 containing the sequence shown in SEQ ID NO: 12, VH CDR2 containing the sequence shown in SEQ ID NO: 13, and VH CDR3 containing the sequence shown in SEQ ID NO: 14, and a human IgG1 HC region having the P329R mutation and the E345R mutation, wherein the amino acid residues are numbered according to Eu numbering.

[0186] In some embodiments, the present invention also relates to a delivery vehicle comprising a nucleic acid sequence encoding the light chain of an antibody according to the present invention. Thus, in one embodiment, the nucleic acid sequence may encode a light chain comprising a VL region comprising VL CDR1 comprising the sequence shown in SEQ ID NO: 16, VL CDR2 comprising the sequence shown in DAS, and VL CDR3 comprising the sequence shown in SEQ ID NO: 17.

[0187] The present invention also relates to a mixture of delivery vehicles, comprising a delivery vehicle comprising a nucleic acid sequence encoding the heavy chain of an antibody according to the present invention, and a delivery vehicle comprising a delivery vehicle comprising a nucleic acid sequence encoding the light chain of an antibody according to the present invention. Accordingly, in one embodiment, the mixture of delivery vehicles comprises a delivery vehicle comprising a nucleic acid sequence encoding a heavy chain, comprising a VH region comprising VH CDR1 comprising the sequence shown in SEQ ID NO: 12, VH CDR2 comprising the sequence shown in SEQ ID NO: 13, and VH CDR3 comprising the sequence shown in SEQ ID NO: 14, and a human IgG1 CH region having P329R mutations and E345R mutations, wherein the amino acid residues are numbered according to Eu numbering; and a delivery vehicle comprising a nucleic acid sequence encoding a light chain comprising VL region, comprising VL CDR1 comprising the sequence shown in SEQ ID NO: 16, VL CDR2 comprising the sequence shown in DAS, and VL CDR3 comprising the sequence shown in SEQ ID NO: 17.

[0188] In some embodiments, the delivery vehicle includes a nucleic acid sequence or combination of nucleic acid sequences encoding the heavy chain and nuclear light chain of the antibody according to the present invention.

[0189] Accordingly, in one embodiment, the delivery vehicle may include a heavy chain comprising a VH region comprising a VH CDR1 comprising the sequence shown in SEQ ID NO: 12, a VH CDR2 comprising the sequence shown in SEQ ID NO: 13, and a VH CDR3 comprising the sequence shown in SEQ ID NO: 14, and a human IgG1 CH region having mutations P329R and E345R, wherein the 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 shown in SEQ ID NO: 16, a VL CDR2 comprising the sequence shown in DAS, and a VL CDR3 comprising the sequence shown in SEQ ID NO: 17.

[0190] Therefore, the nucleic acid sequences encoding the heavy and light chains of the antibody according to the present invention are located within a single (same) nucleic acid molecule.

[0191] In another embodiment, the delivery vehicle may comprise a nucleic acid sequence encoding a heavy chain containing the sequence shown in SEQ ID NO: 9; and a nucleic acid sequence encoding a light chain containing the sequence shown in SEQ ID NO: 10.

[0192] Therefore, the nucleic acid sequences encoding the heavy chain and light chain of the antibody variant according to the present invention reside on separate nucleic acid molecules or on different nucleic acid molecules.

[0193] In some embodiments, the delivery vehicle may be a lipid formulation. The lipids of the formulation may be particles such as lipid nanoparticles (LNPs). The nucleic acid sequence or combination of nucleic acid sequences of the present invention may be encapsulated within the particles, for example, within the LNPs.

[0194] Various lipid formulations suitable for administering nucleic acids to subjects for in vivo expression are well known to those skilled in the art. For example, such lipid formulations may typically include lipids, ionizable aminolipids, PEG-lipids, cholesterol, or any combination thereof.

[0195] Various forms and methods of preparing lipid formulations suitable for administering nucleic acid sequences to targets for the expression of 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).

[0196] The present invention also provides isolated nucleic acid sequences and vectors encoding 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 include, but are not limited to, those known in the Art and described in the Examples. In embodiments in which the variant antibody comprises HC and LC, which are separate polypeptides rather than being contained in a single polypeptide (for example, in an scFv-Fc fusion protein), the nucleotide sequences encoding the heavy and light chains may be present in the same or different nucleic acids or vectors.

[0197] Accordingly, in one aspect, the present invention provides an isolated nucleic acid sequence or combination of nucleic acid sequences encoding an antibody according to any aspect or embodiment described herein. The present invention also provides a nucleic acid sequence encoding a VH region comprising VH CDR1 comprising the sequence shown in SEQ ID NO: 12, VH CDR2 comprising the sequence shown in SEQ ID NO: 13, and VH CDR3 comprising the sequence shown in SEQ ID NO: 14.

[0198] Furthermore, the present invention provides a nucleic acid sequence that encodes a VL region comprising VL CDR1 containing the sequence shown in SEQ ID NO: 16, VL CDR2 containing the sequence shown in DAS, and VL CDR3 containing the sequence shown in SEQ ID NO: 17.

[0199] Furthermore, the present invention provides a nucleic acid sequence encoding a VH region containing the amino acid sequence shown in SEQ ID NO: 20. The present invention also relates to a nucleic acid sequence encoding a VL region containing the amino acid sequence shown in SEQ ID NO: 21.

[0200] In one aspect of the present invention, the nucleic acid sequence or combination of nucleic acid sequences is RNA or DNA. In one aspect of the present invention, the nucleic acid sequence or combination of nucleic acid sequences is mRNA.

[0201] The present invention further provides an expression vector comprising a nucleic acid sequence or a combination thereof in any aspect or embodiment described herein.

[0202] In another aspect, the present invention relates to nucleic acid sequences or combinations of nucleic acid sequences as described herein for use in expression in mammalian cells.

[0203] In a further embodiment, the present invention relates to recombinant host cells that produce antibodies as defined herein, wherein the host cells optionally include the expression vector described above. In a particular embodiment, the recombinant host cells are eukaryotic or prokaryotic cells.

[0204] In another aspect, the present invention relates to a method for producing an antibody according to any aspect or embodiment described herein, comprising the steps of culturing recombinant host cells as described above in a culture medium under conditions suitable for antibody production, and optionally purifying or isolating the antibody from the culture medium.

[0205] In one aspect, the present invention is (i) A nucleotide sequence encoding the heavy chain sequence of an antibody according to any one of the embodiments disclosed herein; (ii) A nucleotide sequence encoding the light chain sequence of an antibody according to any one of the embodiments disclosed herein; or (iii) Both (i) and (ii) This relates to nucleic acids or expression vectors, including those mentioned above.

[0206] In one aspect, the present invention relates to a nucleic acid or expression vector comprising a nucleotide sequence encoding a heavy chain sequence of an antibody variant according to any one of the embodiments disclosed herein.

[0207] In one aspect, the present invention relates to a nucleic acid sequence or expression vector comprising a nucleotide sequence encoding a heavy chain sequence and a light chain sequence of an antibody according to any one of the embodiments disclosed herein.

[0208] In one aspect, the present invention relates to a combination of a first nucleic acid and a second nucleic acid, or a combination of a first expression vector and a second expression vector, in optionally the same host cell, wherein the first comprises a nucleotide sequence according to (i), and the second comprises a nucleotide sequence according to (ii).

[0209] Expression vectors in the context of the present invention may be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, nucleic acids are expressed in a naked DNA or RNA vector containing, for example, linear expression elements (as described, e.g., Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), compacted nucleic acid vectors (as described, e.g., US 6,077,835 and / or WO 00 / 70087), plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119, or "midge" minimal-size nucleic acid vectors (as described, e.g., Schakowski et al., Mol Ther 3, 793 800 (2001)), or precipitated nucleic acid vector constructs, e.g., CaP04-precipitated constructs (as described, e.g., WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, These are included as described in 725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0210] In one embodiment, vectors are suitable for antibody expression in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989)), and pET vectors (Novagen, Madison WI).

[0211] The expression vector may, or alternatively, be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors containing constitutive or inductive promoters such as alpha factor, alcohol oxidase, and PGH (as outlined 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)).

[0212] The expression vector may also be a vector suitable for expression in mammalian cells, such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175, which contains glutamine synthetase as a selectable marker.

[0213] Nucleic acids and / or vectors may also contain nucleic acid sequences encoding secretory / localization sequences that can target polypeptides, such as nascent polypeptide chains, into the periplasmic space or cell culture medium. Such sequences are known in the art and include secretory leader peptides or signal peptides.

[0214] Expression vectors may contain, or may be associated with, any suitable promoters, enhancers, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer, as well as the RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). Nucleic acids may also contain inductive promoters, as opposed to constitutive promoters such as CMV IE.

[0215] In one embodiment, an expression vector encoding an antibody may be placed and / or delivered to a host cell or host animal via a viral vector.

[0216] The present invention also provides recombinant host cells that produce antibodies as disclosed herein, optionally comprising isolated nucleic acids or vectors according to the present invention. Typically, the host cells are transformed or transfected with nucleic acids or vectors. The recombinant host cells of the claims may be, for example, eukaryotic cells, prokaryotic cells, or microbial cells, such as transfectomas. In certain embodiments, the host cells are eukaryotic cells. In certain embodiments, the host cells are prokaryotic cells. In some embodiments, the antibodies are heavy-chain antibodies. However, in most embodiments, the antibodies contain both heavy and light chains, and therefore the host cells express both heavy-chain encoding constructs and light-chain encoding constructs, either on the same vector or on different vectors.

[0217] 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 lymphocytes. In one embodiment, the host cell is a CHO (Chinese hamster ovary) cell. For example, in one embodiment, the host cell may contain a first nucleic acid construct and a second nucleic acid construct stably integrated into the cellular genome, where the first encodes the heavy chain of an antibody variant as disclosed herein, and the second encodes its light chain. In another embodiment, the present invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising the first and second nucleic acid constructs as specified above.

[0218] In one embodiment, the host cell is a cell capable of protein Asn-linked glycosylation, such as a eukaryotic cell, such as a mammalian cell, such as a human cell.

[0219] In one embodiment, the host cell is a host cell that cannot 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 (incorporated herein by reference) lists numerous such antibody-producing systems, e.g., Sp2 / 0, NS / 0, or transgenic mammary gland (goat), which only achieve partial removal of the C-terminal lysine. In one embodiment, the host cell is a host cell in which the glycosylation mechanism has been altered. Such cells have been described in the Art and can be used as host cells expressing the variant of the present invention to produce antibodies with altered glycosylation. For example, see Shields, RL et 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 manipulated glycoforms are known in the art, including 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 technology (GLYCART biotechnology AG, Zurich, Switzerland); US This includes, but is not limited to, what is described in 20030115614;Okazaki et al., 2004, JMB, 336: 1239-49, as well as what is described in WO2018 / 114877, WO2018 / 114878, and WO2018 / 114879.

[0220] In a further aspect, the present invention relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of human heavy chains and human light chains, wherein the animal or plant produces antibodies as disclosed herein.

[0221] In one embodiment, an antibody obtained or obtainable by the above method is provided.

[0222] 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 P329R mutations and E345R mutations into the antibody at amino acid residues corresponding to E345 and P329 in the Fc region of human IgG monohelic acid, numbered according to Eu numbering.

[0223] Therefore, in a particular embodiment, a method is provided for increasing the effector function of a parent antibody, such as Fc-mediated effector function, or for increasing the biological activity of an antibody, such as OX40 agonism, wherein the parent antibody comprises an Fc region and an antigen-binding region that binds to OX40, the method comprising the step of introducing P329R mutations and E345R mutations into the Fc region of a human IgG1 heavy chain at amino acid residues corresponding to P329 and E345 in the Fc region, where the amino acid residues are numbered according to Eu numbering; and the antigen-binding region comprises VH CDR1 containing the sequence shown in SEQ ID NO: 12, VH CDR2 containing the sequence shown in SEQ ID NO: 13, VH CDR3 containing the sequence shown in SEQ ID NO: 14, VL CDR1 containing the sequence shown in SEQ ID NO: 16, VL CDR2 containing the sequence shown in DAS, and SEQ ID NO: Includes VL CDR3 with the sequence shown in 17.

[0224] In another particular embodiment, a method is provided for reducing the effector function of a parent antibody, such as C1q binding or FcγR binding, comprising a VH CDR1 comprising the sequence shown in SEQ ID NO: 12, a VH CDR2 comprising the sequence shown in SEQ ID NO: 13, a VH CDR3 comprising the sequence shown in SEQ ID NO: 14, a VL CDR1 comprising the sequence shown in SEQ ID NO: 16, a VL CDR2 comprising the sequence shown in DAS, and a VL CDR3 comprising the sequence shown in SEQ ID NO: 17, and further comprising an E345R amino acid substitution in the Fc region of a human IgG1 heavy chain, the amino acid residues being numbered according to Eu numbering, the method comprising the step of introducing a further P329R amino acid substitution into the Fc region of a human IgG1 heavy chain, numbered according to Eu numbering, thereby reducing or completely eliminating the effector function of the parent antibody, such as C1q binding or FcγR binding.

[0225] In one embodiment of any of the methods described above, the enhanced effector function includes the OX40 agonism.

[0226] In one of the methods described above, the effector function is a C1q coupling.

[0227] In one embodiment of any of the methods described above, the effector function is an FcgR coupling.

[0228] In one embodiment of any of the methods described above, the reducing effector function includes both C1q bonds and FcγR bonds.

[0229] In one embodiment, the effector function is C1q binding. This describes an embodiment in which the antibody according to the present invention having the E345R mutation and the P329R mutation has reduced C1q binding when compared to an antibody having the E345R mutation but not the P329R mutation.

[0230] In one embodiment, the reduced effector function is the binding to FcγR bonds. Thus, in one embodiment, it is the binding to FcγR such as FcγRIa, FcγRIIa, FcγRIIb, and / or FcγRIIIa. In one embodiment, the binding to FcγRIa is reduced. In one embodiment, the binding to FcγRIIa is reduced. In one embodiment, the binding to FcγRIIb is reduced. In one embodiment, the binding to FcγRIIIa is reduced.

[0231] This describes an embodiment in which the antibody according to the present invention, having the E345R mutation and the P329R mutation, has a reduced FcγR when compared to an antibody that has the E345R mutation but does not have the P329R mutation.

[0232] In one embodiment of the method described above, the Fc region includes the E345R mutation and the P329R mutation.

[0233] In any embodiment of the method described above, the Fc region of the antibody is, apart from the listed mutations, the Fc region of human IgG1, more specifically, human IgG1m(f), IgG1m(a), human IgG1m(x), or human IgG1m(z). Optionally, it includes one Fc region from the sequences indicated as SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61. In a preferred embodiment, the Fc region of the antibody is the human IgG1m(z) Fc region. For example, the antibody may be a human full-length IgG1 antibody, and optionally, a human monoclonal full-length bivalent IgG1,κ antibody. Additionally, the antibody may be a monospecific antibody or a bispecific antibody, for example, a monospecific antibody.

[0234] The Fc region of the antibody may be a human IgG1 isotype, but may also be any naturally occurring human IgG1 allotype sequence, e.g., human IgG1m(f), human IgG1m(a), human IgG1m(x), or human IgG1m(z), which may also be written as human IgG1mf, IgG1ma, human IgG1mx, or human IgG1mz, where, in some embodiments, the Fc region of the antibody includes one or more further mutations as described elsewhere herein.

[0235] The present invention also relates to antibodies obtained or obtainable by any of the methods described above.

[0236] The present invention also relates to a composition comprising an antibody according to the present invention, a nucleic acid according to the present invention, an expression vector according to the present invention, or a host cell according to the present invention.

[0237] In a further embodiment, the composition according to the present invention is a pharmaceutical composition that typically comprises a pharmaceutically 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.

[0238] In a further embodiment, the present invention is: - Antibodies as defined in any aspect and aspect disclosed herein, and - Pharmacologically acceptable carriers This relates to pharmaceutical compositions including [the specified substance].

[0239] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0240] The present invention also relates to a kit of parts, such as a kit for identifying within a patient population patients who are likely to respond to treatment with an antibody as defined herein, for use as a companion diagnostic, the kit of parts comprising an antibody as defined in any aspect or embodiment disclosed herein, and instructions for using the kit.

[0241] The present invention also relates to a kit of parts for therapeutic use comprising an antibody according to the present invention, or a composition comprising an antibody according to the present invention, wherein the kit of parts optionally contains more than one dose of the antibody.

[0242] In one embodiment, the kit of parts comprises such antibody or composition in one or more containers, such as vials.

[0243] In one embodiment, the kit of parts comprises such antibodies or compositions for simultaneous, separate, or sequential use in a therapeutic setting.

[0244] The antibodies of the present invention have numerous therapeutic uses, including the treatment of diseases and disorders that can be treated by activating immune cells expressing OX40. For example, the antibodies may be administered to cells in culture, for example in vitro or ex vivo, or to human subjects, for example in vivo, to treat or prevent a variety of disorders and diseases. Where used herein, the term “subject” is intended to include human and non-human animals that may benefit from or respond to the antibody. Subjects include, for example, CD4 + T cells and / or CD8 + This may include human patients with diseases or disorders that can be corrected or improved by modulating OX40 function so that T cell populations expand and proliferate. Therefore, antibodies may be used to induce the proliferation of T cell populations, such as T helper cells and cytotoxic T cells, in vivo or in vitro.

[0245] Therefore, in one aspect, the present invention relates to antibodies according to the present invention, nucleic acids or combinations of nucleic acids according to the present invention, delivery vehicles according to the present invention, expression vectors according to the present invention, host cells according to the present invention, compositions according to the present invention, or pharmaceutical compositions according to the present invention, for use as pharmaceuticals.

[0246] In one aspect, the present invention relates to the use of antibodies, nucleic acids or combinations of nucleic acids, delivery vehicles, expression vectors, host cells, compositions, or pharmaceutical compositions according to the present invention in the preparation of pharmaceuticals for treating or preventing diseases or disorders.

[0247] In one aspect, the present invention relates to a method for treating a disease or disorder, comprising the step of administering an antibody according to the present invention, a nucleic acid or 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 according to the present invention, a composition according to the present invention, or a pharmaceutical composition according to the present invention to a subject in need thereof.

[0248] In one aspect, the present invention relates to an antibody in any aspect or form for use as a pharmaceutical.

[0249] In one aspect, the present invention relates to the use of antibodies in any aspect or manner in the preparation of pharmaceuticals for treating or preventing diseases or disorders.

[0250] In one aspect, the present invention relates to an antibody in any aspect or manner for use in the treatment or prevention of a disease or disorder.

[0251] In one aspect, the present invention relates to an antibody in either aspect or form for use in diagnostics or for use in diagnostic methods.

[0252] In one aspect, the present invention relates to a method for treating a disease or disorder, comprising the step of administering an antibody in any aspect or manner to a subject in need, typically in a therapeutically effective amount and / or for a period of time sufficient to treat the disease or disorder.

[0253] In one aspect, the present invention relates to a pharmaceutical composition comprising an antibody in any aspect or embodiment for use as a pharmaceutical.

[0254] In one aspect, the present invention relates to a pharmaceutical composition comprising an antibody in any aspect or manner for use in the treatment or prevention of a disease or disorder.

[0255] In one aspect, the present invention relates to a method for treating a disease or disorder, comprising the step of administering a pharmaceutical composition comprising an antibody in any aspect or manner to a subject in need, typically in a therapeutically effective amount and / or for a period of time sufficient to treat the disease or disorder.

[0256] In one aspect, the present invention relates to a method for treating a disease or disorder, comprising the following steps: • The process of selecting subjects suffering from a disease or disability, The step of administering to the subject an antibody, or a pharmaceutical composition containing an antibody, in any form or manner, typically in a therapeutically effective amount and / or for a period of time sufficient to treat the disease or disorder.

[0257] In one embodiment, the disease or disorder is a tumorigenic disorder, such as cancer, i.e., a blood cancer or a malignant solid tumor. In another embodiment, the disease or disorder is an inflammatory and / or autoimmune disease or disorder.

[0258] In one embodiment of the present invention, the antibody is administered in a dose of 1 to 20 mg / kg. In another embodiment of the present invention, the antibody is administered in a dose of 1 mg / kg. In another embodiment of the present invention, the antibody is administered in a dose of 2.5 mg / kg. In another embodiment of the present invention, the antibody is administered in a dose of 5 mg / kg. In another embodiment of the present invention, the antibody is administered in a dose of 10 mg / kg. In another embodiment of the present invention, the antibody is administered in a dose of 20 mg / kg. In a further aspect, the present invention relates to an antibody comprising at least one antigen-binding region capable of binding to OX40, i.e., an anti-idiotype antibody that binds to the antibody according to the present invention as described herein. In a particular embodiment, the anti-idiotype antibody binds to an antigen-binding region capable of binding to OX40 as described herein. In a preferred embodiment, the anti-idiotype antibody binds to an antibody designated by a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 19.

[0259] Anti-idiotype (Id) antibodies are antibodies that recognize specific determinants commonly associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing animals of the same species and genotype as the source of anti-OX40 monoclonal antibodies with the monoclonal antibody against which the anti-Id is prepared. The immunized animals can typically recognize and respond to the idiotype determinants of the immunized antibody by producing antibodies against these idiotype 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.

[0260] Anti-Id antibodies can also be used as "immunogens" to induce an immune response in yet another animal to produce so-called anti-anti-Id antibodies. These anti-anti-Id antibodies may be epitopeically identical to the original monoclonal antibody that induced the anti-Id antibody. Therefore, by using antibodies against the idiotype determinants of the monoclonal antibody, it is possible to identify other clones expressing antibodies of the same specificity. Anti-Id antibodies may be modified (to produce anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to the OX40-specific antibodies of the present invention. For example, a 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 will typically contain anti-anti-Id antibodies with similar binding properties, if not identical, to the original / parental anti-OX40 antibody.

[0261] The Fc region may have lysine at its C-terminus. The origin of this lysine is a naturally occurring sequence found in humans from which these Fc regions originate. During cell culture production of recombinant antibodies, this terminal lysine may be cleaved by proteolysis 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 to produce antibodies without lysine. Antibodies produced from nucleic acid sequences that either encode or do not encode terminal lysine are substantially identical in sequence and function, for example, when using antibodies produced in a CHO-based production system, because the degree of processing of terminal lysine is typically high (Dick, LW et al. Biotechnol. Bioeng. 2008;100: 1132-1143). Therefore, it is understood that proteins according to the present invention, such as antibodies, can be produced with or without encoding or having terminal lysine. It is also understood in accordance with the present invention that sequences having terminal lysine, such as constant region sequences having terminal lysine, can be understood as corresponding sequences without terminal lysine, and sequences without terminal lysine can also be understood as corresponding sequences having terminal lysine.

[0262] (Table 3) Sequence List TIFF2026516142000003.tif234169TIFF2026516142000004.tif244169TIFF202 6516142000005.tif244169TIFF2026516142000006.tif235169TIFF2026516142 000007.tif235169TIFF2026516142000008.tif239169TIFF2026516142000009. tif244169TIFF2026516142000010.tif246169TIFF2026516142000011.tif20169 [Examples]

[0263] Example 1: Generation of anti-human OX40 antibody and its Fc variant As described in WO2016 / 110584A1, nine rabbits were immunized with recombinant human OX40 (CD134) fused to the Fc portion of human IgG1 (Adipogen, catalog no. AG-40B-0014; MAB Discovery GmbH) to produce anti-human OX40 antibodies. Blood samples were collected at four different time points after a series of immunizations, and B cells were enriched. Individual B cells were sorted by flow cytometry, and single clones were cultured at 37°C for 7 days to expand and grow. After 7 days, the culture supernatant was collected to evaluate the production of human OX40-specific antibodies, and the B cells were frozen and stored at -80°C until further use.

[0264] V bonded to OX40 H and V L The identified unique sequences of the combination were transiently co-expressed in HEK293 cells to generate rabbit-human chimeric antibodies. Based on the results of a screening assay evaluating the antibody's ability to bind to human OX40 and its agonist activity, 30 chimeric antibodies were selected for further testing. For this purpose, the V of these antibodies was used. H Region and V LThe region was synthesized and cloned upstream of the human IgG1 constant region. Subsequently, antibodies were produced in HEK293 cells and purified. The antibodies were first tested for binding to human OX40 and cynomolgus monkey OX40 by flow cytometry. Extracts of these antibodies were produced using the IgG1-P329R-E345R-K409R (IgG1-RR-K409R) skeleton, and their agonist activity was evaluated using the OX40 bioluminescence reporter assay (Promega) and the PBMC proliferation assay. Based on the results of this second round of screening, three chimeric antibody clones (IgG1-CD134-003-P329R-E345R-K409R [IgG1-CD134-003-RR-K409R], IgG1-CD134-007-RR-K409R, and IgG1-CD134-012-RR-K409R) were selected as the most promising candidates, and two of them were subsequently humanized (IgG1-CD134-003-RR-K409R and IgG1-CD134-012-RR-K409R). For humanization, a combination of CDR transplantation and amino acid point mutations was used (Abzena), resulting in five humanized V per clone. L Chains (LC1-LC5) and 6 humanized V H I designed the chain (HC1~HC6). V H Chain and V L DNA plasmids encoding all possible chain combinations (30 pairs in total per clone) and a constant IgG1 region with an RR mutation in the heavy chain were transfected into Expi293F cells for antibody production. Antibody binding affinity to human OX40 and cynomolgus monkey OX40 was evaluated using the supernatant of Expi293F cell cultures containing individual humanized OX40 antibody variants by biolayer interferometry, and antibody binding affinity to activated primary human T cells (derived from five healthy donors) was evaluated by flow cytometry.

[0265] Since the binding properties of all humanized antibody variants derived from IgG1-CD134-003-RR and IgG1-CD134-012-RR were very similar to those of the parent antibodies, a further selection of 24 antibodies was made based on the highest sequence similarity to the nearest human homolog and purified for comparison of their ability to bind to activated primary human T cells, induce OX40 signaling, and enhance T cell proliferation. One humanized OX40-binding antibody (IgG1-CD134-003-HC6LC2-RR) was selected and further characterized in experiments described in the following examples, along with chimeric antibodies IgG1-CD134-003, IgG1-CD134-007, and IgG1-CD134-012, which have various mutations in the Fc domain.

[0266] The sequences of the anti-human OX40 antibodies used herein were obtained as follows: IgG1-CD134-Hu106 (WO2020 / 030570A1, SEQ ID NO: 5 and 11), IgG1-CD134-A4453 (WO2019 / 223733, SEQ ID NO: 26 and 28), IgG1-CD134-MEDI0562 (INN 10420, tavolimab), IgG1-CD134-ABBV368 (INN 11242, levdofirimab), IgG1-CD134-IBI101 (INN 11200, cudalolimab), IgG1-CD134-GBR830 (INN 11273, telazorlimab), IgG1-CD134-INCAGN1949 (US10259882B2, SEQ ID NO: 61 and 20), IgG1-CD134-SF2 and IgG2-CD134-SF2 (US2014 / 0377284A1, VL1VH2, SEQ ID NO: 78 and 80), IgG1-CD134-h3C8 (WO2016 / 164480A1, SEQ ID NO: 118 and 119), IgG1-CD134-RG7888 (INN 10272, vonlerolizumab), and IgG1-CD134-49B4 (WO2019 / 086497A2, SEQ ID NO: 40 and 41). V of b12, an HIV1 gp120 specific antibodyH / V L Human IgG1 antibodies containing [specific antibody] were used as a negative control (Barbas et al., J Mol Biol. 1993 Apr 5;230(3):812-2).

[0267] Example 2: Binding affinity of anti-human OX40 antibody to recombinant human OX40 and cynomolgus monkey OX40 The binding affinity of anti-human OX40 antibodies IgG1-CD134-003, IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), IgG1-CD134-007, IgG1-CD134-012, and 12 anti-human OX40 IgG1 antibodies to recombinant human OX40 protein and cynomolgus monkey OX40 protein was measured using label-free biolayer interferometry with an Octet HTX instrument (Sartorius).

[0268] The experiment was performed at 30°C with shaking at 1,000 RPM. To measure the affinity of the OX40 antibody against human OX40 and cynomolgus monkey OX40, an anti-human IgG Fc Capture (AHC) biosensor (Sartorius, catalog no. 18-5060) was pre-prepared by exposure to 10 mM glycine (Sigma-Aldrich, catalog no. 15527) buffer pH 1.7 for 5 seconds, followed by neutralization in sample diluent (Sartorius, catalog no. 18-1104) for 5 seconds; both steps were repeated 5 times. Next, the antibody (1 μg / mL in sample diluent) was loaded into the AHC sensor for 600 seconds. After baseline measurement (100 seconds) in the sample dilution, the binding (200 seconds) and dissociation (1,000 seconds) of human OX40 (Acro Biosystems, catalog number OX0-H5224) and cynomolgus monkey OX40 (Acro Biosystems, catalog number OX0-C5220) were measured using a concentration range of 0.78 to 800 nM at the 2-fold dilution stage.

[0269] The theoretical molecular weight of the antigen, based on its amino acid sequence, was used in the calculations. For each antibody, a reference sensor incubated with sample diluent instead of the antigen was used. The AHC sensor was regenerated by exposure to 10 mM glycine buffer pH 1.7 for 5 seconds, followed by neutralization in sample diluent for 5 seconds; both steps were repeated twice. The antibody was then reloaded into the sensor for kinetic measurements in the next cycle.

[0270] Data was acquired using Data Acquisition Software v12.0 (Sartorius) and analyzed using Data Analysis Software v12.0 (Sartorius). Data traces were corrected by subtracting the reference sensor for each antibody. The Y-axis was aligned to the last 10 seconds of baseline, and Interstep Correction alignment and Savitsky-Goley filtering for dissociation were applied. Data traces with a response of less than 0.05 nm were excluded from the analysis. K below 50 nM D For antibodies with values, data traces at concentrations higher than 100 nM were excluded from the analysis. The data were fitted to a 1:1 model using a 200-second binding period and dissociation time windows of interest set at 50, 200, and 1,000 seconds. The dissociation time was R 2 The selection was based on the values, visual inspection of the curves, and at least 5% signal attenuation during the dissociation phase.

[0271] The affinity for human OX40 can be accurately measured for 14 OX40 antibodies (Table 4; all antibodies tested except IgG1-CD134-SF2 and IgG1-CD134-49B4-G236R-E345R-K439E[RRE] for suboptimal curve fitting), and K D The values ​​were mostly in the nanomolar concentration range.

[0272] The affinity for cynomolgus monkey OX40 was accurately measured for eight OX40 antibodies (IgG1-CD134-RG7888, IgG1-CD134-11D4, IgG1-CD134-003, IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007, IgG1-CD134-A4453, IgG1-CD134-MEDI0562, and IgG1-CD134-ABBV368). The K values ​​measured for these antibodies were accurately determined. D Most of the values ​​were within the nanomolar concentration range (Table 4).

[0273] The antibody IgG1-CD134-RG7888 showed the highest affinity for both human OX40 and cynomolgus monkey OX40. Antibodies IgG1-CD134-007, IgG1-CD134-012, and IgG1-CD134-003-HC6LC2-RR showed higher affinity for human OX40 than antibody IgG1-CD134-h3C8. For antibody IgG1-CD134-SF2, a reliable interpretation of its binding properties to human OX40 and cynomolgus monkey OX40 could not be determined.

[0274] (Table 4) Binding affinity of anti-human OX40 antibodies to human OX40 and cynomolgus monkey OX40 TIFF2026516142000012.tif160165* Binding was observed, but K D , k on , and k dis This value is unreliable due to suboptimal curve fitting, resulting in unreliable interpretations using a 1:1 model. ND: Not detectable.

[0275] Example 3: Evaluation of cross-blockage of anti-human OX40 IgG1 antibody using biolayer interferometry. Antibody cross-blockage analysis of IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), IgG1-CD134-007, IgG1-CD134-012, and 12 anti-human OX40 IgG1 antibodies was performed using biolayer interferometry with an Octet HTX instrument (Sartorius). The experiment was conducted at 1,000 RPM and 30°C with shaking.

[0276] Amine Reactive 2 nd Generation (AR2G) biosensors (Sartorius, catalog no. 18-5092) were activated for 300 seconds with a solution of 20 mM EDC (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; Sartorius, catalog no. 18-1033) and 10 mM s-NHS (N-hydroxysulfosuccinimide sodium salt; Sartorius, catalog no. 18-1067). The activated AR2G sensors were loaded with 20 μg / mL of a primary antibody diluted in 10 mM acetate pH 5.0 (Sartorius, catalog no. 18-1069) or pH 6.0 (Sartorius, catalog no. 18-1070) for 600 seconds and quenched for 300 seconds with 1 M ethanolamine pH 8.5 (ETA; Sartorius, catalog no. 18-1071). After a 50-second baseline measurement in sample diluent (Sartorius, catalog no. 18-1104), human OX40 fused to a His tag (diluted in sample diluent to 100 nM; Acro Biosystems, catalog no. OX0-H5224) was loaded onto an AR2G biosensor containing immobilized antibody for 300 seconds. Binding of a second antibody (10 μg / mL in sample diluent) (300 seconds) was measured. The sensor was regenerated by three cycles of 5-second exposure to 10 mM glycine (Sigma-Aldrich, catalog no. 15527) buffer pH 2.5, followed by 5-second neutralization in sample diluent.

[0277] Data were acquired using Data Acquisition Software v12.0 (Sartorius) and analyzed using Data Analysis HT Software v12.0 (Sartorius). Data traces were corrected by subtracting a reference curve (sample diluent instead of second antibody) to correct for antigen dissociation from the immobilized first antibody. The Y-axis was aligned to the start of the binding phase, and Savitsky-Goley filtering was applied. The corrected binding response of the second antibody was plotted in matrix format (Table 5). Responses greater than 0.1 nm were considered non-blocking antibody pairs, while responses less than 0.1 nm were considered blocking antibody pairs. For some antibody pairs, the second antibody showed a decrease in signal compared to the buffer control. This was thought to be antibody substitution, i.e., the second antibody superseding the interaction between the first antibody and the antigen (Abdiche et al., PLoS ONE 2017 Jan 6;12(1): e0169535).

[0278] The results of the cross-blocking analysis are summarized in Table 5. The data show that IgG1-CD134-003-HC6LC2-RR blocks the binding of IgG1-CD134-003, IgG1-CD134-012, and antibody IgG1-CD134-11D4 to human OX40, while not blocking the binding of antibodies IgG1-CD134-MEDI0562, IgG1-CD134-SF2, IgG1-CD134-RG7888, IgG1-CD134-ABBV368, IgG1-CD134-INCAGN1949, IgG1-CD134-49B4-RRE, IgG1-CD134-Hu106, IgG1-CD134-h3C8, IgG1-CD134-GBR830, IgG1-CD134-IBI101, and IgG1-CD134-A4453. Subtle substitution behavior was observed between IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-007. IgG1-CD134-007 did not block the binding of IgG1-CD134-012 to human OX40, but it did block the binding of antibodies IgG1-CD134-ABBV368, IgG1-CD134-49B4-RRE, IgG1-CD134-Hu106, IgG1-CD134-h3C8, IgG1-CD134-GBR830, and IgG1-CD134-IBI101. Of the antibodies tested, only IgG1-CD134-11D4 was blocked by IgG1-CD134-012.

[0279] The antibodies IgG1-CD134-ABBV368, IgG1-CD134-INCAGN1949, IgG1-CD134-49B4-RRE, IgG1-CD134-Hu106, IgG1-CD134-h3C8, IgG1-CD134-GBR830, and IgG1-CD134-IBI101 all cross-blocked each other. Similarly, the antibodies IgG1-CD134-MEDI0562, IgG1-CD134-SF2, IgG1-CD134-RG7888, IgG1-CD134-ABBV368, and IgG1-CD134-INCAGN1949 all cross-blocked each other. The antibody IgG1-CD134-A4453 did not cross-block any of the other antibodies tested.

[0280] (Table 5) Antibody cross-blockage was determined using biolayer interferometry. The immobilized first antibody is shown vertically, and the second antibody is shown horizontally. The corrected binding response of the second antibody is shown. A response of less than 0.1 nm was considered a blocking antibody pair (indicated by a gray square), and a response of 0.1 nm or more was considered a non-blocking antibody pair (unmarked). For some antibody pairs, the second antibody showed a subtle decrease in signal compared to the buffer control, indicating that antibody substitution had occurred (indicated by bold numbers). In some cases, the antibody interaction was manually adjusted to non-blocking after visual inspection of the sensorgram (indicated by underlined numbers). TIFF2026516142000013.tif163165

[0281] Example 4: Conjugation of anti-human OX40 antibody to human OX40 and cynomolgus monkey OX40 expressed on the cell surface. The binding of anti-human OX40 antibodies IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), IgG1-CD134-007-RR, IgG1-CD134-012-RR-K409R, and 12 anti-human OX40 antibodies (11 IgG1 antibodies and 1 IgG2 antibody) to human OX40 and cynomolgus monkey OX40 expressed on the cell surface was analyzed by flow cytometry using transiently transfected FreeStyle 293-F suspension (HEK293F) cells. The unbound antibody IgG1-b12-RR was used as a negative control antibody.

[0282] HEK293F cells (ThermoFisher, catalog number R79007) were transiently transfected with Opti-MEM® I Reduced Serum Medium with Glutamax (ThermoFisher, catalog number 51985034), which was supplemented with 293fectin Transfection Reagent (ThermoFisher, catalog number 12347019) and 50 units of penicillin and 50 units of streptomycin (pen / strep;Lonza, catalog number 17-603E), according to the manufacturer's instructions, using mammalian expression vector pSBs encoding full-length human OX40 or cynomolgus monkey OX40 (SEQ ID NO: 52 and SEQ ID NO: 51, respectively).

[0283] Transfected cells were seeded in 96-well plates (30,000 cells / well; ThermoFisher, catalog no. 163320) for continuous incubation, with washing steps in between using fluorescence-activated cell sorting (FACS) buffer 1 supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, catalog no. 03690) (phosphate-buffered saline [PBS; Lonza, catalog no. BE17-517Q] supplemented with 0.1% bovine serum albumin [BSA; Roche, catalog no. 10735086001] and 0.02% sodium azide [NaN3; Bio-World, catalog no. 41920044-3]). Cells were sequentially incubated at 4°C for 30 minutes with 50 μL series dilutions of anti-human OX40 antibody (final concentration range of 0.005 to 50 μg / mL at 10-fold dilutions in EDTA-supplemented FACS buffer 1), and then with 50 μL of R-phycoerythrin (R-PE) conjugate goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog no. 109-116-098, 1:200 dilution) in EDTA-supplemented FACS buffer 1 at 4°C for 30 minutes. The cells were then washed with EDTA-supplemented FACS buffer 1, resuspended in 30 μL of viability marker ToPro-3 (Invitrogen, catalog no. T3605, 1:10,000 dilution) in EDTA-supplemented FACS buffer 1, and subsequently analyzed using an iQue® 3 flow cytometer (Satorius). The data was analyzed using FlowJo software and visualized using GraphPad Prism.

[0284] All tested antibodies showed dose-dependent binding to human OX40 and cynomolgus monkey OX40 (Figure 1A, B). The half-effect concentrations (EC2) of IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R against human OX40 and cynomolgus monkey OX40 were measured. 50) was comparable to that of most of the other antibodies tested (Figure 2). Compared to IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R, the antibodies IgG2s-CD134-SF2-E345R, IgG1-CD134-GBR830, IgG1-CD134-Hu106, and IgG1-CD134-MEDI0562 showed higher EC values ​​for binding to human OX40. 50 IgG2s-CD134-SF2-E345R, IgG1-CD134-GBR830, and IgG1-CD134-h3C8-E345R have higher EC2 binding to cynomolgus monkey OX40. 50 The antibodies IgG1-CD134-49B4-E345R and IgG1-CD134-ABBV368 had slightly lower EC values ​​for binding to both human OX40 and cynomolgus monkey OX40 than IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-007-RR, and IgG1-CD134-012-RR-K409R. 50 He possessed it.

[0285] For most antibodies, the EC for binding to human OX40 50 The results were not significantly different from those regarding binding to cynomolgus monkey OX40, with the exception of the antibodies IgG1-CD134-h3C8-E345R and IgG1-CD134-GBR830, both of which showed higher EC values ​​for cynomolgus monkey OX40 than for human OX40. 50 It had a value.

[0286] Example 5: Determination of domains important for the binding of anti-human OX40 antibodies to human OX40 using domain-shuffled OX40 molecules. The OX40 protein contains three complete cysteine-rich domains (CRDs; CRD1, CRD2, and CRD4) as well as a cleaved CRD (CRD3; Willoughby et al. Mol Immunol. 2017 Mar; 83:13-22). OX40L binding spans CRD1-3. OX40L is a trimer, with each trimer associating with three OX40 molecules on the T cell surface. The formation of a trimer receptor-ligand complex ensures clustering of the cytoplasmic domains of OX40, containing the QEE motif characteristic of many TNFR family members, which creates a docking site for TNFR-related factor (TRAF) adapter proteins. These interactions link receptor ligation to the activation of various signaling pathways, for example, through the activation of NF-κB and PI3K / AKT (Willoughby et al. Mol Immunol. 2017 Mar; 83:13-22).

[0287] To determine which CRDs are important for the binding of anti-human OX40 antibodies to human OX40, DNA shuffling was performed between human OX40 and mouse OX40. Four shuffle constructs were prepared by replacing individual CRDs with mouse CRD analogs from the DNA encoding human OX40 (Figure 3). In this way, the following constructs were generated: human OX40 (SEQ ID NO: 52), mouse OX40 (SEQ ID NO: 53), human OX40 with mouse CRD1 (SEQ ID NO: 54), human OX40 with mouse CRD2 (SEQ ID NO: 55), human OX40 with mouse CRD3 (SEQ ID NO: 56), and human OX40 with mouse CRD4 (SEQ ID NO: 57). The homology between human OX40 and mouse OX40 is limited (Figure 4). Therefore, if the CRD in human OX40 is important for the binding of the anti-OX40 antibody, then the binding will be lost when that domain is replaced with a mouse analog. Conversely, the retention of binding of the OX40 antibody to a domain-shuffled OX40 molecule indicates that the shuffled domain of human OX40 is not important for binding.

[0288] Four shuffle constructs, as well as wild-type human OX40 and mouse OX40, were transiently expressed on ExpiCHO-S cells using the ExpiFectamine® CHO Transfection Kit (ThermoFisher, catalog number A29131) according to the manufacturer's protocol. To determine the shuffle construct expressed on the cell surface and its binding to wild-type human OX40 and mouse OX40, transfected cells (30,000 cells / well) were incubated in 96-well plates (ThermoFisher, catalog no. 163320) with 50 μL series dilutions of each OX40 antibody in FACS buffer 1 (final concentration range of 0.005 to 50 μg / mL at 10-fold dilutions) for 30 minutes at 4°C. This was done to determine the binding of the anti-human OX40 antibodies IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), IgG1-CD134-007-RR, IgG1-CD134-012-RR-K409R, and 11 additional OX40 antibodies (10 IgG1 antibodies and 1 IgG2 antibody) to wild-type human OX40 and mouse OX40. Cells were washed twice with FACS buffer 1, and then incubated in 50 μL of secondary antibody R-PE conjugate goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog no. 109-116-098; 1:200 dilution) in FACS buffer 1 for 30 minutes in the dark at 4°C. Next, the cells were washed twice with FACS buffer 1, resuspended in 30 μL of viability marker ToPro-3 (Invitrogen, catalog no. T3605, 1:10,000 dilution) in FACS buffer 1 supplemented with 2 mM EDTA, and analyzed using iQue Screener Plus (Intellicyt Corporation, USA). Data were analyzed using FlowJo software and visualized using GraphPad Prism. The non-target binding antibody IgG1-b12-RR was included as a negative control antibody.

[0289] All of the tested anti-human OX40 antibodies showed binding to wild-type human OX40 (Figures 5-9), but with the exception of the antibody IgG1-CD134-RG7888 at the highest concentration, none of the tested antibodies showed binding to wild-type mouse OX40 (Figure 6A).

[0290] The antibody IgG1-CD134-A4453 was the only antibody that showed loss of binding to human OX40 with mouse CRD4 (Figure 7B, F), and was therefore used to normalize the binding data of the other antibodies in order to combine the results of the two individual experiments (Figure 10). The combined normalized data shows that IgG1-CD134-003-HC6LC2-RR, like IgG1-CD134-012-RR-K409R and antibody IgG1-CD134-11D4, lost binding to human OX40 with mouse CRD1 (Figure 10B), but retained binding to all other shuffle constructs (Figures 10C-E). IgG1-CD134-007-RR, along with antibodies IgG1-CD134-h3C8-E345R, IgG1-CD134-ABBV368, IgG1-CD134-GBR830, IgG1-CD134-Hu106, IgG1-CD134-IBI101, and IgG1-CD134-INCAGN1949, showed loss of binding to human OX40 with mouse CRD2 (Figure 10C). Antibodies IgG1-CD134-RG7888, IgG2s-CD134-SF2-E345R, IgG1-CD134-INCAGN1949, and IgG1-CD134-MEDI0562 showed loss of binding to human OX40 with mouse CRD3 (Figure 10D).

[0291] Combined, these data indicate that CRD1 is important for the binding of IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-012-RR-K409R to human OX40, but for all tested antibodies except IgG1-CD134-11D4, other CRDs are important for human OX40 binding (Table 6). For IgG1-CD134-007-RR and IgG1-CD134-h3C8-E345R, CRD2 appears to be important for binding to human OX40, while for IgG2s-CD134-SF2-E345R and IgG1-CD134-RG7888, CRD3 appears to be important.

[0292] (Table 6) Binding regions important for the binding of anti-human OX40 antibodies to human OX40 TIFF2026516142000014.tif86159

[0293] Example 6: Agonist activity of anti-human OX40 antibody in cell-based OX40 reporter assay The OX40 agonist activity of various anti-human OX40 antibodies possessing the hexamerization-enhancing mutation (E345R) and further comprising either an active or inactive Fc skeleton was measured using Jurkat cells transfected with human OX40 (OX40 Bioassay; Promega, catalog number JA2191). These cells express the firefly luciferase gene under the control of the NF-κB response element and constitutively express human OX40, resulting in luciferase production in response to OX40 agonism. +Jurkat cells were thawed and incubated overnight at 37°C / 5% CO2 in a white, opaque 96-well flat-bottom culture plate (30,000 cells / well; PerkinElmer, catalog no. 6005680) in assay buffer (RPMI1640 medium [Promega, catalog no. G708A] supplemented with 5% heat-inactivated fetal bovine serum [FBS; catalog no. J121A]). The following day, antibody dilution series (final concentrations of 0.000026-10 μg / mL [Figure 11A-D] or 0.00064-50 μg / mL [Figure 11E] in 5-fold dilutions in assay buffer) were added to the wells and incubated at 37°C / 5% CO2 for 5 hours. The test antibodies were IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), IgG1-CD134-007-RR, IgG1-CD134-012-RR, and antibodies IgG1-h3C8-K322A-E345R, IgG1-CD134-h3C8-E345R-L234A-L235A-P329G (IgG1-CD134-h3C8-E345R-LALAPG), IgG1-CD134-RG7888-K322A-E345R, IgG1-CD134-RG7888-E345R-LALAPG, and IgG2s-CD134-SF2-E345R. The unbound antibody IgG1-b12-RR was used as a negative control. After incubation, the Bio-Glo Luciferase Assay Reagent, prepared by mixing Bio-Glo Luciferase Assay Buffer (Promega, catalog number G719A) and Bio-Glo Luciferase Assay Substrate (Promega, catalog number G720A) according to the manufacturer's instructions, was subjected to OX40. + Jurkat cells and antibody dilution series were added in a 1:1 ratio to each well and incubated in the dark at RT for 5-10 minutes. Luminescence was measured using an EnVision Multilabel Reader (PerkinElmer) and presented as relative luminescence (RLU) in a bar graph generated using GraphPad Prism software. 50The values ​​were calculated using GraphPad Prism.

[0294] IgG1-CD134-003-HC6LC2-RR induced stronger activation of human OX40-expressing reporter cells than two hexamerization-enhanced (E345R) variants of IgG1-h3C8 antibody having either an inactive Fc backbone (LALAPG) or an Fc backbone (K322A) that does not show C1q binding and shows reduced FcγR binding (Figure 11A). This is indicated by the higher maximal levels of cell activation induced by IgG1-CD134-003-HC6LC2-RR compared to the IgG1-h3C8 variants, the higher maximal RLU signal observed for the former antibody (Figure 11A), and the slightly lower EC of IgG1-CD134-003-HC6LC2-RR. 50 As reflected in the values ​​(Figure 12), IgG1-CD134-003-HC6LC2-RR was superior to two hexamerization-enhanced variants of the IgG1-RG7888 antibody (IgG1-CD134-RG7888-E345R-LALAPG and IgG1-CD134-RG7888-K322A-E345R) in inducing reporter cell activation (Figures 11B, 12). IgG1-CD134-003-HC6LC2-RR also induced higher maximal activation levels than the IgG1-RG7888 variants (Figure 12). Furthermore, IgG1-CD134-003-HC6LC2-RR exhibited higher maximal RLU signals and lower EC. 50 It induced stronger reporter cell activation than the Fc-inactive variant of IgG2-CD134-SF2 (IgG2s-CD134-SF2-E345R; Figure 11C, 12) possessing a hexamerization-enhancing mutation, as reflected by the value. Both IgG1-CD134-007-RR and IgG1-CD134-012-RR induced reporter cell activation, but IgG1-CD134-012-RR induced higher maximal activation, which was comparable to IgG1-CD134-003-HC6LC2-RR (Figure 11D, 12). Reporter cell activation was not observed for the antibody IgG1-CD134-11D4 (Figure 11E).

[0295] In conclusion, IgG1-CD134-003-HC6LC2-RR demonstrated greater potency in inducing activation of human OX40-overexpressing reporter cells than antibodies IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2-CD134-SF2, which possess the E345R hexamerization-enhancing mutation and consist of either an inactive Fc skeleton or an Fc skeleton lacking C1q binding and exhibiting reduced FcγR binding. Furthermore, IgG1-CD134-007-RR and IgG1-CD134-012-RR induced activation of reporter cells, but no activity was detected for antibody IgG1-CD134-11D4 in this assay. Non-humanized variants of 007 and 012 were tested here along with the humanized 003 clone. In the initial PBMC proliferation experiments, clone 007 was functionally less active than clones 003 and 012. Clones 012 lost functional activity in T cell proliferation assays after humanization.

[0296] Example 7: Ability of anti-human OX40 antibody to enhance T cell proliferation To investigate the ability of IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR) to induce T cell proliferation, and to compare this ability with that of antibodies possessing the hexamerization-enhancing mutation (E345R) and further comprising either an active Fc skeleton, an inactive Fc skeleton (L234A-L235A-P329G; LALAPG), or an Fc skeleton (K322A) that does not show C1q binding and shows reduced FcγR binding, a polyclonal T cell proliferation assay was performed using human peripheral blood mononuclear cells (PBMCs) from healthy donors.

[0297] PBMCs were obtained from healthy donor Buffycoat (Transfusionszentrale, University Hospital, Mainz, Germany) by Ficoll-Paque density gradient separation (GE Healthcare, catalog number 17-1440-03). CD4 was used where applicable. + T cells or CD8 + T cells were depleted from PBMCs using either CD4 microbeads or CD8 microbeads (Miltenyi Biotec GmbH, catalog numbers 130-097-48 and 130-045-201), respectively. The isolation procedure was generally carried out according to the manufacturer's instructions, with minor modifications (reduction of microbead volume), and CD4 - PBMC and CD8 - The isolation of PBMCs was essentially the same. Briefly, after determining the cell number, the cell suspension was centrifuged and the supernatant was discarded. The cells were then placed in MACS buffer (DPBS [Thermo Fisher Scientific, catalog no. 14190250], 5 mM ethylenediaminetetraacetic acid [EDTA; Sigma-Aldrich, catalog no. 03690], 1% human albumin [CSL Behring, catalog no. PZN-00504775]) at a rate of 1 × 10⁶ per 80 μL of buffer. 7 The solution was resuspended in individual living cells. 10 712 μL of CD4 or CD8 microbeads were added per cell. To ensure homogeneous labeling, the cells and beads were thoroughly mixed twice: before and during incubation at 2–8°C for 15 minutes. The cell suspension was then washed with MACS buffer (8 min, 300 × g, RT) and filtered through a 30 μm cell strainer (BD Biosciences, catalog no. 340626). An LS column (Miltenyi Biotec GmbH, catalog no. 130-042-401) was placed on a QuadroMACS separator on a MACS MultiStand (Miltenyi Biotec GmbH) and equilibrated with MACS buffer. Bead-labeled cells were loaded onto the column, and the suspension was flowed through by gravity to retain the bead-labeled cells in the column. The column was then washed three times with MACS buffer. Bead-labeled CD4 + Cells and CD8 + The column containing the cells was discarded. Unlabeled CD4 in flowthrough. - PBMC and CD8 - PBMCs were centrifuged (8 minutes, 300 x g, RT), resuspended in DPBS, and counted.

[0298] Each PBMC sample was labeled with CellTrace® Violet (Thermo Fisher Scientific, catalog number C34557) according to the manufacturer's instructions.

[0299] CellTrace® Violet-labeled PBMCs were placed in a 96-well round-bottom plate and 0.3 μg / mL of soluble anti-human CD3 antibody (STEMCELL) in Iscove's Modified Dulbecco's Medium GlutaMAX (Thermo Fisher Scientific, catalog number 31980030) containing 5% pooled human serum (One Lambda Inc., catalog number A25761). The antibodies were incubated in the presence of a series of dilutions of the following antibodies (technologies, catalog number 60011): IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-h3C8-K322A-E345R, IgG1-CD134-h3C8-E345R-L234A-L235A-P329G(E345R-LALAPG), IgG1-CD134-RG7888-K322A-E345R, IgG1-CD134-RG7888-E345R-LALAPG, IgG2s-CD134-SF2-E345R, or the unbound negative control antibody IgG1-b12-RR (final antibody concentration range of 0.00098 to 10 μg / mL at 2-fold or 10-fold dilutions). Four days after culturing, cells were stained with viability dye (Thermo Fisher Scientific, catalog number 65-0865-14, 1:1,500 dilution), as well as fluorescently labeled antibodies against human CD4 (eBioscience, catalog number 17-0048-42, 1:100 dilution), CD8 (BD Biosciences, catalog number 564116, 1:400 dilution), CCR7 (BioLegend, catalog number 353206, 1:50 dilution), and CD45RA (BD Biosciences, catalog number 560675, 1:100 dilution). T cell proliferation was measured using a BD FACSCelesta® flow cytometer (BD Biosciences) to detect CD4 + T cells and CD8 + The growth index was evaluated by flow cytometry analysis of CellTrace® Violet dilution in T cells. The growth index was calculated using a formula incorporated into the growth modeling tool in the FlowJo software. In addition, CD4+ T cells and CD8 + The percentage of central memory cells (CCR7 + CD45RA - ) in T cells was evaluated.

[0300] IgG1-CD134-003-HC6LC2-RR enhanced CD4 + T cell proliferation and CD8 + T cell proliferation with high potency and dose-dependently (Figure 13). IgG1-CD134-h3C8-E345R-LALAPG also enhanced CD4 + T cell proliferation and CD8 + T cell proliferation but showed reduced potency compared to IgG1-CD134-003-HC6LC2-RR (Figure 13A, B). In contrast, IgG1-CD134-h3C8-K322A-E345R, IgG1-CD134-RG7888-K322A-E345R, IgG1-CD134-RG7888-E345R-LALAPG, IgG2s-SF2-E345R, or IgG1-b12-RR did not enhance CD4 + T cell proliferation or CD8 + T cell proliferation (Figure 13A, B). When the ability of IgG1-CD134-003-HC6LC2-RR to enhance CD4 + T cell proliferation and CD8 + T cell proliferation was tested in a larger cohort of healthy donor PBMCs, a consistent dose-dependent enhancement of CD4 + T cell proliferation and CD8 + T cell proliferation was observed and its EC 50 could be calculated (n = 17; Table 7).

[0301] (Table 7) CD4 + T cell proliferation and CD8 + T cell proliferation enhanced by IgG1-CD134-003-HC6LC2-RR in a polyclonal T cell proliferation assay, based on results from 17 healthy human donors, and its EC 50 value TIFF2026516142000015.tif150140

[0302] CD4 + Increased proliferation of T cells due to IgG1-CD134-003-HC6LC2-RR, CD8 + To evaluate whether it depends on the presence of T cells, and vice versa, a polyclonal T cell proliferation assay was used, CD8 + T cells or CD4 + The study was conducted using PBMC samples in which T cells were depleted. The increased T cell proliferation induced by IgG1-CD134-003-HC6LC2-RR was observed in CD8 + CD4 in the absence of T cells + T cells were retained, but CD4 + CD8 in the absence of T cells + T cells were not retained (Figure 13C, D). As a result, CD4 + The presence of T cells is induced by IgG1-CD134-003-HC6LC2-RR in a polyclonal T cell proliferation assay. + This suggests that it is essential for T cell proliferation.

[0303] Furthermore, IgG1-CD134-003-HC6LC2-RR treatment is effective for CCR7 + CD45RA - Central Memory CD4 + T cells and CD8 + It dose-dependently increased the percentage of T cells (Figure 14). IgG1-CD134-h3C8-E345R-LALAPG also increased the central memory CD4 + While it dose-dependently increased the percentage of T cells, the efficacy was reduced compared to IgG1-CD134-003-HC6LC2-RR. In contrast, treatment with IgG1-CD134-h3C8-K322A-E345R, IgG1-CD134-RG7888-K322A-E345R, or IgG1-CD134-RG7888-E345R-LALAPG increased the central memory CD4 + No significant difference was observed in the percentage of T cells (Figure 14).

[0304] Example 8: Conjugation of anti-human OX40 antibody to human and cynomolgus monkey Fc gamma receptors. The binding of various anti-human OX40 antibodies to the human Fcγ receptor (FcγR) by antibodies possessing a hexamerization-enhancing mutation (E345R) and further comprising either an active Fc skeleton, an inactive Fc skeleton (L234A-L235A-P329G; LALAPG, or P329R), or an Fc skeleton (K322A) that does not show C1q binding and shows reduced FcγR binding, was analyzed using two methods: surface plasmon resonance (SPR) and flow cytometry analysis of anti-human OX40 antibody binding to ExpiCHO-S cells transfected with FcγRIa.

[0305] For the former method, the binding of OX40-specific antibody variants IgG1-CD134-003-HC6LC2, IgG1-CD134-003-HC6LC2-E345R, IgG1-CD134-003-HC6LC2-P329R-E345R (IgG1-CD134-003-HC6LC2-RR), and a selection of other OX40-specific antibodies (see Figures 15-16) to human FcγR variants was analyzed using the Biacore SPR system and compared with an anti-HIV gp120 antibody (IgG1-b12) containing a wild-type Fc domain as a reference sample. Biacore Series S Sensor Chips CM5 (Cytiva, catalog number 29104988) were covalently coated with an anti-His antibody using an amine coupling and His capture kit (Cytiva, catalog numbers BR100050 and 29234602) according to the manufacturer's instructions. Next, His-tagged FcγRIa, FcγRIIa (167-His[H] and 167-Arg[R]), FcγRIIb, or FcγRIIIa (176-Phe[F] and 176-Val[V]) (Sino Biological, catalog numbers 10256-H08S-B, 10374-H08H1, 10374-H27H, 10259-H27H-B, 10389-H27H, and 10389-H27H1-B, respectively) were reversibly captured on the anti-His chip surface in HBS-EP+ buffer (Cytiva, catalog number BR100669) until a capture response of 400 RU was reached. After three startup cycles of HBS-EP+ buffer, antibody samples were injected over 12 cycles per sample to generate binding curves using antibody concentration ranges of 0–3,000 nM for FcγRIa and 0–10,000 nM for other FcγRs. Each sample analyzed on an FcγR-coated surface (active surface) was also analyzed on a parallel flow cell without FcγR (reference surface), and this was used for background correction.Dissociation from the anti-His coated surface was performed by surface regeneration using 10 mM glycine-HCl pH 1.5 (Cytiva, catalog no. BR100354). Sensorgrams were generated using Biacore Insight Evaluation software (Cytiva), and the binding of individual human OX40-specific antibodies to a reference sample (IgG1-b12) was calculated by applying a 4-parameter logistic (4PL) fit.

[0306] For the latter method, human FcγRIa was transiently transfected into ExpiCHO-S cells (ThermoFisher, catalog number A29127) as follows: ExpiCHO-S cells (approximately 3.0 × 10⁻⁶) 6Cells (at / mL; ThermoFisher Scientific, catalog number A29133) were cultured in ExpiCHO® Expression Medium (ThermoFisher Scientific, catalog number A2910001). For each transfection, 10 μg of DNA encoding FcγR1a was mixed in 0.39 mL of cold OptiPro® Serum-Free Medium (OptiPro® SFM, ThermoFisher Scientific, catalog number A29131), and concurrently, 0.03 mL of ExpiFectamineCHO® reagent (ThermoFisher Scientific, catalog number A29131) was added to 0.37 mL of cold OptiPro® SFM. Then, 0.4 mL of the latter mixture was added to the DNA / OptiPro mixture and incubated at RT for 1–5 minutes. Next, this mixture was added dropwise to the cultured ExpiCHO-S cells. After 18–22 hours of incubation, 0.06 mL of ExpiCHO® Enhancer (ThermoFisher Scientific, catalog number A29131) and 2.4 mL of ExpiCHO Feed (ThermoFisher Scientific, catalog number A29131) were added to each culture flask. Following transfection, cells were incubated at 37°C, 70% humidity, and 8% CO2 for 24 hours with shaking, and then frozen in ExpiCHO® Expression Medium supplemented with 10% DMSO (Sigma-Aldrich, catalog number D2438).

[0307] Transfected cells were plated in 96-well U-bottom plates (20,000 cells / well; ThermoFisher, catalog no. 163320) in culture medium (RPMI1640 [Capricorn Scientific, catalog no. RPMI-HA] containing L-glutamine and 25 mM HEPES, supplemented with 50 units penicillin and 50 units streptomycin [pen / strep; Lonza, catalog no. 17-603E] and 10% Donor Bovine Serum with Iron [DBSI; Gibco, catalog no. 20371-030]). Next, the cells were incubated for 30 minutes at 4°C with 25 μL series dilutions of OX40-specific antibodies IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-HC6LC2, IgG1-CD134-003, or other OX40-specific antibodies (variants) in FACS buffer 1 (see Figure 17; final concentration range of 0.00051 to 10 μg / mL at 3-fold dilutions). After incubation, the cells were washed twice with FACS buffer 1 and then incubated for 30 minutes at 4°C with 50 μL of R-PE labeled F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific (Jackson ImmunoResearch, catalog no. 109-116-097, 1:200 dilution) in FACS buffer 1. Next, the cells were washed twice with FACS buffer 1, and incubated with 30 μL of the viability marker ToPro-3 (Invitrogen, catalog number T3605, 1:10,000 dilution) in FACS buffer 1 supplemented with 2 mM EDTA for 20 minutes in the dark at RT. All samples were measured using an iQue® 3 flow cytometer (Satorius). The data were analyzed using FlowJo software and visualized using GraphPad Prism.

[0308] In SPR measurements, IgG1-CD134-003-HC6LC2-RR showed low residual binding to the high-affinity receptor FcγRIa, but binding by this antibody was not observed against FcγRIIa (H and R variants), FcγRII2b, or FcγRIIIa (F and V variants; Figures 15-16). IgG1-CD134-h3C8-K322A-E345R and IgG1-CD134-RG7888-K322A-E345R showed binding to all FcγR tested, although slightly less than the positive control IgG1-b12 (Figures 15-16). In contrast, the latter antibody variant with the E345R-LALAPG Fc skeleton showed no binding to any of the FcγR tested. Using a similar method, low residual binding of IgG1-CD134-003-HC6LC2-RR to cynomolgus monkey FcγRIa was observed at high antibody concentrations (above 100 nM [14.9 μg / mL]).

[0309] ExpiCHO-S cells transfected with FcγRIa and flow cytometry analysis showed that IgG1-CD134-003-HC6LC2-RR did not bind to FcγRIa (Figure 17A). Binding to FcγRIa was observed for antibody variants possessing the wild-type Fc domain, including IgG1-CD134-003-HC6LC2, IgG1-CD134-003, IgG1-CD134-11D4, IgG1-CD134-INCAGN1949, IgG1-CD134-IBI101, IgG1-CD134-h3C8, IgG1-CD134-h3C8-E345R, IgG1-CD134-RG7888, and IgG1-CD134-RG7888-E345R, as well as for variants of IgG1-CD134-h3C8 and IgG1-CD134-RG7888 with the E345R-K322A mutation (Figure 17A, C-E). Binding to FcγRIa was not observed for the IgG2 antibodies IgG2-SF2 and IgG2s-SF2-E345R, nor for the variants of IgG1-h3C8 and IgG1-RG7888 with the E345R-LALAPG mutation (Figure 17B, D-E).

[0310] In conclusion, IgG1-CD134-003-HC6LC2-RR shows minimal binding to the human IgG Fc gamma receptor (FcγRIa) or no binding at all (FcγRIIa, FcγRIIb, and FcγRIIIa).

[0311] Example 9: Activated original CD4 + T cells and CD8 + Antibody binding ability of IgG1-CD134-003-HC6LC2-RR to T cells Activated original human CD4 + T cells and CD8 +The number of binding sites on the cell surface of T cells was quantitatively determined 24, 48, and 72 hours after activation. First, whole human T cells were enriched according to the manufacturer's instructions by direct negative selection from a buffy coat using RosetteSep® Human T Cell Enrichment Cocktail (StemCell Technologies, catalog no. 15021), followed by density centrifugation on a Ficoll gradient (PromoCell, catalog no. C-44010 or Corning, catalog no. 25-072-CI). The enriched T cells were washed once in PBS (GE Healthcare, catalog no. SH3A3830.03 or Capricorn Scientific, catalog no. SP-2121-500 mL), centrifuged at 300 × g for 3 minutes, and resuspended in RPMI-1640 medium [Lonza, catalog no. BE12-115F / Capricorn, catalog no. RPMI-HA] supplemented with 10% thermally inactivated FBS [ATCC, catalog no. 30-2020], 1% penicillin / streptomycin, and 1% L-glutamine [Gibco, catalog no. 25030-081]. The enriched T cells were counted using a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Biosciences) with Cellometer ViaStain® AOPI solution (Nexcelom Bioscience, catalog no. CS2-0106) in PBS to distinguish live cells from dead cells. The cells were pelletized and resuspended in activated culture medium.

[0312] To induce T cell activation, anti-CD3 / CD28 beads (Dynabeads® Human T-Activator CD3 / CD28; ThermoFisher Scientific, catalog no. 11131D) were washed with PBS, and the T cells and beads were resuspended in activation medium at a 1:2 bead-to-cell ratio. Next, 300,000 T cells (150 μL) with beads were plated per well in a round-bottom 96-well plate (ThermoFisher Scientific, catalog no. 163320) and incubated at 37°C, 5% CO2 for 1, 2, and 3 days. The beads were then removed using a Dynal® bead separator (Invitrogen, catalog no. 3019669), and the T cells were washed in activation medium. This bead removal process was repeated twice. Cells were counted using AOPI solution to distinguish live cells from dead cells.

[0313] The number of IgG1-CD134-003-HC6LC2-RR binding sites was analyzed using the Human IgG Calibrator Kit (Biocytex, catalog number CP010) and flow cytometry, according to the manufacturer's instructions. Bead-activated T cells were plated into a round-bottom 96-well plate (50,000 cells / well; ThermoFisher Scientific, catalog number 163320). The cells were washed once with PBS and once with FACS buffer 1, and incubated with IgG1-CD134-003-HC6LC2-RR diluted in FACS buffer 1 at a saturated antibody concentration of 1 μg / mL, for 30 minutes at 4°C. Next, separately from the cells, calibration beads (included in the kit) containing a sufficiently specified number of human IgG monoclonal antibodies per bead were plated into FACS buffer 1 (15 μL of beads per well). After washing with FACS buffer 1, cells and calibration beads were incubated with the antibody panel listed in Table 8 in FACS buffer 1 at 4°C for 30 minutes, protected from light. Both the cell suspension and bead suspension were washed twice and resuspended in 80 μL of ToPro-3 viability marker (1:10,000; Invitrogen, catalog no. T3605) diluted in FACS buffer 1, and measured using a BD FACSCelesta Cell Analyzer. Data were analyzed using FlowJo software. Compensation was performed using UltraComp eBEADS (Thermo Fisher Scientific, catalog no. 01-2222-42). The antibody binding ability (sABC) of IgG1-CD134-003-HC6LC2-RR, corresponding to the number of IgG1-CD134-003-HC6LC2-RR binding sites per cell, was determined by interpolation from a standard curve using GraphPad Prism software.

[0314] (Table 8) Activated CD4 + T cell subsets and CD8 + Antibody panel for analyzing IgG1-CD134-003-HC6LC2-RR binding sites on T cell subsets TIFF2026516142000016.tif31166

[0315] The number of IgG1-CD134-003-HC6LC2-RR binding sites is equal to the number of CD4 + T cells and CD8 + For both types of T cells, the levels were higher after stimulation on day 2 and day 3 compared to day 1 (Figure 18). Furthermore, the number of IgG1-CD134-003-HC6LC2-RR binding sites (i.e., sABC) was higher across all time points than CD8 + CD4 + On T cells, the levels were approximately 4.5 to 8.5 times higher (for stimulation on day 1 and day 3, respectively, CD4: 11,285 and 21,682 mean sABC; CD8: 1,322 and 4,816 mean sABC).

[0316] Example 10: Binding of anti-human OX40 antibody to activated T cells expressing OX40. As described in Example 9, OX40 expression on T cells can be induced upon T cell activation, with maximum expression observed 2-3 days after activation. Here, the binding of anti-human OX40 antibody to T cells activated by anti-CD3 / CD28 antibody was evaluated using flow cytometry.

[0317] Healthy human donor PBMCs were purified essentially as described in Example 7. The cell concentration was set to 2 × 10⁶ in assay medium. 6 The solution was adjusted to the final concentration of cells / mL and plated into a 6-well plate (Greiner, catalog no. 657160). PBMCs were stimulated with 0.3 μg / mL anti-CD3 antibody (STEMCELL Technologies, catalog no. 60011) and 0.5 μg / mL anti-CD28 antibody (BioLegend, catalog no. 302934) and cultured at 37°C and 5% CO2 for 2 days. Next, PBMCs were collected, counted, and 2 × 10⁶ cells were collected in FACS buffer 1 (DPBS containing 2% thermally inactivated FBS and 2 mM ethylenediaminetetraacetic acid [EDTA]). 6The concentration was adjusted to cells / mL. OX40 expression on stimulated T cells was confirmed by staining with a commercially available anti-OX40 antibody (data not shown). Activated PBMCs were placed in a 96-well round-bottom plate at a rate of 1 × 10⁶ per well. 5 Cells were transferred and incubated with either IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-IBI101, IgG1-CD134-11D4, IgG1-CD134-RG7888, or the unbound control antibody IgG1-b12-RR (final concentration: 0.00061-10 μg / mL at 4-fold dilution). After 60 minutes of RT, the cells were prepared for flow cytometry analysis. For this purpose, the cells were washed twice with 150 μL of FACS buffer 1, and 30 μL / well of a cell surface antibody mix (Table 9) in FACS buffer 1 containing an antibody against human Fcγ antibody (Table 9), and fixable viability dye eFluor780 (1:1,500) were added. The staining procedure was performed in the dark at 4°C for 15-20 minutes. Stained cells were washed twice with 120–150 μL of FACS buffer 1 (5 minutes, 460 × g, RT), and then resuspended in 60–100 μL of FACS buffer 1 for direct flow cytometry analysis. Flow cytometry data were acquired using FACSymphony A3 and analyzed with FlowJo software.

[0318] (Table 9) Antibody panel used to evaluate the binding of anti-human OX40 antibodies to activated human T cells. TIFF2026516142000017.tif38159

[0319] All of the tested anti-human OX40 antibodies were stimulated CD4 from all donors tested. + T cells and CD8 + It bound to T cells in a dose-dependent manner (representative donors are shown in Figure 19). EC of IgG1-CD134-003-HC6LC2-RR binding to stimulated T cells. 50 The values ​​are in the high picomolar concentration range, and the average EC 50 The value is 0.140 nM / 0.021 μg / mL (CD4 +T cells; Table 10) and 0.159 nM / 0.024 μg / mL (CD8 + T cells (Table 10) were the EC cells of other anti-human OX40 antibodies. 50 The values ​​were in a similar range (IgG1-CD134-11D4), slightly higher (IgG1-CD134-RG7888), or significantly higher (IgG1-CD134-IBI101), but IgG1-CD134-RG7888 reached a higher maximum binding plateau.

[0320] (Table 10) Activated human CD4 + T cells and CD8 + EC on the binding of anti-human OX40 antibodies to T cells 50 Value. SD: Standard deviation. TIFF2026516142000018.tif72166

[0321] These results indicate that IgG1-CD134-003-HC6LC2-RR bound to activated human T cells expressing OX40 with apparent affinity similar to that of reference OX40 agonist antibodies (IgG1-CD134-11D4) or higher (IgG1-CD134-RG7888, IgG1-CD134-IBI101).

[0322] Example 11: Binding of IgG1-CD134-003-HC6LC2-RR to human OX40 in the presence of soluble OX40L. OX40 and OX40L exist in soluble forms (sOX40 and sOX40L) in addition to their membrane-bound forms, and these have previously been linked to autoimmune diseases. The physiological activity of these soluble forms is supported by reports that sOX40 can compete with membrane-expressed OX40 for OX40L interaction. This can suppress inflammatory responses and thus mimic Treg function (Laustsen et al. Arthritis Res Ther. 2014 Oct 30;16(5):474).

[0323] Most agonist OX40 antibodies currently in clinical development bind to the same region as OX40L, the natural ligand for OX40, and antibody binding to this region is associated with strong OX40 agonist and antitumor activity (Zhang et al. 2019. Ligand-Blocking and Membrane-Proximal Domain Targeting Anti-OX40 Antibodies Mediate Potent T Cell-Stimulatory and Anti-Tumor Activity. Cell Rep 27: 3117-3123 e3115). The binding of IgG1-CD134-003-HC6LC2-RR to human OX40 expressed on activated T cells was tested in the presence of soluble OX40L (sOX40L), and vice versa.

[0324] Activated human CD4 + T cells and CD8 +To define the saturation concentration of sOX40L for binding to T cells, and to evaluate the binding of IgG1-CD134-003-HC6LC2-RR in the presence of this saturated concentration of sOX40L, human PBMCs were stimulated with anti-CD3 / CD28 beads for 2 days (essentially as described in Example 9, except that IMDM medium containing a 6-well plate [Greiner, catalog no. 657160] and 5% pooled human serum [PHS, One Lambda Inc., catalog no. A25761] was used). The stimulated PBMCs were pelletized and resuspended in FACS buffer 2 (DPBS containing 2% thermally inactivated FBS [Sigma-Aldrich, catalog no. F7524] and 2 mM EDTA [Sigma-Aldrich, catalog no. 03690]) and seeded in a round-bottom 96-well plate (VWR International GmbH, catalog no. 734-197) (100,000 cells / well). Next, the cells were incubated for 30 minutes at RT with either mouse Fc-tagged human sOX40L (Sino Biological, catalog number 13127-H04H) at a final concentration ranging from 0.00017 to 30 μg / mL (3-fold dilution in FACS buffer 2 for stimulated cells, or 30 μg / mL for unstimulated cells) to determine the saturated binding concentration of sOX40L, or with either IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR antibody at a final concentration ranging from 0.00046 to 10 μg / mL (10-fold dilution in FACS buffer 2, followed by a 3-fold dilution) in or without 2 μg / mL of sOX40L to determine binding competition.Next, the cells were washed twice with 150 μL of FACS buffer 2 and incubated with 30 μL of APC-conjugated goat anti-mouse IgG F(ab')2 secondary antibody (1:1,000 dilution in FACS buffer 2; Jackson ImmunoResearch, catalog no. 115-135-164) alone or with AF488-conjugated goat anti-human IgG F(ab')2 secondary antibody (1:500 dilution in FACS buffer 2; Jackson ImmunoResearch, catalog no. 109-546-098) for 20 minutes at 4°C, protected from light. Then, the cells were washed twice with FACS buffer and incubated with 30 μL of the antibody panel listed in Table 11, diluted in 1:1,500 FACS buffer 2 containing Fixable Viability Stain eFluor 780, for 20 minutes at 4°C, protected from light. Subsequently, the cells were washed twice with 120-150 μL of FACS buffer 2, measured using a BD FACSCelesta flow cytometer with a BD High Throughput sampler, and then analyzed using FlowJo software.

[0325] (Table 11) CD4 + T cells and CD8 + Antibody panel used to stain T cells TIFF2026516142000019.tif20164

[0326] The saturation concentration of sOX40L was determined to be 2 μg / mL. Maximum binding of IgG1-CD134-003-HC6LC2-RR to OX40 was not inhibited in the presence of 2 μg / mL of sOX40L (Figure 20A), but CD4 + T cells and CD8 + EC binding to T cells 50The values ​​were approximately three times higher in the presence of sOX40L (Table 12). Conversely, sOX40L binding was dose-dependently lost in the presence of IgG1-CD134-003-HC6LC2-RR (Figure 20B), indicating that IgG1-CD134-003-HC6LC2-RR was able to block the binding of sOX40L. This suggests that OX40L and IgG1-CD134-003-HC6LC2-RR bind to overlapping domains on OX40, but the presence of OX40L has minimal effect on the binding of IgG1-CD134-003-HC6LC2-RR (but the reverse is not true).

[0327] (Table 12) Binding of IgG1-CD134-003-HC6LC2-RR to activated human T cells in the presence of soluble OX40L, and vice versa. TIFF2026516142000020.tif51160

[0328] Example 12: Agonist activity of anti-human OX40 antibody in the absence and presence of Fcγ receptor-expressing cells. In Example 6, we described an assay performed using Jurkat cells transfected with human OX40 to investigate the efficacy of various anti-human OX40 antibodies. Here, using the same assay, the efficacy of inducing OX40 agonist activity was measured in both the absence and presence of FcγR-expressing cells for IgG1-CD134-003-HC6LC2-RR and its variant with an active Fc skeleton (IgG1-CD134-003; SEQ ID NO: 9 and 10), or its variant with an Fc skeleton having the hexamerization-enhancing mutation E345R as indicated for the constant region indicated by SEQ ID NO: 2, or its variant with an Fc skeleton having the Fc inactivating mutations L234F, L235E, and D265A in addition to the F405L mutation that promotes heterodimerization of the half-half with another half-half having the K409R mutation as indicated for the constant region indicated by SEQ ID NO: 62 (Labrijn et al. Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. PNAS 2013; The study investigated the antibody 110(13):5145-50). In a separate set of experiments, the efficacy of IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-11D4, IgG1-RG7888, and IgG1-CD134-IBI101 in inducing OX40 agonist activity was evaluated. In all experiments described herein, the unbound control antibody IgG1-b12-RR was used as a negative control.

[0329] The assay was carried out essentially as described in Example 6, with a few exceptions: here, OX40-expressing Jurkat cells were stimulated with an anti-human OX40 antibody either in the absence or in the presence of CHO-K1 cells (Promega, catalog no. JA2255; FcγRIIb-CHO K1 cells) transfected to express human Fcγ receptor 2B. The vials of FcγRIIb-CHO K1 cells were thawed, and the cells were suspended in 14.5–29 mL of RPMI 1640 medium (Promega kit, catalog no. JA2191) supplemented with 5% fetal bovine serum (FBS, Promega kit, catalog no. JA2191). The cells were plated in 96-well white flat-bottom assay plates (Fisher Scientific, catalog no. 10072151) at 100 μL per well. After 5-6 hours of incubation (37°C, 5% CO2), the culture medium was discarded from the wells, and OX40-expressing Jurkat effector cells were added at 60 μL per well (with or without FcγRIIb-CHO K1 cells) and incubated for 16-20 hours (37°C, 5% CO2). In addition, luminescence was measured using a CLARIOstar® Plus microplate reader (BMG Labtech). The data were fitted to the sigmoid, 4PL, X = log(concentration) equation and presented as RLU on a line graph generated using GraphPad Prism software. EC 50 The values ​​were derived from the fitted curve.

[0330] IgG1-CD134-003-HC6LC2-RR induced potent dose-dependent OX40 agonist activity independently of Fc-mediated involvement via FcγRIIb-expressing cells (Figure 21A). In the absence of FcγRIIb-expressing cells, strong OX40 agonist activity was observed only for anti-human OX40 antibodies containing hexamerization-enhancing mutations (IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-003-E345R), while anti-human OX40 antibodies without hexamerization-enhancing mutations (IgG1-CD134-003 and IgG1-CD134-003-FEAL) induced only very weak OX40 signaling. The presence of FcγRIIb-expressing cells clearly enhanced OX40 signaling activity for FcγR-conjugated anti-human OX40 antibodies (IgG1-CD134-003 and IgG1-CD134-003-E345R), while the addition of FcγRIIb-expressing cells did not increase the activity of anti-human OX40 antibodies with an inactive Fc scaffold (IgG1-CD134-003-HC6LC2-RR and IgG1-CD134-003-FEAL), or only minimally increased it.

[0331] In contrast to IgG1-CD134-003-HC6LC2-RR, the anti-human OX40 antibodies IgG1-CD134-11D4, IgG1-RG7888, and IgG1-CD134-IBI101 induced OX40 agonist activity only in the presence of FcγR-expressing cells in the described assay, and not in the absence of FcγR (Figure 21B).

[0332] In conclusion, IgG1-CD134-003-HC6LC2-RR induced potent dose-dependent OX40 agonist activity independently of FcγR-expressing cells, in contrast to other anti-human OX40 antibodies lacking hexamerization-enhancing mutations.

[0333] Example 13: Expression of T cell activation marker during incubation with IgG1-CD134-003-HC6LC2-RR Using the T cell proliferation assay described in Example 7, the expression of T cell activation-related cell surface expression markers after incubation of healthy human donor PBMCs with IgG1-CD134-003-HC6LC2-RR was examined. Furthermore, the efficacy of IgG1-CD134-BMS986178, IgG1-CD134-RG7888, and IgG1-CD134-IBI101, all OX40 agonist reference antibody analogs, in enhancing the expression of T cell activation-related cell surface expression markers was evaluated. CD4 + T cells and CD8 + To measure the expression levels of 4-1BB, CD25, HLA-DR, and PD-1 on T cells, cells were washed and stained in 96-well round-bottom plates (VWR International, catalog no. 734-1797) with titrations of antibody (Table 13), Viability Dye eFluor 780 (1:1,500; ThermoFisher Scientific, catalog no. 65-0865-14), and brilliant stain buffer plus (1:10; BD Biosciences, catalog no. 566385) in 30 μL of FACS buffer for CD4, CD8, 4-1BB, CD25, HLA-DR, PD-1, and viability. The staining procedure was performed in the dark for 15 minutes at 4°C. The cells were then washed twice with 150 μL of FACS buffer and resuspended in 60–100 μL of FACS buffer for flow cytometry analysis. Flow cytometry data was acquired using a BD FACSymphony A3 flow cytometer with a BD High Throughput sampler. CD4 + T cell population and CD8 + The percentage of cells expressing the activation markers 4-1BB, CD25, HLA-DR, and PD-1 within the T cell population was determined using FlowJo and presented as a linear curve using GraphPad Prism.

[0334] (Table 13) Antibody panel used to evaluate the expression levels of T cell activation-related markers TIFF2026516142000021.tif51160

[0335] The expression of the activation marker was analyzed 2 and 5 days after IgG1-CD134-003-HC6LC2-RR incubation. In all donors, IgG1-CD134-003-HC6LC2-RR was evaluated on day 5, as shown on CD4 + T cell proliferation and CD8 + It dose-dependently increased T cell proliferation (data not shown). IgG1-CD134-003-HC6LC2-RR expressed 4-1BB, CD25, and HLA-DR in most donors analyzed. + T cells and CD8 + A dose-dependent increase in the percentage of T cells was induced on either day 2, day 5, or both, but PD-1 was not induced (Figure 22A-H).

[0336] In contrast to IgG1-CD134-003-HC6LC2-RR, the tested OX40 agonist reference antibody analogs IgG1-CD134-RG7888, IgG1-CD134-BMS986178, and IgG1-CD134-IBI101 expressed CD4 4-1BB, CD25, or HLA-DR. + T cells and CD8 + It did not increase the percentage of T cells (Figure 22I-P).

[0337] Example 14: IgG1-CD134-003-HC6LC2-RR enhances cytokine secretion in a polyclonal T cell proliferation assay using healthy human donor PBMCs. From a polyclonal T cell proliferation assay (general assay setup as described in Example 7; cells were stained with either CellTrace Violet [CellTrace® Violet Kit, Thermo Fisher Scientific, catalog number C34557] or CFSE [Vybrant CFDA SE Cell Tracer Kit, Life Technologies, catalog number V12883]), cytokine concentrations in the supernatant collected at 1, 2, 3, 4, and / or 6 days of culture were measured using the V-Plex Proinflammatory Panel 1 Human Kit (10-plex: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL10, IL12p70, IL-13, and TNF-α; MSD, catalog number K15049D-2), or V-PLEX Human Proinflammatory Panel I Measurements were performed by multiplex ECLIA using the Kit (4-plex: IFN-γ, IL-1β, IL-6, and TNF-α, MSD, catalog number K15052D-2) according to the manufacturer's protocol. Briefly, MSD plates were washed with 150 μL of PBST (DPBS containing 0.05% Tween® 20 [Sigma-Aldrich, catalog number P7949]), then standards or samples (1:2 to 1:200 dilutions) were added to the wells (50 μL / well), and the plates were incubated at RT for 2 hours with continuous shaking. The plates were washed three times with PBST, and detection antibodies (25 μL / well) were added. After incubation at RT for 2 hours with continuous shaking, the plates were washed three times with PBST. Subsequently, reading buffer was added (150 μL / well), and the plate was immediately analyzed with a MESO QuickPlex SQ 120 reader. In addition, CD4 was analyzed according to the procedure described in Example 7. + T cells or CD8 +Cytokine concentrations were measured in the supernatant collected from the polyclonal T cell proliferation assays mentioned, four days after depletion of any of the T cells in culture. The ability of IgG1-CD134-003-HC6LC2-RR to induce cytokine secretion was also compared to that of anti-human OX40 antibodies IgG1-h3C8-K322A-E345R, IgG1-CD134-h3C8-E345R-LALAPG, IgG1-CD134-RG7888-K322A-E345R, IgG1-CD134-RG7888-E345R-LALAPG, and IgG2s-CD134-SF2-E345R, four days after culture. The unbound antibody IgG1-b12-RR was used as a negative control. Results are presented as line graphs using GraphPad Prism.

[0338] In all donors, IgG1-CD134-003-HC6LC2-RR was found in CD4 + T cell proliferation and CD8 + It dose-dependently increased T cell proliferation (data not shown). In kinetic studies, IgG1-CD134-003-HC6LC2-RR enhanced cytokine secretion at the highest concentrations tested (0.2 and 2 μg / mL), enhancing TNFα after 3–4 days (Figure 23A), enhancing IL-2 after 2 days, followed by a relatively rapid decrease from 3–6 days (Figure 23B), and enhancing IFNγ (Figure 23C) and IL-13 (Figure 23D) after 2 days.

[0339] IgG1-CD134-003-HC6LC2-RR was found on day 4, CD8 + In PBMCs with depleted T cells, increased secretion of TNFα, IFNγ, and IL-13 was induced, and these cytokines were found to be CD4 + In PBMCs with depleted T cells, secretion was either absent or minimal (Figure 24).

[0340] IgG1-CD134-003-HC6LC2-RR induced higher cytokine levels of TNFα, IL-2, and IL-13 compared to variants of IgG1-CD134-h3C8 and IgG1-CD134-RG7888 (having K322A-E345 mutations or E345R-LALAPG mutations), as well as IgG2s-CD134-SF2-E345R. A similar trend was observed for IFNγ, although the latter showed relatively large fluctuations between consecutive measurements (Figures 25, 26, 27).

[0341] In summary, the results described here indicate that IgG1-CD134-003-HC6LC2-RR enhances the release of pro-inflammatory cytokines in a polyclonal T cell proliferation assay using healthy human donor PBMCs, and this is CD4 + This was shown to be dependent on the presence of T cells. Furthermore, IgG1-CD134-003-HC6LC2-RR more potently enhanced cytokine secretion in this assay than other hexamerized enhanced anti-human OX40 antibodies tested, namely variants of IgG1-CD134-h3C8, IgG1-CD134-RG7888, and IgG2s-SF2.

[0342] Example 15: In an antigen-specific T cell proliferation assay, IgG1-CD134-003-HC6LC2-RR was found to be CD8 + Ability to enhance T cell proliferation IgG1-CD134-003-HC6LC2-RR is CD8 + The ability to increase T cell proliferation was investigated by flow cytometry using T cells stimulated with its congener antigen. Human CD8 cells with electroporated TCRs that recognize CLDN6-derived peptides presented by OX40 and major histocompatibility type I (MHC-I) molecules were also examined. + T cells were co-cultured with immature autologous cellular cells (iDCs) that had been electroporated with full-length human CLDN6.

[0343] PBMCs were isolated from buffy coats obtained from healthy human donors by Ficoll-Paque density gradient separation, essentially as described in Example 7, and used for cell separation. Prior to cell separation, PBMCs were confirmed to be HLA-A*02 positive by flow cytometry. MACS columns or AutoMACS Pro Separators (both from Miltenyi Biotec GmbH) were used for cell separation, depending on availability. CD14 magnetic microbeads (Miltenyi Biotec GmbH, catalog no. 130-050-201) were used to extract CD14 from the newly isolated PBMCs. + Positive selection of monocytes, CD14 - Peripheral blood lymphocytes (PBLs) were negatively selected. CD8 magnetic microbeads were used to extract CD8 from previously frozen PBLs. + T cells were isolated. The isolation procedure was generally carried out according to the manufacturer's instructions, with minor modifications (reducing the volume of microbeads; 10 7 Perform the procedure with 12 μL of beads per cell, and CD14 + / CD14 - Cell isolation and CD8 + The isolation of T cells was, in principle, the same.

[0344] Isolated CD14 + Monocytes were differentiated into iDCs. For this purpose, a maximum of 40 × 10¹⁶ cells were cultured per T175 suspension flask (Greiner Bio-One GmbH, catalog number 661195). 6 CD14 +Monocytes were cultured for 5 days in a DC medium (RPMI 1640, 5% PHS, 1× Minimum Essential Medium Non-Essential Amino Acids [MEM-NEAA; Life Technologies GmbH, catalog number 11140-035], 1 mM sodium pyruvate [Life Technologies GmbH, catalog number 11360-039]) containing 200 ng / mL GM-CSF (Miltenyi Biotec GmbH, catalog number 130-093-868) and 200 ng / mL IL-4 (Miltenyi Biotec, catalog number 130-093-924) in an incubator (37°C, 5% CO2). After 3 days of culture, half of the medium per flask was replaced. The culture medium taken from the flask contained non-adherent monocytes, so it was centrifuged (8 min, 300 × g, RT), the supernatant was discarded, the cell pellet was resuspended in fresh DC medium, and then returned to the original flask with 200 ng / mL GM-CSF and 200 ng / mL IL-4 (final concentration). After 5 days, adherent cells were detached from the cell culture flask by incubation at 37°C for 10 minutes with 10 mL of DPBS containing 2 mM EDTA, and collected together with non-adherent cells before further use. The iDCs were washed with DPBS (8 min, 300 × g, RT), counted, and either cryopreserved in FBS (Sigma-Aldrich, catalog number F7524) containing 10% DMSO (AppliChem GmbH, catalog number A3672, 0100) or resuspended in X-VIVO 15 medium for direct electroporation.

[0345] CD8 isolated from PBL + T cells and iDCs were electroporated using the ECM 830 Electroporation System. CD8 +T cells were electroporated with RNA encoding the α and β chains of the CLDN6-specific TCR (10 μg each, TCR#12α and TCR#12β), as well as 10 μg of RNA encoding OX40. iDCs were electroporated with 2 μg of RNA encoding CLDN6, or electroporated without RNA as a mock control (Table 14).

[0346] (Table 14) RNA used for electroporation TIFF2026516142000022.tif30131

[0347] iDC electroporation typically involves 5 × 10⁶ units in 250 μL of X-VIVO 15 medium. 6 The procedure was performed using cells. T cells were divided into 250 μL of X-VIVO 15 medium at a ratio of 10-15 × 10⁶. 6 Cells were electroporated at a higher cell density. Cells were pipetted into cuvettes (4.0 mm gap size; VWR International, catalog no. 732-0023) at RT. RNA was added to the cells and mixed by pipetting. Immediately after mixing, electroporation was performed at 500 V, 3 ms, and 1 pulse for T cells, and at 300 V, 12 ms, and 1 pulse for iDCs. Immediately after electroporation, 750 μL of pre-warmed assay medium (IMDM, 5% PHS) was added to the cells. To evaluate electroporation efficiency, 2.5 × 10⁶ cells per well were added. 5 iDCs and 2-3 x 10⁶ wells per well 5 CD8 +T cells (unelectroporated and electroporated cells stained with CFSE, see below) were cultured in 150 μL of assay medium in a 96-well round-bottom plate. The remaining iDCs were transferred to a 6-well plate (Greiner, catalog no. 657160), cultured overnight in 3 mL of assay medium per 6 wells, and electroporated (37°C, 5% CO2). The electroporated T cells were transferred to a 15 mL tube and incubated for at least 2 hours before labeling with CFSE (37°C, 5% CO2).

[0348] Electroporated CD8 + T cells were labeled with CFSE using the Vybrant CFDA SE Cell Tracer Kit (Life Technologies GmbH, catalog number V12883). CFSE was dissolved in DMSO at a stock concentration of 9 mM and stored in aliquots at -20°C. PBMCs were washed with DPBS (8 min, 300 × g, RT). The pellet was divided into 20 × 10⁻⁶ units. 6 The cells were resuspended in DPBS at a concentration of cells / mL. An equal volume of 1.6 μM CFSE solution (diluted in DPBS from stock) was added. The cells were incubated in an incubator (37°C, 5% CO2) for 10 minutes. The labeling reaction was stopped by adding a 2:1 excess volume of FBS (Sigma-Aldrich, catalog no. F7524), followed by resting at RT for 2 minutes. To wash the cells, assay medium was added over the volume of the cell suspension, followed by centrifugation (8 min, 300 × g, RT). After CFSE labeling, the cells were resuspended in 3 mL of assay medium per electroporation, transferred to a 6-well plate, and incubated overnight (37°C, 5% CO2).

[0349] Electroporated iDC and CFSE-labeled CD8 + T cells were collected after O / N incubation and counted using a C-Chip cell counting chamber and erythrosine B solution. The concentration was 1.5 × 10⁻⁶ in assay medium. 6T cells / mL and 1.5 × 10 5 Adjusted to iDC / mL. CD8 + T cells and iDCs were seeded in a 10:1 ratio in 96-well round-bottom plates (7.5 × 10⁶ cells per well). 4 Individual T cells and 7.5 × 10 3 (1 iDC). For dose-response analysis of IgG1-CD134-003-HC6LC2-RR, serial dilutions of IgG1-CD134-003-HC6LC2-RR (0.0003 to 10 μg / mL, final concentration) were prepared. IgG1-b12-RR was used as an unbound control antibody at the same concentration as IgG1-CD134-003-HC6LC2-RR. The diluted antibody was added to seeded cells. Assay medium was added to each well to reach a total volume of 150 μL. The plate was gently shaken on a vibrating platform (150 RPM, 1 min) and cultured for 4 days (37°C, 5% CO2). Cell proliferation was analyzed by flow cytometry as described below.

[0350] For proliferation analysis, cultured cells were stained in 96-well round-bottom plates with CD8 antibody (BD Biosciences, catalog no. 564116; 1:400 dilution) and Fixable Viability Dye eFluor780 (ThermoFisher Scientific, catalog no. 65-0865-14; 1:1,500 dilution) in 30 μL of FACS buffer. The staining and washing procedures were performed as described in Example 10. Flow cytometry data were acquired using a BD FACSCelesta flow cytometer with a BD® High Throughput sampler. The flow cytometry data were analyzed using FlowJo software.

[0351] The growth index values ​​were plotted against each antibody concentration using GraphPad Prism software, and the data were fitted to the equation "Sigmoid, 4PL, X is log(concentration)". EC 50 The values ​​were derived from the fitted curve.

[0352] IgG1-CD134-003-HC6LC2-RR was co-cultured for 4 days with iDCs electroporated with the autologous CLDN6, and purified CD8 electroporated with CFSE-labeled OX40. + T cells and CD8 cells electroporated with CDLN6-TCR + It induced a dose-dependent increase in T cell proliferation (Figure 28A). IgG1-CD134-003-HC6LC2-RR only in the presence of antigen stimulation, and CD8 + It was confirmed that IgG1-CD134-003-HC6LC2-RR increased T cell proliferation, that TCR activation is a prerequisite for OX40 co-stimulation, and that IgG1-CD134-003-HC6LC2-RR did not induce the proliferation of resting T cells (Figure 28B).

[0353] Example 16: Binding of IgG1-CD134-003-HC6LC2-RR to human monocyte-derived M2c-like macrophages expressing FcγRIa. To assess whether the minimal binding of IgG1-CD134-003-HC6LC2-RR to FcγRIa as described in Example 8, as detected by SPR, is biologically relevant, the binding of IgG1-CD134-003-HC6LC2-RR to FcγRIa physiologically expressed by human monocyte-derived M2c-like macrophages was evaluated by flow cytometry.

[0354] For this purpose, human peripheral blood mononuclear cells (PBMCs) were purified from healthy human donor buffy coat (Sanquin blood supply foundation, the Netherlands) in LeucoSep® tubes (Greiner, catalog no. 227290) by density gradient centrifugation (800 × g for 20 minutes, low brake) in lymphocyte isolation medium (Promocell, catalog no. C-44010) according to the manufacturer's instructions. The PBMC layer was carefully transferred to a 50 mL tube and washed with excess volume of PBS (HyClone, catalog no. SH3A3830.03). The purified PBMCs were pelletized by centrifugation (300 x g for 10 minutes), washed and resuspended in PBS, and counted using a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience) with Cellometer ViaStain® AOPI Staining Solution (Nexcelom Bioscience, catalog number CS2-0106) in PBS to distinguish live cells from dead cells.

[0355] Human monocytes were purified from PBMCs using positive selection with CD14 MicroBeads (Miltenyi Biotec, catalog number 130-050-201) according to the manufacturer's instructions. + The cells were counted and placed in CellGenix® GMP DC medium (CellGenix, catalog number 20801-0500) supplemented with 50 ng / mL of macrophage colony-stimulating factor (M-CSF; Gibco, catalog number PHC9501), with 1.0 × 10⁶ cells added. 6 The cells were resuspended at a density of cells / mL.

[0356] To polarize monocytes towards M2c-like macrophages, purified monocytes were placed in CellGenix GMP DC medium (CellGenix, catalog number 20801-0500) supplemented with M-CSF, using a 100 mm UpCell® Surface. 2Plating on a Nunc(trademark) dish (1.0 x 10 6 8 × 10 at a cell / mL density 6 Cells / dish (Thermo Fisher Scientific, catalog number 174902). Cells were cultured in the above medium for 7 days (37°C / 5% CO2), followed by 3 days of culture in CellGenix GMP DC medium supplemented with 50 ng / mL M-CSF, 50 ng / mL interleukin (IL)-4 (R&D Systems, catalog number 204-IL), and 50 ng / mL IL-10 (R&D Systems, catalog number 1064-IL / CF). After 10 days of culture, macrophages were detached from the culture dish surface by leaving the dish at RT for 40-60 minutes. The detached macrophages were pelletized by centrifugation (300×g for 5 minutes), counted, and added to CellGenix GMP DC medium in a 1.5×10⁶ pellet. 6 The cells were resuspended at a density of cells / mL.

[0357] The M2c-like phenotype of monocyte-derived macrophages and their associated FcγRIa expression were confirmed by flow cytometry. Cells were plated into 96-well round-bottom plates (75,000 cells / well; Thermo Fisher Scientific, catalog no. 163320), centrifuged, and washed twice with FACS buffer. Next, the cells were incubated in FACS buffer with 50 μL of FITC-labeled anti-human CD64 (FcγRIa) antibody (BioLegend, catalog no. 305006; 1:25 dilution) or a mixture of antibodies for human M2c macrophage characterization (Table 15) at 4°C for 30 minutes in the dark, and then washed twice with FACS buffer.

[0358] (Table 15) Antibody panel used to confirm the M2c phenotype TIFF2026516142000023.tif23160

[0359] Next, cells incubated with the antibody for M2c characterization were resuspended in 100 μL of FACS buffer supplemented with the viability marker 7-AAD (7-aminoactinomycin D; BD Pharmingen, catalog no. 68981E; 1:240 dilution) and measured using a BD FACSymphony® Cell Analyzer (BD Biosciences). Cells incubated with the FcγRIa antibody were resuspended in 100 μL of FACS buffer supplemented with the viability marker DAPI (4',6-diamidino-2-phenylindole; BD Pharmingen, catalog no. 564907; 1:5,000 dilution) and measured using a BD LSRFortessa® Cell Analyzer.

[0360] To evaluate the binding of IgG1-CD134-003-HC6LC2-RR, monocyte-derived macrophages were plated in 96-well round-bottom plates (75,000 cells / well), washed twice with FACS buffer, and incubated with IgG1-CD134-003-HC6LC2-RR, IgG1-b12, or IgG1-b12-RR (final antibody concentration of 10 μg / mL) in 50 μL of CellGenix GMP DC medium at 37°C / 5% CO2 for either 15 minutes or 24 hours. The cells were then washed twice with FACS buffer and incubated with R-PE conjugate goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog no. 109 116 097; 1:200 dilution) in 50 μL of FACS buffer at 4°C for 30 minutes. After washing twice in FACS buffer, the cells were resuspended in 100 μL of FACS buffer supplemented with the viability marker DAPI (BD Pharmingen, catalog no. 564907; 1:5,000 dilution), and then measured using a BD LSRFortessa Cell Analyzer.

[0361] IgG1-b12 with a wild-type Fc domain was included as a positive control for binding to M2c-like macrophages, and Fc-inactive IgG1-b12-RR was included as a negative control. IgG1-b12 showed efficient binding to M2c-like macrophages expressing FcγRIa after 15-minute and 24-hour incubation, but no binding was observed for IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR in any of the three donors tested at any time point (Figure 29). In addition, no binding by IgG1-CD134-003-HC6LC2-RR was observed to cynomolgus monkey FcγR1a expressed by cynomolgus monkey monocytes (data not shown). Therefore, it is considered that IgG1-CD134-003-HC6LC2-RR cannot bind to FcγRIa in a physiological setting.

[0362] Example 17: Binding of IgG1-CD134-003-HC6LC2-RR to the neonatal Fc receptor The binding of IgG1-CD134-003-HC6LC2-RR to immobilized neonatal Fc receptor (FcRn) was evaluated in vitro at pH 6.0 and pH 7.4 by SPR. Aliquots of recombinant His-tagged FcRn protein (SinoBiological, catalog no. CT009-H08H-B) were diluted in PBS-P+ buffer pH 7.4 (Cytiva, catalog no. 28995084) or PBS-P+ buffer adjusted to pH 6.0 (by adding hydrochloric acid [Sigma-Aldrich, catalog no. 30721-M]) and used to capture FcRn protein onto the surface of an anti-His antibody-coated sensor chip using a flow rate of 10 μL / min and a contact time of 60 seconds. This resulted in capture levels ranging from 35 to 60 RU.

[0363] After three startup cycles in PBS-P+ buffer at pH 6.0 or pH 7.4, antibody concentration series (6.25–100 nM at 2-fold dilution in PBS-P+ buffer at pH 6.0 or pH 7.4) were injected to generate binding curves. Each sample analyzed on the surface with captured FcRn (active surface) was also analyzed on a parallel flow cell without captured FcRn (reference surface), and this was used for background correction. The signal from the third startup cycle containing HBS-EP+ as a (mock) analyte was subtracted from the other sensorgrams to produce dual-reference data. At the end of each cycle, the surface was regenerated using 10 mM glycine HCl pH 1.5 (Cytiva, catalog no. BR100354). The data were analyzed using the predefined "Multicycle Kinetics with Capture" evaluation method in Biacore Insight Evaluation software (Cytiva).

[0364] Dose-dependent binding of IgG1-CD134-003-HC6LC2-RR to FcRn was observed at pH 6.0 (Figures 30A-E), but no FcRn binding was observed at pH 7.4 (Figure 30F). These results indicate that, as expected for the IgG1 molecule, IgG1-CD134-003-HC6LC2-RR binds to FcRn at pH 6.0 but not at pH 7.4.

[0365] Example 18: Evaluation of C1q binding to IgG1-CD134-003-HC6LC2-RR bound to a membrane. To confirm the lack of complement binding to the Fc domain of IgG1-CD134-003-HC6LC2-RR, C1q binding to IgG1-CD134-003-HC6LC2-RR bound to OX40 expressed on the membrane of activated human T cells was measured by flow cytometry. IgG1-CD52-E345R (VH: SEQ ID NO: 64, VL: SEQ ID NO: 68; constant region: SEQ ID NO: 2), which has the same hexamerization-enhancing mutation as IgG1-CD134-003-HC6LC2-RR but lacks the Fc-inactivating mutation, was included as a positive control for C1q binding. The Fc-inactivating, unbinding control antibody IgG1-b12-RR was included as a negative control.

[0366] Human T cells were purified from buffy coat (Sanquin blood supply foundation, the Netherlands) obtained from healthy volunteers by negative selection using RosetteSep® Human T Cell Enrichment Cocktail (Example 9), followed by density centrifugation (800 × g, 20 min, low brake) in lymphocyte isolation medium (Corning, catalog no. 25-072-CI), all according to the manufacturer's instructions.

[0367] Purified T cells were washed in PBS (HyClone), pelletized, and resuspended in RPMI 1640 [Lonza, catalog no. BE12-115F] containing 25 mM HEPES and L-glutamine, supplemented with 10% FBS [Sanquin, catalog no. K1146], 50 units of penicillin and 50 μg / mL streptomycin [Lonza, catalog no. DE17-603E], and 1% L-glutamine [Lonza, catalog no. BE17-605E]. Next, the T cells were counted using a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience) with Cellometer ViaStain® AOPI Staining Solution (Nexcelom Bioscience, catalog no. CS2-0106) to distinguish live cells from dead cells. The cells were then plated into a round-bottom 96-well plate (Thermo Fisher). Scientific, catalog number 170189; 3 × 10 in 150 μL 5 (Cells / well). To induce OX40 expression, T cells were activated by incubation at 37°C for 72 hours with anti-CD3 / CD28 beads (Dynabeads® Human T-Activator CD3 / CD28; Thermo Fisher Scientific, catalog no. 11132D) in a 1:2 bead-to-cell ratio. After incubation, the beads were removed using a magnet, the cells were pooled, washed once in PBS, and counted.

[0368] To evaluate the binding of C1q to IgG1-CD134-003-HC6LC2-RR bound to cells, human T cells activated with CD3 / CD28 were plated in round-bottom 96-well plates (50,000 cells / well), and the antibodies were bound to the cells by incubation at 37°C for 15 minutes in 80 μL of sequential dilutions of IgG1-CD134-003-HC6LC2-RR, IgG1-CD52-E435R, or IgG1-b12-RR in activation medium (final concentrations of 0.00051 to 30 μg / mL at 3-fold dilutions). Next, 20 μL of normal human serum (NHS; Sanquin; final concentration of 20%) was added as a source of C1q, and the mixture was incubated on ice for 45 minutes. The cells were washed twice with cold FACS buffer, and then incubated with 50 μL of a mixture of fluorescein isothiocyanate (FITC) conjugate rabbit anti-human C1q antibody (DAKO, catalog no. F0254; 1:100 dilution; final concentration 20 μg / mL) in FACS buffer and antibodies for human T cell characterization (Table 16) at 4°C for 30 minutes, protected from light.

[0369] (Table 16) Antibody panel used for T cell characterization TIFF2026516142000024.tif19160

[0370] Next, the cells were washed twice with cold FACS buffer, pelletized, and resuspended in 80 μL of FACS buffer supplemented with TO-PRO®-3 Iodide (Invitrogen, catalog number T3605; 1:10,000 dilution), a viability marker. C1q binding was analyzed by flow cytometry using a BD FACSCelesta® Cell Analyzer (BD Biosciences). Binding curves were analyzed using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism software. Binding of OX40 antibody to activated T cells was evaluated after incubation with serial dilutions of the antibody. Cells were washed twice with cold FACS buffer and incubated with 50 μL of a mixture of R-phycoerythrin (R-PE) conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog no. 109-116-098; 1:200 dilution) in FACS buffer and an antibody for human T cell characterization (Table 16) at 4°C for 30 minutes in the dark. Next, the cells were washed twice with cold FACS buffer, pelletized, and resuspended in 80 μL of FACS buffer supplemented with the viability marker TO-PRO-3 iodide (1:10,000 dilution). Antibody binding was analyzed by flow cytometry using a BD FACSCelesta Cell Analyzer. Binding curves were analyzed using nonlinear regression analysis (sigmoid dose-response with variable gradient) with GraphPad Prism software.

[0371] Activated CD4 + T cells and CD8 +The binding of IgG1-CD134-003-HC6LC2-RR and IgG1-CD52-E345R to T cells was confirmed by flow cytometry (Figures 31A-C). C1q efficiently bound to membrane-bound IgG1-CD52-E345R, but binding of C1q to membrane-bound IgG1-CD134-003-HC6LC2-RR was not observed at any of the antibody concentrations tested (Figures 31D, E). No binding of C1q was observed to cells incubated with IgG1-b12-RR. These results indicate that C1q cannot bind to membrane-bound IgG1-CD134-003-HC6LC2-RR.

[0372] To confirm that IgG1-CD134-003-HC6LC2-RR does not induce target-independent liquid-phase complement activation, IgG1-CD134-003-HC6LC2-RR was incubated in normal human serum (NHS), and C4d production was measured by ELISA as a measure of complement activation. IgG1-CD134-003-HC6LC2-RR did not induce liquid-phase complement activation in vitro (data not shown).

[0373] Example 19: Evaluation of monovalent vs. divalent binding of IgG1-CD134-003-HC6LC2-RR and its variants. To determine whether IgG1-CD134-003-HC6LC2-RR can bind to OX40 monovalently or bivalently, the binding of IgG1-CD134-003-HC6LC2-RR to activated T cells was compared to the binding of a functionally monovalent OX40-specific antibody (BsIgG1-b12-RR-F405L×CD134-003-RR-K409R; generated through controlled Fab-arm exchange of the parent antibody IgG1-b12-RR-F405L [composed of SEQ ID N0: 78, 75, and 71] and IgG1-CD134-003-RR-K409R [composed of SEQ ID NO: 63, 21, and 20]). Healthy donor T cells were stimulated in vitro with anti-CD3 / CD28 beads for 3 days to induce OX40 expression. Subsequently, activated CD4 + T cells and CD8 + The binding of IgG1-CD134-003-HC6LC2-RR, the monovalent antibody BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, the chimeric control antibody IgG1-CD134-003, or the negative control antibody IgG1-b12-RR to T cells was analyzed by flow cytometry.

[0374] Whole human T cells were enriched by negative selection directly from the buffy coat as described in Example 18. The enriched T cells were washed once or twice in PBS (Capricorn Scientific, catalog number SP-2121-500 mL) or in PBS supplemented with 2% DBSi (Gibco, catalog number 20371-030) and 2 mM EDTA (Sigma-Aldrich, catalog number 03690). The purified cells were counted using a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Biosciences) with Cellometer ViaStain® AOPI solution (Nexcelom Bioscience, catalog number CS2-0106) in PBS to distinguish live cells from dead cells. The T cells were pelleted and resuspended in activation medium.

[0375] T cells were activated by 72 hours of CD3 and CD28 stimulation using anti-CD3 / CD28 beads (Dynabeads® Human T-Activator CD3 / CD28 as described in Example 9; ThermoFisher Scientific, catalog no. 11131D). The activated T cells were either used directly or cryopreserved for further use in a 1:1 mixture of IMDM medium containing L-glutamine and HEPES (Lonza, catalog no. 12-115F) and cryoprotection freezing medium (Lonza, catalog no. 12-132A).

[0376] Activated T cells were seeded (50,000 cells / well) in a round-bottom 96-well plate (Thermo Scientific, catalog no. 163320) and washed once with PBS (GE Healthcare, catalog no. SH3A3830.03) and once with FACS buffer. Subsequently, the cells were resuspended in 50 μL of primary antibody (IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, or the control antibody IgG1-CD134003-RR-K409R, IgG1-b12-RR, and IgG1-CD134-003) diluted in FACS buffer at a 3-fold dilution stage to concentrations ranging from 0.0005 to 10 μg / mL. The cells were incubated at 4°C for 30 minutes and measured by flow cytometry using a BD FACSymphony A1 Cell Analyzer (BD Biosciences), and analyzed using FlowJo software.

[0377] The monovalent OX40-specific antibody BsIgG1-b12-RR-F405L×CD134-003-RR-K409R is activated CD4 + T cells and CD8 + It shows dose-dependent binding to T cells, and compared to IgG1-CD134-003-HC6LC2-RR, as defined by the gMFI Y-span, CD4 + T cells and CD8+ It showed higher maximum binding to both T cells (Figure 32). The chimeric control antibody IgG1-CD134-003 was OX40 + CD4 + T cells and CD8 + It showed similar binding to IgG1-CD134-003-HC6LC2-RR to T cells, indicating that the higher maximum binding was driven by the monovalent nature of BsIgG1-b12-RR-F405L×CD134-003-RR-K409R rather than CDR chimerism or functionally unrelated mutations in different Fc scaffolds. These data suggest that IgG1-CD134-003-HC6LC2-RR may also bind in a bivalent manner, rather than exclusively in a monovalent manner.

[0378] Example 20: Functional comparison of IgG1-CD134-003-HC6LC2-RR and its monovalent variant As described in Example 19, IgG1-CD134-003-HC6LC2-RR can bind in a bivalent form as well as exclusively in a monovalent form. Here, the functional activity of IgG1-CD134-003-HC6LC2-RR and the monovalent binding variant BsIgG1-b12-RR-F405L×CD134-003-RR-K409R was compared in two separate assays: a cell-based OX40 reporter assay and a polyclonal T cell proliferation assay using healthy human donor PBMCs.

[0379] To measure OX40 agonist activity in a cellular reporter assay, the OX40 Bioassay Kit (Thaw and Use Kit; Promega, catalog number JA2191) was used essentially as described in Example 6. In short, OX40 +Jurkat cells were thawed and plated at 60 μL per well in a new 96-well white flat-bottom assay plate (PerkinElmer, catalog no. 6005680), and incubated for 16–20 hours (37°C, 5% CO2). The following day, dilutions of IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, and the control antibodies IgG1-CD134-003-RR-K409R, IgG1-CD134-003, IgG1-CD134-003-FEAL, and IgG1-b12-RR were prepared in RPMI 1640 medium containing 5% FBS (final concentrations of 0.000457–1 μg / mL at 3-fold dilutions). Only 20 μL of the prepared antibody dilution series or culture medium was added to each well, and the assay plate was incubated for 5 hours (37°C, 5% CO2). After incubation, the assay plate was equilibrated to RT. Then, 80 μL of Bio-Glo® Luciferase Assay substrate was added to each well, and the plate was incubated for 10 minutes with continuous shaking and in the dark. Luminescence was measured using an EnVision Multiplate Reader (PerkinElmer). The data were presented as relative luminescence (RLU) on a line graph generated using GraphPad Prism software. The data were fitted to the sigmoid, 4PL, X = log(concentration) equation.

[0380] To evaluate the capabilities of IgG1-CD134-003-HC6LC2-RR, BsIgG1-b12-RR-F405L×CD134-003-RR-K409R, and the negative control antibody IgG1-b12-RR (concentration range of 0.0015 μg / mL to 30 μg / mL) in a polyclonal T cell proliferation assay, the procedure described in Example 7 was followed.

[0381] In a cell-based OX40 reporter assay, BsIgG1-b12-RR-F405L×CD134-003-RR-K409R induced OX40 agonist activity with approximately 5 times lower potency than IgG1-CD134-003-HC6LC2-RR, indicating that the optimal agonist activity of IgG1-CD134-003-HC6LC2-RR is achieved through its ability to bind to both Fab arms (Figure 33A). In a polyclonal T cell proliferation assay, BsIgG1-b12-RR-F405L×CD134-003-RR-K409R induced CD4 + T cell proliferation was enhanced (Figure 33B), but a higher concentration was required to achieve the maximum effect compared to IgG1-CD134-003-HC6LC2-RR.

[0382] Combined, these results indicate that the optimal biological activity of IgG1-CD134-003-HC6LC2-RR is achieved through its ability to bind to OX40 in its two Fab arms.

[0383] Example 21: OX40 expression and OX40 shedding of T cells in response to incubation with IgG1-CD134-003-HC6LC2-RR in vitro IgG1-CD134-003-HC6LC2-RR CD4 in response to treatment + T cells and CD8 +Changes in OX40 expression levels on T cells were analyzed using polyclonal T cell proliferation assays with PBMCs obtained from 10 healthy human donors (general assay setup as described in Example 7; PBMCs were stained with either cell trace violet using CellTrace® Violet Kit [Thermo Fisher Scientific, catalog number C34557; see Example 7 for staining instructions] or CFSE using Vybrant CFDA SE Cell Tracer Kit [Life Technologies, catalog number V12883; see Example 15 for staining instructions]). For seven of the tested donors, OX40 expressed on the membrane was measured by flow cytometry after 2 and 5 day incubations with IgG1-CD134-003-HC6LC2-RR as described in Example 13 (antibody staining: APC-labeled CD4 antibody [ThermoFisher Scientific, catalog no. 17-0048-42; dilution 1:100], BV605-labeled CD8α antibody [BD Biosciences, catalog no. 564116; dilution 1:400], PE-labeled OX40 antibody [BD Biosciences, catalog no. 340420; dilution 1:80]). For the remaining three donors, expression was evaluated after 1-4 and 6 day incubations (antibody staining: PerCP-eFluor710-labeled CD4 antibody [ThermoFisher [Scientific, catalog number 46-0047-42; dilution 1:100], PE-Cy7-labeled CD8α antibody [TONBO, catalog number 60-0088-T100; dilution 1:100], BV421-labeled OX40 antibody (BD Biosciences, catalog number 744881; dilution 1:80)). CD4 + T cells and CD8 + OX40 expression on T cells was evaluated using a one-dimensional histogram based on geometric mean fluorescence intensity (gFMI) values.

[0384] Soluble OX40 (sOX40) has been detected in the serum of both healthy donors and patients with autoimmune diseases and cancer (Taylor and Schwarz. Identification of a soluble OX40 isoform: development of a specific and quantitative immunoassay. J Immunol Methods 255: 67-72), and is thought to act as a decoy receptor by blocking the binding of OX40 ligand (OX40L) to membrane-bound OX40. OX40 shedding in response to IgG1-CD134-003-HC6LC2-RR in vitro treatment was evaluated by ECLIA on the day OX40 expression was measured. sOX40 concentrations in the supernatant collected from polyclonal T cell proliferation assays were measured by multiplex ECLIA using the U-PLEX Human OX40 / TNFRSF4 Assay (MSD, catalog number K151T7K-2) according to the manufacturer's protocol. In short, 25 μL of biotinylated antibody diluted in Diluent 100 was added to each well of an MSD Gold Small Spot Streptavidin plate and incubated at RT for 1 hour. The plate was washed three times with PBST, and a standard or sample (sample diluted 1:8 in Diluent 58) was added to each well (total 50 μL / well). The plate was incubated at RT for 2 hours with continuous shaking. The plate was washed three times with PBST, and a detection antibody diluted in Diluent 3 was added (50 μL / well). The plate was incubated at RT for 1 hour with continuous shaking, and the plate was washed three times with PBST. Then, Gold Read Buffer B was added (150 μL / well), and the plate was immediately analyzed using a MESO QuickPlex SQ 120 imager (MSD).

[0385] IgG1-CD134-003-HC6LC2-RR (concentration of 0.01 μg / mL or higher) showed CD4 in 3 out of 10 donors after 2 days of incubation. +The expression of OX40 induced by CD3 on T cells was enhanced, and CD4 was increased in 6 out of 10 donors after 3-6 days of incubation or 5 days of incubation, respectively. + T cells and CD8 + CD8 on T cells, and after incubation on days 2 and 3. + CD3-induced expression of OX40 on T cells was enhanced (as illustrated in Figure 34A for one responsive donor out of a total of three donors; donor variability was observed in additionally tested donors). In parallel, in all three donors tested for sOX40 release, IgG1-CD134-003-HC6LC2-RR treatment dose-dependently increased the concentration of sOX40 in the supernatant over time, indicating that IgG1-CD134-003-HC6LC2-RR induces continuous release of sOX40, which begins on day 3 and continues to increase until day 6 (Figure 34B). Partial interference of IgG1-CD134-003-HC6LC2-RR with the sOX40 detection ECLIA kit was observed, but an increase in sOX40 levels was clearly detected upon treatment with IgG1-CD134-003-HC6LC2-RR at concentrations of 0.2 and 2 μg / mL.

[0386] These data suggest that IgG1-CD134-003-HC6LC2-RR is associated with high donor variability, but CD4 + T cells and CD8 + This suggests that it may enhance OX40 expression on the membrane of T cells, and in parallel, enhance OX40 shedding from the plasma membrane.

[0387] Example 22: Evaluation of interference of soluble OX40 in IgG1-CD134-003-HC6LC2-RR agonist activity. In Example 21, IgG1-CD134-003-HC6LC2-RR was found to be CD4 + T cells and CD8 +It has been described that sOX40 can enhance OX40 membrane expression on T cells and, in parallel, enhance OX40 shedding from the plasma membrane. Here, we investigated the effect of sOX40 on the observed ability of IgG1-CD134-003-HC6LC2-RR to induce OX40 agonism in a cell-based OX40 reporter assay (see Example 6) and to increase T cell proliferation in a polyclonal T cell proliferation assay (see Example 7).

[0388] To investigate whether the presence of sOX40 interferes with IgG1-CD134-003-HC6LC2-RR-mediated OX40 agonist activity, assays were performed without FcγRIIb-CHO K1 cells as described in Example 13. Final concentrations of IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR in the range of 0.0024 to 40 μg / mL (1:4 series dilution) were added with or without sOX40 (human OX40, AcroBiosystems, catalog no. OX0-H5224) in the range of 1 to 1,000 ng / mL (1:10 series dilution), and luminescence was measured using a CLARIOstar Plus microplate reader. To investigate whether the presence of sOX40 interferes with T cell proliferation enhanced by IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-HC6LC2-RR or IgG1-b12-RR (1:4 series dilution) in concentrations of 0.002 to 40 μg / mL was added either with or without sOX40 (0.1 ng / mL to 1,000 ng / mL; 1:10 series dilution), and CD45RA staining and CCR7 staining were omitted, following the method described in Example 7.

[0389] The dose-dependent OX40 agonist activity induced by IgG1-CD134-003-HC6LC2-RR was retained in the presence of low concentrations of sOX40, but a decrease was observed at 1,000 ng / mL of sOX40, and less at 100 ng / mL of sOX40 (Figure 35). Similarly, the CD4 agonist activity induced by IgG1-CD134-003-HC6LC2-RR was also observed. + T cell proliferation and CD8 + The dose-dependent increase in T cell proliferation was maintained in the presence of soOX40 at concentrations up to 10 ng / mL, but the reduced increase was observed in CD4 + T cell proliferation and CD8 + In both T cell proliferations, sOX40 interference was observed at 100 ng / mL and 1,000 ng / mL concentrations of sOX40 for IgG1-CD134-003-HC6LC2-RR concentrations up to 0.625 μg / mL (Figure 36). No interference with sOX40 was observed in PBMC samples incubated with higher tested IgG1-CD134-003-HC6LC2-RR concentrations (≧2.5 μg / mL).

[0390] In summary, interference of sOX40 with IgG1-CD134-003-HC6LC2-RR-induced OX40 agonism in reporter cells and polyclonally activated T cells was observed only at concentrations considerably higher than those expected to be clinically relevant.

[0391] Example 23: Preclinical immunogenicity evaluation of IgG1-CD134-003-HC6LC2-RR The immunogenicity of the heavy chain (HC) and light chain (LC) of IgG1-CD134-003-HC6LC2-RR was evaluated in silico using the augmented intelligence (AI) platform iTope-AI and TCED (Abzena). The potential immunogenicity of IgG1-CD134-003-HC6LC2-RR was assessed by co-culturing human CD4 with IgG1-CD134-003-HC6LC2-RR and previously incubated MoDC. +Further in vitro analysis was performed using the EpiScreen DC:T cell proliferation assay (Abzena) by measuring T cell proliferation.

[0392] Nine-mer peptides overlapping by eight amino acids across the HC and LC protein sequences of IgG1-CD134-003-HC6LC2-RR were generated in silico. Using iTope-AI technology, favorable interactions between the amino acid side chains of each nine-mer and the 46 most common human leukocyte antigen (HLA) alleles (HLA-DR, HLA-DP, and HLA-DQ allotypes) found worldwide were predicted. For peptides showing favorable interactions, the P1 anchor position indicates the first amino acid of its sequence. Each individual peptide was assigned a binding score from 0 (no binding) to 3 (strong binding) for each of the 46 allotypes, and these scores were summed for all allotypes to provide an overall risk score per nine-mer called the Position Risk Score. Promiscuous peptides (position risk score > 0) were considered weak, moderate, or strong MHC class II binders when their position risk scores reached 1–2, 3–5, or 6+, respectively. The total score for all test protein sequences was calculated by adding the positional risk scores obtained for all individual peptides. Since germline sequences are less likely to have immunogenic potential for T cell tolerance, nine MARPeptides that are completely homologous to human proteome-derived sequences were excluded from the analysis. The total score for IgG1-CD134-003-HC6LC2-RR could then be compared to the total scores observed for reference mAbs, e.g., mouse mAbs, chimeric mAbs, humanized mAbs, and human mAbs.

[0393] To assess the immunogenicity risk of the HC and LC sequences of IgG1-CD134-003-HC6LC2-RR, identified promiscuous peptides were matched against peptide sequences in TCED using the basic local alignment search tool (BLAST) to identify high sequence homology with over 10,000 peptides (i.e., T cell epitopes) derived from unrelated proteins and antibodies that stimulated T cell responses in Abzena's previous ex vivo EpiScreen study.

[0394] Using the EpiScreen DC:T cell assay, CD4 is the primary driver of memory-based immunogenicity. + The potential immunogenicity of IgG1-CD134-003-HC6LC2-RR was evaluated in vitro by measuring the T cell response. First, PBMCs were isolated from healthy donor leukocyte cones (within 24 hours of collection) obtained with consent from the UK National Health Service transfusion service. Donors were characterized by HLA-DR haplotypes using sequence-specific oligonucleotide (SSO) HLA typing (VHBio). A cohort of 50 healthy PBMC donors was selected to represent all known HLA alleles, excluding HLA-DP, which was not considered in selection due to its low prevalence and potentially low expression levels.

[0395] IgG1-CD134-003-HC6LC2-RR was diluted to 0.75 μM (112.5 μg / mL) in MoDC culture medium (RPMI 1640 [ThermoFisher, catalog number 21875-034] supplemented with human serum [HS;VWR, catalog number 21001PM], 2-mercaptoethanol [2ME;Sigma, catalog number M3148], L-glutamine [ThermoFisher, catalog number 25030-024], penicillin / streptomycin [ThermoFisher, catalog number 15140-122], interleukin (IL)-4 [Peprotech, catalog number 200-04], and granulocyte-macrophage colony-stimulating factor [GM-CSF;Peprotech, catalog number 300-03]) before use. As a positive control, the T cell response to the nascent antigen keyhole limpet hemocyanin (KLH) was also measured. KLH (Pierce Life Technologies, catalog no. 77600) was stored at -20°C as a 10 mg / mL stock solution in distilled water. Aliquots of KLH were thawed immediately before dilution to 1 mg / mL in MoDC culture medium. Herceptin® (Roche, catalog no. HERC / 150 / 1 / BG) was used as a reference antibody known for its low clinical immunogenicity, stored at -80°C as a 20 mg / mL stock solution, thawed immediately before use, and diluted to 125 μg / mL in MoDC culture medium.

[0396] For the preparation of MoDC, CD14 + Monocytes were purified from donor PBMCs using a negative human monocyte purification kit (StemCell Technologies, catalog no. 19058RF) and an automated cell purification system (RoboSep® StemCell Technologies) according to the manufacturer's instructions. The monocytes were resuspended in MoDC culture medium and placed in a low-binding 24-well plate at a rate of 1.5 × 10⁶ per well in 2 mL of culture medium (final volume). 6Cells were plated individually and incubated at 37°C. On day 2, cells were nourished by replacing 1 mL of MoDC medium with fresh medium. On day 4, cells were again nourished with 1 mL of MoDC culture medium, which also contained the following reagents (final concentrations): 0.3 μM (45 μg / mL) IgG1-CD134-003-HC6LC2-RR, 0.3 μM (45 μg / mL) Herceptin (reference antibody), or 100 μg / mL KLH (positive control). Untreated control wells were treated in the same way as reagent-containing wells, except for the addition of reagents. Cells were incubated for 1 hour (37°C / 5% CO2). After incubation, 0.01 μg / mL of lipopolysaccharide (LPS; Sigma, catalog number L4391) was added to induce MoDC maturation. On day 5, mature MoDCs were collected and counted, and their viability was evaluated using trypan blue (Sigma, catalog number T8154) stain exclusion. Viability was expressed as the percentage of cells that did not stain with trypan blue out of the total number of cells. Cell viability evaluation confirmed that IgG1-CD134-003-HC6LC2-RR, as well as KLH control and Herceptin control, did not affect the viability of MoDCs used in the EpiScreen analysis. Subsequently, mature MoDCs were irradiated with gamma rays (40 Gy) before use in the proliferation assay. Finally, autologous CD4 + T cells, human CD4 + T cells were purified by negative selection from PBMCs of the same donor using a T cell enrichment kit (StemCell Technologies, catalog number 19052) and an automated cell purification system.

[0397] After counting and evaluating cell viability, 1 × 10 6 A CD4+ T cell is divided into 1 × 10⁶ cells. 5Cells were co-cultured in 24-well plates for 12 days in culture medium (RPMI 1640 supplemented with human serum, 2-mercaptoethanol, and L-glutamine) with a gamma-ray irradiated MoDC at a T:DC ratio of 10:1. On days 9, 10, 11, and 12, the culture was gently resuspended, and 3 × 100 μL aliquots were transferred to each of the 96 round-bottom wells for pulse labeling. 3 The culture medium containing 100 μL of [H]-thymidine (Perkin Elmer) was pulsed, incubated at 37°C for 6 hours, and then collected on a filter mat using a TomTec Mach III cell harvester. For each sample, counts per minute (CPM) were measured using MeltiLex® (Perkin Elmer) scintillation counting with a MicroBeta Microplate Beta Counter in ParaLux® low background count mode. Immunogenicity was expressed as a Stimulation Index (SI) for each sample, defined as the mean CPM of the wells containing compound-treated T cells / mean CPM of the baseline (untreated control well). Stimulation Index (SI) = (Mean CPM (treated wells)) / (Mean CPM (untreated control wells))

[0398] Based on previous analyses by Abzena, an empirical threshold of SI ≥ 1.90 was established as the minimum signal-to-noise threshold enabling maximum sensitivity without detecting a large number of false-positive responses or omitting subtle immunogenic events. Samples inducing a response above this threshold are scored as positive for induction of a T cell immune response in the test donor. For each sample at a given time point, a positive proliferation response is scored using an unpaired two-sample Student's t-test against a mature DC sample loaded with reagents and incubated CD4 + Triple CPM of T cells incubated with mature MoDC cultured in culture medium alone and CD4 +The results were considered statistically significant (P<0.05) by comparing them with triple CPM of T cells. Donors who were positive (SI≧1.90, p<0.05) at at least one time point during the time-course assay were scored as positive donors, and the mean magnitude SI was calculated from the mean positive donor response.

[0399] iTope-AI analysis of the IgG1-CD134-003-HC6LC2-RR HC sequence with P329R and E345R Fc mutations predicted a total of 12 non-germline promiscuous peptides in the HC constant region, which were classified as strong (2), moderate (2), or weak (8) MHC class II binding agents (Table 17). The P329R mutation was located at two of the identified weak binding agents (P1 anchor positions C317 and K318; Table 17), which did not correspond to any known T cell epitopes in TCED. The E345R mutation was not present in any of these peptides. In addition, 10 of the 12 non-germline promiscuous peptides were predicted in the HC variable domain (VH), of which 2 were predicted as moderate binding agents and 2 as strong binding agents (Table 17). The peptide identified in Framework (Fw)3 (location risk score: 1) was found to be homologous to the peptide in TCED. In total, the iTope total score for IgG1-CD134-003-HC6LC2-RR HC was 45.

[0400] (Table 17) Non-germ cell lineage promiscuous IgG1-CD134-003-HC6LC2-RR HC peptides identified by iTope-AI and TCED analysis. Each of the nine MEP peptides is represented by its P1 anchor position. Variable regions were numbered according to Kabat numbering, followed by linear numbering of the constant region. The positional risk score of the promiscuous binding peptides was divided into weak (1-2), moderate (3-5), and strong (6+) affinity binding agents. Homologous peptides identified from TCED are listed. TIFF2026516142000025.tif100161a The first amino acid of the 9-mer peptide. b The location of P1 follows Kabat numbering for the variable region and linear numbering for the steady region. c Underlined residues indicate their position in IgG1-CD134-003-HC6LC2-RR VH CDR as defined according to the IMGT definition. d R=IgG1-CD134-003-HC6LC2-RR P329R mutation in the Fc region; location follows Eu numbering. e The total score for a test sequence is calculated by adding up the positional risk scores obtained for each individual peptide. f An overview of HC TCED matching of non-germ cell lineage binding peptides. Homologous peptides identified from TCED and their matching P1>P9>P7 anchor positions are listed. A hierarchy exists in the contribution of specific "anchor" positions to MHC class II binding, with the greatest effect observed in P1>P9>P7≧P6≧P4.

[0401] iTope-AI analysis predicted the presence of three strongly bound, two moderately bound, and ten weakly bound non-germline promiscuous MHC class II binding peptides in the LC of IgG1-CD134-003-HC6LC2-RR, all located in the variable domain of the LC(VL) sequence (Table 18). Homologous peptides in TCED were identified for three of the predicted peptides (two weakly bound peptides and one moderately bound peptide). The iTope total score for the IgG1-CD134-003-HC6LC2-RR LC sequence was 74. The total score for IgG1-CD134-003-HC6LC2-RR, which is the sum of the HC total score and the LC total score, was 119, which is consistent with the reference humanized mAb.

[0402] (Table 18) Non-germ cell lineage promiscuous IgG1-CD134-003-HC6LC2-RR LC peptides identified by iTope-AI and TCED analysis. Each of the nine MEP peptides is represented by its P1 anchor position. Variable regions were numbered according to Kabat numbering, followed by linear numbering of the constant region. The positional risk score of the promiscuous binding peptides was divided into weak (1-2), moderate (3-5), and strong (6+) affinity binding agents. Homologous peptides identified from TCED are listed. TIFF2026516142000026.tif119160 a The first amino acid of the 9-mer peptide. b The location of P1 follows Kabat numbering for the variable region and linear numbering for the steady region. c Underlined residues indicate their position in the IgG1-CD134-003-HC6LC2-RR VL CDR as defined according to the IMGT definition. d The total score for a test sequence is calculated by adding up the positional risk scores obtained for each individual peptide. e Summary of LC-TCED matching of non-germ cell lineage binding peptides. Homologous peptides identified from TCED and matching P1>P9>P7≧P6≧P4 anchor positions are listed.

[0403] Mature MoDCs loaded with IgG1-CD134-003-HC6LC2-RR induced a low frequency of positive T cell proliferation (SI ≥ 1.90) in 8% (4 / 50) of the donor cohort (Table 19). This is within the same range as Herceptin, which is known to exhibit low clinical immunogenicity (proliferation induced in 8% [4 / 50] of the donor cohort), and is below the 10% threshold set by Abzena based on historical EpiScreen data for proteins considered to have an increased immunogenicity risk in the clinic. The nascent antigen KLH, included as a positive control for immunogenicity, showed a positive response rate of 88%. The mean size SI (mean SI of positive donor response) for IgG1-CD134-003-HC6LC2-RR and Herceptin were comparable (SI=2.66±0.6 and SI=2.64±0.7, respectively), and lower compared to KLH (SI=4.6±2.54).

[0404] (Table 19) CD4 induced by mature MoDC loaded with IgG1-CD134-003-HC6LC2-RR, Herceptin, or KLH + Overview of the extent of the T cell proliferation response TIFF2026516142000027.tif23160 1 The percentage of donors who had a T-cell proliferative response at at least one of the time points (SI ≥ 1.90, p < 0.05, where the mean size SI was calculated from the mean positive donor response).

[0405] Taken together, these data indicate that IgG1-CD134-003-HC6LC2-RR has a low risk of clinical immunogenicity, meaning it falls within the range of therapeutic antibodies known to exhibit low immunogenicity in clinical settings.

[0406] Example 24: Pharmacokinetic analysis of IgG1-CD134-003-HC6LC2-RR in mice in the absence of target binding. The pharmacokinetic (PK) characteristics of the anti-human OX40 antibody IgG1-CD134-003-HC6LC2-RR in the absence of target binding were analyzed in mice and compared with two chimeric control antibodies (IgG1-CD134-003-FEAL and IgG1-CD134-003-RR-K409R, respectively) that either contain the inactive mutations L234F-L235E-D265A and DuoBody mutation F405L, or the same RR Fc mutation as IgG1-CD134-003-HC6LC2-RR in addition to DuoBody mutation K409R. IgG1-CD134-003-HC6LC2-RR did not bind to mouse OX40 as shown in Example 5 and Figure 5A; therefore, the experiment was designed to test the in vivo pharmacokinetic behavior of the anti-human OX40 antibody mentioned in the absence of target binding. Furthermore, IgG1-CD134-003-FEAL and IgG1-CD134-003-RR-K409R also do not bind to mouse OX40 (data not shown). Female severe combined immunodeficiency mice (SCID mice, Envigo;CB-17 / IcrHan®Hsd Prkdcscid) were housed in sterile individual ventilation cages (IVCs) at the Central Laboratory of Laboratory Animals (Gemeenschappelijk Dierenlaboratorium, GDL) of Utrecht University, with 5 mice per cage and free access to sterile food and water. The mice received tail tattooing for identification purposes upon arrival at GDL. The animal experiments were conducted in compliance with the Dutch Animal Protection Act (WoD) and the Code of Practice "Animal Experiments for Cancer Research" (Inspection V&W, Zutphen, The Netherlands, 1999), translated from Directive (2010 / 63 / EU), and were approved by the Utrecht Ethics Committee.The animals were kept and handled in animal facilities (GDLs) accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and ISO 9001:2018, in accordance with good animal practice as defined by the Federation of European Laboratory Animal Science Associations (FELASA).

[0407] At 10 weeks of age, female SCID mice (3 mice per group) were intravenously injected into the tail vein with 60 μL of a solution containing either the mentioned test antibody or control antibody, corresponding to approximately 0.125, 1.25, and 12.5 mg of antibody per kg of body weight. 40 μL of blood samples were collected at 10 minutes, 4 hours, 1 day, 2 days, 8 days, 14 days, and 21 days after antibody administration. Plasma was collected from the blood samples and stored at -65°C until total human IgG concentration was measured by ELISA. To obtain plasma, blood was collected in Microvette® CB 300 K2 EDTA vials (Sarstedt, catalog no. 16.444.100) and centrifuged at 14,000 × g for 10 minutes without thermal inactivation. A 96-well MULTI-ARRAY Standard plate (MSD, catalog number L15XA-3) was coated with 2 μg / mL mouse anti-huIgG capture antibody (IgG2amm-1015-6A05; Genmab, batch number 3093-008-EP) in PBS for 16–24 hours at 2–8°C. The plate was washed with PBS-T (PBS-Lonza supplemented with 0.05% Tween 20 - Sigma, catalog number P1379, catalog number BE17-156Q) to remove unbound antibody. Unoccupied surfaces were then blocked with 3% (w / v) BSA for 60±5 minutes using MSD Blocker A in PBS-T, followed by washing with PBS-T. Calibration samples for creating reference curves were prepared by spiking a dilution series of IgG1-CD134-003-HC6LC2-RR, IgG1-CD134-003-FEAL, or IgG1-CD134-003-RR-K409R (final antibody concentration range of 0.156 to 20 μg / mL) into 2% Pooled Mouse Plasma K2EDTA (BIOIVT, catalog number MSE00PLK2PNN) in assay buffer.Quality control samples used to validate individual assay runs were prepared by spiking undiluted Pooled Mouse Plasma K2EDTA with three antibody concentrations covering the range of the reference curve: 0.5 μg / mL (low-concentration quality control; LQC), 1.75 μg / mL (medium-concentration quality control; MQC), and 15 μg / mL (high-concentration quality control; HQC).

[0408] Next, the coated plates were incubated for 90 ± 5 minutes at RT with 50 μL of mouse plasma samples, calibration samples, and quality control samples (all single samples) diluted in assay buffer (PBS-T [0.05% Tween 20 - Sigma, catalog number P1379 supplemented with PBS-Lonza, catalog number BE17-156Q], 1% [w / v] MSD Blocker A [Meso Scale Discovery-MSD-, catalog number R93AA-1]), calibration samples, and quality control samples. After washing with PBS-T, the plates were incubated with SULFO-TAG conjugate anti-huIgG detection antibody (IgG2amm-1015-4A01-ST; Genmab, batch number 210329_PSM_0053#001; IgG2amm-1015-4A01 antibody labeled with MSD GOLD SULFO-TAG NHS-Ester [MSD, catalog number R91AO-1]) for 90±5 minutes at RT. After washing with PBS-T, electrochemiluminescence (ECL) signals were generated by adding tripropylamine (TPA)-containing Read Buffer (MSD GOLD Read Buffer, catalog number R92TG-2) to electrochemically stimulate the SULFO-TAG on the antibody immobilized on the electrode surface of the plate. The ECL signal was measured at 620 nm using a MESO Sector S 600 plate reader (MSD), and the data was processed using SoftMax® Pro GxP Software (Molecular Devices).

[0409] The predicted IgG concentration-time curve for WT huIgG in mice in the absence of target binding was defined based on a two-compartment PK model of a WT huIgG antibody with linear clearance described in the literature (Bleeker WK, et al. 2001. Accelerated autoantibody clearance by intravenous immunoglobulin therapy: studies in experimental models to determine the magnitude and time course of the effect. Blood 98: 3136-3142). This model adequately explained the PK profiles observed in previous in-house studies with other (unbound) huIgG molecules in mice.

[0410] PK parameters were derived using the Phoenix WinNonlin software (Certara) via a non-compartmental method for IV (or extravascular, if applicable) drug administration. The following parameters were calculated: ·C max - Maximum observed antibody concentration (μg / mL), defined as the highest antibody concentration observed in a blood sample after antibody injection. ·t max - C max The time of sample collection (h) when the phenomenon was observed. ·t 1 / 2 - Terminal disappearance half-life (h), determined by linear regression of at least three data points in the terminal phase of the log(concentration) vs. time plot (d). AUC inf - Area under the plasma concentration-time curve (h*μg / mL) from time zero (t=0) to infinity. 最後 The area between =21 d) was calculated using the linearly increasing-logarithmic linear decreasing trapezoidal method. 最後 The area from infinity to zero was calculated using the estimated efflux rate (λz) between 3 and 21 days after antibody injection. ·CL - Dose / AUC infThis is calculated as whole-body plasma clearance (mL / h / kg). For mice with an extravascular PK profile, the calculated CL is implicitly normalized by apparent bioavailability (F).

[0411] The PK parameters were summarized by median and range. max Except for one exception, the data were summarized by mean ± standard deviation (SD) per treatment group. The results were visualized using GraphPad Prism.

[0412] IgG1-CD134-003-HC6LC2-RR exhibited consistent and typical PK characteristics with non-target-binding human IgG antibodies administered intravenously in mice, with maximum exposure at the earliest tested post-injection time point, followed by a decrease in phase 2 (Figure 37). IgG1-CD134-003-HC6LC2-RR had slightly lower total CL and slightly longer t than the two chimeric OX40-targeting control antibodies. 1 / 2 This was shown. C max The results were similar for all three antibodies across the tested dose range (Figure 38, Table 20).

[0413] (Table 20) Antibody PK parameters per treatment group. Unless otherwise specified, CL and t of the antibody were measured for 3 mice per treatment group. 1 / 2 AUC inf , and C max Mean ± SD, and antibody t max This shows the median with a range. TIFF2026516142000028.tif89166 a The mean and standard deviation are based on samples from two mice.

[0414] Combined, these data demonstrate that the PK properties of IgG1-CD134-003-HC6LC2-RR are comparable to those of WT huIgG in the absence of target binding.

[0415] Example 25: In vivo antitumor efficacy of IgG1-CD134-003-HC6LC2-RR As demonstrated in Example 5, due to the lack of binding to mouse OX40 (mOX40), the antitumor activity of IgG1-CD134-003-HC6LC2-RR was evaluated in immunocompetent C57BL / 6 mice (hOX40 knock-in [KI] mice) genetically engineered to express the extracellular domain of human OX40 together with the intracellular domain of mOX40. The in vivo antitumor activity of IgG1-CD134-003-HC6LC2-RR was evaluated in hOX40 KI mice inoculated with syngeneic MC38 mouse colon cancer cells. In addition, pharmacodynamic changes in response to treatment with IgG1-CD134-003-HC6LC2-RR were examined in the peripheral blood of MC38-carrying hOX40 KI mice.

[0416] hOX40 KI mouse (C57BL / 6-Tnfrsf4 strain) tm1(TNFRSF4) Bcgen (catalog number 110014) was obtained from Beijing Biocytogen Co., Ltd., which features a humanized drug target (i.e., OX40) in immunocompetent mice. Mice were bred at Biocytogen Co., Ltd. and transferred to Crown Bioscience, Inc. All animal experiments were conducted at Crown Bioscience, Inc. and approved prior to execution by its Institutional Animal Care and Use Committee (IACUC). Animals were housed and handled in accordance with good animal practices as stipulated by the regulations of the International Association for Accreditation and Evaluation of Laboratory Animal Care (AAALAC). All experimental data control and reporting procedures strictly followed applicable Crown Bioscience, Inc. guidelines and standard operating procedures. 1 × 10⁶ cells were placed in the lower ventral region of 8-9 week old female hOX40 KI mice. 6 Individual MC38 cells (FuDan IBS Cell Center, catalog number 1101MOU-PUMC000523, 100 μL in PBS) were injected subcutaneously (SC). Tumor growth was evaluated three times a week using calipers. Tumor volume (mm) 3The length is calculated from the caliper measurement as ([length] × [width] × [width]) / 2, where the length is the longest tumor dimension and the width is the longest tumor dimension perpendicular to that length.

[0417] The study consisted of an antitumor activity test and a pharmacodynamic (PD) test. Mouse randomization was performed based on tumor volume using the Matched Distribution method (StudyDirector® software). From all 84 animals inoculated with MC38 cells, the initial sample size was 58.2 mm. 3 Forty mice with an average tumor volume of 63.6 mm were enrolled in four groups of 10 mice / group, and their antitumor activity was evaluated (Table 21). In addition, the remaining 44 animals were randomized and evaluated. 3 Sixteen of these mice, with an average tumor volume, were enrolled in four groups of four mice / group for PD analysis (referred to as PD Part 1) (Table 22). Due to clot formation in some tubes during blood collection from these 16 animals (PD Part 1), it was decided to include 16 additional mice as Part 2 of the study (PD Part 2). For this purpose, a third randomization was performed on the remaining 28 mice at 7 days, with a tumor volume of 146.81 mm. 3 This resulted in four groups of four mice, each with an average tumor volume.

[0418] (Table 21) Treatment group and dose level (antitumor activity part) of hOX40 KI mice TIFF2026516142000029.tif63160

[0419] (Table 22) Treatment group and dose level of hOX40 KI mice (PD Parts 1 and 2) TIFF2026516142000030.tif108160

[0420] Treatment was initiated on the day of randomization (day 0). Mice were administered via IP injection of IgG1-CD134-003-HC6LC2-RR (1, 5, or 20 mg / kg) or IgG1-b12-FEAL control antibody (20 mg / kg). The dose volume was adjusted relative to body weight for each mouse (10 μL / g in PBS). Tumor growth was assessed three times per week after the start of administration. To evaluate antitumor activity over time, animals were administered twice per week for three weeks (2QW × 3). To evaluate pharmacodynamics in PD parts 1 and 2, animals were administered twice on days 0 and 3.

[0421] Tumor growth inhibition (TGI) is determined based on the difference between the average tumor volume on day 0 (start of administration) and the average tumor volume on the final day when all groups have completed the treatment. This difference is calculated for each treatment group using the following formula: TIFF2026516142000031.tif17128 Here, Tt = mean tumor volume of the treatment group on the last day all groups completed, Ct = mean tumor volume of the control group on the last day all groups completed, T0 = mean tumor volume of the treatment group on day 0, and C0 = mean tumor volume of the control group on day 0. The classification of TGI responses for this trial was categorized as detailed in Table X.

[0422] (Table 23) Classification of tumor growth inhibition TIFF2026516142000032.tif27160

[0423] The experiment involved individual mice with a tumor volume of 1,500 mm³. 3 Treatment was terminated when the following conditions were met, or when the animal reached another humane endpoint (e.g., more than 20% weight loss compared to the first day of administration [i.e., day 0], tumor ulceration with an ulcer diameter greater than 5 mm, or observation of severe clinical signs). Any animal exhibiting ulcerated or necrotic tumors was immediately isolated and housed separately.

[0424] For the analysis of progression-free survival, 500 mm 3 A tumor volume cutoff of 500 mm was applied.3 The time points of tumor progression were calculated in GraphPad Prism using nonlinear regression curve fitting of individual tumor growth curves, assuming an exponential growth equation, and plotted as Kaplan-Meier curves. Progression-free survival was compared between the treatment group and the control group using nonparametric log-rank Mantel-Cox analysis and subsequent pairwise comparisons using SPSS software.

[0425] To evaluate the pharmacodynamic effects of IgG1-CD134-003-HC6LC2-RR treatment, mice were euthanized after two doses and CD4 + T cells and CD8 + Changes in the peripheral blood immune cell population, including T cell proliferation and activation, were analyzed on day 5. Approximately 50–100 μL of whole blood was collected via cardiac puncture under isoflurane anesthesia on day 5 of the experiment and transferred to K2-EDTA tubes (BD, catalog no. 36597499). For cell surface staining, whole blood samples (both PD Part 1 [9 animals] and PD Part 2 [14 animals]; whole blood samples with blood clots were excluded from analysis; 50–100 μL) were incubated with 1 μg / mL Mouse BD Fc Block (trademark) (catalog no. 553141; diluted in PBS + 0.09% sodium azide) in the dark at 4°C for 10 minutes. Cells were then stained with either an antibody panel for general immunophenotyping (Table 24) or an antibody panel for T cell characterization (Table 25).

[0426] For general immunophenotyping, each sample was stained at 4°C for 30 minutes with the antibody mixture detailed in Table 24, excluding the anti-FOXP3 antibody, diluted in Fc blocking buffer. Cells were lysed by adding 2 mL of room temperature erythrocyte lysis buffer (1×; BioGems, catalog no. 64010-00100) to each tube, incubated in the dark at RT for 10 minutes, and gently vortexed. Samples were washed twice with PBS by centrifugation at 300×g for 5 minutes and discarding the supernatant. After resuspending the cell pellet, 200 μL of fixation / permeabilization buffer (eBioscience, catalog no. 00-5523) was added, mixed by pulse vortexing, and incubated in the dark at RT for 30 minutes. Cells were washed twice with 1× permeabilization buffer (eBioscience, catalog no. 00-5523; diluted in distilled H2O), followed by centrifugation and decantation of the supernatant. FOXP3 antibody was added to each sample in 100 μL of permeabilization buffer and incubated in the dark at RT for 30 minutes. Cells were washed twice with 2 mL of PBS, resuspended in 250 μL of PBS, and analyzed using a BD LSRFortessa® X-20 cell analyzer (BD Biosciences). Data were analyzed using Kaluza Analysis Software. Absolute counts were calculated using 123 count eBeads (ThermoFisher Scientific, catalog number 01-1234-42). 100 μL of beads were added to each sample to be counted. The following equation was used to calculate the absolute count. TIFF2026516142000033.tif10128

[0427] For T cell characterization, ADPGK tetramers were added to each sample after blocking the cells, gently vortexed, and incubated in the dark at 4°C for 30 minutes. Next, the antibody mixture, detailed in Table 25 except for Ki67, diluted in Fc blocking buffer, was added to each sample and stained at 4°C for 30 minutes. The cells were lysed and washed as described above. After resuspending the cell pellet, the cells were intracellularly stained for Ki67 according to the method described above. The cells were washed twice with 2 mL of PBS, resuspended in 250 μL of PBS, and analyzed using a BD LSRFortessa X-20 cell analyzer (BD Biosciences). The data were analyzed using Kaluza Analysis Software. Absolute counts were calculated using 123 count eBeads as described above.

[0428] (Table 24) Flow cytometry panel for immunophenotyping TIFF2026516142000034.tif132156

[0429] (Table 25) Flow cytometry panel for T cell characterization TIFF2026516142000035.tif135161

[0430] Plasma samples were obtained from mice in PD Parts 1 and 2 on days 0, 2, and 5. 50 μL of blood per mouse was collected via the mandibular vein (days 0 and 2) or via cardiac puncture on day 5 and collected in EDTA anticoagulant tubes (BD, catalog number 365974). The tubes were thoroughly mixed to ensure complete contact of the blood sample with the anticoagulant, and then centrifuged (8000 RPM, 4°C for 5 minutes). After centrifugation, the supernatant was transferred to a 1.5 mL Eppendorf tube.

[0431] Levels of mouse cytokines (IFNγ, IL-2, IL-4, IL-10, TNFα, IP-10, MCP-1, IL-27p28) in plasma samples were measured using a MESO QuickPlex SQ 120 instrument (MSD, LLC., catalog number AI0AA-0) with the V-PLEX Proinflammatory Panel 1 Mouse kit (MSD LLC, catalog number K15048D-2) according to the manufacturer's instructions, in a multiplex ECLIA (Electron-Clinical Lithography) configuration. Cytokine concentrations were calculated using standard curve data fitted with a 4-parameter logistic (4PL) fit using MSD Workbench software and plotted using GraphPad Prism software.

[0432] The treatment was well tolerable, and no reports of clinical signs of disease or observation of weight loss in response to antibody treatment were observed in any of the mice in these studies. Treatment with 5 mg / kg IgG1-CD134-003-HC6LC2-RR significantly delayed tumor growth compared to IgG1-b12-FEAL treatment (Mann-Whitney, P=0.0011) when measured 14 days after the start of treatment, which was the last day all treatment groups had completed, resulting in an intermediate response based on TGI (50.0%; Figure 39A, B; Table 26). Treatment with low (1 mg / kg) and high (20 mg / kg) doses of IgG1-CD134-003-HC6LC2-RR did not result in significantly smaller tumor volume compared to the control group at day 14 (Mann-Whitney, P=0.3527 and P=0.0753, respectively). Progression-free survival was significantly longer in IgG1-CD134-003-HC6LC2-RR treated mice (5 mg / kg or 20 mg / kg) compared to the IgG1-b12-FEAL treated control group (Mantel-Cox, P<0.001 and P=0.031, respectively; Figure 39C and Table 27).

[0433] (Table 26) Mann-Whitney analysis of tumor volume in hOX40 KI mice at treatment. Tumor volume in the IgG1-CD134-003-HC6LC2-RR treatment group was compared to that of the IgG1-b12-FEAL control group using Mann-Whitney analysis on the last day (day 14) after all groups had completed treatment. The TGI value per treatment group was calculated based on the tumor volume on day 14. **=P<0.01. TIFF2026516142000036.tif54160

[0434] (Table 27) Mantel-Cox analysis of progression-free survival in hOX40 KI mice at the time of treatment. Progression-free survival (500 mm) 3 Based on tumor volume cutoffs, the overall differences in survival between groups were analyzed by Mantel-Cox analysis, followed by pairwise comparisons between groups (SPSS). *=P<0.05, and ***=P<0.001. TIFF2026516142000037.tif85160

[0435] Tumor-specific CD8 after treatment + The percentage of T cells was measured using MHC class I ADPGK tetramers.

[0436] Compared to mice treated with 20 mg / kg of IgG1-b12-FEAL, mice treated with 1, 5, and 20 mg / kg of IgG1-CD134-003-HC6LC2-RR showed a lower CD4 count. + A significant increase in the percentage of T cells was observed in the peripheral blood of MC38 tumor-carrying hOX40 KI mice (Figure 40A). CD4 + The absolute number of T cells did not change, which is probably because of CD4 + The increased frequency of cells is CD8 + This may be due to a decrease in T cells (Figure 40B, D). Treatment of hOX40 KI mice with IgG1-CD134-003-HC6LC2-RR resulted in the expression of the proliferation marker Ki67, as well as the activation markers CD25, IA / IE, PD-1, and 4-1BB in CD4. +Peripheral blood CD4, as indicated by a significant increase in the percentage of T cells. + This induced T cell proliferation and activation (Figure 41A-E). Furthermore, IgG1-CD134-003-HC6LC2-RR treatment, compared to IgG1-b12-FEAL treatment, resulted in CD4 cells expressing OX40, likely due to OX40 shedding. + This resulted in a significantly lower percentage of T cells (data not shown). CD4 + All observed effects on T cells were dose-independent, with the exception of the upregulation of 4-1BB, which showed dose-dependent effects (Figure 41E).

[0437] During treatment with IgG1-CD134-003-HC6LC2-RR at 1, 5, and 20 mg / kg, CD8 + A decrease in the percentage and absolute number of T cells is associated with the proliferation of CD8 cells in peripheral blood. + Ki67 + Increased percentage of T cells (1, 5, and 20 mg / kg), and tumor-specific (ADPGK) + )CD8 + This was observed along with increases in the percentage and absolute number of T cells (1 and 5 mg / kg; Figures 40C, D, and 42).

[0438] IgG1-CD134-003-HC6LC2-RR treatment targets CD8 expressing CD25 (all IgG1-CD134-003-HC6LC2-RR doses), IA / IE (1 and 5 mg / kg), PD-1 (all IgG1-CD134-003-HC6LC2-RR doses), and 4-1BB (5 and 20 mg / kg; Figure 43). + CD8 in peripheral blood, as detected by a significant increase in the percentage of T cells + T cell activation was induced. Mice treated with 1 or 20 mg / kg of IgG1-CD134-003-HC6LC2-RR showed increased CD8 in peripheral blood compared to IgG1-b12-FEAL control mice, potentially due to OX40 shedding. + OX40 +It showed a significant reduction in T cells (data not shown).

[0439] The concentrations of IFNγ, IP-10, IL-2, IL-4, IL-10, MCP-1, IL-27p28, and TNFα were measured by ECLIA in plasma samples collected from mice on day 0 (pre-treatment) and on days 2 and 5 (two days after the first and second treatments with IgG1-CD134-003-HC6LC2-RR or IgG1-b12-FEAL). Increased plasma concentrations of IFNγ, IP-10, IL-2, IL-4, IL-10, MCP-1, IL-27p28, and TNFα were observed on day 5 after treatment with IgG1-CD134-003-HC6LC2-RR compared to treatment with IgG1-B12-FEAL (Figure 44). No clear dose-response relationships were observed in these cytokine secretion profiles.

[0440] In conclusion, 5 mg / kg of IgG1-CD134-003-HC6LC2-RR exhibited significant antitumor activity in MC38 tumor-carrying hOX40 KI mice compared to an unbound control antibody. In addition, treatment with IgG1-CD134-003-HC6LC2-RR increased the expression of several activation markers and tumor-specific CD8 + CD8 in peripheral blood, as demonstrated by the relative increase in T cells + T cells and CD4 + This resulted in T cell activation. Finally, increased concentrations of IFNγ, IP-10, IL-2, IL-4, IL-10, MCP-1, IL-27p28, and TNFα were measured in mouse plasma samples after treatment with IgG1-CD134-003-HC6LC2-RR.

[0441] Example 26: Quantitative immunohistochemical analysis of tumor tissue from mice treated with IgG1-CD134-003-HC6LC2-RR As described in Example 25, IgG1-CD134-003-HC6LC2-RR exhibited antitumor activity in MC38 tumor-bearing hOX40 KI mice compared to an unbound control antibody. Here, quantitative immunohistochemistry (IHC) was performed to analyze the presence of T cell populations, T cell phenotype, and tumor growth index in tumor tissue from MC38 tumor-bearing hOX40 KI mice treated with 1, 5, and 20 mg / kg of IgG1-CD134-003-HC6LC2-RR or 20 mg / kg of IgG1-b12-FEAL as a control.

[0442] Tumors derived from hOX40 KI mice treated as described in Example 25 were collected 5 days after treatment, dissected, fixed in formalin, and sectioned (4 μm) using paraffin embedding. For histological evaluation, tumor sections were deparaffinized and stained with Tissue-Tek Prisma H&E Stain Kit (Sakura, catalog no. 6190) using a Tissue-Tek Prisma Plus Automated Slide Stainer (Sakura). To evaluate marker-positive cells within the tumor, sections were deparaffinized, antigens were activated with CC1 buffer (Roche, catalog no. 950-124), followed by quenching of endogenous peroxidase (Dako Agilent, catalog no. S2003) and blocking of nonspecific binding sites with blocking buffer (Roche, catalog no. 05268869001) using the Roche Ventana Discovery (DISC) automated staining platform. Sections were incubated with primary antibodies (Table 28), and these were detected using an anti-rabbit immunohistochemistry detection kit. CD4 and granzyme B were detected using only anti-rabbit DISC Omnimap (Roche, catalog number 05269679001), while CD8 and OX40 were detected by sequential amplification using DISC anti-rabbit HQ (Roche, catalog number 07017812001) and anti-HQ HRP Multimer (Roche, catalog number 06442544001). HRP was visualized using 3,3'-diaminobenzidine (ChromoMap DAB; Roche, catalog number 05266645001) according to the manufacturer's instructions.

[0443] (Table 28) Primary antibodies used for quantitative immunohistochemical analysis. N / A: Not applicable; conc: Concentration; hOX40: Human OX40; RTU: Ready to use (pre-diluted by the supplier). TIFF2026516142000038.tif67160

[0444] In all assays, the nuclei were counterstained by incubation with Meyer hematoxylin. Staining specificity was adjusted by incorporating isotype, positive and negative control stains into sequential tissue sections. Stained slides were subjected to full slide imaging using AxioScan Z1 (Zeiss).

[0445] Multiplex immunofluorescence assays were performed to co-stain CD3 (RTU assay, clone 2GV6, catalog no. 790-4341, Roche), Ki-67 (RTU assay, clone 30-9, Roche, catalog no. 790-4286), and pancytokeratin (polyclonal, 50-fold dilution, ThermoFisher, catalog no. PA1-27114). Standard thickness (4 μm) IHC tissue sections were subjected to a standard protocol for deparaffinization and antigen retrieval (Tris-EDTA buffer pH 7.8). For multiplex immunofluorescence, fluorescent-conjugated secondary antibodies were used (CD3 was labeled with FAM [Discovery-FAM kit, Roche, catalog number 7988150001], Ki-67 with Rhodamine 6G [Discovery-Rhodamine 6G kit, Roche, catalog number 7988168001], and pancytokeratin with Cy5 [Discovery-Cy5 kit, Roche, catalog number 7551215001]). Staining specificity was adjusted by incorporating isotype control stains into consecutive tissue sections. Tissues were counterstained with DAPI. Sections were mounted using ProLong Gold Antifade Mountant (ThermoFisher, catalog number P10144). All tissue slides were scanned with AxioScan Z1 (Zeiss) using a standardized scan profile.

[0446] Digital images were analyzed using HALO software (Indica Labs) with pre-programmed software analysis algorithms (CytoNu...

Claims

1. a. An antigen-binding region comprising a heavy chain variable (VH) region in which CDR1, CDR2, and CDR3 contain sequences as shown in SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable (VL) region in which CDR1, CDR2, and CDR3 contain sequences as shown in SEQ ID NO: 16, DAS, and SEQ ID NO: 17, respectively, and b. Human IgG1 Fc regions containing P329R and E345R mutations, wherein the amino acid positions are numbered according to Eu numbering. An antibody that can bind to human OX40, including [specific component].

2. The antibody according to claim 1, which can bind to human OX40 having sequence SEQ ID NO:

52.

3. The antibody according to any one of the claims, which can bind to the CRD1 domain of human OX40.

4. 3 x 10 -9 M~4×10 -9 M's binding affinity K to human OX40 D The antibody according to any one of the above claims, having the following characteristics.

5. 3.4 × 10 -9 M's binding affinity K to human OX40 D The antibody according to any one of the above claims, having the following characteristics.

6. The antibody according to any one of the claims, which can bind to cynomolgus monkey OX40 having sequence SEQ ID NO:

51.

7. The antibody according to any one of the claims, which is a humanized antibody or a chimeric antibody.

8. The antibody according to any one of the claims, comprising a VH region having the sequence shown in SEQ ID NO: 20 and a VL region having the sequence shown in SEQ ID NO:

21.

9. The antibody according to any one of the claims, wherein the Fc region is a human IgG1mf, human IgG1ma, human IgG1mx, or human IgG1mz allotype.

10. The antibody according to any one of the claims, wherein the Fc region is a human IgG1mf allotype.

11. The antibody according to any one of the claims, wherein the Fc region comprises the sequence shown in SEQ ID NO:

3.

12. The antibody according to any one of the above claims, which is bivalent.

13. The antibody according to any one of the claims, which is a single-specificity antibody, a bispecificity antibody, or a multispecificity antibody.

14. The antibody according to any one of the above claims, which is a full-length antibody.

15. The antibody according to any one of the claims, having a heavy chain constant region comprising a sequence selected from the group including SEQ ID NO: 58, 59, 60, and 61.

16. The antibody according to any one of the claims, having a heavy chain constant region including the sequence shown in SEQ ID NO:

58.

17. The antibody according to any one of the claims, comprising a heavy chain (HC) as shown in SEQ ID NO: 18 and a light chain (LC) as shown in SEQ ID NO:

19.

18. The antibody according to any one of the claims, which is an agonist.

19. The antibody according to any one of the claims, having increased agonist activity compared to a wild-type parent antibody that does not have the P329R mutation and the E345R mutation.

20. An antibody according to any one of the claims, which induces increased T cell proliferation.

21. The antibody according to any one of the claims, which induces increased T cell proliferation compared to a parent antibody that does not have the P329R mutation and the E345R mutation.

22. When assayed as described in Example 7 of this specification, human T cells, for example, CD4 + T cells and CD8 + The antibody according to any one of the claims, which can induce the proliferation of T cells, such as T helper cells and cytotoxic T cells.

23. Compared to a parent antibody that does not have the P329R and E345R mutations, the antibody is modified to induce one or more Fc-mediated effector functions to a lesser extent in the heavy chain constant region. The antibody according to any one of the claims, comprising:

24. The antibody according to claim 23, wherein the one or more Fc-mediated effector functions are 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%.

25. The antibody according to claim 23 or 24, which does not induce one or more Fc-mediated effector functions.

26. The one or more Fc-mediated effector functions are grouped as follows: Complement-dependent cytotoxicity (CDC), complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, and FcγR binding An antibody according to any one of claims 23 to 25, selected from the above.

27. The antibody according to any one of claims 25 or 26, which does not induce C1q binding.

28. A composition comprising an antibody as defined in any one of claims 1 to 27.

29. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

30. An antibody as defined in any one of claims 1 to 27, for use as a pharmaceutical.

31. An antibody for use as a pharmaceutical, according to claim 30, for use in the treatment of a disease.

32. The antibody for pharmaceutical use according to claim 31, wherein the disease is cancer.

33. The antibody for pharmaceutical use according to claim 32, wherein the cancer is a solid tumor or a hematological cancer.

34. A method for treating a disease, comprising the step of administering an antibody as defined in any one of claims 1 to 27, a composition as defined in claim 28, or a pharmaceutical composition as defined in claim 29 to a subject in need thereof.

35. The method according to claim 34, for the treatment of cancer.

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

37. The VH region contains VH CDR1 with the sequence shown in SEQ ID NO: 12, VH CDR2 with the sequence shown in SEQ ID NO: 13, and VH CDR3 with the sequence shown in SEQ ID NO:

14. A nucleic acid sequence that codes for [something].

38. The VL region contains VL CDR1 with the sequence shown in SEQ ID NO: 16, VL CDR2 with sequence DAS, and VL CDR3 with the sequence shown in SEQ ID NO:

17. A nucleic acid sequence that codes for [something].

39. A nucleic acid sequence according to any one of claims 36 and 37, encoding a VH region having the amino acid sequence shown in SEQ ID NO:

20.

40. A nucleic acid sequence according to any one of claims 36 and 38, encoding a VL region having the amino acid sequence shown in SEQ ID NO:

21.

41. A nucleic acid sequence encoding the heavy chain of an antibody according to any one of claims 1 to 27.

42. A nucleic acid sequence encoding the light chain of an antibody according to any one of claims 1 to 27.

43. The nucleic acid sequence according to any one of claims 37, 39, and 41, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

18.

44. The nucleic acid sequence according to any one of claims 38, 40, and 42, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

19.

45. The nucleic acid sequence or combination of nucleic acid sequences according to any one of claims 36 to 44, wherein the nucleic acid is DNA or RNA, for example, mRNA.

46. An isolated nucleic acid sequence, as described in any one of claims 37 to 45.

47. An expression vector comprising a nucleic acid sequence or combination thereof according to any one of claims 36 to 46.

48. A nucleic acid sequence or combination of nucleic acid sequences according to any one of claims 37 to 47, for use in expression in mammalian cells.

49. Recombinant host cells that produce antibodies as defined in any one of claims 1 to 27, and optionally include the expression vector described in claim 47.

50. The recombinant host cell according to claim 49, which is a eukaryotic cell or a prokaryotic cell.

51. A pharmaceutical composition comprising a nucleic acid sequence according to any one of claims 37 to 46 or an expression vector as defined in claim 47, and a pharmaceutically acceptable carrier.

52. A method for producing an antibody according to any one of claims 1 to 27, comprising the steps of culturing recombinant host cells according to claim 49 or 50 in a culture medium under conditions suitable for producing the antibody, and optionally purifying or isolating the antibody from the culture medium.

53. A kit of parts, such as a kit for use as a companion diagnostic / for identifying patients in a patient population who are likely to respond to treatment with an antibody as defined in any one of claims 1 to 27, the kit of parts comprising an antibody as defined in any one of claims 1 to 27 and instructions for using the kit.

54. An antigen-binding region capable of binding to OX40, as defined in any one of claims 1 to 27. An anti-idiotype antibody that binds to [a substance].

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